Fire Sessions 13 Posters 22

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2 INDEX 2

3 WELCOME 4 ORGANIZATION 5 JANSSEN PRIZE 6 TRAVEL GRANTS 7 SPONSORS 8 PROGRAM 9 Fire Sessions 13 Posters 22 PLENARY LECTURES 29 ORAL COMMUNICATIONS 36 Symposium I Neurodevelpment 37 Symposium II Communication in the nervous system 44 Symposium III Cognition and behaviour 51 Symposium IV New targets for nervous system therapy 58 FIRE SESSIONS I 65 Neurogenesis and neural cell differentiation 66 Cell signaling I 79 Neurodegeneration and neuroprotection I 92 FIRE SESSIONS II 105 Neural systems I 106 Cell signaling II 119 Neurodegeneration and neuroprotection II 132 FIRE SESSIONS III 145 Neural systems II 146 Neuroinflammation 159 Neurodegeneration and neuroprotection III 172 POSTERS 185 Neurodegeneration and neuroprotection 186 Cell signaling 224 Neurogenesis and neural cell differentiation 247 Neuroinflammation 254 Neural systems 259 3

4 Caros Sócios da SPN, Sejam bem-vindos à XIII Reunião da Sociedade Portuguesa. A Comissão Organizadora, a quem desde já agradeço, preparou um excelente programa de trabalhos, que seguramente estimulará uma profícua troca de ideias entre todos. E todos, desta feita, serão cerca de 300 delegados, com mais de 80 posters e de 100 fire-talks com posters. A reforçar esta participação, é com enorme alegria que vos informo do aumento exponencial do número de sócios, o que bem demonstra a dinâmica da nossa comunidade de neurocientistas. Estão, portanto, reunidas as condições para uma ótima reunião. Aproveitem e Divirtam-se! Abraço Nuno Sousa 4

5 Organizing Committee: Ana Luísa Carvalho (CNC, University of Coimbra) Ana Paula Silva (IBILI, University of Coimbra) Ana Raquel Santiago (IBILI, University of Coimbra) A. Francisco Ambrósio (IBILI, University of Coimbra) Cláudia MF Pereira (CNC, University of Coimbra) Dan Brudzewsky (IBILI, University of Coimbra) Elisabete Ferreiro (CNC, University of Coimbra) João Malva (University of Coimbra, Coimbra) Jorge Valero (CNC, University of Coimbra) Liliana Bernardino (HealthSciencesResearchCenter, University of Beira Interior) Maria H. Madeira (IBILI, University of Coimbra) Paula Moreira (CNC, University of Coimbra) Sandra M Cardoso (CNC, University of Coimbra) Advisory Board: Alexandre Ribeiro (University of Lisbon) António Freire (University of Coimbra) Arsélio Pato de Carvalho (University of Coimbra) Catarina Oliveira (University of Coimbra) Deolinda Lima (University of Porto) Domingos Henrique (University of Lisbon) Isabel Santana (University of Coimbra) José Castro Lopes (University of Lisbon) Zachary Mainen (Champalimaud Foundation, Lisbon) Scientific Committee: Alexandre Castro Caldas (University of Lisbon) Ana Cristina Rego (University of Coimbra) Ana Sebastião (University of Lisbon) Carlos Jorge Duarte (University of Coimbra) Cecília Rodrigues (University of Lisbon) Graça Baltazar (University of Beira Interior) Inês Araújo (University of Algarve) Isaura Tavares (University of Porto) João Laranjinha (University of Coimbra) João Relvas (University of Porto) Luís Almeida (University of Coimbra) Miguel Castelo-Branco (University of Coimbra) Mónica Sousa (University of Porto) Nuno Sousa (University of Minho) Odete Cruz e Silva (University of Aveiro) Patrícia Maciel (University of Minho) Rodrigo Cunha (University of Coimbra) Rui Costa (Champalimaud Foundation, Lisbon) Tiago Outeiro (University of Lisbon) 5

6 JANSSEN PRIZE 6

7 TRAVEL GRANTS Ana M Oliveira (University of Heidelberg, Germany) Ana Saavedra (University of Barcelona, Spain) Bruno Godinho (University College Cork, Ireland) Mónica Santos (Center for Genomic Regulation, Barcelona, Spain) Raquel Pinho (University Medical Center Göttingen, Germany) Rita Teodoro (Harvard Medical School, Boston, USA) Rui Peixoto (Harvard Medical School, Boston, USA) Susana da Silva (Harvard Medical School, Boston, USA) 7

8 SPONSORS 8

9 PROGRAM 9

10 May 30 13h45 Registration 14h45 15h00 Opening Session 15h00 16h00 Plenary Lecture I Chairperson: Catarina Resende Oliveira PL1 Miguel Castelo Branco (IBILI, Coimbra, PORTUGAL) Systems Neurobiology: New Trends in Translational Neuroscience 16h00 18h30 Symposium I Neurodevelopment Chairpersons: João Relvas and Patrícia Maciel 16h00 SI1 Susana da Silva (Harvard Medical School, Boston, USA) Searching the source of topography in the somatosensory system. Role of periphery TGF β signaling in barrelette development 16h30 SI2 Pedro Brites (IBMC, Porto, PORTUGAL) Ether phospholipid functions on Schwann cell differentiation and myelination 17h00 SI3 Domingos Henrique (IMM, Lisbon, PORTUGAL) Understanding cell fate decisions in the embryonic neural retina 17h30 SI4 Márcia Almeida Liz (IBMC, Porto, PORTUGAL) Neuronal deletion of GSK3β increases microtubule dynamics in the growth cone enabling axonal regeneration 18h00 SI5 Joana Paes de Faria (IBMC, Porto, PORTUGAL) Profilin1 regulates PNS myelination 18h15 SI6 Joana M. Marques (Instituto de Neurociencias de Alicante, SPAIN) CRMP2 tethers kainate receptor activity to cytoskeleton dynamics during development 18h30 18h45 Coffee Break 18h45 19h45 Fire Session I Neurogenesis and neural cell differentiation / Cell signaling I / Neurodegeneration and neuroprotection I 19h45 20h05 Prémio Janssen Neurociências Reception 10

11 May 31 09h00 11h30 Symposium II Communication in the nervous system Chairpersons: Ana Sebastião and Carlos Duarte 09h00 SII1 Ana Saavedra (University of Barcelona, SPAIN) BDNF regulates striatal enriched protein tyrosine phosphatase levels in cortical neurons 09h25 SII2 Rita Teodoro (Harvard Medical School, Boston, USA) Ral mediates activity dependent growth of postsynaptic membranes via recruitment of the exocyst 09h50 SII3 Rui Peixoto (Harvard Medical School, Boston, USA) Trans synaptic signaling by cleavage of Neuroligin 1 10h15 SII4 Ana Luísa Carvalho (CNC, Coimbra, PORTUGAL) Modulation of hippocampal glutamatergic synapses by ghrelin 10h45 SII5 Luísa V. Lopes (IMM, Lisbon, PORTUGAL) Clues to the early aging of the hippocampus Impact on cognition 11h15 SII6 Diogo Rombo (IMM, Lisbon, PORTUGAL) Adenosine A2A receptors mediate hippocampal network disinhibition specifically through parvalbumin expressing interneurons 11h30 12h00 Coffee Break 12h00 13h00 Plenary Lecture II Chairperson: Ana Luísa Carvalho PL2 Daniel Choquet (Institut Interdisciplinaire de Neuroscience, Bordeaux, FRANCE) A nanoscale view into the dynamic of AMPA receptor organization in synapses 13h00 15h00 Lunch/Posters 15h00 17h30 Symposium III Cognition and behaviour Chairpersons: Alexandre Castro Caldas and Rui Costa 15h00 SIII1 Mónica Santos (Center for Genomic Regulation, Barcelona, SPAIN) Hippocampal hyperexcitability and impaired ERK signaling underlie enhanced and extinction resistant fear memories in a mouse overexpressing TrkC, a model of panic disorder 15h30 SIII2 Ana M. Oliveira (University of Heidelberg, GERMANY) Rescue of aging associated decline in Dnmt3a2 expression restores cognitive abilities 16h00 SIII3 Tiago V. Maia (University of Lisbon, PORTUGAL) Reinforcement learning in avoidance, habits, and tics 16h30 SIII4 Marta Moita (Champalimaud Foundation, Lisbon, PORTUGAL) Listening to the sound of silence 17h00 SIII5 Henrique Sequeira (University of Lille, FRANCE) Affective brain networks: a neurodynamic approach 17h15 SIII6 Jorge Valero (CNC, Coimbra, PORTUGAL) Long term effects of an acute administration of lipopolysaccharide on spatial memory and adult neurogenesis in the triple transgenic mouse model of Alzheimer's disease 17h30 18h00 Coffee Break 18h00 18h30 18h30 19h00 19h00 20h00 20h00 Dinner Prémios Artigo Destaque SPN_2012 & SPN_2013 Sponsored by Bayer Horizon 2020 and Neuroscience Research FCT SPN General Assembly 11

12 June 1 09h00 10h00 Plenary Lecture III Chairperson: Nuno Sousa PL3 Óscar Marín (Instituto de Neurociencias de Alicante, SPAIN) Neurodevelopment and Nervous System Diseases: Molecular mechanisms controlling the development of cortical interneurons 10h00 11h00 Fire Session II Neural systems I / Cell signaling II / Neurodegeneration and neuroprotection II 11h00 11h30 Coffee Break 11h30 12h30 Fire Session III Neural systems II / Neuroinflammation/ 12h30 14h30 Neurodegeneration and neuroprotection III Lunch/Posters 14h30 17h00 Symposium IV New targets for nervous system therapy Sponsored by the Portuguese Biochemical Society (SPB) Chairpersons: João Laranjinha and Tiago Outeiro 14h30 SIV1 Juan P. Bolaños (University of Salamanca, SPAIN) Bioenergetics and Antioxidant Coupling in the Nervous System 15h00 SIV2 Raquel Pinho (University Medical Center Göttingen, GERMANY) Understanding the role of alpha synuclein in the nucleus: a new concept in synucleinopathies 15h30 SIV3 Patrícia Maciel (ICVS, Braga, PORTUGAL) Pharmacotherapy for Machado Joseph disease: from worm to mouse to human? 16h00 SIV4 Ana I. Duarte (CNC, Coimbra, PORTUGAL) Insulin/IGF 1/estrogen signalling protects against lipid oxidation and neurodegenerative events in type 2 diabetic female rat brain 16h30 SIV5 Teresa Summavielle (IBMC, Porto, PORTUGAL) The role of acetyl L carnitine in the brain glucose uptake under methamphetamine 16h45 SIV6 Bruno M.D.C. Godinho (University College Cork, IRELAND) A Nanotoxicological Appraisal of Non viral vectors for RNA Interference in the CNS 17h00 18h00 Awards & Closing 12

13 Fire Sessions Fire Sessions I Neurogenesis and neural cell differentiation May 30 Bussaco room Chairperson: Jorge Valero 18:45H FIA1 Santos AI, Carreira BP, Ribeiro LF, Morte MI, Carvalho CM, Martínez-Ruiz A, Araújo IM. p21ras: a target for NO to enhance neurogenesis. 18:50H FIA2 Xavier JM, Morgado AL, Solá S, Rodrigues CMP. p53 Modulates Differentiation-induced Mitochondrial Damage and Promotes Neurogenesis. 18:55H FIA3 Morgado AL, Xavier JM, Solá S, and Rodrigues CM. mir-34a Regulates Autophagy and Neuronal Differentiation During Neural Stem Cell Differentiation. 19:00H FIA4 Barateiro A, Domingues H, Miron V, Tukey RH, Santos S, ffrench-constant C, Relvas J, Fernandes A, Brites D. Unconjugated bilirubin dysregulates oligodendrocyte development and myelinogenesis. 19:05H FIA5 Xapelli S, Agasse F, Ribeiro FF, Bernardino L, Schitine CS, Heimann A., Ferro ES, De Melo Reis RA, Sebastião AM, Malva JO. Hemopressin stimulates oligodendrogenesis in neural stem cell cultures from the subventricular zone. 19:10H FIA6 Domingues HS, Faria JP, Relvas J. The Processossome : identification and characterization of proteins promoting oligodendrocyte process outgrowth and (re)myelination. 19:15H FIA7 Falcão AS, Gomes C, Santos G, Brites D. The Dark Side of Neural Precursor Cells: New Cues for Gliomagenesis. 19:20H FIA8 Patrício P, Pinheiro A, Alves ND, Morais M, Santos AR, Bessa J, Irmler M, Beckers J, Sousa N, Pinto L. Genome-wide characterization of the adult hippocampal neurogenic niche regulators in depression and by antidepressant drugs: Cellular and molecular. 19:25H FIA9 Mateus-Pinheiro A, Patrício P, Morais M, Alves ND, Monteiro S, Almeida OFX, Bessa J, Sousa N, Pinto L. The interplay between dendritic plasticity and cell genesis in depression. 19:30H FIA10 Mendonça L, Nóbrega C, Kaspar B, Pereira de Almeida L. Neural stem cells survive, differentiate and alleviate the motor phenotype of Machado-Joseph disease transgenic mice upon cerebellar transplantation. 19:35H FIA11 Ferreiro E, Valero J, Bernardino L, Pereira C, Agasse F. Imbalance in the stem/progenitor cells properties leads to the suppression of neurogenesis in the triple transgenic mouse model of Alzheimer s disease. 19:40H FIA12 Neto E, Aguiar P, Almeida R, Lamghari M. Peripheral nervous system and bone: Advanced tools to study an essential interaction. 13

14 Cell signaling I May 30 Plátano room Chairperson: Liliana Bernardino 18:45H FIB1 Fernandes D, Ribeiro L, Santos S, Carvalho AL. Contactinassociated proteins 1 and 2 (Caspr1 and Caspr2): novel players in the activity-dependent regulation of synaptic AMPA receptors. 18:50H FIB2 Dias RB, Rombo DM, McGarvey J, Henley JM, Sebastião AM. Erythropoietin modulates activity deprivation-induced regulation of synaptic transmission: a role for a hormone in homeostatic plasticity? 18:55H FIB3 Monteiro FA, Monteiro CB, Raposo A, Clavairoly A, Rebelo S, Matos M, Reguenga C, Parras C, Guillemot F, Castro DS. Transcriptional programs regulated by Prrxl1 in developing dorsal root ganglia-dorsal spinal cord nociceptive circuit. 19:00H FIB4 Martins L, Pinto MJ, Coelho MI, Ryu HR, Jeon NL, Almeida RD.. Role of local protein synthesis in presynaptogenesis. 19:05H FIB5 Batalha VL, Hamdane M, Buée L, Blum D, Sadri-Vakili G, Lopes LV. Modulation of Glucocorticoid Receptors by adenosine A2A receptor activation: from gene to protein level. 19:10H FIB6 Conde-Sousa E, Aguiar P. Beyond synaptic plasticity - storing information in the intrinsic excitability properties of neurons. 19:15H FIB7 Tomé A, Silva CG, Fazeli AW, Métin C, Machado NJ, Darmopil S, Launay P, Ghestem A, Nesa M. Caffeine exposure during pregnancy interferes with adenosine A2A receptors affecting hippocampal GABAergic networks in offspring resulting in memory impairment in adulthood. 19:20H FIB8 Correia-de-Sá P, Noronha-Matos JB, Timóteo MA, Ferreirinha F, Marques P, Soares AM, Carvalho C, Cavalcante WLG, Gallacci M. Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals radiochemical and real-time video microscopy studies. 19:25H FIB9 Martins J, Elvas F, Brudzewsky D, Kolomiets B, Picaud S, Cavadas C, Castelo-Branco M, Santiago AR, Ambrósio AF. Neuropeptide Y modulates retinal ganglion cell activity and protects retinal ganglion cell against excitotoxicity. 19:30H FIB10 Rial D, Silva HB., Chen J, Prediger RDS, Cunha RA. The adenosine facilitation of synaptic plasticity in cortico-striatal synapses involves astrocytic A2AR receptors. 19:35H FIB11 Covita J, Rombo D, Dias RB, Sebastião AM, Diógenes MJ. Effect of BDNF on synaptic and extrasynaptic NMDA currents. 19:40H FIB12 Pedro JR, Leitão L, Almeida RD. The role of β-actin mrna translation in axonal outgrowth. 14

15 Neurodegeneration and neuroprotection I May 30 Auditorium Chairperson: Sandra M Cardoso 18:45H FIC1 Dionísio PA, Amaral JD, Lo AC, D Hooge R and Rodrigues CMP. Short-term treatment with TUDCA attenuates the amyloidogenic processing of amyloid precursor protein in APP/PS1 mice. 18:50H FIC2 Silva MF, d Almeida O, Mateus C, Castelo-Branco M. The effect of development and normal aging in cortical/retinal asymmetries: lowlevel evidence for the HAROLD aging model. 18:55H FIC3 Anjo S, Carvalho, Mendes VM, Grãos M, Manadas B. DJ-1 neuroprotection in oxidative stress: insights into Parkinson's Disease pathogenesis. 19:00H FIC4 Pousinha P, Raymond E, Duplan E, Checler F, Marie H. Is APP intracellular domain, AICD, a key player in Alzheimer s disease? 19:05H FIC5 d Almeida OC, Mateus C, Castelo-Branco M. Structural plasticity on visual cortical representations induced by silent visual loss in a subtle ganglion cell degeneration model. 19:10H FIC6 Carvalho C, Santos MS, Oliveira CR, Moreira PI. Mitochondrial dysfunction in diabetes and Alzheimer s disease: the missing link? 19:15H FIC7 Cardoso A, Silva D, Magano S, Pereira PA, Paulo J. Chronic food restriction increases the number of neuropeptide Y-containing neurons in the hippocampal dentate gyrus of aged rats. 19:20H FIC8 Jerónimo-Santos A, Vaz SH, Parreira S, Rapaz-Lérias S, Caetano AP, Buée-Scherrer V, Castrén E, Valente CA, Blum D, Sebastião AM, Diógenes MJ. Amyloid-β peptide dysregulates TrkB receptors and BDNF function by a calpain-dependent mechanism. 19:25H FIC9 Lopes A, Leite-Almeida H, Pinto V, Carlos S, Lopes S, Pinto- Ribeiro F, Silva S, Carvalho A, Clara A, Fernandes R, Almeida A, Sousa S, Sotiropoulos I. Altered nociception by Tau depletion: implication for Alzheimer s disease 19:30H FIC10 Porciúncula LO, Silva CG., Canas PM, Marques JM, Andrade GM, Oses JP, Gachet C, Cunha RA, Rodrigues RJ. P2Y1 receptor is involved in Abeta-induced synaptic and memory loss. 19:35H FIC11 Plácido AI, Oliveira CR, Pereira CMF, Moreira PI. Intracellular accumulation of amyloid beta is associated with ER stress in endothelial cells 19:40H FIC12 Naia L., Rosenstock TR, Oliveira-Sousa SI, Caldeira GL, Laço MN, Hayden MR, Rego AC. Sirtuin modulators alter acetylation and mitochondrial function in in vitro and in vivo Huntington s disease models. 15

16 Fire Sessions II Neural systems I June 1 Bussaco room Chairperson: João Malva 10:00H FIIA1 Simões AP, Kaster M, Machado N, Gonçalves N, Nunes A, Goosens KA, Cunha RA. Role of adenosine A2A receptors in the amygdala: implications for mood disorders. 10:05H FIIA2 Cunha C-S, Coimbra B, Borges S, Rodrigues AJ, Sousa N. Prenatal glucocorticoid exposure disrupts Pavlovian-instrumental transfer: role of D2 dopamine receptor. 10:10H FIIA3 Ribeiro MJ, Inês R. Violante IR, Castelo-Branco M. The electrophysiological signature of response caution. 10:15H FIIA4 Venâncio C, Lobo A, Fernandes S, Magalhães A, Peixoto F, Antunes L, Summavielle T. Chronic low doses of ketamine induce persistent region specific neuronal changes in an adult rat model. 10:20H FIIA5 French CA, Feliciano C, Paixão VB, Jin X, Fisher SE, Costa RM. Dissecting Foxp2 Function in Motor-Skill Learning Circuits. 10:25H FIIA6 Lopes S, Bessa JM, Fernandez P, Castelhano-Carlos M, Morais M, Vieira MJ, Cerqueira JJ, Almeida OFX, Sousa N, Sotiropoulos I. Is depression a neurodegenerative disorder? an in-vivo link between depression, antidepressant action and Alzheimer s disease. 10:30H FIIA7 Machado NJ, Gonçalves N, Souza CM, de Almeida LP, Cunha RA. Adenosine A2A receptors in the amygdala control behavioral modifications in rats subject to repeated restraint stress. 10:35H FIIA8 Santos FJ, Costa RM. Cortical and striatal dynamics during operant motor skill learning. 10:40H FIIA9 Borges S, Coimbra B, Cunha C, Pego JM, Sousa N, Rodrigues AJ. Dopaminergic modulation of affective and social deficits induced by prenatal glucocorticoid exposure. 10:45H FIIA10 Cardoso-Cruz H, Dourado M, Lima D, Galhardo V. Reduced hippocampus-prefrontal cortex connectivity in rats performing a spatial working memory task under neuropathic pain condition. 10:50H FIIA11 Bernardino I, Castelhano J, Farivar R, Castelo-Branco M. Neural correlates of 3D coherent visual perception in Williams syndrome: electrophysiological evidence. 10:55H FIIA12 Ventura-Silva AP, Melo A, Ferreira AC, Carvalho MM, Campos FL, Sousa N, Pêgo JM. Excitotoxic lesions in the central nucleus of the amygdala attenuate stress-induced anxiety behavior. 16

17 Cell signaling II June 1 Plátano room Chairperson: Ana Luísa Carvalho 10:00H FIIB1 Gonçalves FQ, Rial D, Silva HB, Cunha RA. Inhibition of ecto- 5 -nucleotidase activity facilitates short-term and inhibits long-term plasticity in two different mouse brain regions. 10:05H FIIB2 Ferreira J, Rio P, Li KW, Craig AM, Carvalho AL. Changes in the proteome of the postsynaptic densities of cortical neurons from mice lacking the GluN2B subunit of NMDA receptors. 10:10H FIIB3 Lima A, Reis M, Oliveira AF, Sardinha VM, Mota C, Pinto L, Marques F, Cerqueira JJ, Sousa N, Oliveira JF. Glial pathology in the prefrontal cortex affects the cognitive function of the rat. 10:15H FIIB4 Aroeira RI, Sebastião AM, Valente CA. BDNF decreases glycine uptake in brain synaptosomes by reducing GlyT2 membrane insertion. 10:20H FIIB5 Beleza OB, Ferreira S, Alçada-Morais S, Marques JM, Simões AP, Cunha RA, Rodrigues RJ. Purinergic signaling is essential for proper neuronal polarization. 10:25H FIIB6 Gonçalves S, Outeiro TF. Elucidating the effect of alphasynuclein aggregation modulators in vesicular trafficking. 10:30H FIIB7 Matos M, Augusto E, Wei CJ, Schwarzschild MA, Agostinho P, Cunha RA, Chen J-F. Deletion of adenosine A2A receptors from astrocytes alters glutamatergic synapses and triggers enhanced psychomotor responses and memory deficits characteristic of schizophrenia. 10:35H FIIB8 Silva M, Tavares I, Morgado C. Serotonin elicits pronociception in diabetic neuropathic pain through the activation of spinal 5-HT3 receptor. 10:40H FIIB9 Silva HB, Real J, Chen J-F, Cunha RA, Tomé AR. Enhanced density and greater impact of adenosine A2A receptors in the control of cortico-striatal plasticity in pre-symptomatic Huntington s disease R6/2 mice. 10:45H FIIB10 Esteves SL, Fernandes EF, da Cruz e Silva O, Fardilha M. PPP1 a putative Synphilin-1A PhosphoProtein Phosphatase/ Implications for Lewy Bodies formation. 10:50H FIIB11 Augusto E, Matos M, Sévigny J, El-Tayeb A, Müller CE, Chen J-F, Cunha RA. Ecto-5 -nucleotidase (CD73)-mediated formation of ATP-derived adenosine is critical for the striatal adenosine A2A receptor functions. 10:55H FIIB12 Mota SI, Ferreira IL, Ferreiro E, Carvalho AL, Oliveira CR, Rego AC. Altered NMDA receptor and Src related signaling in Alzheimer s disease. 17

18 Neurodegeneration and neuroprotection II June 1 Auditorium Chairperson: Cláudia MF Pereira 10:00H FIIC1 Lemos C, Gutierres J, Silva H, Agostinho P, Oliveira CR, Carvalho RA, Cunha RA. Intracerebroventricular streptozotocin induces cognitive and metabolic dysfuntion accompanied by synaptic modifications. 10:05H FIIC2 Silva R, Henriques AG, Oliveira JM, Carvalho L, Rosa I, Delgadillo I, Cruz e Silva OAB., Nunes A. Identification of Alzheimer biomarkers with mid-infrared spectroscopy exploratory study. 10:10H FIIC3 Carmo MRS, Canas PM, Menezes APF, Nunes ACL, Queiroz F, Rolo AP, Palmeira CM, Cunha RA, Andrade GM. Impact of a p2x7 (atp) receptor antagonist on 6-ohda-induced neurotoxicity in vivo and in vitro. 10:15H FIIC4 Nascimento-Ferreira I, Nóbrega C, Vasconcelos-Ferreira A, Onofre I, Albuquerque D, Aveleira C, Déglon N, Almeida L. Beclin-1 mitigates motor and neuropathological deficits in genetic mouse models of Machado-Joseph disease. 10:20H FIIC5 Naffah-Mazzacoratti MG, Simões PSR, Visniauskas B, Yacubian EMT, Centeno R, Canzian M, Lopes-Cendes I, Morelli CVM, Cavalheiro EA, Chagas JR. Expression and activity of thimet oligopeptidase (TOP) are modified in the hippocampus in subjects with temporal lobe epilepsy (TLE). 10:25H FIIC6 Canas PM, da Silva BS, Cunha RA. Up-regulation of A2AR density in postmortem prefrontal tissue of individuals who committed suicide. 10:30H FIIC7 Rego AC, Mota SI, Costa RO, Caldeira GL, Santana I, Padovano C, Fonseca AC, Cunha C, Letra L, Oliveira CR, Ferreira IL, Pereira CMF. Oxidative and ER stress in early stages of Alzheimer s disease. 10:35H FIIC8 Cardoso S, Santos MS, Moreira PI. Brain insulin signaling pathway and synaptic integrity under insulin-induced recurrent hypoglycemia and long-term hyperglycemia. 10:40H FIIC9 Martins T, Elvas F, Galvão J, Ambrósio AF, Santiago AR. Activation of adenosine A3 receptor protects retinal ganglion cells from ischemia-reperfusion injury. 10:45H FIIC10 Simões AT, Gonçalves N, Nobre RJ, Duarte CB, de Almeida LP. Orally-administered calpain inhibitor BDA-410 reduces ataxin-3 cleavage and alleviates neuropathology in a lentiviral mouse model of Machado-Joseph disease. 10:50H FIIC11 Amorim RP, Araújo MGL, Canzian M, Ariza CB, Porcionatto M, Cavalheiro EA, Ulrich H, Carrete Jr.H, Centeno RS, Yacubian EM, Fernandes MJS. Expression of purinergic receptors as phenotypic changes in human temporal lobe epilepsy. 10:55H FIIC12 Perfeito R, Ribeiro M, Stefanis L, Cristina Rego AC. Alphasynuclein overexpression modifies the antioxidant response in SH- SY5Y neuroblastoma cells. 18

19 Fire Sessions III Neural systems II June 1 Bussaco room Chairperson: António Francisco Ambrósio 11:30H FIIIA1 Gonçalves N, Machado NJ, Souza CM, de Almeida LP, Cunha RA. Impact of ventro-medial striatal adenosine A2A receptors on moodand anxiety-related behavioural modifications in rats subjected to repeated restraint stress. 11:35H FIIIA2 Quiñones-Correa MJ; Villegas EM, Álvarez-Otero R. Calciumbinding proteins in the brain and retina of a teleost fish. 11:40H FIIIA3 Rodrigues AJ, Bessa C, Marques F, Vasconcelos B, Pereira F, Miranda A, Maciel P. GABAergic and cholinergic circuit defects in a C. elegans mutant of an intellectual disability-associated gene. 11:45H FIIIA4 Duarte J, Raimundo M, Castelo-Branco M. Cortical responses during parametric visual speed discrimination in healthy and diabetic patients. 11:50H FIIIA5 Martins GJ, Vaz A, Costa RM. Motor and instrumental learning in mice with interneuron NMDA receptor deletion. 11:55H FIIIA6 Coimbra B, Soares-Cunha C, Borges S, Sousa N, Rodrigues AJ. From novelty-seeking to impulsive behaviour: effects of prenatal exposure to glucocorticoids. 12:00H FIIIA7 Rodrigues AP, Rebola J, Jorge H, Moreno J, Pereira M, Festas I, Castelo-Branco M, van Asselen M. Noise exclusion processing is impaired in developmental dyslexia: an eye movement study. 12:05H FIIIA8 Real JI,Gonçalves N, Cunha RA. Adenosine A2A receptors control the modification caused by repeated restraint stress of synaptic plasticity in the pre frontal cortex from rat brain slices. 12:10H FIIIA9 Novais A, Ferreira C, Marques F, Campos F, Palha J, Sousa N, Dalla C, Kokras N, Sousa JC. Neudesin abblation induces an anxiety-like phenotype. 12:15H FIIIA10 da Silva JA, Tecuapetla F, Paixão V, Costa RM. Exploring the role of SNC dopaminergic neurons in the initiation of self paced actions. 12:20H FIIIA11 Simões M, Amaral C, Carvalho P, Castelo-Branco M. ERP responses to morphed facial expressions in virtual reality environments. 12:25H FIIIA12 Pinto V, Costa J, Morgado P, Mota C, Miranda A, Bravo FV, Oliveira TG, Cerqueira JJ, Sousa N. Chronic stress has different outcomes in the structure and function of the dorsal and ventral hippocampus. 19

20 Neuroinflammation June 1 Plátano room Chairperson: Ana Paula Silva 11:30H FIIIB1 Cristóvão G, Pinto MJ, Gomes CV, de Almeida R, Cunha RA. Neuron-microglia interactions in the developing CNS: focus on the role of microglia before synapse formation. 11:35H FIIIB2 Caldeira C, Frederico A, Oliveira AF, Lidónio G, Vaz A, Fernandes A, Brites D. Cell ageing effects on microglia response to Aβ. 11:40H FIIIB3 Monteiro S, Ferreira F, Pinto V, Roque S, Calçada D, Correia- Neves M, Cerqueira JJ. Absence of IFN-γ leads to an enhanced cognitive phenotype. 11:45H FIIIB4 Vaz AR, Barbosa M, Ferreira A, Cunha C, Brites D. Exploring anti-inflammatory strategies on motor neuron degeneration in ALS. 11:50H FIIIB5 Mesquita SD, Falcão AM, Ferreira AC, Sousa JC, Sousa N, Marques F, Palha JA. Lipocalin 2 is produced in response to amyloid beta. 11:55H FIIIB6 Saraiva T, Rocha S, Esteves M, Cristóvão AC, Baltazar G, Alves G, Bernardino L. Histamine modulates dopaminergic neuronal survival by boosting microglial activity. 12:00H FIIIB7 Elvas F, Madeira M, Martins T, Santiago AR, Cavadas C, Ambrósio AF. Neuropeptide Y receptor Y1 modulates microglia activation in the rat retina. 12:05H FIIIB8 Cristóvão G, Viegas M, Vieira O, Cunha RA, Gomes CV. Microglia phagocytic ability is preserved irregardless changes in ATP levels and depends upon adenosine A2AR tonic activation. 12:10H FIIIB9 Cunha C, Vaz AR, Brites D. Motor neuron signaling to microglia: a clue for ALS pathogenesis. 12:15H FIIIB10 Madeira MH, Elvas F, Ambrósio AF, Santiago AR. Adenosine A2A receptor modulates LPS-induced neuroinflammation in the retina. 12:20H FIIIB11 Martins I, Carvalho P, Westerink BH, Wilson SP, Tavares I. GABA enhances pain facilitation from the brain during inflammatory pain. 12:25H FIIIB12 Miranda C, Santos NC, Sousa N, Correia-Neves M, Roque S. Can distinct cognitive performances among aged individuals be related with distinct immune system profiles? 20

21 Neurodegeneration and neuroprotection III June 1 Auditorium Chairperson: Paula Moreira 11:30H FIIIC1 Esteves M, Saraiva T, Rocha S, Baltazar G, Ferreira L, Bernardino L. Retinoic acid-loaded polymeric nanoparticles induce neuroprotection in a mouse model of Parkinson s disease. 11:35H FIIIC2 Vieira M, Fernandes J, Carreto L, Santos M, Duarte CB, Carvalho AL, Santos AE. Necroptotic neuronal death of hippocampal neurons submitted to an ischemic stimulus. 11:40H FIIIC3 Brudzewsky D, Elvas F, Martins J, Santiago AR, Woldbye DPD, Ambrósio AF. Retinal ganglion cells express neuropeptide y and npy receptors. 11:45H FIIIC4 Morgado C, Silva M, Amorim D, Almeida A, Pinto-Ribeiro F, Tavares I. Diabetes alters the activity of rostroventromedial medulla (RVM) ON-like and OFF-like cells: a role in diabetic neuropathic pain? 11:50H FIIIC5 Silva DF, Esteves AR, Cardoso SM. Mitochondrial dysfunctiondependent microtubule depolarization drives autophagic-lysosomal pathway impairment in MCI and AD cybrids. 11:55H FIIIC6 Ciaccioli G, Martins A, Rodrigues C, Vieira H, Calado P. A Powerful Yeast Model to Investigate the Synergistic Interaction of a- Synuclein and Tau in Neurodegeneration. 12:00H FIIIC7 Esteves AR, Cardoso SM. LRRK2 as a player on mitochondrial and microtubule dynamics impairment in sporadic Parkinson s disease. 12:05H FIIIC8 Gomes E, Samy S, Carvalho MM, Teixeira FG, Leite-Almeida H, Reis RL, Sousa N, Silva NA, Salgado AJ. Functional Gellan Gum Hydrogels and Cell based Therapies A Novel Therapeutic Approach for Spinal Cord Injury Regeneration. 12:10H FIIIC9 Ferreira DG, Coelho JE, Batalha VL, Albino-Teixeira A, Outeiro TZ, Lopes LV. A2AR blockade prevents ltp impairment induced by alpha-synuclein. 12:15H FIIIC10 Fernandes J, Vieira M, Carreto L, Santos M, Duarte CB, Carvalho AL, Santos AE. In vitro ischemia in hippocampal neurons triggers a transcriptional response to down-regulate synaptic proteins. 12:20H FIIIC11 Oliveira AM; Cardoso SM; Ribeiro M; Seixas RSGR, Silva AMS; Rego AC. Luteolin and 3-alkyl-luteolin derivatives: potential neuroprotective agents in Huntington s disease striatal cells. 12:25H FIIIC12 Candeias E, Duarte AI, Carvalho C, Correia S, Fernandes MA, Seiça R, Santos MS, Oliveira CR, Moreira PI. Adult female rat brain is less prone to Alzheimer disease hallmarks than male brain: the role of insulin/igf-1 signaling. 21

22 Posters Cruzeiro room Neurodegeneration and neuroprotection P1. Pereira PA, Vilela M, Sousa A, Cardoso A, Madeira MD. Effects of Chronic Ethanol Treatment and Withdrawal on the Neuropeptide Y Content and Cholinergic Innervation of the Rat Cerebral Cortex. P2. Pinheiro S, Silva J, Pinto V, Sousa N, Sotiropoulos I. Structural dynamics of synapse: the role of TAU and its vulnerability to stress hormones. P3. Bessa C, Pereira F, Vasconcelos B, Marques F, Miranda A, Maciel P*, Rodrigues AJ*. Neurobehavioral deficits in C. elegans mutants of neurodevelopmental disorders. P4. Correia SC, Santos MS, Moreira PI. Mitoplasticity during hypoxic preconditioning: the brain battles to survive! P5. Tak H, Durán R, Alfonso M, Faro LRF. Effects of the cholinesterase inhibitors galantamine and tacrine on in vivo release of dopamine from rat striatum. P6. Lopes JP, Rial D, Canas PM, Rodrigo A. Cunha RA. The β-amyloid (1-42) peptide-induced impairment of hippocampal LTP is eliminated in adenosine A2A receptor knock-out mice. P7. Naia L, Ferreira IL, Cunha-Oliveira T, Ribeiro M, Duarte AI, Ribeiro MJ, Oliveira CR, Saudou F, Humbert S, Cristina Rego A. Activation of IR,IGF-1R signaling pathways promote mitochondrial function and energy metabolism in Huntington s disease human lymphoblasts. P8. Caetano AP, Jerónimo-Santos A, Vicente H, Outeiro TF, Sebastião AM, Diógenes MJ. Unravelling the effect of different Abeta species on BDNF effects upon hippocampal synaptic plasticity. P9. da Silva AC, Jones J, Rego AC, Cunha RA. Circadian oscillation of memory performance, hippocampal long-term potentiation and cortical mitochondrial function in a mouse model of Alzheimer s disease. P10. Júlio F, Caetano G, Januário C, Castelo-Branco M. Neuropsychological and Saccadic Measures in Premanifest and Manifest Huntington s Disease. P11. Sousa RM, Carvalho LS, Morais M, Pinho D, Tavares I, Albino-Teixeira A. Spinal redox status in peripheral neuropathic pain. P12. Mota C, Neves S, Martins S, Sousa JC, Sousa N, Cerqueira JJ. Heterogeneity in cognitive aging: looking for structural correlates. P13. Martins-Araujo M, Teixeira-Castro A, Jalles A, Santos L, Maciel P. Establishing a fast-throughput system for the identification of therapeutic compounds in a C. elegans model of Machado-Josep disease (MJD). 22

23 P14. Caetano G, Júlio F, Januário C, Castelo-Branco M. Oculomotor and Executive Function in Huntington s Disease: an fmri study. P15. Caetano G, Leitão R, Júlio F, Cunha G, Januário C, Castelo-Branco M. Underpinning alterations in Cortical thickness and Basal Ganglia Volumetric measures in Huntington s Disease. P16. Alfonso M, Oliveira IM, Faro LRF, Durán R. The role of Gluathione and Dithiothreitol in protecting against Clothianidin Dopamine induced release in Striatum of freely moving rats. P17. Cousiño LAJ, Blanco DF, Kim HT, Barbosa RD. Effects of isatin (an endogenous MAO inhibitor) on release of dopamine and its metabolites in experimental Parkinsonian rats induced by reserpine. An in vivo study by brain microdialysis. P18. Blanco DF, Cousiño LAJ, Kim HT, Pallares MA. Mechanisms underlying paraoxon-induced dopamine release from rat striatum: an in vivo microdialysis study. P19. Gouveia TLF, Almeida SS, Araujo R, Iha HA, Silva Jr, JA, Casarini DE, Pesquero JB, Pesquero J, Cavalheiro EA, Mazzacoratti MG. Angiotensin II overexpression in the hippocampus may be responsible for the death occurred during pilocarpine induced status epilepticus. P20. Scorza FA, Lopes MD, Colugnati DB, Lopes AC, Scorza C, Cavalheiro EA, Cysneiros RM. Omega-3 fatty acid supplementation reduces resting heart rate of rats with epilepsy. P21. Faro LRF, Alfonso M, Cousiño LAJ, Durán D. Role of glutamate receptors and nitric oxide on the effects of glufosinate ammonium, an organophosphate pesticide, on in vivo dopamine release from rat striatum P22. Neves-Carvalho A, Martins AM, Freitas A, Logarinho E, Duarte-Silva S, Heutink P, Relvas J, Maciel P. Ataxin-3 deubiquitylase contributes to neuronal differentiation through the modulation of alpha-5 integrin subunit levels. P23. Araújo MGL, Amorim RP, Fernandes MJS. P2X7 receptor blockade promotes neuroprotection in different subregions of the hippocampus of rats subjected to pilocarpine-induced status epilepticus. P24. Santos-Carvalho A, Elvas F; Álvaro AR; Ambrósio AF, Cavadas C. Neuropeptide Y receptors activation protects rat retinal cells against glutamate excitotoxicity. P25. Lopes C, Neto E, Sampaio P, Pêgo AP. Imaging axonal transport of nanoparticles functionalized with the tetanus toxin fragment HC. P26. Ferreira IL*, Ferreiro E*, Mota SI, Cardoso JM, Pereira CMF, Oliveira CR, Cristina Rego AC. In situ mitochondrial dysfunction evoked by immediate exposure to amyloid-beta 1-42 and NMDA receptor activation involving calcium release from endoplasmic reticulum. 23

24 P27. Videira R, Saavedra A, Baltazar G. STEP expression in the nigrostriatal pathway: effect of the dopaminergic lesion. P28. Neves J, Gonçalves N., Cavadas C, de Almeida LP. Neuroprotective role of Neuropeptide Y in a lentiviral mouse model of Machado-Joseph Disease. P29. Esteves S, Teixeira-Castro A, Fernandes AS, Maciel P. Citalopram as a candidate therapeutic agent for Machado-Joseph Disease. P30. Tralhão P, Bóia R, Brudzewsky D, Ambrósio AF, Santiago AR. Effect of caffeine administration in the electrophysiological response of the retina in a model of retinal ischemia-reperfusion. P31. Henriques AG, de Oliveira JM, Gomes B, Ruivo R, da Cruz e Silva EF* and da Cruz e Silva OAB. Abeta disaggregating proteins counteract Abeta induced effects on intracellular sapp accumulation by remodeling actin network. P32. dos Santos JB, Herrera F, Outeiro TF. The Role of N-terminal Phosphorylation on Mutant Huntingtin Oligomerization, Aggregation and Toxicity. P33. Duarte-Silva S, Silva-Fernandes A, Neves-Carvalho A, Amorim M, Soares- Cunha C, Oliveira P, Teixeira-Castro A, Maciel P. 17-DMAG induces autophagy and delays disease progression in a mouse model of Machado- Joseph disease. P34. Pliássova A, Oliveira CR, Cunha RA, Agostinho P. Synaptic Localization of the Secretases Involved in the Metabolism of Amyloid Precursor Protein. P35. Santos RX, Correia SC, Santos MS, Moreira PI. Effects of type 1 diabetes and insulin therapy on brain mitochondrial dynamics and autophagy. P36. Durán R, Lilian R Ferreira Faro. Brenda V,Ferreira Nunes F, Alfonso M Protective effects of antioxidants on the glufosinate ammonium-induced striatal dopamine release in vivo P37. Matos CA, Nóbrega N, Vigorita M, Cortes L, de Almeida LP, de Macedo- Ribeiro S, Carvalho AL. Ataxin-3 phosphorylation and its effects on protease activity and Machado-Joseph disease mechanisms. Cell signaling P38. David-Pereira A, Puga S, Almeida A, Pinto-Ribeiro F. Potential role for mglur5 in pain inhibition from the infralimbic cortex in rodents. P39. Ribeiro A, Roque S, Neves MC, Sousa N, Sotiropoulos I. BACE-ics in thymocyte differentiation: implications for Alzheimer s disease therapeutics. P40. Teixeira-Castro A, Jalles A, Morimoto R, Maciel P. A whole organism screen identifies serotonin signaling as modulator of Machado-Joseph disease pathogenesis. 24

25 P41. Bastos A.EP, Mondragão M, Costa G, Costa PF, Lima PA. A new understanding of the interplay insulin-glucose in neurons from the rat hippocampus. P42. Castro Neto EF, Cunha RH, Silveira DX, Yonamine M, Lima CAS, Gouveia TLF, Cavalheiro EA, Amado D, Naffah-Mazzacoratti MG. Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion. P43. Patrício C, Coelho JE, Sebastião AM, Lopes LV, Lima PA. Differential expression of the Kv1.3 channel/current throughout the rat hippocampus. P44. Szabó EC, Tomé AR, Cunha RA. The chronic consumption of caffeine leads to enhanced plasma levels of caffeine and the appearance of both demethylated and deaminated metabolites in mouse tissues. P45. Leffa D, Machado NJ, Gonçalves N, Souza CM, da Rosa F, Souza DO, Lopes LV, Pandolfo P, Cunha RA. Adenosine A2A receptors in the prefrontal cortex regulates impulsive behavior in rats. P46. Ribeiro FF, Santos T, Sousa M, Sebastião AM. Adenosine A2A receptor activation promotes microtubule dynamics. P47. Bravo FV, Castro A, Chan RB, Paolo GP, Sousa N, Oliveira TG. Characterization of the role of phospholipase D in Caenorhabditis elegans. P48. Mouro F, Dias RB, Rombo D, Sebastião AM. Modulation of NMDA receptor activity through adenosine A2A receptors in the hippocampus. P49. Silva I, J Correia, F Ferreirinha, M Silva-Ramos, J Sévigny, Correia-de-Sá P. Adenosine, acting on A1 receptors, negatively modulates acetylcholine and ATP release from cholinergic nerves of the bladder of patients with benign prostatic obstruction. P50. Carvalho P, Martins I, Wilson SP, Tavares I. Opioidergic modulation of a pain facilitatory area of the brain using gene transfer. P51. Messias JP; Paula JR; Grutter A; Oliveira R; Bshary R; Soares MC. The role of dopaminergic system in the modulation of the Indo-pacific bluestreak cleaner wrasse Labroides dimidiatus cooperative behavior. P52. Paula J, Messias J, Grutter A; Oliveira R; Bshary R; Soares M. Serotonin Neuromodulation of Cooperative Behaviour in a Cleaner Fish. P53. Duarte-Araújo M, Monteiro S, Tavares C, Lima H, Ferreirinha F, Magalhães-Cardoso MT, Sévigny J, Correia-de-Sá P. Differential localization and enzymatic activity of ecto-ntpdases and ecto-5 -nucleotidase in the rat ileum. P54. Esteves SLC, Korrodi-Gregório L, da Cruz e Silva OAB, da Cruz e Silva E, Fardilha M. Protein Phosphatase 1γ Isoform Associated Interactions in Brain. 25

26 P55. Silva MM; Fernandes J; Santos SD; Carvalho AL. Uncovering novel micrornas involved in homeostatic plasticity. P56. Carvalho S, Coelho JE, Ferreira DG, Lopes LV. Subcelular localization of adenosine a2a receptors in hippocampal synapses. P57. Cardoso SC, Paitio JR, Mazzei R, Bshary R, Oliveira RF, Soares MC. Ecological relevance determines arginine vasotocin influence on cleanerfish learning abilities. P58. Silva T, Cunha RA, Agostinho A. Interaction between ecto-5 -nucleotidase and adenosine A2A receptors in nerve terminals of the mouse prefrontal cortex. P59. Ferreira NR, Lourenço CF, Barbosa RM, Laranjinha J. The redox interplay between nitrite, ascorbate and nitric oxide in the brain. Neurogenesis and neural cell differentiation P60. Pires AO, Neves-Carvalho A, Baltazar G, Sousa N, Salgado AJ. Mesenchymal Stem Cells Secretome: a Step Towards Improving Neuronal Survival and Recovery. P61. Mendes-Pedro D, Duarte-Samartinho M, Mendes-Pedro D, Ribeiro FF, Ferreira FF, Sebastião AM, Xapelli S. Modulatory role of Adenosine A2A receptors on brain-derived neurotrophic factor (BDNF)-induced neurogenesis in murine dentate gyrus stem/progenitor cell cultures. P62. Alves ND, Mateus-Pinheiro A, Patrício P, Ninkovic J, Schorle H, Goetz M, Sousa N, Pinto L. The role of the transcription factor AP2γ on the modulation of adult glutamatergic neurogenesis in stress-related disorders. P63. Peteira-Martínez A, Álvarez-Otero R; de Miguel Villegas E. Development of cones in the retina of adult turbots. An electron microscopy study. P64. Fonseca MB, Solá S, Xavier JM, Dionísio PA, Rodrigues CMP. Amyloid β peptides modulate differentiation of mouse neural stem cells by an autophagy-dependent manner. P65. Bento AR, Pêgo AP, Amaral IF. Optimization of cell culture parameters for the 3-D culture of single embryonic stem cell-derived neural stem cells in fibrin. Neuroinflammation P66. Coelho-Santos V, Leitão RA, Cardoso FL, Fontes-Ribeiro C, Brito MA, Silva AP. The role of TNF-α in METH-induced barrier dysfunction. P67. Roque S, Calçada D, Branco C, Rodrigues B, Monteiro S, Sousa S, Palha JA., Correia-Neves M. Mycobacterium avium chronic infection induces alterations in the hippocampal cytokine profile. 26

27 P68. Rocha DN, Barrias CC, Relvas JB*, Pêgo AP. An engineered alginatebased platform to mimic a glial scar. P69. Bóia R, Tralhão P, Madeira M, Elvas F, Tomé AR, Ambrósio AF, Santiago AR. Caffeine inhibits neuroinflammation and cell death in the retina induced by ischemia-reperfusion injury. Neural systems P70. Castelhano J, Duarte C, Rodriguez E, Castelo-Branco M. The sources of gamma-band oscillatory activity during a simultaneous EEG/fMRI ambiguous perception task. P71. Silva JC, Matos G, Andersen ML, Schoorlemmer GHM, Tufik S, Cavalheiro EA. Characterization of the sleep-wake cycle of the neotropical rodent proechimys. P72. Tavares P, Mouga S, Guiomar O, Castelo-Branco M. Configural processing of faces in high-functioning adults with autism: an EEG/ERP study. P73. Pereira AC, Ferreira S, Quendera B, Mateus C, Almeida MR, Silva E, Castelo-Branco M. Cortical structural alterations due to peripheral visual loss in Retinitis Pigmentosa. P74. Ferreira S, Pereira AC, Quendera B; Mateus C; Almeida MR; Silva E; Castelo-Branco M. Peripheral Vision Loss and Visual Cortical Organization in Retinitis Pigmentosa. P75. Carvalho LS, Sousa RM, Patinha D, Pinho D, Tavares I, Albino-Teixeira A. Neuronal activation of the nucleus tractus solitarii (NTS) in spontaneously hypertensive rats with concurrent diabetic neuropathy. P76. Guimarães MR, Martins H, Cerqueira JJ, Sousa N, Almeida A, Leite- Almeida H. Differential impact of right-/left-sided neuropathy in reversal learning: a c-fos expression study in the prefrontal cortex. P77. Leite-Almeida H, Melo AA, Pêgo JM, Bernardo S, Sousa N, Almeida A, Cerqueira JJ. Variable delay-to-signal: a new paradigm for decision and response impulsivity assessment. P78. Camacho M, Fernandes AB, Castro C, Branco T, Martins S, Costa RM, Maia A. Effects of bariatric surgery in gustatory function: exploring preliminary data. P79. Ramalhosa F, Carvalho AF, Sousa N. The role of cannabinoids in the braingut axis. P80. Santos A.*, Mateus-Pinheiro A.*, Patrício P.*, Alves ND., Bessa J., Sousa N, Pinto L. The Sweet Drive Test (SDT): a novel paradigm to measure anhedonic behavior in rodents. P81. Soares M, Cardoso S, Grutter A, Oliveira R, Bshary R. Stress and cooperative levels in a cleaner fish mutualism. 27

28 P82. Ferreira AC, Pinto V, Mesquita SD, Novais A, Sousa JC, Correia-Neves M, Flo TH, Coban C, Sousa N, Palha JA, Marques F. Lipocalin-2 is involved in cognition and emotional behaviors. P83. de Souza CM, Canas PM, Yacoubi ME, Vaugeois J, Cunha RA. Different distribution of 5-HT1B and 5-HT2C receptor in two different genetic mouse models of depression. P84. Morais M, Pinheiro A, Patrício P, Alves ND, Pinto L, Sousa N, Bessa J. The impact of antipsychotic drugs in depression: a role of adult neurogenesis? P85. Amorim D, Pereira A, AlmeidaA, Pinto-Ribeiro F. Potential role for galanin in pain-depression comorbidity. P86. Mutlu S, Feliciano C, Martins G, Tachupetla F, Costa RM. Mapping the functional organization of striatal circuits. P87. Lourenço CF, Ledo A, Cateano MA, Laranjinha J, Barbosa RM. Matching oxygen and glucose dynamics during neurovascular coupling in rat hippocampus. 28

29 PLENARY LECTURES 29

30 Miguel Castelo-Branco Short Biosketch MCB obtained his MD PhD in 1999 (supervisors: Wolf Singer, Max-Planck Institute for Brain research, Frankfurt, Germany and José Cunha-Vaz, University of Coimbra, Portugal). He is now Associate Professor at the University of Coimbra, and has held a Professorship in Psychology in 2000 at the University of Maastricht, the Netherlands. Before, he was shortly a Postdoctoral fellow at the Max-Planck-Institute for Brain Research, Germany where he had also performed his PhD work ( ). His achievements are well reflected in publications in top General Journals, such as Nature and PNAS and Top Clinical Translational research journals such as Journal of Clinical Investigation, Brain as well as others in the field of Human Neurophysiology and Neuroscience (The Journal of Neuroscience, The Journal of Neurophysiology, Human Brain Mapping, Neuroimage, Cerebral Cortex, Neuron and others). MCB has made interdisciplinary contributions in the fields of Cognitive Neuroscience, Human and Animal Neurophysiology, Visual Neuroscience, Human Psychophysics, Functional Brain Imaging and translational research in Neurology. His lab has formerly accomplishing tasks in the context of several European Networks, (Evi-Genoret, BACS, two e-rare Consortia, and a European Project on Neurofeedback), and has succeed in collaborating with labs working in other fields of knowledge such as Human Genetics and Clinical Neuroscience. MCB has also been successful in generating interdisciplinary work with scientists working in the field of neuropsychology, neuroinformatics and neuroengineering. This enabled proof of concept publications showing the effectiveness of brain computer interfaces and neurofeedback in normal and neurological populations. MCB has been awarded several National and International Prizes and is the Scientific Coordinator of the National Functional Brain Imaging Scientific initiative. His lab is representing his own host University in this national Imaging Consortium. He is also the Director of ICNAS, a Medical Imaging Infrastructure at the University of Coimbra and IBILI, a Research Unit of our National Scientific System. Under his leadership IBILI was classified as an Excellent Research Unit by international evaluation panels. He also has strong experience in preclinical and clinical research (including clinical trials involving drug therapies). He has also been involved in spin-off initiatives and entrepreneurial consortia between academia and the industry. 30

31 PLENARY LECTURE I May 30 Auditorium 15:00 PL1 s Systems Neurobiology: New Trends in Translational Neuroscience Miguel Castelo-Branco Miguel Castelo-Branco IBILI, Institute for Biomedical Research in Life Sciences, Faculty of Medicine, University of Coimbra & ICNAS, National Brain Imaging Network of Portugal We will address our recent work in fundamental and translational neuroscience ranging from sensory biophysics to visual attention and high level processes in human neurophysiology. In particular, we will address the temporal dynamics of perceptual decision mechanisms and the role of context. Our research involves linking engineering approaches with physiological studies in human health and disease. This is achieved by using statistical classification approaches in the discovery of multivariate biomarkers of diseases of the brain as well as in clinical applications of brain computer interfaces. Also, we discuss model and data driven approaches to understand brain function in health and disease, in particular in which concerns impaired cognitive control and decision making processes. Finally we will address recent trends in translational research in brain development and ageing, with a particular focus on direct links between animal and human research. Human Neuroscience, Perception, Decision Making, Translational Research, Development, Ageing [email protected] 31

32 Daniel Choquet Daniel Choquet obtained an engineering degree from Ecole Centrale (Paris, France) in He then got attracted to neuroscience (Master s degree Paris VI) and completed his Ph.D. in the lab of Henri Korn at the Pasteur Institute (Paris), studying ion channels in lymphocytes. He got appointed tenure Research officer at the CNRS in 1988 in the group of H. Korn. He then performed a postdoctoral/sabbatical at the Duke University (North Carolina, USA) in the laboratory of Michael Sheetz where he studied the regulation of integrin-cytoskeletal linkage by force, and demonstrated that cells can sense and respond to extracellular traction. He then setup his group in Bordeaux (France) at the Institute for Neuroscience where he got a directorship position at the CNRS. He launched an interdisciplinary program on the use of high resolution imaging to study the trafficking of neurotransmitter receptors in neural cells. He is now heading the project of the Institute for Interdisciplinary Neuroscience and the Bordeaux Imaging Center core facility. He develops several research topics, combining neuroscience, physics and chemistry in order to unravel the dynamics of multimolecular complexes and their role in synaptic transmission. He has recently uncovered a new role for the mobility of glutamate receptors in synaptic transmission. He has been the recipient of several awards including the 1990 Bronze medal from the CNRS, the Research prize from the Fondation pour la Recherche Médicale, 1997, the Grand Prix from the French Academy of Sciences, prix du CEA and the 2009 Silver medal from the CNRS. He is a Member of the Institut de France, the French Science Academy since November The leitmotif of his research has always been to build bridges between disciplines in order to explore new scientific territories. 32

33 PLENARY LECTURE II May 31 Auditorium 12:00 PL2 s A nanoscale view into the dynamic of AMPA receptor organization in synapses Daniel Choquet Daniel Choquet Institut Interdisciplinaire de Neuroscience, UMR 5297 CNRS-Université de Bordeaux The spatio-temporal organization of neurotransmitter receptors in the postsynaptic membrane is a fundamental determinant of synaptic transmission and thus information processing by the brain. Ionotropic AMPA glutamate receptors (AMPAR) mediate fast excitatory synaptic transmission in the central nervous system. Using a combination of high resolution single molecule imaging techniques and video-microscopy, we had previously established that AMPARs are not stable in the synapse as thought initially, but undergo continuous entry and exit to and from the post-synaptic density through lateral diffusion. Using three independent super-resolution imaging methods, on both genetically tagged and endogenous receptors, we now demonstrate that, in live hippocampal neurons, AMPAR are highly concentrated inside synapses into a few clusters of around seventy nanometers. AMPAR are stabilized reversibly in these domains and diffuse freely outside them. Nanodomains are themselves dynamic in their shape and position within synapses as they can form and disappear within minutes, although they are for the most part stable for at least up to an hour. These results open the new possibility that glutamatergic synaptic transmission is controlled by the regulation at the nanometer scale of the position and composition of these highly concentrated nanodomains. Glutamate receptors; single molecule imaging; super-resolution imaging; nanodomains [email protected] 33

34 Oscar Marín Oscar Marín was born in Madrid in He graduated in Biological Sciences from the Universidad Complutense (Madrid, Spain) in 1993, where he also obtained his Doctoral Degree in 1997 (Extraordinary award and European mention). He subsequently joined the University of California in San Francisco as a postdoctoral fellow in the laboratory of John L. R. Rubenstein. In 2003, he took a group leader position at the Instituto de Neurociencias in Alicante (Spain), where he is currently a Full Professor for the Spanish National Research Council (CSIC). He has received numerous Awards and recognitions, such as EMBO (European Molecular Biology Organization) Young Investigator (2003), NARSAD Young Investigator (2000 and 2004), and European Young Investigator (EURYI) Awards (2004), Banco Sabadell Award for Biomedical Research (2008), Rey Jaime I Award on Basic Research (2011), and the FENS/EJN Young Investigator Award. Oscar Marín serves in several editorial boards, and he is currently a member of the Board of Reviewing Editors at Science. In 2005, Oscar Marín was selected as one of the 22 founding members of the Scientific Council of the European Research Council, where he served until

35 PLENARY LECTURE III June I Auditorium 9:00 PL3 s Molecular mechanisms controlling the development of cortical interneurons Oscar Marín Oscar Marín Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández Gamma-aminobutyric acid-containing (GABAergic) interneurons play major roles in the function of the cerebral cortex. Through mostly inhibitory mechanisms, interneurons regulate the activity of pyramidal cells, prevent hyperexcitability, and synchronize the rhythmic output of cortical activity. In addition, growing evidence suggest that disruption of interneuron function is common to several psychiatric conditions, such as schizophrenia. Given their extraordinary diversity, understanding cortical interneuron development seems crucial to shed light into their role in cortical processing, both in health and disease. This talk will examine the molecular mechanisms controlling the development of cortical interneurons in the mouse, focusing on thee different problems: how does cortical interneuron diversity arise? What mechanisms control the migration and final allocation of interneurons in the cortex? How does abnormal interneuron development relate to disease? Cerebral cortex, development, interneuron, specification, migration, connectivity, schizophrenia [email protected] 35

36 ORAL COMMUNICATIONS 36

37 Symposium I Neurodevelopment May 30 Auditorium 37

38 16:00 SI1 s Searching the source of topography in the somatosensory system. Role of periphery TGF-β signaling in barrelette development. Susana da Silva Susana da Silva, Hiroshi Hasegawa, Alexandra Scott, Xiang Zhou, Amanda K. Wagner, Bao-Xia Han, Fan Wang Harvard Medical School Mammalian somatosensory topographic maps contain specialized neuronal structures that precisely recapitulate the spatial pattern of peripheral sensory organs. In the mouse, whiskers are orderly mapped onto several brainstem nuclei as a set of modular structures termed barrelettes. Using a dual-color iontophoretic labeling strategy, we found that the precise topography of barrelettes is not a result of ordered positions of sensory neurons within the ganglion. We next explored another possibility that formation of the whisker map is influenced by periphery-derived mechanisms. During the period of peripheral sensory innervation, several TGF-β ligands are exclusively expressed in whisker follicles in a dynamic spatiotemporal pattern. Disrupting TGF- β signaling, specifically in sensory neurons by conditional deletion of Smad4 at the late embryonic stage, results in the formation of abnormal barrelettes in the principalis and interpolaris brainstem nuclei and a complete absence of barrelettes in the caudalis nucleus. We further show that this phenotype is not derived from defective peripheral innervation or central axon outgrowth but is attributable to the misprojection and deficient segregation of trigeminal axonal collaterals into proper barrelettes. Furthermore, Smad4-deficient neurons develop simpler terminal arbors and form fewer synapses. Together, our findings substantiate the involvement of whisker-derived TGF- β/smad4 signaling in the formation of the whisker somatotopic maps. Development, Somatosensory System, TGF- β, Barrellette, Topographic Map [email protected] 38

39 16:30 SI2 s Ether-phospholipid functions on Schwann cell differentiation and myelination Pedro Brites Pedro Brites Nerve Regeneration lab, Instituto de Biologia Molecular e Celular- IBMC, Porto Schwann cells, the myelinating the peripheral nervous system, undergo major cellular changes to recognize and engulf axons in order to accomplish their enwrapment with a myelin sheath. Ether-phospholipids are highly abundant in the nervous tissue, and their deficiency is associated with neurodegenerative disorders. The biosynthesis of etherphospholipids is crucial to attain the high levels of these phospholipids in myelin. Our research shows that lack of ether-phospholipids cause defects in Schwann cell development that impair their ability to interact with axons, initiate myelination, and assemble normal myelin. Mechanistically, we show that a deficiency in etherphospholipids specifically affects AKT activation, which hampers Schwann cell differentiation in a GSK3 -dependent manner. Treatment with the GSK3 inhibitor, lithium chloride, was able to restore the defects in axonal sorting and myelination bypassing the defects in ether-phospholipid levels. Taken together, our research demonstrates that ether-phospholipids are crucial for the correct activation of AKT, which is necessary for the correct and timely differentiation of Schwann cells. Schwann cells; myelin; phospholipids; cell differentiation; neurodegeneration [email protected] 39

40 17:00 SI3 s Understanding cell fate decisions in the embryonic neural retina Domingos Henrique Domingos Henrique, C. Gaspar, A. I. Rosa, S. Ferreira, C. Ramos 1 Unidade Biologia do Desenvolvimento, Instituto Medicina Molecular, Faculdade de Medicina Lisboa, Portugal The adult neural retina consists of seven different neural cell types, born between embryonic day E11.0 and postnatal day P11. All cell types derive from a pool of multipotent retinal progenitor cells (RPCs) in a conserved chronological sequence. Our work aims to unravel the molecular mechanisms underlying the generation of these various cell types in the developing neural retina. The Notch pathway has been associated with cell fate decisions during development, making two cells adopt distinct fates, controlling neurogenesis and creating diversity. In the retina, two Notch ligands (Dll1 and Dll4) are expressed in overlapping patterns and our work has been focused on elucidating the function of these two ligands. To address this, we have been carrying out loss of function studies by conditional deletion of Dll4 and/or Dll1 in the mouse retina during embryogenesis, using a retina specific Cre-driver Chx10:Cre. Our results show that Dll1 and Dll4 exhibit non redundant roles in retinal neurogenesis. Dll1 is mainly involved in the control of RGC generation while Dll4 is necessary for the control of PR, AC and HC fates. We are also addressing which and how proneural bhlh proteins (Math5, NeuroD, Ptf1a, Ngn2, Olig2, bhlhb5) interact to prime multipotent retinal progenitors (RPCs) into the different cell fates. We have found that different combinations of proneural bhlh genes are expressed not only in RPCs but also in differentiating neurons, overlapping with Dll4 expression. Our data supports a model in which is that the simultaneous expression of proneural genes in differentiating retinal neurons is central to their multipotent character, with Dll/Notch signaling acting to generate the observed spatio-temporal pattern of neuronal specification in the retina. Dll1 acts upstream of Dll4 to inhibit RGC fate and control the pool of progenitors while Dll4 acts upon a subset of RPCs to promote the binary fate cell fate decision between PR/AC or HC fates. neurogenese, Notch, cell-fate decisions, proneural genes, neural retina [email protected] 40

41 17:30 SI4 s Neuronal deletion of GSK3β increases microtubule dynamics in the growth cone enabling axonal regeneration Márcia Almeida Liz Liz MA, Mar FM, Pimentel HI, Santos TE, Marques AM, Vieira S, Sousa VF, Woodgett JR, Sousa MM Nerve Regeneration Group, IBMC - Instituto de Biologia Molecular e Celular Regulators of microtubule dynamics are attractive targets to promote axonal regeneration. Here we show that neuronal ablation of glycogen synthase kinase 3β (GSK3β), a key player in microtubule dynamics, increases microtubule growth speed, enabling axonal regeneration through the glial scar following spinal cord injury. Moreover, we demonstrate that decreased GSK3β activity, through decreased levels of P-GSK3βTyr216, increases axonal regeneration. Our data further supports that phosphorylation of certain GSK3β substrates, including microtubule-associated protein 1B, is unnecessary for increasing microtubule dynamics. In summary, we show that neuronal deletion of GSK3β in vivo promotes microtubule dynamics in the growth cone and axonal regeneration. glycogen synthase kinase 3β, microtubule dynamics, axonal regeneration, spinal cord injury [email protected] 41

42 18:00 SI5 s Profilin1 regulates PNS myelination Joana Paes de Faria Laura Montani1,2,*, Tina Thurnherr2,*, Joana Paes de Faria1, Jorge Pereira2, Nuno Dias1, Rui Fernandes1, Atilla Braun3, Yves Benninger2, Klaus-Armin Nave3, Robin JM Franklin4, Dies Meijer6, Reinhard Fässler5, Ueli Suter2, João B. Relvas1,2, 7 1-Instituto de Biologia Molecular e Celular, Porto, Portugal 2- Cell Biology, Institute of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology, ETH Zurich, Switzerland 3-Max-Planck für Experimentelle Medizin, Göttingen, Germany 4- Brain Repair Center, Cambridge University, Cambridge, UK 5- Max-Planck Institut für Biochemie, Martinsried, Germany 6-Erasmus MC, Rotterdam, Netherlands During development, myelin-forming Schwann cells (SC) generate radial lamellipodia to sort and ensheath axons. This process requires integrin-β1 mediated activation of rac1 and negative regulation of rho/rock signaling. The actin-binding protein profilin1 is a ROCK substrate, and we show that in LPA-treated SC or in SC lacking the focal adhesion protein integrin-linked-kinase the activation of rho/rock signaling increased profilin1 phosphorylation and reduced the numbers of radial lamellipodia, independently of integrin-β1 or rac1-activity. Knocking-down profilin1 in SCs impaired lamellipodia formation, suggesting a direct role for this protein in SC myelination. Accordingly, using SC-specific gene ablation in mice we demonstrate essential functions for profilin1 in axon sorting and myelination of axons. Our findings establish rho/rock/profilin1 function as a critical and independent pathway in regulating axon sorting and myelination during PNS maturation. PNS myelination, Schwann cells, radial sorting, lamellipodia, Profilin [email protected] 42

43 18:15 SI6 s CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics During Development Joana M. Marques Joana M Marques*, Ricardo J Rodrigues*, Jose L Rozas, Sergio Valbuena, Sanja Selak, Phillipe Marin, Maria I Aller and Juan Lerma Instituto de Neurociencias de Alicante, CSIC-UMH, Alicante, Spain Kainate receptors (KARs) are highly expressed during development being involved in the morphological guidance for the formation of synaptic connectivity and modulation of the activity of immature network. However, their role in these phenomena remains poorly understood and the mechanisms underlying KAR activity during development wait to be defined. We now found in mouse dorsal root ganglion neurons (DRG) visualized upon the infection with Sindbis virus expressing GFP and morphologically classifyied into three phenotypes according to their stage of maturation (immature, middle-stage, mature) 24 h after plating, that KARs bidirectionally modulate neuronal maturation and neurite outgrowth. Weak activation of KARs with low concentrations (300 nm) of KA delayed neuronal maturation and enhanced neurite outgrowth, while maturation was promoted by higher concentrations of KA (3 µm), which limited neuritic elongation. This bidirectional modulation is supported by the dual signalling ability of KARs. KAR-stimulated PKC activity through Gi/o-protein activation delays maturation and stimulates neurite outgrowth, whereas strong KAR stimulation promotes neuronal maturation and the arrest of neurite growth through the canonical ionotropic activity. Moreover, the delayed maturation and enhanced neurite elongation are attained by a PKC-driven differential modulation of the activity of a newly found interacting protein of GluK5, the microtubule-associated protein CRMP2. Promotion of neurite elongation involves de-repression of CRMP2 activity by the decrease in the levels of p- CRMP2T514, while the delay in maturation is driven by a PKC-mediated increase in p- CRMP2T555 leading to a down-regulation of CRMP2-mediated Cav2.2 trafficking to the membrane, reducing Ca2+-entry. These findings consign a relevant role to KARs in neuronal development, for the first time providing a link between KAR activation and the modulation of neuronal cytoskeleton dynamics. Kainate receptors, CRMP2, neuronal development, neurite outgrowth. [email protected] 43

44 Symposium II Communication in the nervous system May 31 Auditorium 44

45 9:00 SII1 s BDNF regulates striatal-enriched protein tyrosine phosphatase levels in cortical neurons Ana Saavedra Ana Saavedra, Shiraz Tyebji, Mar Puigdellívol, Albert Giralt, Pradeep Kurup, Jian Xu, Silvia Ginés, Jordi Alberch, Paul J. Lombroso, and Esther Pérez-Navarro Departament de Biologia Cel lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that has an important role in synaptic plasticity and neuronal function through the modulation of key signalling molecules (ERK1/2, p38, Fyn, NMDA and AMPA receptors). Brain-derived neurotrophic factor (BDNF) also contributes to the regulation of synaptic plasticity in the brain. Our results from BDNF knockout mice indicate a regulation of STEP levels by BDNF during postnatal development in the striatum and cortex. To explore this mechanism, we treated cortical primary neurons with BDNF (10 ng/ml; 5 min up to 6 h) and we found a significant reduction of STEP levels compared to control cultures. This effect was prevented by the tyrosine kinase inhibitor K252a, but not by the MAPK inhibitor PD98059, the calpain inhibitor ALLN or the PI-3K inhibitor wortmannin. In contrast, by inhibiting the PLCγ pathway (U73122), PKC (chelerythrine chloride) or the ubiquitin-proteasome system (MG-132) we were able to block the reduction of STEP levels in BDNF-treated cultures. In this line, STEP ubiquitination was higher in cortical neurons exposed to BDNF than in control cultures. To investigate the impact of BDNF-induced STEP degradation, we analyzed the phosphorylation level of NR2B (Tyr1472), a STEP substrate. The levels of pnr2b (Tyr1472) increased in cultures exposed to BDNF, and prevention of STEP degradation using MG-132 partially decreased BDNF-induced NR2B phosphorylation. In conclusion, our results show that STEP degradation takes part in BDNF effects. STEP, ubiquitin-proteasome system, PLCgamma, PKC, NR2B [email protected] 45

46 9:25 SII2 s Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst Rita Teodoro Rita Teodoro, Gulçin Pekkurnaz, Abdullah Nasser, Misao Higashi-Kovtun and Thomas Schwarz F.M. Kirby Neurobiology Center, Children s Hospital Boston and Dep. of Neurobiology, Harvard Medical School Remodeling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signaling pathway by which synaptic activity and its consequent Ca2+ influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones in Drosophila. In accordance with the ability of the exocyst to direct delivery of post-golgi vesicles, constitutively active Ral expressed in muscle enlarges the membrane folds of the postsynaptic plasma membrane (called subsynaptic reticulum, SSR), but not in sec5-/- muscles. Chronic changes in synaptic activity control the plastic growth of this membrane and SSR growth requires Ral and the exocyst. Thus Ral regulation of the exocyst represents a control point for postsynaptic plasticity and one that may also function in mammals as expression of activated Ral in hippocampal neurons increases dendritic spine density. Drosophila Neuromuscular Junction, Membrane trafficking, Postsynapse, Ral GTPase, Synaptic Activity [email protected] 46

47 9:50 SII3 s Trans-synaptic signaling by acute cleavage of Neuroligin-1 Rui Peixoto Rui Peixoto, Portia Kunz, Hyungbae Kwon, Angela Mabb, Ben Philpot, Bernardo Sabatini, Michael Ehlers Harvard Medical School Adhesive contact between pre- and postsynaptic neurons initiates synapse formation during brain development and provides a natural means of transsynaptic signaling. Numerous adhesion molecules and their role during synapse development have been described in detail. However, once established, the mechanisms of adhesive disassembly and its function in regulating synaptic transmission have been unclear. Here, we report that synaptic activity induces acute proteolytic cleavage of neuroligin-1 (NLG1), a postsynaptic adhesion molecule at glutamatergic synapses. NLG1 cleavage is triggered by NMDA receptor activation, requires Ca2+ /calmodulin-dependent protein kinase, and is mediated by proteolytic activity of matrix metalloprotease 9 (MMP9). Cleavage of NLG1 occurs at single activated spines, is regulated by neural activity in vivo, and causes rapid destabilization of its presynaptic partner neurexin-1β (NRX1β). In turn, NLG1 cleavage depresses synaptic transmission by abruptly reducing presynaptic release probability. Thus, local proteolytic control of synaptic adhesion tunes synaptic transmission during brain development and plasticity. Synaptic Plasticity, Matrix Metalloprotease-9, Neuroligin, Neurexin, Trans-synaptic [email protected] 47

48 10:15 SII4 s Modulation of hippocampal glutamatergic synapses by ghrelin Ana Luísa Carvalho Luís F Ribeiro1,2, Tatiana Catarino1, Sandra D Santos1, J Fiona van Leeuwen1,2, José A Esteban3, Ana Luísa Carvalho1,2 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2Department of Life Sciences, University of Coimbra, Portugal; 3Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad Autónoma de Madrid, Madrid, Spain Ghrelin is a peptide mainly produced by the stomach and released into circulation, affecting energy balance and growth hormone release. These effects are guided largely by the expression of the ghrelin receptor (GHS-R1a) in the hypothalamus and pituitary. However, GHS-R1a is expressed in other brain regions, including in the hippocampus, where its activation enhances memory retention. Herein, we explore the molecular mechanism underlying the hippocampal action of ghrelin. Our data show that GHS-R1a is localized in the vicinity of hippocampal excitatory synapses, and that its activation increases delivery of AMPA receptors (AMPARs) to synapses. These changes are paralleled by functional modifications at excitatory synapses. Moreover, GHS-R1a activation enhances two different paradigms of long-term potentiation in the hippocampus and increases AMPARs and stargazin phosphorylation. These results indicate that GHS-R1a activation enhances excitatory synaptic transmission in the hippocampus by regulating AMPARs trafficking. synaptic plasticity, hippocampus, ghrelin, AMPA receptors; memory [email protected] 48

49 10:45 SII5 Clues to the early-aging of the hippocampus impact on cognition Luísa V. Lopes s Luísa V. Lopes, Vânia L. Batalha (1), Diana G. Ferreira (1,3,4,5), Jorge S. Valadas (1), Sara Carvalho (1), Joana E. Coelho (1), Rui Gomes (1, 2) (1)Instituto de Medicina Molecular, Faculty of Medicine Lisbon, and (2) Faculdade de Ciências, Universidade de Lisboa; (3) Department of Neurodegeneration and Restorative Research, University Medizin Göttingen Germany, (4) Instituto de Farmacologia e Terapêutica, Faculdade de Medicina, Universidade do Porto, (5) IBMC, Universidade do Porto; PORTUGAL Aging is associated with cognitive decline in both humans and animals. Among brain structures, the hippocampus appears to be particularly vulnerable to senescence and degeneration. Importantly, aeing is the main risk factor for Alzheimer s disease (AD) which targets primarily the temporal lobe and hippocampal formation. A2A receptors (A2ARs) are one of the main brain target of the homeostatic neuromodulator adenosine. A2ARs are constitutively activated G-protein coupledreceptors, preferentially expressed by the striatopallidal medium spiny striatal neurons. They exhibit however a very distinct pattern of expression in the hippocampus and cortex where their expression is very low in physiological conditions. Our team and others have found compelling evidence of cortical and hippocampal upsurge of A2AR expression/function associated to cognitive deficits. This is accompanied by clear behavioural deficits in hippocampal-dependent tasks, such as spatial memory in rats. Recently, we particularly evidenced adult hippocampal Long-Term Potentiation (LTP) and cognitive/memory impairments in a chronic stress ageing-like model, generated by maternal separation during the early post-natal period, in association with increased A2AR hippocampal expression. Strikingly, we observed, in adults, a normalization of synaptic and cognitive dysfunctions following A2AR blockade with the selective antagonist KW6002. This suggests an instrumental role of A2A receptors dysregulation in the genesis of synaptic dysfunctions underlying cognitive impairments. However the exact mechanism leading to A2AR dysfunction is still unknown. We will share our most recent data evaluating epigenetic changes, subcelular localization and receptor signalling underlying this age-related shift in A2AR function. This will hopefuly provide novel clues to the potential use of A2AR blockade as cognitive enhancer. Funding: FCT, EMBO, BIAL, NESTEC A2A receptors,hippocampus,ltp,stress,aging [email protected] 49

50 11:15 SII6 Adenosine A2A receptors mediate hippocampal network disinhibition specifically through parvalbumin expressing interneurons Diogo M. Rombo s Diogo M. Rombo (1,2), Kathryn Newton (3), Karri Lamsa (3), Ana M. Sebastião (1,2) (1) Instituto de Farmacologia e Neurociêcias, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal; (2) Unidade de Neurociências, Instituto de Medicina Molecular, Lisboa, Portugal; (3) Department of Pharmacology, University of Oxford, Oxford, United Kingdom. Glutamatergic principal cell excitability in the hippocampus is regulated by local circuit neurons that release inhibitory neurotransmitter GABA. These GABAergic interneurons exhibit vast structural, physiological and biochemical diversity. They innervate both excitatory principal cells and other interneurons, hence a tuned modulation of inhibitory circuits is of great importance in the control of network function. Adenosine, acting through high affinity A1 and A2A receptors, is a modulator of glutamatergic cells excitability. However, its role in modulating inhibitory transmission is much less known. We used patch-clamp recordings associated with targeted optogenetic stimulation of molecularly-defined interneuron populations to evaluate A2A receptor actions on parvalbumin (PV+) or cholecystokinin (CCK+) expressing presynaptic terminals to both pyramidal cells and other interneurons. We also tested A2A receptor actions on network hippocampal excitability by repetitive optogenetic stimulation of Schaffer Collateral fibers. When recording from PV+ to interneuron connections, A2A receptor caused a significant increase in the amplitude of IPSCs and decrease in paired-pulse ratio indicating a presynaptic increase in GABA release. In contrast, PV+ terminals to CA1 pyramidal cells and CCK+ terminals to both pyramidal cells and interneurons were not affected by adenosine A2A receptor activation. We also got evidence that A2A receptor activation causes a depression of polysynaptic inhibitory inputs to pyramidal cells during repetitive activation of Schaffer Collaterals that is accompanied by an increase in glutamatergic transmission onto these cells. Together, data suggests that adenosine A2A receptors modulate inhibitory synaptic transmission by acting specifically on GABA release from PV+ terminals onto other interneurons causing a weakening of inhibitory inputs to pyramidal cells and increasing excitability. Supported by FCT, NENS and MRC UK Hippocampus, adenosine, interneurons, network inhibition. [email protected] 50

51 Symposium III Cognition and behaviour May 31 Auditorium 51

52 15:00 SIII1 s Hippocampal hyperexcitability and impaired ERK signaling underlie enhanced and extinction-resistant fear memories in a mouse overexpressing TrkC, a model of panic disorder Mónica Santos Mónica Santos, Davide D'Amico, Alejandro Amador-Arjona, Oliver Stork, Mara Dierssen Center for Genomic Regulation, Barcelona, Spain Genetic and functional studies have shown that variation in the expression of the neurotrophin-3 receptor gene (NTRK3) may have a role in the pathophysiology of anxiety disorders and specifically of panic disorder. In mice, dysregulated expression of the tropomyosin receptor kinase C (TrkC) encoded by NTRK3 in brain (TgNTRK3), recapitulates panic disorder phenotypes. In particular, TgNTRK3 mice showed enhanced and sustained hippocampal long-term potentiation that may relate to the context-related emotional learning dysfunction in panic disorder. Our study examines pathological fear and the brain fear network, which critically involves the hippocampus. TgNTRK3 showed increased fear memories but also impaired extinction of these memories that was congruent with an altered activation pattern of the amygdala hippocampus medial prefrontal cortex fear network. We also detected an unbalanced excitation to inhibition ratio in the hippocampal CA3-CA1 sub-circuit. The resulting hyperexcitability may be responsible for the behavioral differences found, as supported by the efficacy of tiagabine, which increases GABAergic neurotransmission, to rescue the fearful phenotype. Further, we identified alterations in hippocampal extracellular signal-regulated kinase signaling that may explain the resistance to fear extinction of TgNTRK3 mice. We conclude that TrkC plays a role in hippocampal-dependent fear memories supporting the previous reports that associate NTRK3 with panic disorder. This fearful phenotype appears to be the result of hippocampal hyperexcitability and aberrant fear circuit activation. neurotrophins, panic disorder, cognition, c-fos, VGLUT1 [email protected] 52

53 15:30 SIII2 s Rescue of aging-associated decline in Dnmt3a2 expression restores cognitive abilities Ana MM Oliveira Ana MM Oliveira, Thekla J Hemstedt, Hilmar Bading Department of Neurobiology, Interdisciplinary Centre for Neurosciences (IZN), University of Heidelberg, Heidelberg DNA methylation, an epigenetic mechanism catalyzed by DNA methyltransferases (DNMTs), is important for many cellular processes and has recently been implicated in memory formation. In aged animals, the degree of DNA methylation decreases in many tissues including the nervous system. In this study we investigated a possible causal link between DNA hypomethylation and aging-related cognitive decline. We found that the expression levels of Dnmt3a1 and Dnmt3a2 are reduced in the hippocampus and cortex of aged mice. We showed that Dnmt3a2, but not Dnmt3a1, is regulated by neuronal activity, both in vitro and in vivo, and behaves like an immediate early gene. We demonstrated that increasing the levels of Dnmt3a2 in the aged mouse hippocampus rescues the cognitive ability. Conversely, reducing the levels of Dnmt3a2 in the hippocampus of young adult, cognitively normal mice generated an aged-like cognitive phenotype with impaired long-term memory formation. Furthermore, we showed that the plasticity-related genes, Arc and Bdnf, require the activity of Dnmt3a2 for activity-dependent induction. In this study we have established that the levels of Dnmt3a2 in the hippocampus determine the ability to form long-term memories, and that an aging-related decrease in Dnmt3a2 expression is causally linked to cognitive decline. Epigenetics, DNA methylation, Memory, Ageing, Hippocampus [email protected] 53

54 16:00 SIII3 Reinforcement learning in avoidance, habits, and tics Tiago V. Maia s Tiago V. Maia University of Lisbon and Columbia University Humans and other animals often learn which actions to perform on the basis of the consequences of those actions i.e., on the basis of whether those actions lead to positive or negative outcomes. This process is termed reinforcement learning, and we now understand a fair amount about its neural, psychological, and computational underpinnings. In the first part of the talk, I will present a computational reinforcementlearning model that suggests that learning to avoid negative outcomes involves a similar neuronal signal as learning to obtain a positive outcome does. More specifically, learning to avoid negative outcomes involves a positive prediction error, signaled by the bursting of dopamine neurons. I will present evidence about the patterns of dopamine release when rats perform an avoidance-learning task that is consistent with the predictions of the model. In the second part of the talk, I will present evidence that habit learning in humans engages a region of the putamen analogous to that implicated in habit learning in other animals. Furthermore, the functional connectivity between this region and sensory and motor cortical regions changes during learning in a way that is consistent with the predictions of computational reinforcement-learning models. Finally, I will show that patients with Tourette syndrome exhibit hyperactivity in this same region of the putamen, and in the regions connected with it, when having tics, suggesting that tics may be maladaptive, exaggerated motor habits. Reinforcement learning, dopamine, basal ganglia, Tourette syndrome, avoidance [email protected] 54

55 16:30 SIII4 s Listening to the sound of silence Marta A. Moita Marta A. Moita, Ana Pereira, Andreia Cruz, Susana Lima Champlaimaud Neuroscience Programme Learning to fear cues that are associated with an experienced aversive event allows animals to avoid the same threat in the future and is arguably the best understood of all forms of memory in the mammalian brain. In addition to learning signs associated with specific threats, animals are able to detect danger using cues from the social environment. This allows animals to avoid the necessity of learning through a possibly fatal encounter. We have recently shown that rats use freezing as a signal of threat. We trained one rat, the demonstrator, to fear a tone cue and the next day tested their fear of the cue in the presence of the cage-mate, the observer. We found that observer rats perceive the cessation of movement-evoked sound (caused by freezing of the demonstrator) as a signal of danger and its resumption as a signal of safety. Using the recorded sound of a rat exploring a box, we found that silence is necessary and sufficient to induce freezing in experienced rats. In our study it is the defensive behavior itself that, while providing safety to the threatened animal, signals other individuals at no added cost. As freezing is a pervasive fear response across several animal taxa, silence may constitute a true public cue that rapidly spreads across animals within an ecosystem. Fear, social behavior, rodents [email protected] 55

56 17:00 SIII5 Affective brain networks: a neurodynamic approach H. Sequeira s H. Sequeira, J. Honoré, F. D'Hondt Functional Neurosciences and Pathologies, University of Lille, France. Emotion is a major component of human mind and thus contributes to regulate most behaviors. Indeed, the affective value associated to individuals, objects and environments largely contributes to orient our life goals. In particular, some authors revived the idea that emotion is a major interface between brain and body and then could contribute to explain mental, somatic and visceral pathologies. Thus, in spite of several decades during which medical and life sciences have been prejudiced against the possibility of a scientific study of emotion, the investigation of the neural basis of emotion is now one of the most active research areas in neuroscience. From this increased interest, emotion appears to be critical to general health and mental or physical diseases. In this context, it was shown that emotion is not a single response but rather a group of complex cognitive, physiological and behavioral components. However, how these components are integrated together remains to be understood. Consequently, the aim of presented researches was to identify the neurodynamic of affective brain networks and its impact at body level. We developed an integrated approach conjugating cerebral, physiological and behavioral responses evoked by emotional stimulations. Thus, in successive and different researches, we recorded brain (evoked-related magnetic fields, ERFs) and body (skin conductance; indices of behavioral reactivity) markers of cerebral neurodynamic of emotional impact. In brief, the original conjugation of neuromagnetic and somatic signals allowed us to identify early and hemispherical focused effects of emotional information. These studies open original issues for a new brain architecture of emotion and may ultimately show how emotions can guide cognitive and behavioral priorities*. [*D Hondt et al., Emotions guide us: behavioral and MEG correlates, Cortex, in press]. Emotion, Cognition, Behaviour, MEG, Autonomic [email protected] 56

57 17:15 SIII6 Long-term effects of an acute administration of lipopolysaccharide on spatial memory and adult neurogenesis in the triple transgenic mouse model of Alzheimer's disease Jorge Valero s Jorge Valero(1), Ismael Neiva(1), Giorgia Mastrella(1) and João Malva(1, 2) (1) Center for Neuroscience and Cell Biology, (2) Faculty of Medicine, University of Coimbra, Coimbra, Portugal Increasing evidences highlight a role for neuroinflammation and neurogenesis during Alzheimer's disease (AD) progression. The aim of this work is to reveal a possible impact of neuroinflammatory conditions in memory and adult neurogenesis in wild type (WT) and triple transgenic mouse model of AD (3xTg-AD). 4 month-old mice housed in mild-enriched cages (containing a malleable paper bag) were intraperitoneally injected with saline or lipopolysaccharide (LPS) and their performance on spatial memory analyzed with the Morris water maze test (MWM) after 6 weeks. Only 3xTg-AD mice performing similarly to WT mice during the last-day MWM trial session were selected for this study (~50%). By analysing MWM performance, we observed that 3xTg-AD mice used a less direct strategy to reach the goal position (Wishaw's index), when compared to WT mice. Furthermore, LPS-treated 3xTg-AD mice showed a more "classical" memory deficit (quadrant permanency and platform crossings). 3xTg-AD mice also showed a decrease in the number, volume and branching of doublecortin positive (Dcx+) cells (newlygenerated neurons) in the dentate gyrus. Moreover, the branching of mid-size Dcx+ cells was slightly-reduced after LPS treatment in 3xTg-AD mice while increased in WT mice. Interestingly, a decrease in the number and volume of Dcx+ cells was also observed in LPS-treated WT mice. Indeed, when tested in a more demanding MWM protocol, LPStreated WT mice showed memory deficits similar to those observed in 3xTg-AD mice. These data indicate that LPS-induced neuroinflammation causes a long-term detrimental effect on hippocampal neurogenesis and spatial memory performance, more prominent in 3xTg-mice. Our results suggest that acute and sporadic neuroinflammatory events may influence the production of new hippocampal neurons and favour the development of strong memory deficits associated to Alzheimer's disease pathology. Work supported by Fundação para a Ciência e a Tecnologia (FRH/BPD/68950/2010). Adult neurogenesis, Alzheimer's disease, Hippocampus, Neuroinflammation, Spatial memory, [email protected] 57

58 Symposium IV New targets for nervous system therapy Sponsored by the Portuguese Biochemical Society (SPB) June 1 Auditorium 58

59 14:30 SIV1 s Bioenergetics and Antioxidant Coupling in the Nervous System Juan P. Bolaños Juan P. Bolaños Institute of Functional Biology and Genomics, University of Salamanca-CSIC We are interested in understanding the molecular mechanisms underlying the energetic and redox homeostasis in the central nervous system. Despite the enormous energy demand that the neurotransmission process requires, neurons modestly utilize glucose as an energetic fuel in contrast to the vast majority of other cells due to ubiquitylation and proteasomal degradation of PFKFB3 (6-phosphofructo-2-kiinase/fructose-2,6- bisphosphatase-3), which catalyzes the biosynthesis of fructose-2,6-bisphosphate, a potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK1). However, glucose metabolism in neurons is very active towards the pentose-phosphate pathway, which serves to conserve the redox energy of glucose as NADPH, an essential cofactor in the regeneration of antioxidants, such as glutathione. Here, it will be discussed the mechanisms responsible for the metabolic adaptation of neurons to this status, including proteins that regulate the energy and antioxidant homeostasis. Neurons, astrocytes, energy metabolism, glutathione, glycolysis, pentose-phosphtae pathway [email protected] 59

60 15:00 SIV2 s Understanding the role of alpha-synuclein in the nucleus: a new concept in synucleinopathies Raquel Pinho Raquel Pinho1,2,3, Lilach Soreq4, Hermona Soreq5, Luís Fonseca6, Markus Zweckstetter6, Kristina Gotovac7, Fran Borovecki7, Joaquim Feirreira8, Ana Cristina Rego2, Tiago F. Outeiro1 1Department of NeuroDegeneration and Restorative Research, University Medical Center Göttingen, 2Center for Neuroscience and Cell Biology, Coimbra, 3University of Medicine of Porto, Porto, 4Department of Medical Neurobiology, Hebrew University, 5The Life Sciences Institute, Hebrew University, 6MPI for Biophysical Chemistry, Max Planck Society,Gõttingen, 7Department for Functional Genomics, University of Zagreb, Croatia, 8Neurological Clinical Research Unit, Instituto de Medicina Molecular,Lisboa Alpha-synuclein (asyn), a common player in sporadic and familiar forms of Parkinson s disease (PD) was first described as a pre-synaptic and nuclear protein. Although the presence and role of asyn in the nuclear compartment remains controversial, it has been suggested that it may modulate histone acetylation and interfer with transcriptional (de)regulation. Here, we explore both transcriptional changes in PD and the role of nuclear asyn. Using gene expression profiling, we analyzed two groups of PD patients displaying different clinical progression of the disease (slow versus fast progression patients). We selected 5 genes for further validation by qpcr, in a subset of patients. Interestingly, we found differences in the expression of genes whose function is linked to transcription regulation, ubiquitin-proteasome system, membrane trafficking and cytoskeleton dynamics. We next investigated the presence of asyn in the nucleus in various cell and animal models. Our results demonstrate that asyn is present in nuclear fractions of embryonic mice (E14), A30P mice midbrain and neuroblastoma cells expressing human asyn. We also found that oxidative stress promotes the translocation of A30P and E46K asyn mutants to the nucleus, 24h after treatment with H2O2. To further investigate the interaction of asyn with DNA and to identify novel promoter binding regions we performed NRM experiments, genome-wide chromatin immunoprecipitation (ChIP)- sequencing and dual-luciferase assays. Our preliminary results indicate that asyn interacts with DNA and, ChIP-sequencing data, show that asyn can bind to several gene promoter regions, such as NEDD4 and SLC4A5. Furthermore, dual-luciferase assays confirmed that asyn overexpression increases SLC4A5 promoter activity. Our results highlight transcriptional deregulation and nuclear asyn as important players in synucleinopathies and identify novel genes that can be used as biomarkers of disease progression or therapeutic targets. Parkinson s disease, Alpha-synuclein, Transcription deregulation, Nucleus [email protected] 60

61 15:30 SIV3 s Pharmacotherapy for Machado-Joseph disease: from worm to mouse to human? Patrícia Maciel Sara Duarte-Silva*, Anabela Silva-Fernandes*, Andreia Teixeira-Castro*, Ana Jalles, Sofia Esteves, Andreia Neves-Carvalho, Marina Amorim and Patrícia Maciel *these authors contributed equally to this work. Life and Health Sciences Research Institute (ICVS) ICVS/3B s PT Government Associate Laboratory School of Health Sciences, University of Minho Machado-Joseph disease or spinocerebellar ataxia type 3 is a late-onset neurodegenerative disorder caused by expansion of a polyglutamine tract within the coding region of the protein ataxin-3. We have developed new transgenic animal models for the study of this disorder, in the mouse and in the nematode C. elegans, which express the mutant human ataxin-3 cdna. These models show marked neuronal dysfunction, with loss of motor coordination, and the typical pathological feature of ataxin-3 aggregation, and can be used both to study the mechanisms of disease and to test therapeutic strategies. We have identified several genetic modifiers of disease severity and progression in the C. elegans model, which pinpointed key pathways for pathogenesis. Interestingly, several of these pathways are related to aging and may explain the late onset of this disorder. These modifiers have also provided clues to possible therapeutic targets, which we have validated in C. elegans and are now testing in the mouse model. In this presentation I shall discuss our recent findings using a novel transgenic mouse model of MJD (CMVMJD135), which replicates many clinical and pathologic features of the disease. I will discuss the results of clinical trials with candidate drugs targeting different pathways in our C. elegans model, and the validation of some of these results in the mouse. The results of these preclinical studies inform us on useful therapeutic targets but also provide important clues to the key aspects of pathogenesis that are amenable to therapy. Neurodegeneration, ataxia, polyglutamine, therapy, preclinical trials, transgenic models [email protected] 61

62 16:00 SIV4 Insulin/IGF-1/estrogen signalling protects against lipid oxidation and neurodegenerative events in type 2 diabetic female rat brain Ana I. Duarte s A.I. Duarte1,2, E. Candeias1,2, S. Correia1,2, C. Carvalho1,3, M. A. Fernandes3,4, R. Seiça5, M.S. Santos1,3, C.R. Oliveira1,6, P.I. Moreira1,5 1Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, Coimbra, Portugal. 2Institute for Interdisciplinary Research (IIIUC), University of Coimbra, Coimbra, Portugal. 3Life Sciences Department, University of Coimbra, Coimbra, Portugal. 4IMAR Instituto do Mar, Life Sciences Department, University of Coimbra, Coimbra, Portugal. 5Laboratory of Physiology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Increasing evidence suggests that aged diabetic brain is more prone to cognitive dysfunction and Alzheimer disease (AD). Although chronic hyperglycemia and/or insulin (INS) resistance have been involved herein, the precise molecular mechanisms and gender effects remain unclear. We hypothesized that INS-/IGF-1-/estrogen (EST)- mediated signalling is involved on gender effects in type 2 diabetic (T2D) rat brain. Thus, we analysed INS, IGF-1 and EST-mediated signalling in brain cortical homogenates from adult (8-month) male and female T2D Goto-Kakizaki (GK) rats. Peripheral glycemia was measured using a glucometer. INS, IGF-1, cholesterol, DHEA, testosterone, EST and amyloid beta (Abeta1-42) levels were determined by ELISA and the density of pivotal signalling molecules by immunoblotting. Although peripheral glycemia remained unchanged, steroid hormone metabolism was impaired in female GK rat brains (given by cholesterol, DHEA, testosterone and EST levels). Surprisingly, despite their lower brain INS and IGF-1 levels, the density of INS, IGF-1 and EST receptors was higher in T2D females. Accordingly, downstream phospho-akt was significantly increased in T2D female brains and this may underlie the decrement in Abeta1-42 and lipid oxidation in GK females. Altogether, T2D female rat brains may be protected against lipid oxidation and neurodegenerative events via stimulation of INS/IGF-1/EST receptors density and subsequent signalling. Supported by Fundação para a Ciência e a Tecnologia (FCT), Ministério da Educação e Ciência, POPH, QREN, COMPETE, FEDER and European Social Fund (projects: PTDC/SAU-NEU/103325/2008, PTDC/SAU-NMC/110990/2009, PTDC/SAU- TOX/117481/2010 and Pest/SAU/LA0001/2011; fellowships: SFRH/BPD/26872/2006, PTDC/SAU-NMC/110990/2009; SFRH/BPD/84163/2012; SFRH/BD/43965/2008). Brain cortex, Estrogen/IGF-1/Insulin signalling, Gender, Neuroprotection, Type 2 diabetes [email protected] 62

63 16:30 SIV5 The role of acetyl-l-carnitine in the brain glucose uptake under methamphetamine Teresa Summavielle s Lídia Cunha (1,2), Joana Bravo (1), Ana Magalhães (1,2), Sílvia Fernandes (1,2), Krisztian Szigeti (3), Domokos Máthé (3), Teresa Summavielle (1,2) (1) IBMC-Instituto de Biologia Molecular e Celular (UP); (2) ESTSP-IPP, Porto; (3) Nanobiotechnology & In Vivo Imaging Center, Semmelweis University, Budapest, Hungary Acetyl-L-Carnitine (ALC) has been implicated in metabolic compromising diseases, especially those related to the mitochondria, but its molecular mechanisms of action remain elusive. ALC is commercially available in several forms, typically used to promote weight loss, and is easily accessible without any medical prescription. In patients with compromised cognitive function, ALC promoted an effective recovery of the cerebral capacity. Work from our lab has also demonstrated that ALC confers effective neuroprotection against 3,4-methylenedioximethamphetamine (MDMA), preventing mitochondrial oxidative damage and the typical MDMA-induced loss of serotonin. Carnitine participates also in the glucose metabolism and seems to have beneficial effects through the modulation of insulin-like growth factors (IGFs). Here, we used positron emission tomography (PET/MRI scanner nanoscan, Mediso, Hungary) to evaluate changes in regional glucose uptake in the brains of control and ALC-preconditioned C57BL/6J mice exposed to METH. Our results show a significant increase in glucose uptake in ALC-preconditioned mice exposed to METH (ALC/METH) compared to other experimental and control mice. Such increase was associated with increased Igf1R transcript levels, impaired reference memory in the water maze test and, in PC12 cells, to decreased cellular viability, reduced ATP synthesis, increased ROS, loss of mitochondrial membrane potential and translocation of active Bax to the nucleus. As ALC by itself also had a detrimental effect on behavior performance, our data indicate a potential risk in using ALC to improve cognitive function in healthy subjects that warrants further investigation. Carnitine, methamphetamine, glucose metabolism, mitochondria, Bax. [email protected] 63

64 16:45 SIV6 A Nanotoxicological Appraisal of Non-viral vectors for RNA Interference in the CNS Bruno M.D.C. Godinho s Bruno M.D.C. Godinho, David J. McCarthy, Aoife Quinlan, Caroll Beltrán, Julien R. Ogier, Raphael Darcy, Caitriona M. O Driscoll Univeristy College Cork, School of Pharmacy and Dept. Anatomy and Neuroscience Therapeutic gene silencing through the RNAi pathway offers great promise for the treatment of neurological and neurodegenerative disorders. However, there remains a great need to develop more effective and less toxic carriers for sirna delivery to the CNS. Modified cyclodextrins (CD), based on naturally occurring oligosaccharide molecules, are promising nucleic acid carriers that have been shown to bind and complex sirna. The aim of the present study was to investigate the toxicity profile of modified amphiphilic β-cd in comparison with other commercial non-viral vectors used for delivery of sirna to the CNS. BV2 murine microglia and U87 human astroglioma cells, were transfected with nonsilencing sirnas. To this end we used amphiphilic cationic β-cds and commercially available cationic vectors: LipofectamineTM2000 (Lf2000), INTERFERin and Superfect. Trypan blue assays and MTT assays were used to determine cell viability and mitochondrial metabolic activity respectively. LDH release and nitric oxide release were assessed in cell culture supernatants. Stereotaxic injections of nanoparticle.sirna complexes into the striatum of C57/BL6 mice were carried out. Brain tissue was harvested after 24 hours and gene expression of TNF-α, IL-1β, IL-6 assessed by RTqPCR. Although reduction in mitochondrial metabolic activity and increase in LDH release to the media was evident for all non-viral vectors used, CD nanoparticles had a better toxicity profile than commercial transfection reagents. Single direct injections into the brain revealed that Superfect significantly increased expression of TNF-α, IL-1β, IL-6 when compared to naked sirna. No significant changes were observed with CD, Lf2000 and INTERFERin. CNS RNAi-based therapeutics requires efficient and safe gene delivery vectors. Modified CDs presented the most favourable and improved toxicity profiles in these studies, thus offering great potential as novel sirna carriers for treatment of neurological disorders. microglia, astroglia, sirna, tranfection, stereotaxic surgery [email protected] 64

65 Fire Sessions I 65

66 Neurogenesis and neural cell differentiation May 30 Bussaco room 66

67 18:45 FIA1 p21ras: a target for NO to enhance neurogenesis Inês M. Araújo s A.I. Santos1,2,3, B.P. Carreira1, L.F. Ribeiro1, M.I. Morte1, C.M. Carvalho1, A. Martínez-Ruiz4 and I.M. Araújo1,2,3 1Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Regenerative Medicine Program, Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal; 3IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine, University of Algarve, Faro, Portugal; 4Servicio de Inmunología, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain. Nitric oxide (NO) enhances proliferation of neural stem cells (NSC) by the activation of the ERK1/2/MAPK pathway (Carreira et al. 2010, Stem Cells, 28, ). Moreover, NO derived from inducible NO synthase (inos) is essential for the neurogenic response in the dentate gyrus (DG) following an excitotoxic brain injury (Carreira et al. 2010). In this pathway, p21ras is a likely target for NO, and the possible first event that triggers proliferation of NSC. p21ras has a cysteine residue (Cys118) that can be modified by NO by S-nitrosylation, resulting in Ras activation. By overexpressing WT or C118S mutant Ras in SVZ-derived NSC, we have observed that NO stimulates cell proliferation in cells that overexpress WT but not C118S Ras, suggesting that the effect of NO on NSC proliferation depends on the presence of Cys118. Current studies aim at identifying Ras S-nitrosylation in this context. Moreover, treatment with a permeable nitrosothiol, S-nitroso-L-cysteine (CysSNO), also stimulates NSC proliferation. In an in vivo acute seizure model that entails increased NO production, we have observed an increase in the cysteine oxidation of p21ras 1 and 2 days after seizures, but not for longer times, as assessed by a biotin switch assay (Izquierdo-Alvarez et al. 2012, J Proteomics, 75, ), suggesting that Ras activation precedes cell proliferation in the DG Overall, the present results suggest p21ras Cys118 as a target of NO-induced modification in the proliferation of NSC. This study helps to better understand the transient activation of p21ras as a target to enhance neurogenesis. This work was supported by FCT, Portugal, COMPETE and FEDER (project PTDC/SAU-NEU/102612/2008), by a Spanish-Portuguese Acção Integrada (PRI- AIBPT /E-10/12), and by ISCIII (Spain, PS09/00101 and PI12/00875). AI Santos, BP Carreira, LF Ribeiro and MI Morte were supported by FCT (fellowships SFRH/BD/77903/2011, SFRH/BPD/78901/2011, SFRH/BD/47879/2008 and SFRH/BD/38127/2007). Neurogenesis, nitric oxide, neural stem cells, p21ras, S-nitrosylation [email protected] 67

68 18:50 FIA2 s p53 Modulates Differentiation-induced Mitochondrial Damage and Promotes Neurogenesis Joana M. Xavier Joana M. Xavier, Ana L. Morgado, Susana Solá, Cecília M. P. Rodrigues Research Institute for Medicines and Pharmaceutical Sciences (imed.ul) and Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Av. Prof Gama Pinto, Lisbon, Portugal Apoptosis-regulatory proteins, including p53, are pivotal in neural differentiation, through mechanisms independent of cell death. Recently, p53 has emerged as a key regulator of mitochondrial survival response, mediating mtdna integrity and oxidative protection. Further, reactive oxygen species (ROS) function as signaling molecules of autophagy, which results in lysosomal degradation of cytoplasmic constituents critical to prevent cell death. Our results showed that ROS production, mitochondrial membrane depolarization and cytochrome c release increase at early stages of mouse neural stem cell (NSC) differentiation, with no evidence of apoptosis. Curiously, mitochondrial mass decreased, while lipidated LC3-II and mitochondria-associated Parkin, increased throughout neural differentiation, suggesting activation of mitophagy. In addition, p53 was actively translocated to mitochondria. To further understand the precise role of p53 in mitochondrial events, we modulated p53 mitochondrial translocation in NSCs undergoing differentiation by transfecting cells with either wild-type RECQL4, an helicase essential for p53 translocation to mitochondria, RECQL4-Δ84, which cannot translocate to mitochondria, and RECQL4-GST, unable to physically interact with p53. Higher levels of mitochondrial p53 were observed in RECQL4-overexpressing cells when compared with control (mock), while mitochondrial oxidative stress, cytochrome c release and mitophagy were markedly reduced. Importantly, p53-mediated mitochondrial alterations were not seen in NSCs transfected with either RECQL4-84 or RECQL4-GST overexpression plasmids. Finally, active mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth. In conclusion, our results reveal that mitochondrial translocation of p53 modulates mitochondrial stress and apoptosis-associated events in the context of neural differentiation, thus promoting neurogenesis. Apoptosis, Mitochondria, Neural stem cell fate, Oxidative stress, p53 [email protected] 68

69 18:55 FIA3 s mir-34a Regulates Autophagy and Neuronal Differentiation During Neural Stem Cell Differentiation Morgado AL Morgado ALa, Xavier JMa, Solá Sa,b, and Rodrigues CMPa,b aresearch Institute for Medicines and Pharmaceutical Sciences (imed.ul), and bdepartment of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, Lisbon, Portugal We have previously demonstrated the involvement of specific apoptosis-associated micrornas (mirs) in mouse neural stem cell (NSC) differentiation. Further, we discovered that tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, is a strong inhibitor of apoptosis. Finally, a growing body of evidence points to a critical role for autophagy during proliferation and differentiation, as a response-survival mechanism to limit oxidative stress associated with these processes. The aim of this study was to investigate the role of mir-34a in NSC fate decision and test the potential modulatory effects of TUDCA. Our results showed that mir-34a expression was markedly downregulated during neural differentiation. TUDCA reverted all mir-34a differentiation-related changes, while maintaining the proliferation potential as demonstrated by increased multipotency marker, Nestin, and BrdU incorporation. Interestingly, synaptotagmin 1, a synaptic-related protein, and ATG9, a protein required for autophagosome formation during autophagy, were shown to be mir-34a targets in this cellular context. In fact, synaptotagmin 1 and ATG9 levels significantly increased during neurogenesis and decreased upon mir-34a overexpression. Further, autophagy inhibition impaired neuronal differentiation, as demonstrated by decreased synaptotagmin 1 levels, and βiii-tubulin-positive cells. Finally, both neuronal differentiation and morphology, including neurite outgrowth, were significantly impaired in mir-34a overexpressing cells. In conclusion, our results demonstrate a pivotal role for mir-34a during neural differentiation. Downregulation of mir-34a at early stages of neural differentiation appears to be required to induce autophagy and elicit neurite outgrowth. In addition, our findings support a role for TUDCA in potentially regulating mir-34a to repress general differentiation and maintain the stemness potential of NSCs. autophagy, mir-34a, neural stem cell fate, TUDCA [email protected] 69

70 19:00 FIA4 Unconjugated bilirubin dysregulates oligodendrocyte development and myelinogenesis s Barateiro A Barateiro, A.1, Domingues, H.2, Miron, V.3, Tukey, R.H.4, Santos, S.2, ffrench- Constant, C.3, Relvas, J.2, Fernandes, A.1, Brites, D.1 1 imed.ul, Faculty of Pharmacy, University of Lisbon, Portugal; 2 MRC Centre for Regenerative Medicine, The University of Edinburgh, UK; 3 IBMC, Faculty of Medicine, University of Porto, Portugal; 4 Department of Pharmacology, University of California, San Diego, La Jolla, USA Oligodendrocytes (OL) are the myelinating CNS cells. Elevated concentrations of unconjugated bilirubin (UCB) may determine brain injury and related sequelae. UCB effects on oligodendrocyte precursor cells (OPC) and myelinogenesis were never explored. Here, we investigated UCB-induced OPC vulnerability and changes in OPC differentiation, neuronal myelination and glia reactivity. We used OPC culture to evaluate the mechanisms of UCB toxicity; OPC differentiation cultures and myelination co-cultures to evaluate changes in OPC differentiation and OL maturation/myelination; organotypic cerebellar slice cultures and an in vivo model of severe hyperbilirubinemia, to reassure the myelination deficits and glial deficits. Reduced OPC survival by UCB involves early ER stress, mitochondrial dysfunction and JNK1/2 activation, and is followed by ROS generation, together with calpain and caspase activation. UCB impaired OL differentiation causing an increase in the number of immature cells and changes in the mrna expression of Olig1 and Olig2 transcription factors. Mature cells showed reduction in diameter and in the number of primary processes due to diminished active Rac1. UCB elicited an abnormal pattern of myelin development by decreasing myelinated internodes and internode length in the co-culture system. Myelin deficits were observed in UCB-treated cerebellar slice cultures, in association with microgliosis and astrogliosis. Increased release of glutamate and proinflammatory mediators were additionally observed. In in vivo studies, we observed a maximal atrophy of cerebellum with massive degeneration of Purkinje cells, along with decreased myelination and increased glial reactivity. It is suggested that UCB impairment of OL development, myelinogenesis delay and reactive gliosis are features of bilirubin encephalopathy Supported by FEDER (COMPETE Programme) and by National funds (FCT - projects PTDC/SAU-NEU/64385/2006 and PEst-OE/SAU/UI4013/2011). Glial cells, hyperbilirubinemia, myelination, neurons, oligodendrocytes [email protected] 70

71 19:05 FIA5 s Hemopressin stimulates oligodendrogenesis in neural stem cell cultures from the subventricular zone Sara Xapelli S Xapelli, F Agasse, FF Ribeiro, L Bernardino, CS Schitine, A. Heimann, ES Ferro, RA De Melo Reis, AM Sebastião, JO Malva Unidade de Neurociências, Instituto de Medicina Molecular, Faculdade de Lisboa, Lisboa, Portugal. Multipotent stem cells are present in the adult mammalian brain in specialized niches. Several recent studies suggest that the subventricular zone (SVZ) stem cell niche can be stimulated by brain lesions or neurodegenerative processes which promotes the differentiation of new neurons or glial cells. In this study we used Hemopressin (Hp), a peptide recently shown to act on cannabinoid CB1 receptor, to promote oligodendrogenesis. In fact, we showed that Hp did not induce proliferation or cell death, but it increased oligodendroglial differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1 3 C57BL/6 mice. Exposure of SVZ cell cultures with Hp for 7 days promoted oligodendrocytic differentiation as shown by an increase in the protein levels of the transcription factor Olig2. Moreover, by monitoring intracellular calcium concentrations ([Ca 2+ ] i ) in single cells following histamine and thrombin stimuli, a method that allows the functional evaluation of oligodendroglial differentiation, we observed an increase in oligodendrocyte-like cells. In fact, exposure of SVZ cultures to Hp resulted in increased differentiation of cells displaying an oligodendroglial profile of [Ca 2+] i responses, compared with the predominant profile of immature cells observed in non-treated cultures. Furthermore, we performed immunocytochemistry for proteolipid protein (PLP) and we observed an increase in the number of PLP-positive cells when the cells were treated with Hp. Taken together, these results show that Hp induces oligodendrogenesis in neonatal SVZ cell cultures of mice, which is important for future development of strategies to treat brain diseases, especially diseases that compromise oligodendrocytes. Support:FCT: SFRH/BPD/34870/2007, PTDC/SAU-NEU/104415/2008, PTDC/SAU- NEU/101783/2008, PTDC/SAU-NEU/68465/2006, Calouste Gulbenkian Foundation (project nº 96542) and CAPES-Grices protocol No Neural stem cells, SVZ, Oligodendrogenesis, endocannabinoids [email protected] 71

72 19:10 FIA6 s The Processossome : identification and characterization of proteins promoting oligodendrocyte process outgrowth and (re)myelination Helena Sofia Domingues Helena Sofia Domingues, Joana Paes de Faria and João Bettencourt Relvas Instituto de Biologia Molecular e Celular Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). Failure to form myelin or to maintain it often leads to neurological deficits, such as those present in multiple sclerosis (MS), the most frequent CNS chronic demyelinating disease. Progression of MS is thought to be caused by the incapacity of oligodendrocyte progenitor cells (OPC) to differentiate into myelin-competent OL within the changing microenvironment of the MS lesion. Our aim is to investigate the mechanisms regulating the formation, maintenance and stability of OL processes, as well as the key events leading to wrapping and myelination of axons. Using an approach previously described for neurons, we established a method similar to a Boyden chamber to physically separate the OPC soma from the process for RNA sequencing. The goal is to identify and validate RNAs mainly present in the process of the OPC, which are the most likely to participate in OPC process extension, wrapping and myelination of axons. Analysis of RNA sequencing data reveal an enrichment in the OPC process of RNAs involved in cytoskeleton remodelling, transport and local translation. We are currently paying particular attention to candidates involved in the cytoskeleton and Rho GTPase signaling pathways, as ongoing research in our lab identified these pathways to be critical for OL process extension and differentiation. This knowledge will allow us to gain valuable insight into the mechanisms that regulate the establishment and stabilization of oligodendrocyte-axon cell cell interactions and how their impairment compromise remyelination in demyelinating diseases such as MS. Oligodendrocyte, process outgrowth, cytoskeleton, RNA sequencing [email protected] 72

73 19:15 FIA7 The Dark Side of Neural Precursor Cells: New Cues for Gliomagenesis Ana Sofia Falcão s Ana Sofia Falcão (1,2), Cátia Gomes (1), Gisela Santos (1), Dora Brites (1,2) (1)Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, Lisbon, Portugal; (2) Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal Neural stem cells (NSC) biology is being applied to the study of brain tumors, since these cells share some common features with glioma cells. Therefore, it has been suggested that neural stem/progenitor cell (NSPC) or early-differentiated cell type lineages are in the origin of gliomas, although it is not known which developmental stage is more susceptible to malignant transformation. We investigated which developmental stage, in the NPC astroglial differentiation process, is the most similar to the glioma phenotype. NPC were obtained from E15 mice brains, grown as neurospheres and astroglial differentiation was induced during 7 days in vitro (DIV), where specific neural cell markers were determined. We also evaluated some tumor-related factors such as the proliferative potential (BrdU), multidrug resistance-associated protein 1 (Mrp1), autophagy (LC3-II/LC3-I), vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2), the matrix metalloproteinases (MMP-9 and 2) activities and the chemokine S100B. These parameters were evaluated in neurospheres, differentiating cells and in GL261 mouse glioma cells. As expected, during differentiation, there was a reduction in the undifferentiated cell markers Sox2 and Nestin (0.5- and 0.2-fold, respectively, p<0.01), and an increase in the astrocytic GFAP and GLAST (3.8- and 2.8-fold, respectively, p<0.01). Along differentiation, the expression of Mrp1 decreased and BrdU staining was dramatically reduced, whereas LC3B increased. VEGF/VEGFR-2 expression, MMP activity and S100B release increased until 2/3 DIV, and decreased thereafter. Moreover, data indicate that the first and second days of NSPC differentiation are the stages that most resemble the GL261 cells phenotype, sustaining immature/early differentiated glia as the most vulnerable to malignancy and providing new cues for gliomagenesis. Funded by FEDER (COMPETE Programme) and National funds (FCT-project PEst- OE/SAU/UI4013/2011 and 2012) gliomagenesis, mouse neural stem cells, mouse glioma cells, neural differentiation, tumor-related factors [email protected] 73

74 19:20 FIA8 Genome-wide characterization of the adult hippocampal neurogenic niche regulators in depression and by antidepressant drugs: Cellular and molecular perspectives. Patrícia Patrício s P. Patrício, A. Pinheiro; N.D. Alves; M. Morais; A. R. Santos; J. Bessa; M. Irmler; J. Beckers, N. Sousa; L. Pinto Life and Health Sciences Research Institute (ICVS), University of Minho Major depression is one of the top ten causes of morbidity and mortality worldwide. However, the mechanisms involved in its aetiology and in the therapeutic actions of antidepressants (ADs) are largely unknown due to its complex nature, in which genetics interacts with environmental factors. Importantly, studies propose that alterations in adult hippocampal cell genesis and synapto-dendritic morphology may be involved in the pathophysiology of depression and in the actions of ADs. Nonetheless, the molecular mechanisms underlying the regulation of these phenomena in depression and by ADs are still poorly understood. In this context, we performed a genome-wide study to identify molecular regulators that are altered in the adult hippocampal neurogenic niche by stress - a precipitating factor for depression - and ADs. First, a behavioral characterization of rats subjected to chronic mild stress (CMS) (to induce a depressive like phenotype) and treated with 4 different ADs - fluoxetine, imipramine, tianeptine and agomelatine was performed; the AD-treated rats recovered from the behavioral and cellular deficits induced by CMS. The genome-wide expression changes in the macrodissected hippocampus were measured using Affymetrix Gene 1.0 ST arrays. RNA profiling disclosed the differential regulation of several genes and pathways among control, CMS-exposed and AD-treated rats. Moreover, functional and canonical pathway categorization revealed important molecules related to metabolism, survival and neuroplasticity in AD-treated animals. These results further correlate with the observed increased neurogenesis and dendritic arborization in the hippocampus of ADtreated compared to CMS-exposed rats. This ongoing study gives us an overview on the molecular pathways responsible for the recovery of adult hippocampal homeostasis after AD treatment emerging as a promising tool for the discovery of targets for disruption and new therapeutic strategies in depression. Adult hippocampal neurogenesis, depression, antidepressants, genome-wide characterization [email protected] 74

75 19:25 FIA9 s The interplay between dendritic plasticity and cell genesis in depression Mateus-Pinheiro A. Mateus-Pinheiro A., Patrício P., Morais M., Alves ND., Monteiro S., Almeida OFX., Bessa J., Sousa N & Pinto L Life and Health Sciences Research Institute (ICVS), University of Minho; ICVS/3B s- PT Government Associate Laboratory The potential to respond to environmental stimuli through dynamic rearrangements of synapto-dendritic networks, as well as by regulating the generation of new neuronal and glial cells, renders the brain highly mutable. These phenomena, collectively known as neuroplasticity, are critical to promote neuronal adaptations; its failure is now increasingly considered to be a major component in many neuropsychiatric conditions. Among these, depressive spectrum disorders are a paradigmatic example of the importance of neuroplastic alterations in the adult brain. Using an animal model of depression, we assessed the role of cell genesis and dendritic plasticity in the precipitation of multi-dimensional depressive-like behavior. Furthermore, the participation of these plastic processes in the immediate and long-term behavioral improvemement triggered by antidepressants was explored. Here, we found that ongoing neurogenesis is essential for the maintenance of behavioral homeostasis and that its arrest precipitates symptoms commonly found in depressed patients. Importantly, the incorporation of newly-born neurons and astrocytes into the pre-existing hippocampal neurocircuitry is shown to be necessary for the spontaneous recovery from stress and for the sustained therapeutic response to antidepressant drugs. Moreover, fast synapto-dendritic alterations are involved in the more immediate moodimproving effects of antidepressant drug. Taken together, our results suggest that the re-establishment of normal neuro-glial networks seems to be achieved in a bi-phasic manner: in a short-term context, ADs actions rely on rapid modulatory effects upon genes involved in the restructuring of the synaptic network; later on, the generation of new fully matured neuronal and glial cells will impact on the long-term remission from emotional and cognitive disabilities manifested during a depressive episode. Depression, Neuroplasticity, Antidepressants [email protected] 75

76 19:30 FIA10 Neural stem cells survive, differentiate and alleviate the motor phenotype of Machado-Joseph disease transgenic mice upon cerebellar transplantation Liliana S. Mendonça s Mendonça L. (1), Nóbrega C. (1), Kaspar B. (2), Pereira de Almeida L. (1,3) 1- Center for Neuroscience and Cell Biology, Vectors and Gene Therapy Group, University of Coimbra, Coimbra, Portugal. 2- The Research Institute at Nationwide Children's Hospital, Center for Gene Therapy, Ohio State University School of Medicine, Columbus, Ohio, USA. 3- Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. Machado-Joseph disease (MJD) is a progressive neurodegenerative disease caused by a mutation involving the over-repetition of the CAG trinucleotide on the ATXN3 gene translating into an expanded polyglutamine tract within the ataxin-3 protein. Mutant ataxin-3 causes neuronal dysfunction and degeneration in specific brain regions leading to severe motor symptoms. Transplantation of neural stem cells (NSC) has shown promising results in animal models of other neurodegenerative diseases. Thus, we investigated whether NSC transplantation would improve the motor phenotype of MJD transgenic mice. NSC were isolated from the cerebellum of postnatal day 1-3 wild type mice and cultured as neurospheres. The multipotency of the NSC was demonstrated by immunocytochemistry of neurospheres, which are positive for the multipotency marker Sox2 and give rise to different neural cells positive for astrocyte (GFAP), oligodendrocyte (NG2) and neuronal (β3-tubulin ) markers, upon differentiation. Moreover, the capacity of NSC to originate functional neurons was demonstrated through single cell calcium imaging. We observed that ± 8.01 % of cells responded to potassium but not to histamine stimulation, which is consistent with a neuronal-like profile. The transplantation of NSC by stereotactic surgery into the cerebella of 6 weeks old MJD mice resulted in a significant improvement of mice motor behavior performance in rotaroad, foot print and beam walking tests as compared to MJD mice injected with HBSS (2.14; 5.26; 1.69 and 2.88 fold improvement, respectively). Moreover, analysis of the mice cerebellum two months after NSC transplantation demonstrated that the injected NSC survived, migrated and underwent differentiation into GFAP, NG2 and β3-tubulin positive cells. In conclusion, this study indicates that NSC, give rise to neural cells that are able to survive, migrate and differentiate promoting a significant improvement on the motor behavior performance of transgenic MJD mice. MJD, NSC, cerebellar transplantation, motor behavioral tests [email protected] 76

77 19:35 FIA11 s Imbalance in the stem/progenitor cells properties leads to the suppression of neurogenesis in the triple transgenic mouse model of Alzheimer s disease Elisabete Ferreiro Elisabete Ferreiro1; Jorge Valero1; Liliana Bernardino1,2; Cláudia Pereira1,3; Fabienne Agasse1,4 1 Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. 2 Health Sciences Research Center, University of Beira Interior, Covilhã, Portugal; 3 Faculty of Medicine, University of Coimbra, Portugal; 4 Institut Curie, Paris, France Alzheimer s disease (AD) is a progressive neurodegenerative disease characterized by neuronal loss, cognitive dysfunction and dementia. In AD, a decrease in neurogenesis has been proven to be an early event in the occurrence of the disease that may play a role in the development of the neurodegenerative process. Neurogenesis occurs in the subgranular zone (SGZ) of hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ) lining the lateral ventricles. The reason why neurogenesis is compromised in AD is poorly understood. We hypothesized that stem cells intrinsic properties are impaired in AD brains. To test this hypothesis, proliferation, cell death, differentiation and stemness were analyzed in SVZ and DG neurospheres derived from P0-3 triple transgenic (3xTg-AD) mice, a model that mimics AD pathology in humans. 3xTg-AD cells, either from SVZ or DG, displayed increased numbers of BrdU- and TUNELpositive cells when compared to NonTg cell cultures. The numbers of NeuN-positive neurons were decreased while levels of GFAP astrocytes increased, suggesting an increment in gliogenesis to the detriment of neurogenesis in 3xTg-AD cell cultures. Regarding the stemness properties we observed the loss of self-renewal capacity of cells isolated from 3xTg-AD DG and SVZ. Moreover, we also observed that the protein levels of Sox2, a transcription factor required for self-renewal and pluripotency of stem cells was decreased in AD SVZ spheres. Finally, we used 3 month-old 3xTg-AD male slices to further explore the stemness properties in an early stage of the progression of the pathology. Our results indicate that there is a reduction in the number of Sox2 positive cells in the SGZ of 3xTg-AD mice. Taken together, these data show that stem/progenitor cell intrinsic properties are impaired in the 3xTg-AD mouse model of AD. The elucidation of the mechanisms underlying changes in neurogenesis may bring new perspectives and possibly new therapeutic targets for AD. Alzheimer s disease, Neurogenesis, Stemness, [email protected] 77

78 19:40 FIA12 Peripheral nervous system and bone: Advanced tools to study an essential interaction Estrela Neto s Estrela Neto 1,2; Paulo Aguiar 3; Ramiro Almeida 4 and Meriem Lamghari 1,5 1 INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal; 2 Faculdade de Medicina, Universidade do Porto, Portugal; 3 Centro de Matemática da Universidade do Porto, Portugal; 4 CNC Center for Neuroscience and Cell Biology, Coimbra, Portugal; 5 ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal Several evidences have showed the presence of nerve fibers in the bone microenvironment. The involvement of the peripheral nervous system (PNS) in the bone homeostasis is evidenced by the expression of different neuropeptides and neurotransmitters, as wells as its receptors, in bone cells [1, 2]. To unravel the cross-talk between bone cells and the PNS, namely nerve fibers within the bone microenvironment, new tools are required. Therefore we established a novel coculture system for osteoblasts (OBs) and dorsal root ganglia (DRGs) based on microfluidic devices. Embryonic or adult primary DRGs and MC3T3-E1 or primary osteoprogenitor cells were used and cell density tests were performed. The time of adhesion and readout of axonal outgrowth was improved by the alignment of DRGs, with the axis of microgrooves, which showed to be a crucial step for the designed experiments. For the first time, cocultures of entire DRGs from adult origin with OBs were performed showing DRGs extended projections towards the axonal compartment, reaching bone cells. Aiming to mimic the properties of tissue extracellular matrices, OBs were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices, where axonal outgrowth was still observed. To analyze and quantify data with reproducible image analysis, a semi-automated algorithm was developed. The spatial profile produces a fingerprint for the extent and properties of the axonal outgrowth (e.g. spatially constant, decay exponentially or even produces large axonal sprouting). It was observed that the collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Nonetheless, axons were able to reach longer distances in the presence of alginate. Overall, the explored methods revealed to be a suitable tool to study the interaction between peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario. Sensory neurons, osteoblastic lineage, non-contiguous coculture, microfluidics [email protected] 78

79 Cell signaling I May 30 Plátano room 79

80 18:45 FIB1 s Contactin-associated proteins 1 and 2 (Caspr1 and Caspr2): novel players in the activity-dependent regulation of synaptic AMPA receptors. Dominique Fernandes Dominique Fernandes (1,2,3), Luís Ribeiro (3), Sandra Santos (3), Ana Luísa Carvalho (3) (1) PDBEB, Doctoral Programme in Experimental Biology and Biomedicine, UC, PT; (2) Institute for Interdisciplinary Research, UC, PT; (3) Center for Neurosciences and Cell Biology & Department of Life Sciences, UC, PT Phenomena such as learning and memory rely on the expression of several forms of plasticity such as long-term potentiation and homeostatic synaptic plasticity. These mechanisms are highly dependent on the regulation of the synaptic availability of glutamate receptors of the AMPA-type (AMPAR), at several levels. Nevertheless, these regulatory mechanisms remain poorly understood. We have recently identified the cell-adhesion molecule Contactin-associated protein 1 (Caspr1) as a novel interactor of AMPARs, capable of regulating the trafficking of the GluA1 subunit into synapses. We now found evidence for an additional posttranscriptional role for Caspr1 in increasing GluA1 mrna and regulating the phosphorylation of the RNA-binding protein Zipcode-binding protein 1 (ZBP1). Our results indicate that ZBP1 binds to GluA1 mrna, and that this interaction is significantly decreased upon chronic changes in neuronal activity, suggesting an activity-dependent release of GluA1 transcripts to undergo translation. Indeed, we show that chronic blockade of activity significantly upregulates not only total mrna levels and surface synaptic puncta of GluA1, but also phosphorylation levels of ZBP1. Additionally, we demonstrate that the expression of Caspr1 is regulated by neuronal activity, since its total protein and mrna levels increase upon homeostatic stimuli. Furthermore, we show that synaptic puncta of another member of the Contactinassociated protein family, Caspr2, significantly increase when neuronal activity is chronically blocked, suggesting a possible function for Caspr2 in this process. Curiously, Caspr2 has been implicated in autism-spectrum disorders and identified as an antigen in autoimmune synaptic encephalitis. Taken together, our results suggest a promising role for Contactin-associated proteins in regulating plasticity phenomena. Synaptic plasticity, AMPA receptors, Contactin-associated proteins, posttranscriptional control, receptor trafficking [email protected] 80

81 18:50 FIB2 Erythropoietin modulates activity deprivation-induced regulation of synaptic transmission: a role for a hormone in homeostatic plasticity? Raquel B Dias s Raquel Baptista Dias 1,2, Diogo M. Rombo 1,2, Jennifer McGarvey 3, Jeremy M. Henley 3 Ana M. Sebastião 1,2 1 Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa 2 Unidade de Neurociências, Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal 3 Medical Research Council Centre for Synaptic Plasticity, School of Biochemistry and School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom Erythropoietin (EPO) is a pleiotropic growth hormone best known for its actions in the hematopoietic system. Other tissues also express EPO receptors, such as the brain, where its anti-apoptotic and anti-inflammatory properties protect from ischemia. Beyond this neuroprotective role, EPO may enhance recovery from ischemic injury through its ability to modulate synaptic plasticity processes such as long-term potentiation. It is unclear whether EPO is also able to affect complementary homeostatic plasticity triggered by activity deprivation following input loss, in stroke. We used an in vitro protocol to simulate activity deprivation and tested the influence of EPO upon excitatory synaptic transmission. Hippocampal slices were incubated with tetrodotoxin (1µM) and the NMDA receptor antagonist DL-APV (50µM) for 3h. Activity-deprived neurons had similar resting membrane potentials and action potential firing thresholds to control neurons (n=3-4, p>0.05), but showed a significant increase (n=6; p<0.05), in both the amplitude and frequency of miniature excitatory postsynaptic currents (mepscs), suggesting that activity deprivation induces a compensatory increase in synaptic transmission. Cotreatment with EPO (2.4 IU/mL) prevented the increase in mepsc frequency (p>0.05, n=4). When acutely applied to non-activity deprived neurons, EPO (2.4 IU/mL, min) decreased mepsc frequency and tended to increase mepsc amplitude (p>0.05, n=3). EPO (2.4 IU/mL) superfusion also caused an increase in surface GluR2 expression compared to vehicle alone (p<0.05, n=4-10), as assessed by live-imaging of neurons expressing Super Ecliptic Phluorin-GluR2. These results suggest that EPO modulates homeostatic plasticity, likely by coordinating pre- and post-synaptic changes of excitatory synaptic transmission that are triggered in response to activity deprivation. Possible implications upon circuit remodeling in physiological and/or pathological conditions await further investigation. Erythropoietin, homeostatic plasticity, excitatory synaptic transmission [email protected] 81

82 18:55 FIB3 Transcriptional programs regulated by Prrxl1 in developing dorsal root gangliadorsal spinal cord nociceptive circuit Filipe Almeida Monteiro s F. A. Monteiro (1), C. B. Monteiro (1), A. Raposo (2), A. Clavairoly (3), S. Rebelo (1), M. Matos (1), M. F. Costa (1), C. Reguenga (1), C. Parras (3), F. Guillemot (4), D. S. Castro (2) and D. Lima (1) (1) Departamento de Biologia Experimental, Faculdade de Medicina do Porto e Morfofisiologia do Sistema Somatosensitivo, IBMC - Instituto de Biologia Celular e Molecular, Portugal, (2) Molecular Neurobiology, IGC - Instituto Gulbenkian de Ciência, Portugal, (3) Développment Oligodendrocytaire et Interactions Neurovasculaires, ICM - Institut du Cerveau et de la Moelle épinière, France, (4) Division of Molecular Neurobiology, NIMR - National Institute for Medical Research, UK Nociceptive system transduces external stimuli into pain sensations which are essential as a warning system. Prrxl1 is a homeodomain transcription factor (TF) expressed in distinct regions of the developing nociceptive system, including the dorsal root ganglia (DRG) and their central synaptic targets at dorsal spinal cord (DSC). Prrxl1 null mice have shown several developmental defects, including migration, differentiation, axon guidance and survival of nociceptive neurons. Although the cellular functions of Prrxl1 in wiring DRG-DSC nociceptive circuit appear well characterized, the target genes downstream are mostly unknown To compare the functions of Prrxl1 in each neuronal lineage, we combined ChIP-seq with an expression microarray analysis comparing wild type and Prrxl1 null embryos. ChIP-seq analysis shows a remarkable different genomic binding profile for Prrxl1 between developing DRG and DSC. DNA motif search identifies, in addition to the homeodomain binding site, a Runx consensus site in vicinity of Prrxl1 binding sites, specifically in the DRG. Given that Runx1 is co-expressed with Prrxl1 in DRG nociceptors and that knockout mice for both genes are functionally related, Runx1 is likely a cofactor for Prrxl1. Association of binding with transcriptional profiling revealed fairly distinct lists of target genes for both regions. According to gene ontology analysis, there is a significant enrichment for target genes with roles in neurodevelopment, including migration, projection morphogenesis, axon guidance and synaptic transmission. A systematic analysis of the expression patterns shows that the majority of target genes are expressed in Prrxl1 expression domains. Our work is providing a molecular framework for the cellular functions of Prrxl1 in developing nociceptive system. Importantly, our work resulted in the identification of tissue specific targets of Prrxl1, and suggests this TF participates in distinct transcriptional networks in DRG and DSC regions. DRG-dorsal spinal cord nociceptive system, Prrxl1, ChIP-seq, microarray [email protected] 82

83 19:00 FIB4 s Role of local protein synthesis in presynaptogenesis Luís Martins Luís Martins, Maria Joana Pinto, Maria Inês Coelho, Hyun R. Ryu, Noo Li Jeon, Ramiro D. Almeida CNC - Center for Neuroscience and Cell Biology, University of Coimbra The mechanism of local protein synthesis in dendrites and axons is currently under intense investigation. Recent studies identified a large number of mrnas localized at distal axons and growth cones, suggesting that local axonal translation may play an important role in different steps of neuronal development. In line with these evidences, early studies in axons demonstrated the requirement of local translation during axon chemotrophic responses to guidance cues. Moreover, it was demonstrated that local axonal translation is required for other neurodevelopmental mechanisms, such as axonal outgrowth, neuronal survival and axon regeneration. Interestingly, recent studies in Aplysia, suggest that local translation might be important for synapse formation. However, the role of local protein synthesis in presynaptic differentiation is largely unknown. The purpose of this study was to determine if local protein synthesis is required for presynaptogenesis. Using a novel platform, a microfluidic chamber system which allows the physical separation of axons from cell bodies and dendrites, we were able to specifically manipulate axons without the cell body contribution. Our results demonstrate that FGF22 induced the clustering of synaptic vesicles, a hallmark of presynaptic assembly, when added specifically to axons. Moreover, FGF22 leads to the phosphorylation of 4E-BP1, a translational repressor, in an asymmetric pattern. Our findings also demonstrate that two mechanistically distinct protein synthesis inhibitors block FGF22-induced presynaptogenesis when applied specifically to axons. Taken together our results show that FGF22 activates cap-dependent translation and that this intra-axonal mrna translation is required for presynaptic differentiation in CNS neurons. This work was supported by FCT and COMPETE (PTDC/SAU-NEU/104100/2008) and by Marie Curie Actions, 7th Famework programme. Local protein synthesis, Presynaptogenesis, FGF22, 4E-BP1 [email protected] 83

84 19:05 FIB5 s Modulation of Glucocorticoid Receptors by adenosine A2A receptor activation: from gene to protein level. Vânia L Batalha1 Vânia L Batalha1, Malika Hamdane2, Luc Buée2, David Blum2, Ghazaleh Sadri- Vakili3 and Luísa V. Lopes1 1IMM, Fac.Med.UL, Portugal; 2Jean-Pierre Aubert Res.Ctre. Univ. Lille-Nord de France,France; 3HMS, Neur. Dep.,Boston,MA,USA Chronic stress induces permanent changes in the central nervous system. We ve shown that in stressed animals the blockade of adenosine A2A receptor (A2AR) can reestablish the decreased glucocorticoid receptor (GR) levels in the hippocampus and, more importantly, the deleterious effects of stress in learning tasks, synaptic plasticity and dendritic retraction (Batalha et al.,2013, Mol.Psychiatry, 18:320). We therefore hypothesized that A2AR have a direct impact on GR activation and/or transcriptional regulation. The effect of A2AR modulation (agonist-cgs21680,30nm or antagonist- SCH58261,50nM) in dexamethasone (100nM) induced GR transcriptional activity was evaluated N1E115 cells transiently transfected with PGL(GRE)3tk vector encoding luciferase under the control of Glucocorticoid-Response-Element (GRE). A2AR activation increased dexamethasone induced GR-dependent transcriptional activity by 41±8% (n=10,p<0.01), whereas blockade decreased it by 28±5% (n=7,p<0.001). Concomitant observations in primary neuronal cultures show that A2AR blockade prevents dexametasone induced GR nuclear translocation. We evaluated A2AR impact in GR gene (Nr3c1) regulation, in the hippocampus of week-old male Wistar rats, by measuring its association with acetylated histone H3. The chronic blockade of A2AR (KW6002; 3mg/Kg/day during 1 month) increased histone H3 acetylation in Nr3c1 non-coding regions (149±47% in CTR vs 353±93% in CTR+KW,P<0.05; and 129±42% in MS vs 379±96% in MS+KW,n=4,P<0.05). Plus, the treatment reverted the stress-induced increase in H3 acetylation of the Nr3c1 coding regions (252±42% in MS to 94±34% in MS+KW,n=5,P<0.05). These results show that A2AR may play a direct role in GR activation. Moreover they provide the first evidence that A2A endogenous activation impacts on chromatin modulation. Overall these observations may reveal part of the mechanism underlying the therapeutic effects achieved by A2AR blockade against stress-induced damage. Stress, Adenosine, Epigenetics, Glucocorticoids, Hippocampus [email protected] 84

85 19:10 FIB6 Beyond synaptic plasticity - storing information in the intrinsic excitability properties of neurons Paulo Aguiar s Eduardo Conde-Sousa (1) and Paulo Aguiar (1,2) (1) Centro de Matematica da Universidade do Porto; (2) Instituto de Biologia Molecular e Celular One of the fascinating features of every nervous system is its plasticity; that is, its ability to store perceptual experiences and use this stored information (memories) in future. Findings by Kandel, Bliss and Lomo, have led to the widely held view that memories are stored as modifications in synaptic strength. Since this outstanding work, many models have successfully used synaptic plasticity as the basis for mechanisms related to learning, memory and development in neural circuits. But synaptic plasticity is not, by far, the only adaptive processes available on the nervous system, with the suitable properties for memory formation. Parallel experimental studies have demonstrated information storage based on non-synaptic processes, with the modulation of membrane conductances being one of the most important among them. These mechanisms remain however quite unexplored. Here we present a model for one-shot short-term memory which relies on the modulation of the intrinsic excitability properties of neurons, instead of synaptic plasticity, to retain novel information for periods of seconds to minutes. We show that ubiquitous types of membrane currents, such as long-lasting calcium currents, calciumactivated nonspecific cation current or persistent sodium currents, can act synergistically to provide short-term memory buffers. Memory is stored for periods of seconds to minutes, not on synapses, but on the intrinsic excitability and activity of neurons. Postulating a specific class of neurons we also show that it is also possible to effectively use this mechanism to store the serial order in a sequence of patterns of activity. Our model exhibits properties which are in very close agreement with experimental results regarding working memory. Namely, the recall process plays an important role in stabilizing and prolonging the memory trace. This means that the stored information is correctly maintained as long as it is being used (see Conde-Sousa and Aguiar, JCNS 2013). non-synaptic plasticity, intrinsic excitability modulation, computational neuroscience, short-term memory, one-shot learning [email protected] 85

86 19:15 FIB7 s Caffeine exposure during pregnancy interferes with adenosine A2A receptors affecting hippocampal GABAergic networks in offspring resulting in memory impairment in adulthood Angelo Tomé Carla G. Silva1,2,3, A. Walid Fazeli4, Christine Métin5,6, Nuno J. Machado1, Sanja Darmopil2,3, Pierre Launay5.6, Antoine Ghestem2,3, Marie-Pascale Nesa2,3, Emilie Bassot2,3, Eszter Szabó1, Younis Baqi7, Christa E. Müller7, Angelo R. Tomé1,8, Anton Ivanov2,3, Dirk Isbrandt4, Yuri Zilberter2,3, Rodrigo A. Cunha1,9, Monique Esclapez 2,3, Christophe Bernard2,3 1Center for Neurosciences of Coimbra, University of Coimbra, Portugal; 2INSERM UMR1106, Inst. Neurosci. Systèmes, Marseille, France; 3Aix-Marseille U., Marseille, France; 4Experimental Neuropediatrics, U. Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5INSERM UMR- S 839, Institut du Fer à Moulin, Paris, France; 6U. Pierre & Marie Curie, Paris, France; 7PharmaCenter Bonn, U. Bonn, Germany; 8Dep Life Sciences, Fac. Sci. & Tech., U. Coimbra, Portugal; 9Fac. Medicine, U. Coimbra, Portugal Psychoactive drug consumption during pregnancy cause deleterious long-term neuropsychiatric consequences. Caffeine, the most commonly used psychoactive substance, is also consumed by pregnant women and may interfere with developmental programs since it crosses the placenta and brain blood barrier. We now tested the impact of caffeine exposure during development on the long term functioning of hippocampal networks. Female mice were exposed to 0.3 g/l caffeine in their drinking water, starting 2 weeks before mating and up to 2 weeks postpartum. We found that caffeine, or the adenosine A2A receptor (A2AR) antagonist KW6002, delayed the migration of GABAergic neurons and their insertion into the hippocampal circuitry, increasing network excitability during the first postnatal week. Early life caffeine exposure also had long-term consequences in adult offspring, typified by impaired behavior in the novel object recognition and modified Y maze tests, with no change in locomotion (open field) or anxiety (elevated plus maze); this was accompanied by a marked neuronal loss (NeuN staining) in the adult stratum oriens, mostly accounted by loss of somatostatincontaining GABA neurons (30% decrease). This translated into a reorganization of the circuitry in adult treated offspring with a 43% increased frequency of sipscs and 46% decreased frequency of sepscs (by 46%) in CA1 pyramidal cells. All the reported long term morphological, electrophysiological and behavioural effects caused by early life caffeine exposure were mimicked by KW6002 (2 mg/kg/day, starting 15 days before mating up to weaning at P15) and the effects of caffeine were wiped out in global A2AR knockout mice. These results show the deleterious effects of maternal caffeine consumption during development, through its action on A2AR, compromising the structural and functional properties of neuronal networks in offspring. (Supported by FCT and DARPA) caffeine, development, adenosine, memory [email protected] 86

87 19:20 FIB8 s Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: radiochemical and real-time video-microscopy studies Paulo Correia-de-Sá Paulo Correia-de-Sá*, José B. Noronha-Matos*, M.Alexandrina Timóteo*, Fátima Ferreirinha*, Patrícia Marques*, Andreimar M. Soares, Cicilia Carvalho, Walter L. G. Cavalcante, Márcia Gallacci *Laboratório de Farmacologia e Neurobiologia / UMIB, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal; Universidade Federal de Rondônia, UNIR, Brazil; Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil. Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalyticallyindependent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 µm) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. Work supported by CAPES (Brazil) and by FCT (Portugal, FEDER funding, PEst- OE/SAU/UI0215/2011). J.B.N.M. is in receipt of a PhD Fellowship from FCT (SRFH/BD/68584/2010). Bothropstoxin-I; [3H]-Acetylcholine release; Real-time transmitter exocytosis; Neuromuscular junction. [email protected] 87

88 19:25 FIB9 Neuropeptide Y modulates retinal ganglion cell activity and protects retinal ganglion cell against excitotoxicity João Martins s João Martins, F. Elvas, D. Brudzewsky, B. Kolomiets, S. Picaud, C. Cavadas, M. Castelo-Branco, A.R. Santiago, A.F. Ambrósio Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra The apoptosis of retinal ganglion cells (RGCs) is a hallmark of several retinal degenerative diseases, particularly glaucoma. Neuropeptide Y (NPY) is a neuromodulator in central nervous system that can exert potent neuroprotective effects. The aims of this work were to investigate the modulatory and neuroprotective potential of NPY on RGCs. RGC spiking activity was recorded in an ex-vivo rat retinal preparation using a multielectrode array (MEA). The application of 1 µm NPY and 1 µm (Leu31,Pro34)- NPY (NPY Y1 / Y5 receptor agonist), for 10 min, decreased NMDA-stimulated RGC spiking activity. In purified RGC cultures, the application of 1 µm NPY for 10 min also reduced the increase in the [Ca2+]i triggered by repeated 30 µm glutamate in 33% of RGCs on average, compared to 17% in basal conditions. The protective effect of NPY against RGC death was assessed in cultured retinal explants using propidium iodide (PI) and TUNEL assays. Exposing retinal explants to 300 µm NMDA for 48 h significantly increased the PI-positive cell ratio (DIV4/DIV2) and the number of TUNEL-positive cells in RGC layer. Pre-treatment with 1 µm NPY prevented the PI ratio and TUNEL increase triggered by NMDA. A pharmacological approach revealed a major contribution of Y1 and Y5 receptors mediating the protective effect of NPY. In conclusion, NPY is able to modulate the activity and [Ca2+]i changes of RGCs. Moreover, NPY exerts a neuroprotective effect against retinal cell death triggered by NMDA, possibly involving Y1 and Y5 receptor activation. Support: FCT: SFRH/BD/44817/2008, Grant PTDC/SAU-NEU/099075/2008, and Strategic Project PEst-C/SAU/UI3282/2011, and COMPETE/FEDER; Portugal. Retina, Neuropeptide Y, Neuroprotection [email protected] 88

89 19:30 FIB10 The adenosine facilitation of synaptic plasticity in cortico-striatal synapses involves astrocytic A2AR receptors Daniel Rial s Daniel Rial1,2, Henrique B. Silva1, Jiang-Fan Chen3, Rui D.S. Prediger1, Rodrigo A. Cunha1,4. 1CNC-University of Coimbra, Portugal; 2Departament of Pharmacology, UFSC;3Neurology Boston USA;4Medicine, Coimbra University Adenosine controls synaptic transmission and plasticity through a balanced activation of inhibitory A1 and facilitatory A2A receptors (A2AR). A2AR are mainly located in striatal medium spiny neurons (MSN), which are activated by cortical projection. We decided to test if the key neurophysiological trait of incoming information to be processed by striatal circuits, i.e. cortico-striatal plasticity, was controlled by the most abundant A2AR located post-synaptically in MSN. We compared the amplitude of longterm potentiation (LTP, triggered by stimulation of cortical afferent with 3 trains at 100 Hz for 1 s with 10 s interval) in cortico-striatal slices, in absence and in presence of the selective A2AR antagonist, SCH58261 (50 nm) in 3 mouse lines: wild type (WT), global A2AR knockout (KO) and striatum-a2ar-ko, where A2AR are selectively deleted from MSN; in WT mice, we also tested the impact of inhibitors of the muscarinic (atropine 1 µm) and astrocytic glutamate uptake systems (DL-TBOA 50 µm). In WT mice, LTP amplitude (after 30 min) was 127+4% of basal and SCH58261 inhibited LTP by 57±4% (n=4). In global-a2ar-ko mice, LTP had similar amplitude, but SCH58261 was devoid of effects (n=4). In striatum-a2ar-ko mice, LTP had an amplitude of 115±5% and SCH58261 inhibited LTP by 58±5% (n=4), ruling out a participation of post-synaptic A2AR in the control of LTP. Atropine by itself did not change LTP amplitude nor did it interfere with the effect of SCH58261 on LTP (n=4), ruling out the participation of the cholinergic system in the A2AR-mediated control of LTP. Interestingly, DL-TBOA was devoid of effects per se, but attenuated the ability of SCH58261 to inhibit LTP (45+12% inhibition alone and 72+10% in the presence of DL- TBOA, n=3). These results suggest that astrocytic A2AR controlling glutamate uptake (Matos et al., 2012, Glia 60: 702) are involved in the adenosine-mediated facilitation of LTP in cortico-striatal synapses. synaptic plasticity, adenosine A2A receptors, astrocytes [email protected] 89

90 19:35 FIB11 s Effect of BDNF on synaptic and extrasynaptic NMDA currents Covita J Covita J, Rombo D, Dias RB, Sebastião AM, Diógenes MJ Unidade de Neurociências do Instituto de Medicina Molecular; Instituto de Farmacologia e Neurociências da Faculdade de Medicina, Universidade de Lisboa N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that can be divided into two distinct populations according to their localization: synaptic and extrasynaptic receptors. While activation of synaptic NMDA receptors promotes neuronal survival and gene expression, extrasynaptic receptors appear to have the opposite action. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that has emerged not only as an essential mediator of neuronal survival and development, but also as an important modulator of synaptic transmission. BDNF has been shown to facilitate synaptic transmission in adult rat hippocampus, an effect dependent on NMDA receptor activation. However, the differential interactions between BDNF and synaptic and extrasynaptic NMDA receptors are yet to be explored. Therefore, the goal of this work was to evaluate the modulatory effects of BDNF on synaptic and extrasynaptic NMDA currents. Hippocampal slices (300 µm) were prepared from adult Wistar rats and patch-clamp recordings were obtained from CA1 pyramidal neurons. NMDA currents were recorded in the presence of picrotoxin (100µM), a GABAA receptor antagonist, CNQX (50µM), an AMPA and Kainate receptor antagonist, and glycine (50µM). While evaluating NMDA extrasynaptic currents, DL-TBOA (50µM), a glutamate transporter inhibitor, was also added to the perfusion medium We found that BDNF (20ng/mL) had a depressing action on NMDA currents (n=5, p<0.05), an effect slightly attenuated in the presence of memantine (1 µm; n=5), an NMDA receptor antagonist with greater affinity for extrasynaptic receptors at the concentration used (n=3; p<0.01). BDNF s inhibitory effects on NMDA currents were significantly greater on extrasynaptic currents when compared to synaptic currents (n=3; p<0.01). The work shows that BDNF preferentially inhibits extrasynaptic NMDA currents, suggesting that BDNF differentially modulates synaptic and extrasynaptic NMDA receptor activity. Support: FCT and Regeneron NMDA receptors, synaptic, extrasynaptic, BDNF [email protected] 90

91 19:40 FIB12 s The role of β-actin mrna translation in axonal outgrowth Joana R. Pedro Joana R. Pedro, Luis Leitão and Ramiro D. Almeida Center for Neuroscience and Cell Biology, Coimbra The growth cone is a highly motility structure present at the tip of the axon. It responds to guidance molecules therefore regulating axon outgrowth, turning and pathfinding. The autonomous and rapid response of the growth cone is dependent on local mrna translation. This is a mechanism by which neurons are able to rapidly supply subcellular compartments with newly synthesized proteins. One of the first transcripts found in distal axons and growth cones was beta-actin mrna. It has been shown that β-actin is locally synthesized in the growth cone, mediating axonal turning. However, the relevance of local β-actin mrna translation in axonal outgrowth is not fully elucidated. This study focused on the role of local mrna translation in NGF-induced axonal outgrowth. We found that global application of NGF increases F-actin levels at the growth cone as measured by phalloidin intensity. Moreover, making of use of microfluidic chambers, which allow a physical and fluidic isolation between axons and cell bodies, we specifically stimulated axons and evaluated cue-induced axonal events. We observed that local application of NGF induces an augment in axonal length and an increase of actin polymerization (F-actin) in growth cones. Additionally, when protein synthesis is inhibited in axons, the NGF-induced increase in F-actin levels is reduced to basal levels. All together these results indicate that local mrna translation is required for actin remolding during NGF-induced axonal outgrowth. Joana R. Pedro is supported by Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BD/77789/2011). This work is funded by FEDER funds through Programa Operacional Factores de Competitividade COMPETE and by National Funds through FCT Fundação para a Ciência e Tecnologia under the project PTDC/SAU- NEU/104100/2008 and by Marie Curie Actions, 7th Framework programme, EU. b-actin, axonal outgrowth, local translation, NGF, [email protected] 91

92 Neurodegeneration and neuroprotection I May 30 Auditorium 92

93 18:45 FIC1 s Short-term treatment with TUDCA attenuates the amyloidogenic processing of amyloid precursor protein in APP/PS1 mice Amaral JD Amaral JD, Dionísio PA, Amaral JD, Lo AC, D Hooge R and Rodrigues CMP Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal and Laboratory of Biological Psychology, University of Leuven, Leuven, Belgium Tauroursodeoxycholic acid (TUDCA) is an anti-apoptotic bile acid with potent neuroprotective properties in several experimental models of Alzheimer s Disease (AD). We have previously reported the therapeutic efficacy of TUDCA in APP/PS1 doubletransgenic mice fed with a diet containing TUDCA, before amyloid plaque deposition. In this study, we evaluated the protective effects of TUDCA when administrated after the onset of amyloid pathology. APP/PS1 transgenic mice with 7 months of age were injected 500 mg/kg bw TUDCA every 3 days for 3 months. Mice were tested for the Morris water maze task at 9 months, and sacrificed at 10 months. One hemisphere of the brain was fixed, paraffin-embedded, sectioned and stained with Thioflavin T, a general marker of amyloid deposits. The other hemisphere was snap-frozen and processed for total protein extracts and Western blot analysis. APP/PS1 mice treated with TUDCA showed a trend to improvement in memory and spatial learning compared to control APP/PS1 mice. A reduction of amyloid plaque number was evident in the hippocampus of TUDCA-treated transgenic mice. In addition, TUDCA treatment modulated the protein levels of several lipid metabolism mediators, including the connective tissue growth factor (CTGF) and sortilin-related receptor with A-type repeats (SORLA). More importantly, COOH-terminal fragments produced by the γ-secretase cleavage of APP (APP-CTF-γ), and the respective soluble APP-β fragments were significantly decreased in TUDCA-treated transgenic mice compared to respective controls, pointing toward a reduction in the amyloidogenic processing of APP by TUDCA. Whether TUDCA is solely modulating the amyloidogenic processing of APP, or is also affecting the clearance rate of APP-processed fragments is still under investigation. These results suggest that TUDCA is a potential therapeutic option not only for prevention, but also for treatment of AD after the onset of the disease. TUDCA, APP processing, APP/PS1 mice [email protected] 93

94 18:50 FIC2 s The effect of development and normal aging in cortical/retinal asymmetries: lowlevel evidence for the HAROLD aging model Maria Fátima Silva Maria Fátima Silva, Otília C. d Almeida, Catarina Mateus, Miguel Castelo-Branco Visual Neuroscience Laboratory, IBILI, Faculty of Medicine, University of Coimbra, Portugal The effects of aging on brain activity during cognitive performance have been conceptualized in terms of a model called hemispheric asymmetry reduction in older adults (HAROLD). This model has been supported by functional neuroimaging and other evidence in various cognitive domains. We have previously documented low-level interhemispheric (left/right), superior/inferior and retinal (nasal/temporal) patterns of asymmetry based on contrast sensitivity (CS) psychophysical measurements. It is well known that aging affects CS, but its relation with visual hemifield asymmetries is still poorly understood. Our main goal was to explore cortical/retinal asymmetries in development and normal aging using low-level CS behavioral tasks, probing distinct spatiotemporal frequency channels. CS was measured using stimuli with distinct properties: intermediate (ISF: 3.5 cycles per degree (cpd) and 0 Hz; 303 eyes; 7-72 years, sampled in five age groups) and low spatial frequency (LSF: 0.25 cpd undergoing 25 Hz counterphase flicker; 311 eyes; years). Using ISF task, a significant cortical hemifield effect was found only for young adults (p=0.002; left hemifield advantage corresponding to right hemisphere). The superior/inferior asymmetry was not present in children but increased with aging, with enhancement of the inferior hemifield advantage (p<0.001). Retinal asymmetries were present with a temporal hemifield advantage. Concerning the LSF task, only adolescents/adults showed cortical/retinal asymmetries (p=0.005; higher CS in left, inferior and nasal hemifields). We conclude that visual asymmetries with a direct ecological meaning (up/down at the highest spatial frequency) emerge during development and aging, whereas retinal forms of anisotropy tend to stabilize or decline. Interhemispheric asymmetries are more specific to young adults accordingly to the age-related hemispheric asymmetry reduction model, yielding a low-level evidence of the HAROLD model. human aging, HAROLD model, contrast sensitivity, spatial vision, visual asymmetries [email protected] 94

95 18:55 FIC3 DJ-1 neuroprotection in oxidative stress: insights into Parkinson's Disease pathogenesis Sandra Anjo s Sandra Anjo1; José Carvalho1, 2; Vera M. Mendes1; Mário Grãos3; Bruno Manadas1, 3 1Center for Neuroscience and Cell Biology, Cantanhede, Portugal; 2University of Coimbra, Coimbra, Portugal; 3Biocant - Biotechno Background and aims: DJ-1 has been pointed as playing major roles in Parkinson s disease. The DJ-1 most relevant function to PD is its role in neuroprotection, most likely by its involvement in protection against oxidative stress, a major player in PD pathogenesis. How this protein exerts this neuroprotection is not fully understood, but mediation of signaling pathways seems to play the most significant role. Moreover, several studies have indicated that oxidative stress is the trigger condition for its neuroprotective function. Therefore, the aim of the present study was to provide a comprehensive characterization of DJ-1 dynamic interactome under oxidative stress conditions, which will help to elucidate the mechanisms through which DJ-1 exerts its response to oxidative challenges. Methods: To identify DJ-1 putative partners, a dynamic interactomics approach was conducted. Experimental conditions were defined based on the kinetics of ERK and PI3- K/Akt pathways activation on SH-SY5Y cells under oxidative stimuli. Immunoprecipitation was used to isolate the endogenous DJ-1 and associated proteins. Isolated complexes were analyzed by gel based liquid chromatography coupled to tandem mass spectrometry approach. Results: A large number of proteins were indentified, including phosphatases, oxidoreductases, and proteins involved in DNA repair, gene expression and protein folding. Most of the proteins were identified as DJ-1 interacting partners for the first time, and present a broad range of biological functions mainly associated with cellular response to stress. Conclusions: This approach led to a characterization of the dynamic interactome of DJ-1 under oxidative stress conditions. The several novel binding partners identified point to new mechanisms for DJ-1-mediated neuroprotection. Finally, many of the proteins identified are well established in distinct cellular functions implicated in PD, thus, these results will also contribute to a better understanding of PD. DJ-1, Interactome, Neuroprotection, Oxidative Stress, Parkinson's Disease [email protected] 95

96 19:00 FIC4 s Is APP intracellular domain, AICD, a key player in Alzheimer s disease? Paula Pousinha Paula Pousinha, Elisabeth Raymond, Eric Duplan, Fréderic Checler and Hélène Marie. Institut de Pharmacologie Moléculaire et Cellulaire - CNRS A strong focus in Alzheimer s disease (AD) research has been to investigate the pathological roles of the Amyloid beta (Ab) peptide and the protein tau. However, it is becoming clear that Ab or tau accumulation per se are unlikely to be the sole causes of AD. The intracellular domain derived from APP proteolysis, AICD, also accumulate in AD patient brains together with Ab, but its pathological effects have yet to be clearly defined. It is therefore crucial to investigate the influence of AICD on brain function. To study the influence of AICD over-expression on hippocampal neuronal excitability and glutamatergic synaptic function, we have used an adeno-associated viruses (AAV) enconding AICD together with GFP to over-express them in rat hippocampal CA1 pyramidal neurones in vivo, by means of stereotaxic surgery. AAV injections in CA1 area were done at P days after infection, hippocampal slices were obtained. Patch-clamp whole cell recordings of infected neurons were performed. AICD-GFP infected neurons showed an impairment in neuronal excitability. The mean frequency of spiking as a response to depolarizing injected currents was decreased in these neurons, when compared to GFP infected neurons; an effect that was neither related to Na+ conductance, since both spike threshold or dv/dt ratio were not different between groups; nor to changes in spiking accomodation, since the Fo/Fss ratio was not modified. AICD-GFP infected neurons showed a significant increase in the action potential s AHP, and also a reduction in the magnitude of Ih. AICD-GFP infected neurons showed a significant decrease in the AMPAR/NMADAR currents ratio an effect that is due to the post-synaptic neuron, since the paired-pulse ratio was similar between groups. In conclusion, these results suggest that some of AD feautures, until now associated with Ab accumulation, can be caused by AICD over-expression, reinforcing the hypothesis that AICD might be a key player in AD pathology. Alzheimer's Disease, AICD, CA1 pyramidal neurons, electrophysiology, AAV [email protected] 96

97 19:05 FIC5 s Structural plasticity on visual cortical representations induced by silent visual loss in a subtle ganglion cell degeneration model Otília C. d Almeida O. C. d Almeida, C. Mateus & M. Castelo-Branco Visual Neuroscience Laboratory, IBILI Faculty of Medicine, University of Coimbra, Portugal Visual loss is often associated with neural atrophy. However, previous studies proposed that primary sensory areas of the human visual cortex retain a certain degree of plasticity through lifespan, and overt retinal lesions may induce visual cortical reorganization. Likewise, developmental plasticity may occur in preclinical stages of retinal degeneration. We studied the cortical impact of non scotomatous silent retinal ganglion cell degeneration, in carriers of the mitochondrial DNA 11778G>A mutation causing Leber s hereditary optic neuropathy. We have tested 15 asymptomatic LHON carriers (age range: 8-47y) and 15 subjects from an age-matched control group (age range: 7-44y). All participants were submitted to a complete ophthalmological examination and to a 3T MRI acquisition. Anatomical MRI images were used to measure cortical thickness by the Laplacian method and fmri retinotopy paradigms were used to define cortical visual areas V1, V2 and V3 in silent carriers and controls. Surprisingly, ANOVA analysis showed that cortical thickness was significantly higher in the LHON carrier group. Setting a developmental age cut-off of 21 years old, we found that mean cortical thickness in LHON group was, on average, higher than in the control group both below and above 21 years. This main effect was more pronounced extrastriate cortex, namely in V2 in younger subjects and in V3 in older subjects, suggesting enhanced plastic developmental mechanisms in neighbouring regions not receiving direct retinocortical input from lateral geniculate nucleus. These results show that sensory deprivation in a preclinical disease model of silent ganglion cell death leads to differential regionally-specific plasticity of regions of human visual cortex. These early effects are age dependent, suggesting that neuroplasticity relies on temporal interactions between neurodevelopmental and silent neurodegeneration processes. Cortical plasticity, retinotopic visual areas, ganglion cell loss, neurodevelopment, mitochondrial gene mutations [email protected] 97

98 19:10 FIC6 s Mitochondrial dysfunction in diabetes and Alzheimer s disease: the missing link? Cristina Carvalho Cristina Carvalho, Maria S. Santos, Catarina R. Oliveira, Paula I. Moreira Center for Neuroscience and Cell Biology, University of Coimbra, Portugal Alzheimer s disease (AD) is the most common form of dementia among people aged 65 and older, affecting more than 35 million people worldwide. Growing evidence highlights a direct relationship between AD and type 2 diabetes (T2D), with T2D patients often suffering from a higher prevalence of mood symptoms and cognitive dysfunctions than non-diabetic age-matched patients. Besides aging, T2D is considered a major risk factor for AD development. We hypothesize that mitochondrial alterations are a common feature between AD and T2D. Therefore, this study was aimed to evaluate and compare the effects of AD and T2D on behavioral and cognitive function, AD neuropathological hallmarks and brain mitochondrial function. For this purpose, we performed studies on the triple transgenic mice of Alzheimer s disease (3xTg-AD) and T2D mice, whose diabetes was induced by high sucrose intake. Wild type (WT) mice were used as control animals. Both T2D and 3xTg-AD showed similar behavioral and cognitive anomalies characterized by increased fear and anxiety and decreased learning and memory abilities. Mitochondria from 3xTg-AD and T2D mice presented a similar impairment of the respiratory chain and phosphorylation system, decreased capacity to accumulate calcium, ultrastructural abnormalities and oxidative imbalance. Interestingly, a significant increase in Aβ levels, a hallmark of AD, was observed in the cortex and hippocampus of T2D mice. Altogether our results show that mitochondrial alterations are a common denominator between T2D and AD. In fact, both pathological conditions present comparable defects that culminate in a similar profile of behavioral and cognitive changes. In conclusion, this study gives support to the idea that T2D may trigger AD-like pathology. Cristina Carvalho has a Investigation fellowship from QREN DoIT, DIAMARKER PROJECT, n.º and had a PhD fellowship from the Fundação para a Ciência e a Tecnologia (SFRH/BD/43965/2008). Mitochondria, Alzheimer Disease, Type 2 diabetes [email protected] 98

99 19:15 FIC7 s Chronic food restriction increases the number of neuropeptide Y-containing neurons in the hippocampal dentate gyrus of aged rats Armando Cardoso Armando Cardoso, Diogo Silva, Sara Magano, Pedro A. Pereira, José Paulo Andrade Department of Anatomy, Faculty of Medicine, University of Porto It is known that food restriction (FR) has several beneficial effects, including increasing of the mean and maximum lifespan and neuroprotection. However, most of the FR studies are realized in relatively young animals and the information of its brain effects in old rats is scarce. Therefore, we have analyzed the effects of FR on the total number of neurons in the hippocampal dentate hilus of 24-month male Wistar rats. Furthermore, we also analyzed the effects of FR on the hilar NPY neurons due to its important roles in the regulation of feeding behavior, emotion and memory. Young control rats (2-month old) were fed for 6 months ad libitum with standard rodent laboratory chow. Aged rats (18-month old) were randomly assigned to an aged control group and to an aged FR group. Aged control rats were fed for 6 months ad libitum with standard rodent laboratory chow and FR rats were fed 60% of the calories consumed by controls for 6 months. After treatments the animals were perfused and processed for glycolmethacylate embedding and immunocytochemistry. The total number of neurons and the number of NPY-immunoreactive (ir) neurons were estimated using stereological methods. It was found that the total number of neurons and NPY-ir neurons in the hilus were decreased in old rats when compared to young controls. Although there were no differences in total number of hilar neurons between aged FR rats and control old rats, the FR treatment increased the number of NPY-ir neurons. Our results corroborate previous works demonstrating that aging induces reduction of the total number of hilar neurons and NPY-ir neurons. Furthermore, the present results also show that FR appears to have an important role in the prevention of aging-related reduction of NPY-ir neurons. Supported by National Funds through FCT Fundação para a Ciência e a Tecnologia within the scope of the Strategic Project Centro de Morfologia Experimental (CME/FM/UP) and Project PEst-OE/SAU/UI0121/2011. Food restriction, aging, neuropeptide Y, hippocampus [email protected] 99

100 19:20 FIC8 s Amyloid-β peptide dysregulates TrkB receptors and BDNF function by a calpaindependent mechanism. André Jerónimo-Santos André Jerónimo-Santos, Vaz SH, Parreira S, Rapaz-Lérias S, Caetano AP, Buée- Scherrer V, Castrén E, Valente CA, Blum D, Sebastião AM, Diógenes MJ Unidade de Neurociências, Instituto de Medicina Molecular, Universidade de Lisboa, Portugal. The brain-derived neurotrophic factor (BDNF), through the activation of its full length receptor (TrkB-FL), plays a central role in the nervous system by providing trophic support to neurons and by regulating synaptic plasticity and memory. BDNF signalling is drastically affected in Alzheimer s disease (AD), a disease involving the accumulation of amyloid-β (Aβ) peptide. Previously, we demonstrated that, Aβ induces a decrease in TrkB-FL and an increase in truncated TrkB-Tc receptors, negative modulators of TrkB- FL signalling, on primary neuronal cultures, in a similar way of what is observed in AD brains, suggesting a direct correlation between Aβ and TrkB receptors alterations. The present work was designed to reveal the mechanisms behind TrkB changes induced by Aβ and the functional impact of this dysregulation. This work shows that exposure of cortical cultures to Aβ (25µM, 24h) increases mrna levels of TrkB-T1 and T2 isoforms by 45±19% and 58±17%, respectively, (n=8, p<0.05) without affecting TrkB-FL mrna levels. Moreover, we found that Aβ promotes a calpain-mediated cleavage of TrkB-FL, downstream Shc binding site, generating a new truncated receptor (TrkB-T ) and a stable intracellular domain fragment (TrkB-ICD) which was also detected in human brain samples. Functionally, we found that Aβ impairs BDNF actions upon neurotransmitter (GABA and glutamate) release on synaptosomes prepared form adult hippocampal slices and impairs hippocampal long-term potentiation (LTP), in a calpain-dependent way. This work shows that Aβ 1) increases mrna levels of TrkB-Tc isoforms without affecting TrkB-FL mrna; 2) induces a calpain-mediated cleavage of TrkB-FL, originating a new truncated receptor and an intracellular fragment and 3) impairs BDNF effects, in a calpain-dependent way, on neurotransmitters release as well as in LTP. TrkB, BDNF, Calpain, Amyloid-β, LTP [email protected] 100

101 19:25 FIC9 s Altered nociception by Tau depletion: implication for Alzheimer s disease André Lopes André Lopes, Hugo Leite-Almeida, Vitor Pinto, Sara Carlos, Sofia Lopes, Filipa Pinto- Ribeiro, Sara Silva, Andreia Carvalho, Daniela Clara, Rui Fernandes, Armando Almeida, Nuno Sousa, Ioannis Sotiropoulos Instituto de Investigação em Ciências da Vida e da Saúde (ICVS) TAU protein has an essential role on neuronal cytoskeleton through its interactions with various structures/proteins such as microtubules and F-actin, while recent evidence involves TAU in synapses where its associates to NMDA receptors. On the other hand, Tau hyperphosphorylation and subsequent malfunction is postulated as crucial mechanism of cytoskeletal pathology underlying Alzheimer s disease (AD). Besides their involvement in AD, some of the above cytoskeletal elements (e.g. microtubules, F- actin) are suggested to play a role in pain mechanisms. While there is no evidence about TAU and its role in nociception, clinical studies demonstrate that AD patients, characterized by Tau hyperphosphorylation, report consistently lower pain pointing towards a potential interaction between TAU and pain mechanisms. Thus, this study aims to clarify the TAU involvement in pain processing and perception. Our findings demonstrate that TAU-KO animals displayed decreased pain-like responses to acute painful stimulation (thermal and neuropathic pain). On the contrary, TAU-KO presented increased pain-related behaviours in the tonic phase on the formalin test which was paralleled by an increased c-fos expression in the dorsal horn. In addition, sciatic nerve ultrastructure analysis revealed decreased density of c-fibers as well as reduced myelination of A-δ-fibers that was confirmed with an electrophysiological approach measuring nerve conduction velocity. Our study reveals for the first time a direct involvement of TAU in nociceptive processing which we postulate to drive both from its role in axonal integrity (acute nociception) and synaptic plasticity (tonic pain). Tau-KO, pain, AD, myelin, nerve [email protected] 101

102 19:30 FIC10 P2Y1 receptor is involved in Abeta-induced synaptic and memory loss Ricardo J. Rodrigues s Lisiane O. Porciúncula, Carla G. Silva, Paula M. Canas, Joana M. Marques, Geanne M. Andrade, J.P.Oses, C. Gachet, Rodrigo A. Cunha, Ricardo J. Rodrigues Center for Neuroscience and Cell Biology of Coimbra, Univ. of Coimbra, Coimbra, Portugal. Early Alzheimer s disease (ead) is characterized by memory impairment associated to a synaptic loss and dendritic atrophy in cortical and hippocampal regions (Science 298,789-91). These early morphological traits, already evident in mild cognitive impairment (MCI), better correlates with the increased levels of Abeta oligomers (Science 297,353-56). Since ATP, which activates P2 receptors (P2R), is a proposed danger signal upon brain insults, we now tested if P2Rs are involved in Abeta-induced synaptic and memory loss. Pharmacological manipulation of P2Rs in hippocampal neurons revealed that the blockade of P2Y1R (MRS um) prevented not only Abeta1-42(0.5 um)-induced neuronal death (24 h) but more importantly the initial synaptotoxicity (MAP2 and synaptophysin labelling at 12 h). Interestingly, we found an increased density of P2Y1Rs in hippocampal terminals of both rats and mice 2 weeks after administration of Abeta1-42 (2nmol, icv) at a time where they displayed a mnemonic deficit (Y maze) and synaptotoxicity (reduced levels of synaptic markers) but no neuronal death (Fluoro-Jade C staining). Indeed, P2Y1R KO mice did not display mnemonic deficit or synaptotoxicity 2 weeks after administration of Abeta1-42. Moreover, CRMP2, a microtubule-associated protein involved in the formation of synaptic connectivity and stabilization, is hiperphosphorylated and hence its function is disrupted in ead. We now observe that the Abeta1-42-induced increase in the phosphorylation of CRMP2 at Thr555 (Brain 131,90-108) is recapitulated by P2Y1R activation. Together, these findings show that P2Y1R is involved in Abeta-induced synaptic and memory loss, suggesting the involvement of P2Y1R-driven phosphorylation of CRMP2, which should be responsible or at least contributing to neuritic cytoskeleton instability leading to early synaptic loss and hence synaptic network dysfunction underlying the cognitive deficits associated to ead. Support: EXPL/NEU-NMC/0671/2012 P2 receptors, CRMP2, synapse, Abeta, memory [email protected] 102

103 19:35 FIC11 s Intracellular accumulation of amyloid beta is associated with ER stress in endothelial cells Ana I. Plácido Ana I. Plácido, Catarina R. Oliveira, Cláudia M.F. Pereira, Paula I. Moreira Center for Neuroscience and Cell Biology & Faculty of Medicine, University of Coimbra, Coimbra, Portugal Objective: Although the cause(s) of sporadic Alzheimer disease (AD) remain(s) unclear, accumulating evidence implicates cerebrovascular deficiencies as contributory agents. In fact, amyloid-beta (Abeta) aggregates are often detected in the walls of cerebral vessels of AD patients, a condition called cerebral amyloid angiopathy (CAA). The endoplasmic reticulum (ER) is a site of Abeta1-42 generation while the Abeta1-40 isoform can be produced in the trans-golgi network. Perturbed ER function leads to a state known as ER stress that can trigger apoptotic cell death. We hypothesize that the loss of protein quality control in the secretory pathway, as a result of ER stress, can lead to brain endothelial cell injury through the enhancement of Abeta production. Methods: Rat brain endothelial cells (RBE4) were treated with the ER stress inducers thapsigargin (TP) or brefeldin A (BA). The protein levels of amyloid precursor protein (APP), beta secretase (BACE1), GRP78 and caspase 12 were assessed by western blotting. Caspase-9-like and caspase-3-like activities were assayed with colorimetric substrates and Abeta levels were analysed by ELISA. Results: In TP- or BA-treated brain endothelial cells, an increase in the levels of the ER stress marker GRP78 was observed concomitantly with activation of apoptosis-related caspases 9, 3 and 12. Under these conditions, increased protein expression levels of APP, followed by Abeta accumulation, were detected. Conclusion: ER stress promotes the accumulation of Abeta in brain endothelial cells, which supports the idea that loss of protein quality control in the secretory pathway may predispose to the development of CAA. Ana I. Plácido has a fellowship from Fundação para a Ciência e a Tecnologia, Portugal (SFRH / BPD / / 2010) Endoplasmic reticulum stress, Abeta peptide, Endothelial cells, cerebral amyloid angiopathy [email protected] 103

104 19:40 FIC12 s Activation of IR,IGF-1R signaling pathways promote mitochondrial function and energy metabolism in Huntington s disease human lymphoblasts Luana Naia Naia L.1,2, Ferreira I.L.1, Cunha-Oliveira T.1, Ribeiro M.1, Duarte A.I.1, Ribeiro M.J.3, Oliveira C.R.1,2, Saudou F.4, Humbert S.4, A. Cristina Rego1,2 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 3CEDOC- Chronic Diseases Research Center, FCM NOVA, Lisbon, Portugal; 4Institut Curie, Orsay, F-91405, France. Huntington s disease (HD) is a neurodegenerative disorder caused by a CAG expansion in HD gene. This trinucleotide expansion encodes for a polyglutamine stretch in the huntingtin protein (Htt), resulting in progressive death of striatal neurons with clinical symptoms of chorea and dementia. Mutant Htt interacts with mitochondria affecting their function and leading to energy failure. In the present work we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin on intracellular signaling pathways, and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways, linked to activation of insulin and IGF-1 receptors (IR, IGF-1R), Akt and ERK was largely prevented by IGF-1 and, at to a less extent, by insulin in HD lymphoblasts. Importantly, both compounds stimulated Htt phosphorylation at Ser421 in HD cells. IGF-1 and insulin also prevented energy depletion in HD peripheral cells, as evaluated through a decrement in ATP and phosphocreatine levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential in HD lymphoblasts, which was correlated with increased cytochrome c protein levels. HD lymphoblasts further exhibited increased intracellular Ca 2+ before and following exposure to hydrogen peroxide (H 2 O 2 ), and decreased mitochondrial Ca 2+ accumulation. The later was recovered by both IGF-1 and insulin in HD lymphoblasts pre-exposed to H 2 O 2. In summary, data support an important role for IR,IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts. This work was supported by FCT, projects reference PTDC/SAU-FCF/66421/2006, PTDC/SAU- FCF/108056/2008 and PEst-C/SAU/LA0001/2011, COMPETE QREN and FCT PhD fellowship SFRH/BD/86655/2012. Huntington's disease, mitochondrial dysfunction, energy metabolism, insulin, IGF-1 [email protected] 104

105 Fire Sessions II 105

106 Neural systems I June 1 Bussaco room 106

107 10:00 FIIA1 Role of adenosine A2A receptors in the amygdala: implications for mood disorders Ana Patrícia Simões s Ana Patrícia Simões 1, Manuella Kaster 1, Nuno Machado 1, Nélio Gonçalves 1, Ana Nunes 1, Ki Ann Goosens 2, Rodrigo A. Cunha 1,3 1 CNC, University of Coimbra; 2 MIT, Massachusetts; 3 Faculty of Medicine, University of Coimbra The amygdala is involved in processing fear responses and abnormal plastic changes of amygdalar circuits are associated with mood related disorders, namely with chronic stress. Since the consumption of caffeine (an adenosine receptor antagonist) attenuates the burden of depression, and adenosine A2A receptors (A2AR) control synaptic plasticity phenomena, we now tested if chronic stress affected the density of A2AR in the amygdala and if A2AR control synaptic plasticity phenomena in amygdalar circuits. Receptor binding assays with the A2AR antagonist 3H-SCH58261 (6 nm) revealed an upregulation of A2AR density in amygdalar nerve terminals of rats subject to repeated restraint stress (4 h/day for 15 days) (31.4±4.1 versus 21.3±2.8 fmol/mg protein in controls, n=4, p<0.05) and in mice subject to fear conditioning (67.0±8.0 versus 26.0±4.2 fmol/mg protein in control, n=5, p<0.05), where caffeine (10 mg/kg) or SCH58261 (0.05 mg/kg) attenuated contextual fear responses (n=9-10). Electrophysiological recording in the lateral amygdala upon stimulation of cortical afferents showed that SCH58261 (50 nm) decreased both short-term potentiation (134.0±3.0% versus 196.5±4.5% in control, n=3, p<0.001), measured within the first 10 min after high frequency stimulation (HFS, 3 pulses of 100 Hz with 5 s interval), and long-term potentiation measured 60 min after HFS (120.5±1.5% versus 148.6±3.5% in control, n=3, p<0.001). These results show that repeated stress and fear responses upregulate A2AR in the amygdala, where they can control synaptic plasticity phenomena. This supports the manipulation of amygdalar A2AR as a promising strategy to manage mood-related disorders. Financial support: DARPA (09-68-ESR-FP-010) and FCT (PTDC/SAU- NSC/122254/2010, PEST-C/SAU/LA0001/2011). adenosine, A2A, amygdala, fear, stress [email protected] 107

108 10:05 FIIA2 s Prenatal glucocorticoid exposure disrupts Pavlovian-instrumental transfer: role of D2 dopamine receptor Carina Soares-Cunha Carina-Soares Cunha, Bárbara Coimbra, Sónia Borges, Ana João Rodrigues, Nuno Sousa Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B s Stress, or elevated levels of glucocorticoids (GCs), has been identified as a triggering factor in the development of reward-associated disorders such as depression, schizophrenia and drug addiction. These pathologies can result from changes in the neural networks responsible for the complex processes that assign value to rewards and associated stimuli, namely the mesocorticolimbic dopaminergic system, that we have shown to be particularly vulnerable to stress/gcs effects. Essential in the understanding on how cues influence our behaviour is the Pavlovian-instrumental transfer (PIT), a phenomenon that refers to the capacity of a Pavlovian stimulus that predicts a reward to elicit/increase instrumental responses for that same reward. In this study, we demonstrated that prenatal exposure to GCs (iugc) impairs both general and selective versions of the PIT paradigm, suggestive of a general incentive motivational deficit. These animals presented reduced levels of dopamine in the nucleus accumbens and in prefrontal and orbitofrontal cortices, key regions involved in the integration of Pavlovian and instrumental stimuli. Normalization of dopamine levels by L-DOPA administration rescued this behavior; and importantly, this process seemed to rely on D2 dopamine receptor activation, since D2 agonist quinpirole had a similar effect in reverting PIT impairment. In summary, iugc exposure programs mesocorticolimbic dopaminergic circuitry, leading to a reduction in the attribution of the incentive salience to food cues, in a dopamine/d2-dependent manner. Importantly, our results are important to understand how GCs shift/biases incentive processes, decreasing wanting for some rewards (food) while increasing it for others (drugs); a fact that is particularly relevant in addiction, where differential attribution of incentive salience has been proposed to occur. early life stress, Glucocorticoids, dopamine, motivation [email protected] 108

109 10:10 - FIIA3 s The electrophysiological signature of response caution Maria J. Ribeiro Maria J. Ribeiro, Inês R. Violante and Miguel Castelo-Branco IBILI, Faculty of Medicine, University of Coimbra In go/no-go tasks, participants are requested to respond to frequent go stimuli, while withholding the response to infrequent no-go stimuli. Different participants adopt different response strategies. More cautious subjects respond slower to go stimuli thereby increasing the chances of successful response inhibition to no-go stimuli. Less cautious, more risk-taking subjects trade accuracy for speed: respond faster to go stimuli and, consequently, present a higher number of errors of commission (failures to inhibit the response to no-go stimuli). Our aim was to characterize the neural correlates of the different response strategies associated with fast or slow responses in go/no-go tasks. For this, we recorded the electroencephalogram (EEG) of healthy participants and individuals with a neurodevelopmental disorder associated with impaired impulse control, Neurofibromatosis type 1 (NF1), during performance of a go/no-go task with constant inter-stimulus intervals. In both groups, faster participants failed more often to inhibit the response to no-go stimuli. In addition, individuals with NF1 responded faster to go stimuli and committed a significantly higher number of errors of commission than control participants. EEG/ERP analysis revealed that faster participants presented stronger motor potentials during the pre-stimulus period indicating that response preparation started before stimulus onset. Accordingly, pre-stimulus motor potentials were enhanced in the NF1 group. In addition, individuals with NF1 showed decreased amplitude of medial frontal ERPs suggesting impaired decision making that could be related to their response style opting for fast rather than accurate responses and thus disregarding adequate stimulus discrimination and response choice. In conclusion, pre-stimulus activation of the motor cortex determines the level of response caution, in both healthy participants and individuals with NF1. Funding: Compete - PTDC/SAU-ORG/118380/2010 EEG, response caution, reaction time, go/no-go task, impulsivity [email protected] 109

110 10:15 - FIIA4 s Chronic low doses of ketamine induce persistent region specific neuronal changes in an adult rat model Andrea Lobo Andrea Lobo, Carlos Venâncio, Andrea Lobo, Sílvia Fernandes, Ana Magalhães, Franscisco Peixoto, Luís Antunes, Teresa Summavielle Instituto Biologia Molecular e Celular Although chronic administration of low-doses of ketamine, a NMDA receptor antagonist, has been used in the management of chronic pain and depression, the mechanisms underlying ketamine chronic use are largely unknown, particularly regarding safety issues. The goal of the present study was to determine the consequences of a chronic ketamine administration pattern mimicking analgesic or antidepressant treatments, using an adult rat model. The effects of ketamine were evaluated at neurochemical and plasticity levels in specific brain regions previously reported as susceptible to ketamine effects. Administration of ketamine in a 5 mg/kg dose increased anxiety-like behavior, accompanied by increased dopamine levels in the amygdala and decreased dopamine levels in the prefrontal cortex. For the 10 mg/kg dose, behavioural changes were less pronounced, probably associated to increased serotonin levels in hippocampus and amygdala, which may be related to an antidepressant and anxiolytic effect. The administration of low-doses of ketamine decreased the levels of GABAergic parvalbumin-expressing neurons in the prefrontal cortex and in the hippocampus, a process also involved in anxiety-like behaviours. No differences were observed in protein levels of PSD-95 and in the phosphorylation of ERK, indicating that ketamine treatment did not induce changes in synaptic plasticity. These findings point out the need for prudence when using low-doses of ketamine to treat chronic conditions. ketamine, chronic, neurochemical, plasticity, anxiety [email protected] 110

111 10:20 - FIIA5 s Dissecting Foxp2 Function in Motor-Skill Learning Circuits Catherine A. French Catherine A. French 1, Cátia Feliciano 1, Vítor B. Paixão 1, Xin Jin 2, Simon E. Fisher 3 and Rui M. Costa 1 1 Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown, Lisbon, Portugal. 2 The Salk Institute for Biological Studies, La Jolla, CA, USA. 3 Language and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, the Netherlands. Disruptions of the FOXP2 gene cause a severe developmental speech and language disorder. This has been well studied in the KE family where a heterozygous FOXP2 mutation is dominantly inherited. Affected individuals have difficulty producing the sequences of orofacial motor movements necessary for fluent speech. This feature has been proposed to be central to the disorder, although other expressive and receptive language problems also exist. FOXP2 encodes a transcription factor that is expressed in the cortico-striatal/ -cerebellar circuits required for sensorimotor integration and motorskill learning, and imaging studies of the KE family have shown structural abnormalities in the caudate nucleus and ventral cerebellum. The FOXP2 protein is also highly conserved in other vertebrate species, with only 3 amino acid changes between humans and mice. Mice carrying the KE-family mutation (Foxp2-R552H/+) have motor-skill learning deficits and lack striatal long-term depression. They also show altered striatal activity in vivo during the learning of a motor task. We are now selectively deleting Foxp2 from the cortex, striatum or cerebellar Purkinje cells of mice. This genetic approach is being combined with an operant motor-sequence learning task which allows us to examine the microstructure of animals' behaviour. In the first phase of training, mice must complete a sequence of 8 lever presses to obtain a food reward. After 12 days a time constraint is added and the sequence must be performed at increasingly high speeds. Surprisingly, the press rate of Foxp2-R552H/+ and heterozygous knockout mice was faster than that of controls. Press sequences were shorter in duration and the time between sequences was also reduced. In contrast cerebellar mutants, and to a lesser extent striatal mutants, produced sequences of a longer duration with increased inter-sequence intervals. These data suggest that Foxp2 function in different subcircuits may affect distinct behavioural processes. speech and language, motor-skill learning, striatum, cerebellum, operant behaviour [email protected] 111

112 10:25 - FIIA6 s Is depression a neurodegenerative disorder? an in-vivo link between depression, antidepressant action and Alzheimer s disease Lopes S Lopes S 1,2, Bessa JM 1,2, Fernandez P 3, Castelhano-Carlos M 1,2, Morais M 1,2, Vieira MJ 1,2, Cerqueira JJ 1,2, Almeida OFX 3, Sousa N 1,2, Sotiropoulos I 1,2 1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 2- ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3- Neuroadaptations Group, Max Planck Institute of Psychiatry, Munich, Germany Clinical and experimental studies suggest a causal role of chronic stress for brain pathology and diseases e.g. depression and Alzheimer s disease (AD) as stress is strongly associated with neuronal and synaptic atrophy/loss resulting in impaired mood and/or cognition. Indeed, synaptic loss is a key underlying pathomechanism in both disorders while growing clinical evidence supports a pathological link between depression and AD pointing to shared neurobiological underpinnings and pathogenic mechanisms e.g. ADrelated mechanisms, such as APP misprocessing, are also found to be affected in depression while depression predisposes individuals to develop AD. Based on the above, our studies have been conceived to contribute towards bridging the current gap monitoring AD-related mechanisms in the CMS (chronic mild stress) animal model of depression before and after antidepressant treatment. We found that depressive status in these animals was accompanied by increased APP misprocessing and tau accumulation as well as neuronal atrophy in hippocampus and prefrontal cortex. Interestingly, antidepressant treatment with two different antidepressants reversed both biochemical and morphometric changes in neurons of the both brain. Furthermore, we demonstrate the blockage of stress-triggered depressive behavior and neuronal atrophy in animals lacking APP misprocessing and generation of amyloid beta, further corroborating the involvement of APP misprocessing in depressive pathology. Thus, this study form the first in vivo approach to clarify the involvement of AD-related tau and APP proteins on stress-driven depressive pathology adding to our knowledge on stress-triggered mechanisms and their role in pathophysiology of depression and its connection to AD. Depression,Alzheimer's Disease, Stress, APP, Tau [email protected] 112

113 10:30 - FIIA7 s Adenosine A2A receptors in the amygdala control behavioral modifications in rats subject to repeated restraint stress Nuno J. Machado Nuno J. Machado, Nélio Gonçalves, Carolina M. Souza, Luís Pereira de Almeida, Rodrigo A. Cunha Center for Neuroscience and Cell Biology, Faculty of Pharmacy, Faculty of Medicine, University of Coimbra, Portugal Chronic stress contributes for depression, the greater burden of disease in Europe. Abnormal amygdala function is paramount in depressive conditions and intake of caffeine (a non-selective antagonist of adenosine A2A receptors - A2AR) correlates inversely with the incidence of depression. Since we have shown that chronic stress enhances A2AR density in the amygdala, we now tested if the genetic ablation of amygdalar A2AR abrogated the behavioral modifications caused by chronic stress. We injected in the amygdala of adult Wistar male rats a lentivirus encoding either for a short hairpin to silence A2AR (sha2ar) or a scrambled control (shctr). After one week, rats were subject to a daily 4-hour period of restraint stress during 15 days (stressed) or simply handled (control) before behavioral evaluation (n=7-10 per group). In the first week, shctr-stressed rats did not gain as much weight (21 g) as shctrcontrol rats (32 g) while both sha2ar-control and sha2ar-stressed rats displayed an increased weight gain (12 g more). shctr-stressed rats also displayed an increased anxiety-like behavior (81% and 54% above control rats) in both the open field test (OFT) and in the elevated plus maze (EPM), while sha2ar-stressed rats were similar to sha2ar-control rats (10% and 6% change). Surprisingly, sha2ar-control rats displayed and anxious profile in EPM (54% above control), but no differences were observed in OFT. shctr-stressed rats also displayed a depressive-like behavior in the forced swimming test (FST, 113% immobility of shctr-control) that was prevented by sha2ar injection (91% immobility). These results support the hypothesis that amygdalar A2AR directly participate in the pathophysiology of chronic stress-induced abnormal behavior and their manipulation may assist managing depressive state upon chronic stress. Supported by DARPA (09-68-ESR-FP-010), FCT (PTDC/SAU-NSC/122254/2010) and CAPES. Depression, stress, adenosine, behavior, amygdala [email protected] 113

114 10:35 - FIIA8 s Cortical and striatal dynamics during operant motor skill learning Fernando J. Santos Fernando J. Santos and Rui M. Costa Champalimaud Foundation - Champalimaud Neuroscience Programme Learning a motor skill is a continuous and dynamic process characterized by initial stages of fast improvements, which evolve to later stages of slower improvements. We designed a task where mice learn a motor skill and reach maximum performance within 4 days. Mice were trained to perform an operant task where they were required to press a lever at increasingly higher frequencies to obtain a reward. In the initial session mice could press freely, but with training the time window was progressively reduced until a frequency of 6Hz was reached. We observed that with training, mice organized their behavior in sequences of presses. Trial-to-trial variability decreased for the task-relevant behavioral features, but this was not observed for other features. To investigate the corticostriatal circuit dynamics, we implanted multielectrode arrays to record simultaneously activity in primary motor cortex (M1) and dorsal striatum (DS). We started recording while animals were still naive to the motor skill, and continuously recorded throughout 4 days of training. Putative single units were classified based on waveform characteristics and firing statistics, and a proportion of them remained stable throughout the continuous recording period. Trial-to-trial variability of neuronal units firing rates increased during the initial sessions but decreased throughout training. Overall firing rates in MI and DS did not change with learning. We went further to investigate the relationship between these dynamics, and observed a positive correlation emerging between the variability of neuronal firing rates and the variability of sequence frequency. Correlations between other behavior features and neuronal data were not observed. This suggests that during the acquisition of a motor skill there is a decrease in both the neuronal discharge variability and behavioral variability, but the correlation between the two increases as the animals learn to perform the task. motor skill learning, basal ganglia, motor cortex, electrophysiology [email protected] 114

115 10:40 - FIIA9 s Dopaminergic modulation of affective and social deficits induced by prenatal glucocorticoid exposure Sónia Borges Sónia Borges, Bárbara Coimbra, Carina Cunha, José Miguel Pego, Nuno Sousa, Ana João Rodrigues ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Stress activates the hypothalamic-pituitary-adrenal (HPA) axis and leads to a controlled release of glucocorticoids (GCs) in the blood stream. Due to their numerous effects, synthetic GCs are often prescribed in clinics, as for example in preterm risk pregnancies in order to accelerate fetal lung maturation. Prenatal stress or in utero exposure to synthetic glucocorticoids (iugc) can potentiate the development of several neuropsychiatric disorders in adulthood, such as depression and anxiety. Previous studies have shown that iugc can lead to a long-lasting impairment of the HPA axis, anxiety and depressive-like behavior. However, little is known about the impact of prenatal GCs exposure in social behavior. In this work we evaluated the impact of prenatal GC exposure in emotional and social behaviors, using different behavioral paradigms in combination with the measurement of ultrasonic vocalizations (USVs). Our results show that prenatal GCs administration potentiates emotional and social deficits at different ages; importantly, this was correlated with decreased emission of positive 50kHz vocalizations, a measure of positive affective status in rodents. This seemed to occur as a consequence of a deficient dopaminergic signaling in the mesolimbic circuitry, since administration of L-DOPA, a dopamine precursor, mitigated most of the behavioral deficits. social behavior, depression, dopamine, mesolimbic circuit, [email protected] 115

116 10:45 FIIA10 s Reduced hippocampus-prefrontal cortex connectivity in rats performing a spatial working memory task under neuropathic pain condition Cardoso-Cruz, Helder Cardoso-Cruz, Helder; Dourado, Margarida; Lima, Deolinda; Galhardo, Vasco Dpto. Biologia Experimental - Fac. Medicina, e IBMC - Grupo de Morfofisiologia, Universidade do Porto, Portugal. Impaired working memory (WM) is observed in chronic pain patients, but the mechanisms and brain areas underlying this cognitive impairment remain elusive. The neuronal populations of the medial prefrontal cortex (mpfc) and dorsal hippocampal CA1 (dca1) are well known to form an interconnected neural circuit that is crucial for correct performance in spatial memory-dependent tasks. In this study, we investigated whether the functional connectivity between these two areas is affected by the onset of an animal model of peripheral neuropathic pain. To address this issue, we chronically implanted two multichannel arrays of electrodes in the mpfc and dca1 hippocampus of adult rats and recorded the neuronal activity during a food-reinforced spatial WM task in a reward-based alternate trajectory maze. Recordings were performed for 3 weeks, before and after the establishment of the spared nerve injury model of neuropathy. Our results show that the nerve lesion caused a clear impairment of WM performance that is temporally correlated with changes in the mpfc populational firing activity patterns when the animals navigated between decision points when memory retention was most needed. Moreover, the activity of both recorded neuronal populations after the nerve injury increased their phase-locking in respect to hippocampal theta rhythm. Finally, our data revealed that chronic pain reduces the overall amount of information flowing in the fronto-hippocampal circuit and induces the emergence of different oscicllation patterns that are well correlated with correct/error performance fo the animal on a trial-by-trial basis. The present results demonstrate that functional disturbances in the mpfc-dca1 connectivity are a relevant cause for pain-related WM deficits. Supported by FCT Grant-SFRH/BD/70522/2010, FCT Project-PTDC/SAU- NEU/100773/2008. Neuropathic pain, impaired spatial memory, hippocampus-prefrontal cortex, connectivity, rat [email protected] 116

117 10:50 FIIA11 s Neural correlates of 3D coherent visual perception in Williams syndrome: electrophysiological evidence Inês Bernardino Inês Bernardino, João Castelhano, Reza Farivar, Miguel Castelo-Branco IBILI - Visual Neuroscience Laboratory, Faculty of Medicine, University of Coimbra, Portugal Williams syndrome(ws) is a model of impaired visual integration associated with dorsal stream vulnerability. However, little is known about the neurophysiological correlates of such impairments. Here, we have used a 3D structure-from-motion (SFM) integrative task to characterize the neuronal underpinnings of 3D perception in WS. Nine WS participants (Fish test; mean age±se=21.44±2.30) and 8 control participants (mean age±se=21.89±2.40) matched for chronological age, gender and handedness were included. In the 3D structure-from-motion (SFM) task, participants viewed movies of SFM defined faces parametrically modulated in 3D depth (three depth levels) and indicated with a button press, after stimulus offset, if they identified a face or not. EEG data were recorded using a 64 channel Quick-Cap and processed offline. Event Related Potentials (ERPs), Independent Component Analysis (ICA), source localization and timefrequency analyses were performed. We found that the electrophysiological (EEG/ERP) correlates of 3D coherent percepts were distinct across groups. ICA revealed a novel component in WS with a late positivity around 200ms that was absent in controls. Source localization analysis of ERP signals showed a posterior occipital shift and reduced parietal dorsal stream sources in WS. Moreover, low gamma-band oscillations (20-40Hz) specifically induced by this 3D integration task were significantly stronger in WS whereas high gamma-band oscillations (60-80Hz) were reduced, as compared to controls. These observations suggest that dorsal stream processing of 3D SFM stimuli has distinct neural correlates in WS and different cognitive strategies are employed by these patients to reach visual coherence. Importantly, we found evidence for the presence of different sub-bands (20-40Hz / 60-90Hz) within the gamma range which can be dissociated concerning the respective role on the coherent percept formation, both in typical and atypical development. Williams syndrome, gamma oscillations, 3D integration, visual coherence, EEG/ERP [email protected] 117

118 10:55 FIIA12 s Excitotoxic lesions in the central nucleus of the amygdala attenuate stress-induced anxiety behavior Ana Paula Ventura-Silva Ana Paula Ventura-Silva, António Melo, Ana Catarina Ferreira, Miguel Murteira Carvalho, Filipa Lopes Campos, Nuno Sousa, José Miguel Pêgo Instituto de Investigação em Ciências da Vida e Saúde (ICVS)/ ICVS/3B s - PT Government Associate Laboratory The extended amygdala, composed by the amygdaloid nuclei and the bed nucleus of the stria terminalis (BNST), plays a critical role in anxiety behavior. In particular, the link between central nucleus of the amygdala (CeA) and the BNST seems to be critical to the formation of anxiety-like behaviors. Stress exposure is recognized as a validated animal model of anxiety and is known to trigger significant morphofunctional changes in the extended amygdala. Quite surprisingly, no study has ever analyzed the role of the CeA in the onset of anxiety- and fear- behaviors associated with chronic stress exposure. To better understand the role of CeA in stress induced anxiety we induced a bilateral excitotoxic lesion in male rats that were subsequently exposed to a chronic stress protocol. We observe that lesioned animals showed an attenuation of anxiety-like behavior in the elevated-plus maze when compared with stressed animals with a sham lesion. In parallel, there is also an attenuation the stress-induced increase in basal levels of corticosteroids after CeA lesion. In opposition, CeA lesions are able to block fearconditioning behavior in control and stressed animals. These results unravel the relevance of the CeA in the regulation of the HPA axis and on the onset of stress-induced anxiety. (Ventura-Silva, in press) BNST, amygdala, anxiety, stress [email protected] 118

119 Cell signaling II June 1 Plátano room 119

120 10:00 FIIB1 Inhibition of ecto-5 -nucleotidase activity facilitates short-term and inhibits longterm plasticity in two different mouse brain regions Francisco Queiroz Gonçalves s Francisco Queiroz Gonçalves, Daniel Rial, Henrique B. Silva, Rodrigo A. Cunha Centre for Neuroscience and Cell Biology (CNC) ATP is stored in synaptic vesicles and released together with neurotransmitters. ATP acts as an extracellular signal, either directly activating P2 receptors or indirectly activating adenosine A1 or A2A receptors (A2AR) after its extracellular catabolism into adenosine by ecto-nucleotidases, the final step being mediated by ecto-5 -nucleotidase (e5 N). A1R inhibit synaptic transmission, whereas A2AR are selectively engaged upon synaptic plasticity. Since it is proposed that e5 N is selectively responsible for the formation of the ATP-derived adenosine activating A2AR, we now tested the impact of e5 N inhibition (with 100 mm a,b-methylene ADP, AOPCP) on synaptic transmission and plasticity in two glutamatergic synapses: cortico-striatal synapses and Schaffer fiber- CA1 pyramid synapses. AOPCP did not change basal synaptic transmission neither in the hippocampus nor in the striatum, whereas removal of all endogenous adenosine with adenosine deaminase (ADA, 2 U/mL) facilitated hippocampal synaptic transmission by 50.4±6.4% (n=5; P<0.05). AOPCP enhanced short-term plasticity (paired-pulse facitation with inter-pulse intervals between 20 and 80 ms) both in the hippocampus (n=5) and in the striatum (n=3). In contrast, AOPCP inhibited long-term potentiation (induced by a 100 Hz train for 1s) by 12.7±2.1% (n=4; P< 0.05) in hippocampus and by 37.0±1.7% (n=3; *P< 0.05) in the striatum. These results suggest that ATP-derived adenosine indeed seems to be preferentially coupled with the activation of A2AR to facilitate long-term potentiation; in contrast, under conditions of short-term plasticity it seems that e5 N mediates the formation of adenosine responsible for the A1R-mediated inhibition of paired pulse facilitation. Finally, it seems that the endogenous extracellular adenosine responsible for the tonic inhibition of synaptic transmission might not be originated from the extracellular catabolism of ATP. Supported by DARPA (09-68-ESR-FP-010), FCT (PTDC/SAU-TOX/122005/2010) ATP, Adenosine, ecto-5 -NT, Synaptic Plasticity [email protected] 120

121 10:05 - FIIB2 s Changes in the proteome of the postsynaptic densities of cortical neurons from mice lacking the GluN2B subunit of NMDA receptors Joana Ferreira Joana Ferreira1,2, Pedro Rio1,2, Ka Wan Li3, Ann Marie Craig4 Ana Luísa Carvalho1,2 1CNC, UC, PT. 2Dep of Life Sciences, UC, PT. 3CNCR, VU, NL. 4BRC, UBC, CA N-methyl-D-aspartate receptors (NMDARs) are unique regulators of glutamatergic synaptic transmission in the central nervous system. These receptors play a crucial role in shaping the strength of synaptic connections, which underlies mechanisms of learning and memory formation. To obtain insight into the role of GluN2B in the synaptic targeting of NMDARs, we have used hippocampal neurons from GluN2B(-/-) mice and found that the synaptic levels of NMDARs are dramatically decreased, while synaptic NMDARs are unaltered in GluN2A(-/-) hippocampal neurons. Additionally, we observed that the synaptic expression of the alpha-amino-3-hydroxy-5-methyl-4- isoxazole receptors (AMPAR) subunits GluA1 and GluA2 is increased in the absence of GluN2B. To determine the composition of the molecular complexes organized by GluN2B we evaluated biochemically the PSDs isolated from cultured cortical neurons of wild-type, heterozygous and GluN2B(-/-) mice. Quantitative analysis of the protein content of the isolated PSDs, by itraq-labeling in conjunction with tandem mass liquid chromatography and mass spectrometry, revealed alterations in several proteins involved in a diversity of signaling processes. Given that the synaptic proteasome is decreased in GluN2B(-/-) neurons, and that we observed an impairment of AMPAR endocytosis, a proteasome-dependent process, we have hypothesized that the proteasome may be involved in the GluN2B-mediated regulation of synaptic AMPAR under basal activity. In fact, neuronal incubation with an enhancer of the proteasome activity rescued GluA1 synaptic levels in GluN2B(-/-) neurons. Our data suggest that GluN2B-containing NMDARs may play an important role in regulating the synaptic proteasome and thereby in regulating basal AMPAR levels. Supported by FCT, Portugal: PTDC/SAU-NEU/099440/2008, PEst- C/SAU/LA0001/2011, SFRH/BD/37522/2007 and SFRH/BPD/90045/2012. Glutamate receptors, Postsynaptic densities, Proteasome, Synaptic Targeting, Endocytosis [email protected] 121

122 10:10.-. FIIB3 s Glial pathology in the prefrontal cortex affects the cognitive function of the rat João Filipe Oliveira Ana Lima, Magda Reis, Ana Filipa Oliveira, Vanessa Morais Sardinha, Cristina Mota, Luísa Pinto, Fernanda Marques, João José Cerqueira, Nuno Sousa, João Filipe Oliveira Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal The arising of the tripartite synapse concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. However, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. The aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. The rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. For that purpose, we used a pharmacological approach in which Wistar-Han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of GFAPpositive cells are observed. This animal model was tested for its cognitive abilities by the attentional set-shifting task (ASST) and water maze-based tests. A clear impairment of cognitive function was observed in the animals treated with aminoadipate. A detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. This seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. astrocyte, GFAP, rat, cognition, aminoadipate [email protected] 122

123 10:15 - FIIB4 s BDNF decreases glycine uptake in brain synaptosomes by reducing GlyT2 membrane insertion Rita I Aroeira Rita I Aroeira, Ana M Sebastião and Cláudia A Valente Institute of Pharmacology and Neurosciences and Neurosciences Unit, Institute of Molecular Medicine, University of Lisbon Glycine transporter 2 (GlyT2) is localized in the nerve terminals of glycinergic neurons, promoting glycine uptake into the neurons and ensuring the refilling of glycinergic vesicles. Brain-derived neurotrophic factor (BDNF) activates its high affinity TrkB receptors, which occur in two isoforms, full length (TrkB-FL) and truncated (TrkB- T1/T2). After BDNF binding to TrkB receptor, several intracellular cascades are triggered, specifically PLC, Akt and MAPK signaling pathways. To determine if BDNF modulates glycine uptake mediated by GlyT2 in synaptosomes obtained from rat hippocampus, [3H]glycine uptake assays were performed. We showed that BDNF decreased glycine uptake mediated by GlyT2. Upon synaptosomes incubation with BDNF, GlyT2 displayed a lower Vmax value and a similar Km constant when compared to BDNF absence. Our results also demonstrated that both TrkB receptor isoforms, TrkB-FL and TrkB-T, are present in synaptosomes. However, we revealed that the BDNF effect is TrkB-FL dependent. Firstly, the tyrosine kinase inhibitor, k252a, was able to restore glycine uptake. Furthermore, the BDNF action was lost when specific inhibitors of TrkB signaling pathways, namely U73122, LY and U0126 (inhibitors of PLC, Akt and MAPK pathways, respectively) were used. Moreover, synaptosomes pre-incubation with monensin, a transporter recycling inhibitor, prevented the BDNF action upon glycine uptake, suggesting that BDNF reduces GlyT insertion in the plasma membrane. In conclusion, our results showed that BDNF effect upon glycine uptake in brain synaptosomes requires the activation of the TrkB-FL receptor and associated signaling pathways and occurs by inhibiting GlyT2 membrane incorporation. Glycine transporter 2, glycine uptake, synaptosomes, BDNF [email protected] 123

124 10:20 - FIIB5 s Purinergic signaling is essential for proper neuronal polarization Rui Beleza Rui O. Beleza, Sofia Ferreira, Sofia Alçada-Morais, Joana M. Marques, Ana P. Simões, Rodrigo A. Cunha, Ricardo J. Rodrigues Center for Neuroscience and Cell Biology of Coimbra, Univ. of Coimbra, Coimbra, Portugal. Neuronal P2 receptors (P2Rs) are ubiquitous in the adult brain, being essentially targeted to synapses, where they participate in neurotransmission and play a prominent neuromodulatory role. Besides their neuromodulatory action on the activity of matured synapses (e.g. J.Neurosci. 25, ), P2Rs are also involved in the morphological guidance for the formation of synaptic connectivity during development, modulating axonal outgrowth of hippocampal neurons in a complex multi-receptor manner (J. Cell Sci. 125, ). We now found that in addition to a modulation of axonal elongation, purinergic signaling is essential for a proper polarization of developing hippocampal neurons. We observed that the removal of extracellular ATP and ADP, ligands of P2Rs, by the presence of apyrase (20 U/mL) from DIV 1 onwards leads to the formation of aberrant axons (Smi-31 positive neurites) (apyrase 1.84±0.08 vs. untreated 1.13±0.03 axons/neuron, n=3) and a correlated reduction in the nº of dendrites (MAP2-positive neurites) in hippocampal neurons at DIV 3. This indicates that purinergic signaling not only regulates axonal outgrowth, but also is essential for a correct axon specification necessary for the establishment of a normal polarization. Moreover, we have found that P2Rs regulate the phosphorylation levels of CRMP2, a microtubule associated protein crucial for axonal outgrowth and axon specification (Nat. Neurosci. 4, 781-2). CRMP2 is negatively regulated by phosphorylation, disrupting CRMP2-tubulin binding, and hence microtubule assembly, resulting in growth cone collapse leading to the arrest of axonal elongation and/or axon formation. We now found that P2Rs differentially modulate the phosphorylation levels of the two regulatory targets, Thr514 and Thr555 of CRMP2, in a receptor dependent-manner. These findings indicate that purinergic signaling is essential for neuronal polarization, most likely through the regulation of CRMP2 function. Support: EXPL/NEU-NMC/0671/2012 ATP, neuronal polarity, P2 receptors, CRMP2 [email protected] 124

125 10:25 FIIB6 s Elucidating the effect of alpha-synuclein aggregation modulators in vesicular trafficking Susana Gonçalves Susana Gonçalves and Tiago Fleming Outeiro Instituto de Medicina Molecular Parkinson s disease is the second most frequent neurodegenerative disorder and is associated with the misfolding and aggregation of alpha-synuclein ( Syn), a pre-synaptic protein whose function is still unclear. There is ample debate as to what the toxic species of Syn are, although it has been postulated that misfolded oligomeric species of Syn constitute the toxic genus. Here, we investigated the molecular mechanisms underlying the initial steps of Syn oligomerization. In an RNAi screen we identified and validated 9 genetic modifiers of Syn oligomerization, including genes involved in intracellular transport/trafficking and in signal transduction pathways and we are currently in the process of validating the hits using secondary assays. Specifically, using living cells, we are investigating the functional role of the identified hits on αsyn oligomerization/aggregation, subcellular localization and secretion. We found that specific hits modulate Syn dimerization and protein levels, and induce changes in the typical subcellular localization of Syn, resulting in the sequestration into protein aggregates. Altogether, our approaches provide novel insights into the biology of Syn in living cells, and may enable the development of novel strategies for therapeutic intervention in synucleinopathies. alpha-synuclein, Parkinson's disease, aggregation, dimerization, vesicular trafficking [email protected] 125

126 10:30 - FIIB7 Deletion of adenosine A2A receptors from astrocytes alters glutamatergic synapses and triggers enhanced psychomotor responses and memory deficits characteristic of schizophrenia Paula Agostinho s Marco Matos 1,2,3, Elisabete Augusto 1,2,3, Catherine J. Wei 3, Michael A. Schwarzschild 4, Paula Agostinho 1,2, Rodrigo A. Cunha 1,2, Jiang-Fan Chen 3 1 Center for Neuroscience and Cell Biology, 2 Faculty of Medicine, University of Coimbra, Portugal; 3 Department of Neurology, Boston University School of Medicine, USA; 4 Molecular Neurobiology Laboratory, Massachusetts General Hospital, Boston, USA. Schizophrenia is an incurable mental disorder encompassing positive symptoms, (delusions, hallucinations), negative symptoms (anhedonia, social withdrawal) cognitive symptoms (working memory and executive deterioration). It has traditionally been considered to result from a dopaminergic imbalance but glutamatergic hypofunction can explain most symptoms. In particular astrocytic glutamate transporters (GLT-1) have been implicated in the pathogenesis of schizophrenia. Since we recently found that astrocytic adenosine A2A receptors (A2AR) control GLT-1 expression and activity (Matos et al., 2012, Glia 60: 702), we now tested how the selective deletion of astrocytic A2AR (i.e. GFAP-A2AR-KO mice) affected endophenotypes characteristic of schizophrenia, namely enhanced psychomotor activity to MK-801 and impaired working memory. After habituation to the activity cages, MK-801 administration (0.5 mg/kg) bolster locomotion to a greater extent (p<0.001) than in WT mice, which also had a superior working memory (Y-maze and 8-radial arm tests) than GFAP-A2AR-KO mice (p<0.01, n=12). GFAP-A2AR-KO mice displayed enhanced density of GLT-1 and of glutamate transport into gliosomes as well as enhanced density of NR-2B subunits and decreased biotinylated levels of GluR1/2 AMPA receptors in synaptosomes. Notably, a GLT-1 inhibitor (dihydrokainate, 10 mg/kg) or a peptide blunting GluR1/2 regulated endocytosis (Tat-Glur23Y, 3 μmol/kg) attenuated the MK-801-induced hyperlocomotion and the impaired working memory in GFAP-A2AR-KO mice. These results indicate that astrocytic A2AR, by controlling GLT-I activity, which prompts an astrocyte-to-neuron adaptation of glutamatergic synapses, involving an NMDA-2Breceptor up-regulation and culminating with an internalization of AMPA receptors, may be critical mediators of the psychomotor and cognitive dysfunctions associated with schizophrenia. Supported by DARPA (09-68-ESR-FP-010) and FCT (PTDC/SAU-NSC/122254/2010). astrocytes, adenosine A2A receptors, glutamatergic dysfunction, cognitive deficits, schizophrenia [email protected] 126

127 10:35 - FIIB8 s Serotonin elicits pronociception in diabetic neuropathic pain through the activation of spinal 5-HT3 receptor Marta Silva Marta Silva, Isaura Tavares, Carla Morgado Department of Experimental Biology of Faculty of Medicine of Porto and IBMC, University of Porto Diabetic neuropathic pain (DNP) can be associated with chronic pain installation. Selective serotonin reuptake inhibitors (SSRIs) are clinically valuable as analgesics, but were shown to elicit low efficacy in DNP. Spinal release of serotonin is known to inhibit locally nociceptive transmission. However, it was recently shown that in traumatic neuropathic pain models serotonin might facilitate pain through its action on spinal 5-HT3 receptor (5-HT3R). As this mechanism is likely to account to DNP, this study aimed to evaluate the role of spinal 5-HT3R in mechanical hyperalgesia in streptozotozin (STZ)- diabetic rats. Diabetes was induced in male Wistar rats by an intraperitoneal injection of STZ (60 mg/kg). Control animals (CTR) received only the vehicle. A catheter was implanted in the lumbar subarachnoid space and at 4 weeks post-injection behavioural evaluation of mechanical nociception was assessed using the paw pressure test in STZ and CTR rats receiving intrathecal infusions of saline or 5-HT3R antagonist (Y-25130). Spinal segments L4-L5 were immunoreacted against 5-HT3R and its expression was quantified in the spinal dorsal horn by densitometry. STZ rats presented significantly increased glycaemia and haemoglobin A1C levels, along with decreased mechanical pain thresholds at 4 weeks post-injection. The intrathecal delivery of 5-HT3R antagonist induced a significant increase in the paw pressure thresholds in the STZ rats whereas no effects were detected in the CTR rats. Spinal 5- HT3R expression was not significantly different between STZ and CTR rats. These results demonstrated that inhibition of spinal 5-HT3R alleviates mechanical hyperalgesia in STZ rats. 5-HT3R activation by increasing serotonin levels upon SSRIs administration may be overlapping the inhibitory actions of serotonin through others 5- HT receptors. Novel pain therapies for DNP should act both on serotonin reuptake and 5- HT3R blockage. Support: FCT/COMPTE project PTDC/SAU-NSC/110954/2009. Diabetes, Pain, Serotonin, 5-HT3 receptor [email protected] 127

128 10:40 - FIIB9 Enhanced density and greater impact of adenosine A2A receptors in the control of cortico-striatal plasticity in pre-symptomatic Huntington s disease R6/2 mice Henrique B. Silva 1 s Henrique B. Silva 1, Joana Real 1, Jiang-Fan Chen 2, Rodrigo A. Cunha 1,3, Angelo R. Tomé 1,4 1 Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2 Department of Neurology, Boston University School of Medicine, USA; 3 Faculty of Medicine, 4 Faculty of Sciences and Technology, University of Coimbra, Portugal. Huntington s disease (HD) is characterized by a progressive motor symptoms associated with the degeneration of striatal neurons. However, motor symptoms are often preceded by cognitive and mood disturbances, reflecting an underlying dysfunction of corticostriatal synapses rather than resulting from neuronal death. Adenosine A2A receptors (A2AR), which are abundantly located in the striatum, have a dual impact on HD, preventing the onset of HD motor symptoms but aggravating its evolution at later stages. Given that A2AR can control synaptic plasticity, we now posted that A2AR blockade should selectively abrogate abnormal plastic changes in motor pre-symptomatic phases of HD. Thus, carried out electrophysiological recordings of synaptic plasticity (LTP and LTD) in cortico-striatal slices from wild type (WT) and R6/2 mice (models of HD) at 5 weeks of age, i.e. before onset of motor symptoms, and tested the impact of the A2AR antagonist SCH58261 (50 nm). High-frequency stimulation (3 trains of 100 Hz for 1 s with a 5 s interval) triggered LTP in WT (43.7±3.4% above baseline, n=6) and R6/2 mice (38.3±9.7%, n=3). SCH58261 inhibited LTP more in R6/2 (51.3±13.8%, n=3) than in WT (32.6±3.3%, n=6). Moderate saturating stimulation (4 trains of 1 Hz for 1 s at maximal intensity with a 10 s interval) triggered LTD in WT mice (-28.4±6.2%, n=5), which was unaffected by SCH58261 (n=5); in contrast, in R6/2 mice, LTD amplitude tended to be larger (36.8±2.9%, n=3) and SCH58261 inhibited LTD amplitude by 48.8±13.2% (n=3). Accordingly, A2AR density (3H-SCH58261 binding) in nerve terminal membranes from R6/2 (952.7±51.3 fmol/mg protein) was larger than in WT mice (694.8±7.6 fmol/mg protein, n=4). These results confirm that metaplasticity is modified in pre-symptomatic HD, where the enhanced A2AR density has greater normalizing impact on metaplasticity, supporting the benefits afforded by blocking A2AR in early HD. Supported by DARPA (09-68-ESR-FP-010) and FCT (PTDC/SAU-NSC/122254/2 Hungtinton's disease [email protected] 128

129 10:45 FIIB10 s PPP1 a putative Synphilin-1A PhosphoProtein Phosphatase/ Implications for Lewy Bodies formation Emanuel Ferreira Fernandes Sara Luisa Esteves, Emanuel Ferreira Fernandes, Odete da Cruz e Silva, Margarida Fardilha Aveiro University One of the major Parkinson s disease (PD) hallmarks is the development of cytoplasmic inclusions, termed Lewy bodies, within surviving neurons mainly in the brainstem of affected patients. Many proteins have been identified in Lewy bodies, but their formation mechanism remains unclear. Among the proteins already identified in the Lewy bodies are synphilin-1, a -synuclein-interacting protein, and synphilin-1a, a synphilin-1 splice variant. Synphilin-1 has been considered a key element in PD as co-expression of synphilin-1 and -synuclein in human embryonic kidney 293 cells leads to cytoplasmic inclusions, resembling Lewy bodies. Moreover, synphilin-1a is even more aggregative, being able to produce inclusion bodies upon overexpression, even without -synuclein co-expression. Therefore, efforts have been made to clarify the regulatory mechanisms behind synphilin-1 and synphilin-1a aggregation, being that protein phosphorylation is one of the most prominent events. Although kinases able to phosphorylate synphilin-1 have been described, there is no specific data concerning the phosphatases responsible for its dephosphorylation. Recently, using the yeast two hybrid system, synphilin-1a was identified as a novel phosphoprotein phosphatase 1-interacting protein in human brain. Hence, in the present work, the interaction between synphilin-1a and phosphoprotein phosphatase 1 is validated by yeast co-transformation and overlay assays. It is also demonstrated that the phosphoprotein phosphatase 1 binding motif RVTF present in synphilin-1a is crucial for the interaction and is conserved in human synphilin-1 and across species. Additionally, upon synphilin-1a-gfp overexpression in African green monkey cells, synphilin-1a and phosphoprotein phosphatase 1 are shown to co-localize in cytoplasmic aggregates. Taken together, these data suggest an interaction between synphilin-1a and PPP1, uncovering a putative phosphatase able to regulate synphilin-1a aggregation. Parkinson's disease, Lewy Bodies, Synphilin-1A, Phosphoprotein Phosphatase 1, Phosphorylation [email protected] 129

130 10:50 FIIB11 s Ecto-5 -nucleotidase (CD73)-mediated formation of ATP-derived adenosine is critical for the striatal adenosine A2A receptor functions Rodrigo A. Cunha Elisabete Augusto(1,2,3), Marco Matos(1,2,3), Jean Sévigny(4), Ali El-Tayeb(5), Christa E. Müller(5),, Jiang-Fan Chen(3), Rodrigo A. Cunha(1,2) (1)Center for Neuroscience and Cell Biology, (2)Faculty of Medicine, University of Coimbra, Portugal; (3)Department of Neurology, Boston University School of Medicine, USA; (4)Departement of Microbiology-Infectiology and immunology, Faculty of Medicine, University of Laval, Canada; (5)Pharma-Zentrum Bonn, Pharmazeutisches Institut, Pharmazeutische Chemie I, University of Bonn, Germany. Adenosine is a neuromodulator acting through inhibitory A1 receptors (A1R) and facilitatory A2AR, which have similar affinities for adenosine. A1R provides a global inhibition of activity whereas A2AR are selectively engaged to assist the implementation of synaptic changes in excitatory synapses. Whereas the activity of intracellular adenosine kinase preferentially controls the activation of A1R, we now tested the hypothesis that ATP-derived adenosine, through the action of ecto-5 -nucleotidase (CD73), would be the source of the adenosine activating A2AR, which are most densely located in the basal ganglia. We now report that CD73 co-localizes with A2AR in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 co-immunoprecipitated with A2AR and proximity ligation assays confirmed the close proximity of CD73 and A2AR in the striatum. Accordingly, the formation of camp in synaptosomes as well as the hypolocomotion induced by PSB (a novel A2AR prodrug that require CD73 metabolization to activate A2AR) were observed in wild type mice, but not in CD73 knockout (KO) mice or A2AR-KO mice. Moreover, CD73-KO mice displayed increased working memory performance (assessed with the Y maze and 8-radial arm maze tests), and a blunted the locomotor sensitization to amphetamine (2.5 mg/kg), mimicking the phenotype of global or forebrain-a2ar-ko mice. These results show that CD73-mediated extracellular formation of ATP-derived adenosine is responsible for the activation of striatal A2AR function. This study paves the way to propose CD73 as a new target that can fine-tune A2AR activity, and a novel therapeutic target to manipulate A2AR-mediated control of striatal function and neurodegeneration. Supported by DARPA (09-68-ESR-FP-010) and FCT (PTDC/SAU- TOX/122005/2010). adenosine, ecto-5'-nucleotidase, working memory, sensitization, striatum [email protected] 130

131 10:55 FIIB12 s Altered NMDA receptor and Src related signaling in Alzheimer s disease Elisabete Ferreiro Sandra I. Mota 1,2 ; Ildete L. Ferreira 1,2 ; Elisabete Ferreiro 1,2 ; Ana L. Carvalho 1,2,3 ; Catarina R. Oliveira 1,2,4 ; and A. Cristina Rego 1,2,4 1 CNC-Center for Neuroscience and Cell Biology, 2 Institute for Interdisciplinary Research (IIIUC), 3 Life Science Department, Faculty of Sciences and Technology, 4 Faculty of Medicine, University of Coimbra. Early cognitive deficits in Alzheimer s disease (AD) are thought to be related to NMDA receptor (NMDAR) dysregulation, related with synaptic dysfunction in response to amyloid beta-peptide accumulation. Thus, we analyzed age-and gender-dependent changes in NMDAR subunit protein levels and activation using cortical and hippocampal homogenates from the 3xTg-AD versus age-matched WT mice. In hippocampus, GluN2B Tyr1472 phosphorylation increased in 3xTg-AD females at 15 months of age, but decreased in 3xTg-AD males was observed at 3 months of age; importantly, the latter was correlated with modified activity of Src Tyr kinase. Moreover, early decreased Src activation in hippocampus and cortex of 3xTg-AD mice was associated with decreased Dab1 activation, a target of Src implicated in cytoskeleton stabilization. Reelin protein levels were also diminished in the cortex of young 3xTg-AD mice. In addition, Dab1 and cortactin, two proteins linked to cytoskeleton stability, were significantly decreased in 3 month-old 3xTg-AD hippocampus and cortex. Our results evidence early reduced Src activity in the hippocampus, which reduces the activation of Dab1 and GluN2Bcomposed NMDARs, favoring modified NMDAR activity in AD. This work was supported by Lundbeck Foundation, Project QREN DO-IT and FCT grants PTDC/SAU-NEU/71675/2006, PEst-C/SAU/LA0001/2011, and fellowships ref.sfrh/bd/43430/2008 and SFRH/BPD/43536/2008. Alzheimer s disease, NMDA receptor, cytoskeleton, 3xTg-AD mice, Src Tyr kinase [email protected] 131

132 Neurodegeneration and neuroprotection II June 1 Auditorium 132

133 10:00 FIIC1 s Intracerebroventricular streptozotocin induces cognitive and metabolic dysfuntion accompanied by synaptic modifications Cristina Lemos Cristina Lemos1,*, Jessié Gutierres1,2,*, Henrique Silva1, Paula Agostinho1,3, Catarina R. Oliveira1,3, Rui A Carvalho1,4, Rodrigo A Cunha1,3 *Equivalent first authors; 1CNC-Centre for Neuroscience and Cell Biology, 2Department of Biochemistry, Federal University of Santa Maria; RS, Brazil, 2Faculty of Medicine, 3Faculty of Sciences and Technology, University of Coimbra, Portugal. Alzheimer s disease (AD) is characterized at its early stages by memory dysfunction. Clinical and experimental evidence show a relation between metabolic dysfunction and neurological symptoms in AD. This lead us to explore a non-transgenic animal model to study AD based on the intracerebroventricular (icv) injection of streptozotocin (STZ, 3mg/kg) to adult male Wistar rats. After 4 weeks, icv-stz rats displayed a higher latency to reach the platform (p<0.05) in a spatial version of the Morris water maze; they also spent less time in the correct quadrant (CTR 23.6±2.5%; STZ 16.0±3.6%; n=8; p<0.05) and performed less crossings of the platform (CTR 3.6±0.7; STZ 1.4±0.5; p<0.05). icv-stz rats also showed a decreased recognition index score in the object recognition test (CTR 0.21±0.09; STZ -0.06±0.10; n=8; p<0.05) and a lower exploration time of an object new localization in the object recognition test (CTR 19.7±2.4; STZ 13.2±2.9; n=8; p<0.05). HRMAS-NMR analysis of cortical tissue showed that icv-stz rats displayed a significant reduction of the lactate/alanine ratio, consistent with an increased oxidative stress. Western blot analysis showed that icv-stz rats had a 48%±13 decrease of synaptophysin density while no differences were observed for postsynaptic marker (PSD-95) in the hippocampus. Electrophysiological extracellular recordings in hippocampal slices of icv-stz rats showed a decrease of high-frequencyinduced long-term potentiation (LTP: CTR 148.4±0.9%; STZ 116.7±0.2%; n=7) in Schaffer fibers-ca1 pyramid synapses. These results indicate that icv-stz triggers a metabolic impairment typified by enhanced oxidative stress that predominantly affects synaptic compartments, leading to a deficits of synaptic plasticity that underlies the memory impairment. Since this icv-stz model recapitulates key features of sporadic AD, it is ideally suited to search for novel neuroprotective strategies. (Supported by FCT grant PTDC/SAU-NMC/114810/2009 and CAPES-FCT) Alzheimer s disease, intracerebroventricular streptozotocin, [email protected] 133

134 10:05 FIIC2 Identification of Alzheimer biomarkers with mid-infrared spectroscopy exploratory study Alexandra Nunes s Rafaela Silva1, Ana Gabriela Henriques1, Joana M. Oliveira1, Liliana Carvalho1, Ilka Rosa1, Ivonne Delgadillo2, Odete A. B. Cruz e Silva1, Alexandra Nunes1 1 Centre for Cell Biology; Biology Department; Health Sciences Department, University of Aveiro, Portugal; 2 QOPNA, Chemistry Department, University of Aveiro, Portugal Absorption of radiation in the mid-infrared spectral region provides extensive information on the chemical composition of biological samples. Most molecular species absorb infrared light, giving rise to characteristic spectral patterns in transmitted light. Pathological conditions are associated with perturbations of metabolism that are reflected in homeostatic changes of molecular components in cells and tissues. The clinical potential of FTIR spectroscopy to detect such changes and its use as a diagnostic tool has received increasing attention and it is a straightforward way to clearly understand dementia at a biochemical level and assist in identifying specific biomarkers. This exploratory study aims to test the ability of FTIR to discriminate between control and Alzheimer s Disease (AD) samples and to identify biomarkers present in serum and plasma in order to provide information that allows the development of objective and sensitive methodology for the diagnosis of this dementia, based on spectroscopy and multivariate analysis. Blood samples were collected and the volunteers provided clinical information and were submitted to several cognitive tests including the Mini Mental State and Clock test. Putative AD positive plasma and serum samples, as well as age and sex matched controls were analyzed by FTIR. Spectra in the cm-1 range were submitted to Principal Component Analysis (PCA) multivariate analysis. With PCA scores it was possible to undoubtedly discriminate between AD samples and corresponding age and sex matched controls. PCA loading profiles allowed for the identification of spectroscopic signals of the functional groups of biomarkers involved in the discrimination between normal and patient samples. After matching all the spectroscopic signals to the corresponding AD biomarkers and gathering the major biomarker responsible for the spectroscopic identification of AD a multivariate classification model will be developed. Acknowledgements: Fundação para a Ciência e Tecnologia, Portuguese Ministry of Science and Technology: JPND BIOMARKAPD - Biomarkers for Alzheimer s disease and Parkinson s disease Alzheimer, Biomarkers, FTIR, mid-infrared spectroscopy [email protected] 134

135 10:10 FIIC3 s impact of a P2X7 (ATP) receptor antagonist on 6-OHDA-induced neurotoxicity in vivo and in vitro Marta R S Carmo Marta R. S. Carmo, Paula M. Canas, Ana Paula F. Menezes, Ana Carla L. Nunes, Francisco Queiroz, Anabela P. Rolo, Carlos M. Palmeira, Rodrigo A. Cunha, Geanne M. Andrade Federal University of Ceará; Center for Neuroscience and Cell Biology, University of Coimbra. Parkinson s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and a concomitant decrease of dopamine (DA) in the striatum, which can be modeled by 6-hydroxydopamine (6-OHDA) administration. Since ATP has been explored as a danger signal acting through its P2X7 receptors (P2X7R), we now tested the ability of a P2X7R antagonist, Brilliant Blue G (BBG), on 6-OHDA-induced neurotoxicity. Administration of BBG (45 mg/kg i.p., every 48 h for 2 weeks) to male adult rats attenuated the 6-OHDA-induced: 1) increase of contralateral rotations in the apomorphine test (sham: 3.429±1.304; 6-OHDA: 237.0±27.55; 6- OHDA+BBG: 137.4±27.38; p<0.05); 2) reduction of DA content in both the striatum (sham: ±349.3; 6-OHDA: 44.9±21.7; 6-OHDA + BBG: ±349.3 ng/mg tissue; p<0.05) and SN; 3) microgliosis and astrogliosis in the striatum. No differences were observed in locomotor activity in animals treated with BBG compared to 6-OHDA or sham group. To grasp the mechanism of action of BBG, we turned to in vitro models exploring synaptotoxicity (using striatal synaptosomes) and neurotoxicity (using a DAdifferentiated human neuroblastoma SH-SY5Y cell line). We first showed the presence of P2X7R in striatal dopaminergic terminals, where BBG (100 nm) prevented the 6- OHDA-induced synaptosomal dysfunction assessed by the Alamar Blue assay. We also showed a co-localization of P2X7R and tyrosine hydroxylase in SH-SY5Y cells; here, 6- OHDA (30 µm) increased the extracellular ATP levels and BBG (100 nm) attenuated the 6-OHDA-induced neurotoxicity, measured as LDH release (% of control: 6-OHDA: 262±27; 6-OHDA+BBG: 182±17). This suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in PD. Supported by FCT - Portugal, CNPq (Edital MCT/CNPq 14/2010) and CAPES Brazil. ATP, P2X7, Parkinson s disease, synaptotoxicity, neuroprotection [email protected] 135

136 10:15 FIIC4 s Beclin-1 mitigates motor and neuropathological deficits in genetic mouse models of Machado-Joseph disease Clevio Nóbrega Isabel Nascimento- Ferreira, Clévio Nóbrega, Ana Vasconcelos-Ferreira, Isabel Onofre, David Albuquerque, Célia Aveleira, Nicole Déglon, Luis de Almeida Center for Neurosciences of Coimbra Machado-Joseph disease or spinocerebellar ataxia type-3, the most common dominantly-inherited spinocerebellar ataxia, results from translation of the polyglutamine-expanded and aggregation prone ataxin-3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs and there is no therapy to delay disease progression. Beclin-1, an autophagy-related protein essential gene for cell survival, is decreased in several neurodegenerative disorders. This study aimed at evaluating if lentiviral-mediated beclin-1 overexpression would rescue motor and neuropathological impairments when administered to pre and post-symptomatic lentiviral-based and transgenic mouse models of Machado-Joseph disease. Beclin-1 mediated significant improvements in motor coordination, balance and gait with beclin-1-treated mice equilibrating longer periods in the rotarod and presenting longer and narrower footprints. Furthermore, in agreement with the improvements observed in motor function beclin-1 overexpression prevented neuronal dysfunction and neurodegeneration, decreasing formation of polyglutamine-expanded aggregates, preserving Purkinje cell arborization and immunoreactivity for neuronal markers. These data show that overexpression of beclin-1 in the mouse cerebellum is able to rescue and hinder the progression of motor deficits when administered to pre and post-symptomatic stages of the disease. Autophagy,Beclin-1,Machado-Joseph disease,spinocerebelar ataxia type 3, neurodegeneration [email protected] 136

137 10:20 FIIC5 Expression and activity of thimet oligopeptidase (TOP) are modified in the hippocampus in subjects with temporal lobe epilepsy (TLE) Maria da Graça Naffah-Mazzacoratti s Naffah-Mazzacoratti MG, Simões PSR; Visniauskas B; Yacubian EMT; Centeno R; Canzian M ; Lopes-Cendes I; Morelli CVM; Cavalheiro EA, Chagas JR Universidade Federal de São Paulo, The Thimet oligopeptidase (TOP) is a metalloprotease and has been described as neuropeptides metabolizing enzyme in the nervous system. Its substrates include several peptides and between them are: bradykinin, beta amyloid (βa) and MHC I peptides. In this study, we evaluated the expression, distribution and activity of TOP through the techniques of RT- PCR, enzymatic activity, Western blotting and immunohistochemistry in the hippocampus of patients with temporal lobe epilepsy (TLE) and control tissues obtained from autopsy. Hippocampi of rats submitted to epilepsy model induced by pilocarpine were also analyzed. The animals were divided according to the periods of the epilepsy model in: acute, silent and chronic phases and their respective controls received saline. We found an increase in mrna expression in tissues of patients, when compared to control. However, protein levels and enzymatic activity proved diminished. This decrease distribution of TOP was also visualized by immunohistochemistry. Similar results were found in rats during acute phase of epilepsy model. These results show that the process of protein synthesis seems to be increased. However, post-translational mechanisms make TOP with lower enzymatic activity being less recognized by the employed antibody. In the silent phase we also found an increase in TOP mrna expression and minor immunoreactivity in the hippocampus. Differently, in the chronic phase, an increase in mrna expression and increased enzymatic activity of TOP were found. Taken together these results show that these alterations could be related to a failure in mechanisms involved in the clearance of inflammatory peptides in the hippocampus, suggesting an accumulation of potentially harmful substances to the nervous tissue, such as βa, bradykinin and antigenic peptides. These accumulations could be linked to hippocampal inflammation found in TLE. Temporal lobe epilepsy, thimet oligopeptidase, hippocampus [email protected] 137

138 10:25 FIIC6 Up-regulation of A2AR density in postmortem prefrontal tissue of individuals who committed suicide Canas P. M. s Canas P.M. 1, da Silva B.S 2, Cunha R.A. 1,3 1 Center for Neurosciences and Cell Biology, Coimbra Portugal; 2 INMLCF, I.P., Coimbra Portugal; 3 FMUC, Coimbra Portugal Chronic caffeine consumption attenuates the burden of repeated stress, the incidence of depression and suicides in humans and prevents phenotypic changes caused by chronic stress in rodents. However, neither the neurobiology of depression nor the mechanisms of adenosine A2A receptor (A2AR) control of mood dysfunction are known. The study of the neurobiology of depression can be bolstered by the study of brain tissue from people who committed suicide (suicidals), since over 90% have clinical signs of depression. We now tested if there is a modification of synaptic markers in areas related with mood disorders and if this is accompanied by an up-regulation of A2AR. We compared postmortem tissue from male suicidals when compared with age-matched controls. Specifically, we evaluated by Western blot analysis total extracts and synaptic fractions (synaptosomes) prepared from Brodmann area 25 (BA25) of suicidals. This revealed a decrease of 41.3±11.0% (n=5, p<0.05) of the density of SNAP-25 (a synaptic marker) in synaptosomes from BA25 of suicidals. In total extracts there was no difference in the astrocytic marker, glial fibrillary acid protein (GFAP), whereas the densities ratio between A2AR and -actin increased by 97±30.5% (n=4, p<0.05) in BA25 of suicidals. Additional studies using purified nerve terminals are on-going to define if the increase of A2AR occurs at the particular synapse or in other compartments or cell types; these studies will also enable to define is any particular type of nerve terminals are affected in BA25 of suicidals and if this also occurs in other brain regions pertinent to mood control. These results will further reinforce the relevance of targeting A2AR to manage depression. Supported by DARPA ESR-FP-010, PTDC/SAU-NSC/122254/201, PEstc/SAU/LA0001/2011) A2AR, suicide, postmortem, prefrontal [email protected] 138

139 10:30 FIIC7 Oxidative and ER stress in early stages of Alzheimer s disease Rego A.C. s Rego A.C.1,2,3, Mota S.I.1,2, Costa R.O.1,2, Caldeira G.L.1,2, Santana I.3,4, Padovano C.1,2, Fonseca A.C.1,2, Catarina Cunha4, Letra L4., Oliveira C.R1,2,3, Ferreira I.L.1,2, Pereira C.M.F1,2,3 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 2Institute for Interdisciplinary Research (IIIUC), 3Faculty of Medicine, University of Coimbra, and 4Neurology Unit of Coimbra University Hospital Center, Coimbra, Portugal. Here we show that oxidative stress involving Nrf2 deregulation and ER stress constitute early events in AD pathogenesis. Increased levels of ROS were observed in human peripheral blood mononuclear cells (PBMCs) isolated from MCI individuals, which correlated with downregulation of Nrf2, a ROS-related transcription factor, and SOD1, a Nrf2 target. Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of oxidative stress in human peripheral cells were corroborated by decreased SOD1 and reduced nuclear Nrf2 activation in young (3 month-old) 3xTg-AD versus age-matched non-transgenic mice male brain cortex; increased ER stress was detected in the brain cortex of young female and old male 3xTg-AD mice. Moreover, decreased nuclear levels and activation of Nrf2 correlated with increased ROS generation in mature rat brain cortical cultures treated with Aβ1-42. Interestingly, treatment of cortical cultures with the N-methyl-D-aspartate receptor antagonist memantine prevented Aβ1-42- induced ROS generation, although did not significantly decrease Nrf2 levels. In conclusion, data largely suggest depletion and decreased nuclear activation of Nrf2 contributing to oxidative stress, which is linked to ER stress, in early stages of AD. This work was supported by Lundbeck Foundation, project QREN DOIT and FCT (Fundação para a Ciência e a Tecnologia, Portugal) grant PEst-C/SAU/LA0001/2011 and by the Programa Operacional Temático Factores de Competitividade (COMPETE), supported by the European community fund FEDER, and by FCT Ph.D. fellowships SFRH/BD/43430/2008, SFRH/BD/28403/2006 and SFRH/BD/47573/2008. Alzheimer disease, oxidative stress, Ca2+ homeostasis, endoplasmic reticulum stress, amyloid-beta peptide [email protected] 139

140 10:35 FIIC8 Brain insulin signaling pathway and synaptic integrity under insulin-induced recurrent hypoglycemia and long-term hyperglycemia Susana Cardoso s Cardoso S1,2, Santos MS1,2, Moreira PI1,3 1Center for Neuroscience and Cell Biology, University of Coimbra, 2Department of Life Sciences Faculty of Sciences and Technol Evidence corroborates that diabetic conditions affect brain function and cognitive performance while alterations of the insulin signaling cascade underlie cognitive decline and the development of several neurodegenerative diseases. The present study was devoted to assess the effects of blood glucose fluctuations on brain cortical insulin signaling pathway and synaptic integrity. To achieve our goal, experiments were performed in streptozotocin-induced diabetic rats treated with twice-daily insulin injections for 2 weeks to induce hypoglycemic episodes, thus mimicking the oscillations of blood glucose levels occurring in type 1 diabetic patients. The levels of some proteins involved in the insulin signaling pathway and the protein levels of synaptophysin, a marker of synaptic integrity, were evaluated in brain cortical homogenates. Recurrent hypoglycemic episodes promoted a decrease in the protein levels of insulin receptor and an increase in insulin-like growth factor-1 receptor (IGF-1R). Further, the evaluation of the downstream phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) insulin-dependent pathways revealed a decrease in P-Akt/Akt and P-ERK/ERK protein levels and an increased activation of glycogen synthase 3-beta (GSK-3beta) in cortical homogenates from recurrent hypoglycemic animals. Likewise, a decrease in synaptophysin protein levels was also found in recurrent hypoglycemic rats. Overall, our results show that blood glucose fluctuations lead to an impairment in the insulin signaling cascade, which might compromise brain cells function and integrity. Susana Cardoso has a PhD fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/43968/2008). brain, hyperglycemia, hypoglycemia, insulin signaling pathway [email protected] 140

141 10:40 FIIC9 Activation of adenosine A3 receptor protects retinal ganglion cells from ischemiareperfusion injury Tiago Martins s Tiago Martins 1,2, Filipe Elvas 1,2, Joana Galvão 1, António F. Ambrósio 1,2, Ana R. Santiago 1,2 1 Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2 AIBILI, Coimbra, Portugal Several ocular diseases lead to optic nerve damage and retinal ganglion cell (RGC) loss. The ultimate cause of vision loss in glaucoma, the second leading cause of blindness worldwide, is the RGC death. Neuroprotection of RGCs is therefore an important goal of glaucoma therapy, and so it is crucial to develop treatment approaches for glaucoma that actively prevent the death of RGCs. Adenosine is a neuromodulator in the central nervous system (CNS) and it is considered one of the most promising neuroprotective agents in CNS. The activation of adenosine A3 receptor (A3R) has been shown to protect against a broad spectrum of insults. Since RGCs express A3R, the main objective of this work was to investigate whether activation of A3R prevents RGCs from death induced by ischemia-reperfusion (I/R) injury in the rat retina. Retinal I/R injury was induced in Wistar rats by elevating the IOP to 80 mmhg for 60 min. The A3R agonist 2Cl-IB-MECA or vehicle solution was injected intravitreally 2 h before the onset of ischemia. At 24 h post ischemia injury, immunoreactivity for A3R and Brn3a, and TUNEL assay, were performed in retinal sections. The immunoreactivity of A3R decreased in the retinas subjected to I/R injury, as compared to control retinas. The administration of 2-Cl-IB-MECA prevented A3R downregulation induced by I/R injury. The number of TUNEL positivecells was significantly increased after I/R injury, and this increase was prevented by 2-Cl-IB- MECA. Also, the intravitreal injection of 2-Cl-IB-MECA prevented the I/R-induced loss of RGCs in the rat retina. Taken together these data demonstrate that activation of A3R protect the retina from I/R injury, particularly RGCs. These results may open the possibility of a new pharmacologic strategy for the treatment of retinal degenerative diseases, like glaucoma. Support: FCT Strategic Project (Ref: PEst-C/SAU/UI3282/2011), Portugal and COMPETE-FEDER. Adenosine A3 receptor, Ischemia-reperfusion, Retina, Retinal ganglion cells [email protected] 141

142 10:45 FIIC10 s Orally-administered calpain inhibitor BDA-410 reduces ataxin-3 cleavage and alleviates neuropathology in a lentiviral mouse model of Machado-Joseph disease Ana T. Simões 1,2 Ana T. Simões 1,2, Nélio Gonçalves 1,2, Rui J. Nobre 1, Carlos Bandeira Duarte 1,3, Luís Pereira de Almeida 1,2 1Center for Neuroscience and Cell Biology 2Faculty of Pharmacy of Coimbra 3Faculty of Sciences and Technology of Coimbra Machado-Joseph disease (MJD) is the most prevalent autosomal dominantly-inherited cerebellar ataxia. It is caused by an expanded CAG repeat in the MJD1 gene, which translates into a polyglutamine tract within the ataxin-3 protein. Present treatments are symptomatic and do not prevent disease progression. As calpain overactivation has been shown to contribute to mutant ataxin-3 proteolysis, translocation to the nucleus, inclusions formation and neurodegeneration, we investigated the potential role of calpain inhibition as a therapeutic strategy to alleviate MJD pathology. For this purpose, we administered orally the calpain inhibitor BDA-410 to a lentiviral mouse model of MJD. Our data provide evidence that not only the fragments are the toxic species, but also that mutant ataxin-3 aggregates of specific size have different toxic properties. Western-blot and immunohistochemical analysis revealed the presence of N- and C-terminal mutant ataxin-3 fragments and the colocalization of large inclusions with cleaved caspase-3 in the mice brain. Furthermore, we show that oral administration of the calpain inhibitor BDA-410 decreased both fragments formation and full-length ataxin-3 levels, reduced aggregation of mutant ataxin-3 and prevented cell injury and striatal degeneration. In conclusion, BDA-410 alleviates Machado-Joseph neuropathology and may therefore be an effective therapeutic option for MJD. This work was supported by the Portuguese Foundation for Science and Technology (PTDC/SAU-NEU/099307/2008), the Richard Chin and Lily Lock Machado-Joseph research fund, the National Ataxia Foundation and the Association Française pour les Myopathies for funding. A.T.S., N.G. and R.J.N. were supported by FCT fellowships BD/38636/2007, BD/33186/2007 and SFRH/BPD/66705/2009. Machado-Joseph Disease, calpains, proteolysis, aggregation [email protected] 142

143 10:50 FIIC11 Expression of purinergic receptors as phenotypic changes in human temporal lobe epilepsy 1Amorim RP s 1Amorim RP; 1Araújo MGL; 2Canzian M; 3Ariza CB; 3Porcionatto M; 1Cavalheiro EA; 4Ulrich H; 5Carrete Jr.H; 5Centeno RS; 5Yacubian EM; 1Fernandes MJS 1Departamento de Neurologia e Neurocirurgia Disciplina de Neurologia Experimental UNIFESP São Paulo, Brazil. 2Departamento de Anatomia Patológica Incor/USP São Paulo, Brazil. 3Departamento de Bioquímica Disciplina de Biologia Molecular UNIFESP São Paulo, Brazil. 4Departamento de Bioquímica Instituto de Química, USP São Paulo, Brazil. 5Departamento de Neurologia e Neurocirurgia Disciplina de Neurologia Clínica UNIFESP São Paulo, Brazil. Objective: Previous studies have shown that purinergic receptors are involved in the pathophysiology of Temporal Lobe Epilepsy (TLE). The aim of this study was to evaluate the expression of the P2 receptors as well as the ectonucleotidases (ectonucleoside triphosphate diphosphohydrolases, ENTPDases) in the hippocampus of patients with TLE. Methods: TLE patients (N = 11) were selected and subjected to detailed anamnesis, EEG and MRI analyses. The hippocampi were processed for P2 receptors and ENTPDases analyses using RT-PCR and differential mrna expression was validated by Western Blot. Non-epileptic autopsy tissue was used as control (N = 12). All procedures were approved by the al Ethics Committee of the Universidade Federal de São Paulo. Results: mrna expression for several subtypes of P2 receptors (P2X4, P2X6, P2X7, P2Y1) and of ENTPDase 1, 3 and ecto-5 -nucleotidase were increased in the hippocampus of patients with TLE. Significant increases were observed in the hippocampus of TLE (>80-fold to P2X4, p<0.05; >25-fold to P2X6, p<0.05; >5-fold to P2X7 and P2Y1, p<0.001 >40-fold to ENTDase 1 and 3, p<0.05; and >15-fold to ecto- 5 -nucleotidase, p<0.05, Student t test) when compared to control tissue. The Western Blot analysis showed an increased expression in P2X4, P2X7 and P2Y1 (>10-fold, p<0.001; >3-fold, p<0.0001; >7-fold, p< , respectively, Student t test) when compared to control sample. Conclusions: This study shows that the purinergic signaling is up regulated in the hippocampus of patients with TLE. The higher expression of P2 receptors and ectonucleotidases indicates that purines could be part of genetic factors involved with hippocampal sclerosis and seizure refractoriness. Financial Support: Cinapce-Fapesp, Fapesp-Fapemig, CNPq, CAPES and INNT/MCT. hippocampus, human brain, purinergic receptor, temporal lobe epilepsy. [email protected] 143

144 10:55 FIIC12 s Alpha-synuclein overexpression modifies the antioxidant response in SH-SY5Y neuroblastoma cells Rita Catarina Gonçalves Perfeito Rita Catarina Gonçalves Perfeito, Márcio Ribeiro, Leonidas Stefanis, A. Cristina Rego Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Alpha-synuclein (alpha-syn) is an abundant neuronal protein that has been linked to neurodegeneration in both sporadic and familial forms of Parkinson s disease (PD). Mutations, duplications and triplications in the gene encoding alpha-syn are responsible for earlier forms of PD. Moreover, alpha-syn post-translational modifications, aggregated alpha-syn, mitochondrial dysfunction and oxidative stress have been also pointed out as key pathogenic events of this disorder. Here, we analyzed the link between oxidative stress and alpha-syn in SH-SY5Y neuroblastoma cell lines conditionally expressing WT alpha-syn in a system regulated by doxycycline ( Dox). Our results showed an increase in reactive oxygen species (ROS) under basal conditions in cells overexpressing alpha-syn, accompanied by a reduction in the activity of mitochondrial complex I, compared to cells expressing the endogenous protein (+Dox). Furthermore, increased ROS levels were closely related with a decrease in the activities and levels of proteins involved in the antioxidant defense pathways, namely SOD1, SOD2 and reduced (GSH) and oxidized (GSSG) glutathione. Accordingly, the levels of the catalytic subunit of glutamate-cysteine ligase (GCLc) were also diminished. Further analysis of nuclear fractions demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2), which activates the gene transcription of these antioxidant proteins following mild intracellular ROS production, showed decreased levels in Dox cells. Preliminary results also demonstrated reduced Nrf2 activity in cells overexpressing alpha-syn. Overall, data suggest that overexpression of wild-type alpha-syn may be involved in modifying the susceptibility to oxidative stress and the antioxidant response, accordingly to the pathogenic mechanisms described in PD. This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, project reference PTDC/SAU-NEU/101928/2008. Parkinson s disease, alpha-synuclein, ROS, antioxidant enzymes, Nrf2 [email protected] 144

145 Fire Sessions III 145

146 Neural systems II June 1 Bussaco room 146

147 11:30 - FIIIA1 s Impact of ventro-medial striatal adenosine A2A receptors on mood- and anxietyrelated behavioural modifications in rats subjected to repeated restraint stress Nélio Gonçalves Nélio Gonçalves (1), Nuno J. Machado (1,2), Carolina M. Souza (1), Luís Pereira de Almeida (1,3), Rodrigo A. Cunha (1,2) 1)CNC-Center for Neuroscience and Cell Biology, 2)Faculty of Medicine and 3)Faculty of Pharmacy, University of Coimbra, Portugal Mood and anxiety disorders are the leading cause of burden of disease in Europe. Subjecting rodents to repeated stress triggers a constellation of behavioral modifications resembling the human situation. We have previously reported that cortical adenosine A2A receptors (A2AR) are up-regulated upon repeated stress (Cunha et al., 2006, Neuroscience 141: 1775) and others have shown that A2AR blockade attenuates learning disabilities caused by chronic stress (Batalha et al. 2013, Mol. Psychiatry 8: 320). Since we also found changes of synaptic markers and a higher density of A2AR in the ventro-medial striatum (VMS) of repeatedly stressed rats, we now probed the specific role of A2AR in the VMS to control stress-induced mood and anxiety abnormalities. Male adult rats (Wistar) were injected bilaterally in the VMS with lentiviral vectors encoding either a short hairpin for A2AR silencing (sha2ar) or a control (shctr). One week after, rats were handled (control) or subjected to 2 weeks of repeated restraint stress (4h daily). All rats (n=8-10 per group) were behaviourly evaluated in reward (anhedonia), despair (forced-swimming test - FST) and anxiety-based tests (elevatedplus maze - EPM). Stressed rats injected with shctr displayed a significant (p < 0.01) reduction of sucrose preference as well as a reduction of the EPM % time in the open arms, with no major modifications either on locomotion or on despair-related behaviour. Lentiviral-mediated A2AR knockdown in VMS recovered rats from stress-induced anhedonia and anxiety. These data is highly suggestive of a crucial role for ventro-medial striatal A2AR on stress-induced behavioural modifications associated with depressive- and anxiety-like conditions. Supported by DARPA (09-68-ESR-FP-010), FCT (PTDC/SAU-NSC/122254/2010, PEst-C/SAU/LA0001/2011) and CAPES. chronic restraint stress, A2A receptors, reward, despair, anxiety [email protected] 147

148 11:35 - FIIIA2 s Calcium-binding proteins in the brain and retina of a teleost fish E. de Miguel Villegas E. de Miguel Villegas, MJ. Quiñones-Correa; E. de Miguel Villegas; R. Álvarez-Otero University of Vigo Calbindin (CB), calretinin (CR) and parvalbumin (PV) are calcium-binding proteins (CaBPs) which serve as intracellular calcium buffers. They are expressed in both the central nervous and sensory organs of teleosts and have been widely used as neuronal markers to identify different neuron types. In this study, we describe the distribution of CB, CR and PV proteins in the brain and retina of a teleost species. Brain and retina were fixed and immunohistochemically processed using antisera for specific binding of these three proteins. Double immunofluorescence methods were used for co-localization studies. Immunoreactive (ir) structures were present in specific neuronal populations and in fibres throughout the brain. CR-ir elements spreaded in the dorsal telencephalon and in the tracts extending towards the diencephalon. CB-ir and CR-ir cells were observed in the diencephalic preoptic area. In addition, neurons containing CB, CR and PV were present in the glomerular nucleus and some cells also showed CB/CR co-localization. In the midbrain, the optic tectum contained CB- and CR-ir cells and fibres. Throughout the mesencephalic and rhombencephalic tegmentum, localization of CB and CR was prominent in the medial longitudinal fascicle. In the cerebellum, Purkinje and eurydendroid cells as well as some fibres showed immunoreactivity for the three CaBPs. In the rhombencephalon, several cells in the reticular formation and the cranial nerve nuclei were also CB-, CR- and PV-ir. In the retina, the most prominent CR-immunolabeling was found in ganglion cells as well as in the optic nerve layer. In addition, CR was also expressed in a subpopulation of amacrine and bipolar cells. A CR-ir varicose band was observed in the inner plexiform layer together with some positive labeling of the outer plexiform layer. In contrast, only a few retinal elements were PV- and/or CB- ir. Therefore, only a few amacrine cells were stained by PV or CB and a few ganglion cells expressed CB. Calcium-binding proteins, brain, retina, teleost [email protected] 148

149 11:40.-. FIIIA3 s GABAergic and cholinergic circuit defects in a C. elegans mutant of an intellectual disability-associated gene Carlos Bessa Carlos Bessa, Ana João Rodrigues*, Carlos Bessa*, Filipe Marques, Bruno Vasconcelos, Filipa Pereira, Adriana Miranda, Patrícia Maciel Life and Health Sciences Research Institute (ICVS), School of Health Science, University of Minho. Intellectual disability (ID) is one of the most frequent and disabling neurological impairments with an estimated prevalence of 1.5-2% in Western countries. Technological advances such as the use of array comparative genomic hybridization (acgh), and more recently, next generation sequencing, have been adopted by several genetic laboratories, in order to identify novel genetic basis of ID. This large-scale analysis originates an incredible number of novel genetic associations every year. However, these associations often lack functional validation, and for several of the identified genes, their function in the nervous system remains undisclosed. This prompted us to use a simple model, the Caenorhabditis elegans (C. elegans), to functionally validate the genetic associations and to better understand the role of a given protein in the nervous system. So far, we have studied 25 mutant strains, that correspond to 18 orthologues of human genes previously linked to ID. One candidate, ID34, presents gross anatomical defects (multivulva, loss of vulva), developmental delay (larval arrest), embryonic lethality and decreased life span. In parallel, this strain presents motility defects (increased uncoordination) and mild chemotaxis defects, suggesting some degree of impairment of neurological function. Crossing ID34 with strains expressing GFP in specific neuronal sub-types, we found that the GABAergic network was strongly affected - abnormal neuronal positioning and migration. The cholinergic circuit also presented some defects, but not so pronounced. Consistently, animals were more sensitive than the wild-type to Pentylenetetrazol (PTZ - GABA antagonist) and to aldicarb, a cholinesterase inhibitor. We are now assessing these mutants in other behavioral paradigms to assess learning and memory. Intellectual disability, C. elegans, GABA [email protected] 149

150 11:45 - FIIIA4 s Cortical responses during parametric visual speed discrimination in healthy and diabetic patients João Duarte João Duarte, Miguel Raimundo, Miguel Castelo-Branco Visual Neuroscience Laboratory, IBILI - Faculty of Medicine, University of Coimbra, Portugal Here we compared the BOLD functional magnetic resonance imaging (fmri) and psychophysical responses of 49 healthy subjects and 36 patients with type 2 diabetes during a speed discrimination visual task. After an individual determination of the psychophysical speed difference threshold the participants detected the faster of two dots, one in each visual hemi-field (moving with 4 possible speed differences). Speed discrimination recruited a visuomotor network including parietal and occipital regions in relation to stimulus physical characteristics and hmt and frontal regions modulated with task difficulty. In healthy participants BOLD responses were parametrically modulated such that conditions with higher speed differences showed higher activation in sensory regions and lower activation regions related to perceptual decision (beta values per condition tested with ANOVA, p<0.05). Furthermore, a contrast comparing the response to speed differences in the same visual hemi-field revealed statistically significant activation in the contra-lateral insula, suggesting its important role in interhemispheric integration of motion information. In diabetic patients we observed significant fmri signal change in some of the same regions identified in the healthy brain. Whereas we found a linear parametric effect of the speed discrimination task difficulty in fmri responses in healthy participants, this was not identified in type 2 diabetic patients. Interestingly, there was a difference in the psychophysical speed discrimination threshold between groups (thrcnt = 1.52; thrdia = 2.80; p = 0.003). These results suggest that a different hemodynamic response function might be the cause of the weak modulation of neuronal responses found in type 2 diabetes during speed discrimination. vision, motion, speed discrimination, functional magnetic resonance imaging, type 2 diabetes [email protected] 150

151 11:50 - FIIIA5 s Motor and instrumental learning in mice with interneuron NMDA receptor deletion Gabriela J. Martins Gabriela J. Martins, Ana Vaz, and Rui M. Costa Champalimaud Neuroscience Programme, Av. de Brasilia, Doca de Pedrouços Lisboa, Portugal The striatum is the major input structure for the basal ganglia, processing inputs from virtually all cerebral cortical areas and from the thalamus, thus modulating motor, emotional, and cognitive behaviors. Proper development of inhibitory neurons is critical for modulating excitatory transmission. Alterations to this balance lead to various longterm neurological disorders. Two key interneuron populations in striatum are the parvalbumin-positive (PV+) and cholinergic (ChAT+) cells, which strongly modulate the activity of striatal projection neurons. Patients with Tourette syndrome, a childhoodonset neuropsychiatric disorder, have a significant reduction in both PV and ChATexpressing cells in the striatum, and show deficits in procedural learning. In this study, we analyzed the loss of the NMDA subunit, NR1, in PV+ (PVCre:NR1fx/fx) and ChAT+ (ChATCre:NR1fx/fx) cells in conditional knockout mice, to assess the role of plasticity at glutamatergic synapses onto interneurons in motor and instrumental learning. Mice tested on the open field both during postnatal development and in adulthood demonstrated proper gross behavioral development. As adults, the loss of the NR1 subunit in PV+ cells appears to impair motor abilities on the rotarod whereas loss of this same subunit in ChAT+ cells increased the latency to fall from the rotarod. Furthermore, these mice were tested for the formation of goal-directed and habitual behaviors in an operant task. Preliminary data suggests that the loss of the NR1 subunit in these two cell populations may have opposing effects on sensitivity to outcome devaluation and that the loss of NR1 in PV+ cells may increase motivation in lever pressing for food. Future studies will aim to target the loss of parvalbumin and ChAT+ cells to the dorsal striatum through the use of the cre-dependent inducible diptheria toxin receptor mechanism. NMDA, Interneuron, motor function, instrumental learning [email protected] 151

152 11:55 - FIIIA6 s From novelty-seeking to impulsive behaviour: effects of prenatal exposure to glucocorticoids Coimbra B. Coimbra B., Soares-Cunha C., Borges S., Sousa N., Rodrigues A.J. ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Prenatal exposure to glucocorticoids (GCs) has been shown to target the mesocorticolimbic system, inducing structural and molecular alterations that may underlie the addictive-like behavior observed in animals exposed to GCs in utero (iugc). Vulnerability for addictive behaviors may be modulated by individual emotional condition and/or specific personality traits. For example, novelty-seeking and impulsivity traits are two behavioral dimensions that contribute substantially to drugseeking behavior. In this work, we evaluated the novelty-seeking and impulsive traits in animals prenatally treated with GCs, using novelty-dependent tests (novelty place preference, novel object recognition/preference) and impulsivity measures (5-Choice Serial Reaction Time Task (5-CSRTT) and Delay Discounting (DD) test). Our results show that iugc treatment does not seem to affect novelty-seeking behavior nor impulsive action, since treated animals did not present major differences in novelty induced locomotor activity, general exploratory behavior or even in 5-CSRTT performance. However, prenatal exposure to GCs does seem to affect the performance in the DD test in a subgroup of iugc animals, a finding that needs to be further explored. Impulsivity, novelty seeking, prenatal glucocorticoids, rat [email protected] 152

153 12:00 - FIIIA7 s Noise exclusion processing is impaired in developmental dyslexia: an eye movement study Ana P. Rodrigues Ana P. Rodrigues, José Rebola, Helena Jorge, Joana Moreno, Marcelino Pereira, Isabel Festas, Miguel Castelo-Branco, Marieke van Asselen IBILI - Instituto Biomédico de Investigação em Luz e Imagem Developmental dyslexia is a highly prevalent neurodevelopmental disorder. It is characterised by a reading and writing deficit despite of a normal level of intelligence. It has been shown that, dyslexics make different eye movements during reading when compared to normal readers. It has also been suggested that dyslexics have deficits in excluding surrounding noise. In this study, we aim to understand the influence of noise in reading deficits by measuring eye movements during word reading under different levels of noise in dyslexic children and normal readers. Two groups of children with (N=10) and without dyslexia (N=14) were included. Regular, irregular and pseudowords were randomly presented in the centre of the screen in three background noise conditions: no noise (white), symbol noise (abstract symbols) and white noise (random bright and dark spots). Subjects had to discriminate between words and pseudowords by pressing a button. Eye movements were measured using a remote eye tracker. A Repeated Measures analysis indicated a main effect for group for the accuracy rate (p<0.05), which was lower for dyslexics. Concerning eye movements analyses, for the number of fixations and fixation duration, a Repeated Measures ANOVA showed a main effect for group (p<0.01) as well as an interaction effect for Group x Noise (p<0.05). As expected, overall, dyslexic children did more and longer fixations than controls. With the increase of noise, controls showed increasingly number and duration of fixations. On the other hand, in the dyslexic group, the duration of fixations was not affected by the noise and the number of fixations was only influenced by the white noise. In addition, overall both groups had equivalent saccadic amplitudes (p>0.05). The different eye movements patterns found in dyslexics suggest a different noise exclusion processing in these children, which may play a role in the reading deficits that characterize this condition. Developmental Dyslexia, noise exclusion, eye movements [email protected] 153

154 12:05 - FIIIA8 Adenosine A2A receptors control the modification caused by repeated restraint stress of synaptic plasticity in the pre frontal cortex from rat brain slices RealJ.I s RealJ.I. 1,GonçalvesN. 1, Cunha,R.A. 1,2; 1CNC Center for Neuroscience and Cell Biology, University of Coimbra; 2Faculty of Medicine, University of Coimbra, Portugal. The prefrontal cortex (PFC) plays an important role in executive functions, involving decision making, attention and working memory. Since stress have a great impact on cognition and adenosine A2A receptors (A2AR) have been shown to control stress induced behavioural modifications caused by chronic stress, we now explored the role of A2AR in the control of PFC synaptic plasticity. We compared long term potentiation (LTP, triggered by a first tetanus of 50 Hz for 1 s followed 15 min later by 4 trains of 50 Hz for 1 s every 10 s) and subsequent depotentiation (triggered by 900 pulses at 1 Hz applied 60 min after LTP) with stimulation applied to layers II/III and recordings done in layer V of the prelimbic area of slices from control and stressed rats (4 h of restraint stress for 15 days), in the absence or in the presence of the A2AR antagonist SCH58261 (50 nm). SCH58261 attenuated the amplitude of LTP (from 51.8±1.6%, n=5, to 14.7±3.5%, n=3; p<0.05) in slices from control rats. Stressed rats displayed lower amplitude of LTP (22.5±4.0%, n=4; p<0.05 vs control), and SCH58261 eliminated LTP (n=4). SCH58261 also attenuated the amplitude of depotentiation in slices from control rats (from 17.8±1.7%, n=5, to 13.8±0.9%, n=3; p=0.53), whereas SCH58261 was devoid of effects in the lower depotentiation (6.4±1.1% in control, n=4; p<0.05 vs control) observed in slices from stressed rats (n=4; p>0.05). These results provide the first demonstration that A2AR control metaplasticity in the PFC; they also show that repeated restraint stress narrows the window of plastic in the PFC and modifies the control by A2AR. It remains to define the relation between the different types of PFC plasticity and behavioral modifications upon stress to define if the A2AR mediated control of metaplasticity is related to the ability of A2AR to control the behavioral modifications caused by chronic stress. Supported by FCT (PTDC/SAU NSC/122254/2010) and DARPA (09 68 ESR FP 010). Adenosine, Pre-frontal cortex, Stress [email protected] 154

155 12:10 - FIIIA9 s Neudesin abblation induces an anxiety-like phenotype. Ashley Novais A. Novais, C. Ferreira, F. Marques, F. Campos, J. Palha, N. Sousa, C. Dalla, N. Kokras, J. C. Sousa Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, ICVS/3B s In this work we aimed at investigating the role of neudesin (nenf), a scarcely studied putative non-canonical neurotrophic factor, in brain function. An extensive behavioral characterization revealed an anxious-like phenotype as assessed in the elevated plus maze, light/dark box and novelty suppress feeding tests in mice with a targeted deletion of the nenf gene. We then analyzed the neuronal morphology in brain regions involved in processing contextual anxiety and observed that dentate gyrus granular neurons in the ventral hippocampus of nenf-null mice display shorter dendritic length and fewer intersections between dendrites; furthermore shorter dendrites were also observed in the bed nuclei of the stria terminalis (BNST). Finally we observed a higher dopaminergic activity in the ventral hippocampus of nenf-null mice. These findings lead us to suggest that neudesin is a relevant player in the maintenance of the anxiety circuitry, specifically at the ventral hippocampus and BNST. Neudesin, Anxiety, ventral hippocampus, dentate gyrus, BNST [email protected] 155

156 12:15 FIIIA10 s Exploring the role of SNC dopaminergic neurons in the initiation of self paced actions Joaquim Alves da Silva Joaquim Alves da Silva, Fatuel Tecuapetla, Vítor Paixão, Rui M. Costa Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown, Lisboa We frequently initiate new actions and transition between actions. Although vital to our survival, this orchestration of action sequences may happen so naturally that we don't even notice it. However for people with Parkinson's disease initiating movement when they want becomes specially daunting. Among other changes in the brain, these patients present with a loss of substantial nigra (SN) dopamine neurons. Recently, phasic activity of these same neurons has been found to be correlated with the start and stop of a learned action sequence. However, it is still not clear if this activity is necessary for action initiation. We used an optogenetic strategy to evaluate the role of SNC dopaminergic neurons in the initiation of self paced movement in mice. We selectively expressed an inhibitory opsin (ArchT) in SN dopaminergic neurons. We used 3-axis accelerometers and video recordings to characterize the behavior of mice freely moving in an open field while intermitently inactivating SN dopaminergic neurons during several periods of 15 seconds. Our results show that inhibiting these neurons with high temporal resolution and a short duration is enough to decrease movement in the open field. This was not true for a control group of mice expressing YFP but not ArchT in SN dopaminergic neurons. A careful analysis of the data revealed that the effect of the inhibition was much more striking when the mice were already in low acceleration/immobile before the inhibition started than when they were moving. Moreover, although there was no apparent effect when the animals were moving in the beginning of inhibition trials, a clear effect emerged after they stopped for the first time. These findings suggest that activity of SN dopaminergic neurons is necessary for the initiation, but less for the ongoing performance, of self paced actions. Dopamine, optogenetics, action intiation. [email protected] 156

157 12:20 FIIIA11 s ERP responses to morphed facial expressions in virtual reality environments Marco Simões Marco Simões, Carlos Amaral, Paulo Carvalho, Miguel Castelo-Branco IBILI, Faculty of Medicine, University of Coimbra, Portugal Visual event-related potentials of facial expressions (FEs) have been studied using usually static stimuli after a nonspecific black screen as a baseline. However, when studying social events, the ecology of the environment and stimuli can be a bias. Virtual reality provides a possible approach to improve ecology while keeping stimulus control. We propose a new approach to study responses to FEs. A human avatar in a virtual environment (a plaza) performs the FEs correspondent of the six universal emotions: happiness, sadness, surprise, fear, disgust and anger. The animations were performed along the time, morphing being the only change in the visual field of the subject. Each animation was composed by the instantaneous morphing of the FE, which was maintained for one second before the 'unmorphing' to the neutral expression. ISI was set to three seconds. The setup consisted of a 3D projection system coupled with a precision-position tracker. Subjects (N=7, mean age=25.6y) wore a 32-channel EEG cap together with 3D glasses and two infrared emitters for position tracking. The environment adapted in real time to subjects' position, giving the feeling of immersion. ERP analysis was conducted: raw EEG was band-pass filtered by Hz. Epochs were created time-locked to the stimulus onset (the morphing of the facial expression). ICA was performed on the epochs and used for artifact correction. After that, epochs containing artifacts were excluded and the ERPs were calculated averaging all the remaining epochs of each FE type. For the occipito-temporal region, we found a asymmetrical negativity [ ]ms after stimulus onset, followed by a positivity on the centro-parietal region at latency [ ]ms. Given the neutral face baseline, these observations suggest the identification of two specific neural processors of facial expressions. This study was conducted under the financial support of FCT grants SFRH/BD/77044/2011 and SFRH/BD/78982/2011. EEG, ERP, Animated Facial Expressions, Emotion, Virtual Reality [email protected] 157

158 12:25 FIIIA12 s Chronic stress has different outcomes in the structure and function of the dorsal and ventral hippocampus. Vítor Pinto Vítor Pinto, Costa J, Morgado P, Mota C, Miranda A, Bravo FV, Oliveira TG, Cerqueira JJ, Sousa N. School of Health Sciences, Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal; ICVS/3B s PT Government Associate Laboratory, Braga/Guimarães, Portugal The hippocampal role in the stress response, through its feedback inhibitory action on the HPA axis, has been extensively documented. However, few studies have focused so far on the intrinsic functional heterogeneity of the hippocampal axis and on the differential responses it might present when challenged with stressful stimuli. In this study, we assessed the functional and structural alterations caused by a chronic unpredictable stress (CUS) paradigm upon the two poles of the hippocampal axis of rats. CUS had opposite outcomes in the structure of the dorsal (DH) and ventral hippocampus (VH). Whereas the DH reduced its volume and presented a lower average value for the length of CA3 and CA1 apical dendrites, the VH showed no changes in volume and presented, instead, an increase in the average length of the apical dendrites of CA3. Functional alterations along the hippocampal axis were assessed by the ability of the DH and VH to express LTP and LTD. Although CUS had no net influence on LTP expression in both regions, there was a decrease in the ability of the VH to express LTD, in accordance with the structural data, and suggesting an overall increase in the activity of the VH when challenged with stressful stimuli. Overall, our work showed that the DH and VH hippocampus have different responses to CUS, suggesting an opposite regulation of these two regions during the stress response. Chronic stress, Hippocampal axis, LTP, LTD, Morphology [email protected] 158

159 Neuroinflammation June 1 Plátano room 159

160 11:30 - FIIIB1 s Neuron-microglia interactions in the developing CNS: focus on the role of microglia before synapse formation Gonçalo Cristóvão Gonçalo Cristóvão (1), Maria Joana Pinto (1), Catarina V. Gomes (1), Ramiro de Almeida (1), Rodrigo A. Cunha (1,2) (1) CNC- Center for Neuroscience and Cell Biology, (2) Faculty of Medicine, University of Coimbra, Portugal Yolk-sac derived macrophages, precursors of microglial cells, begin migrating into and colonizing the brain between embryonic days, E9.5 and E10.5. Due to their unique phagocytic function, microglia has been proposed as the scavenger of the developing brain, able to physically contact and eliminate unwanted synapses, a process termed synaptic pruning. However, the role of microglia after brain colonization and before synapse formation is unclear. We now aimed to evaluate if and how microglia might impact on: (1) synapsin puncta, which are related with the onset of pre-synaptic maturation; (2) the axonal length, which is indicative of axonal growth and branching. To address these questions we used microfluidic chambers that ensure a fluidic isolation between two compartments. These devices are appropriate to culture hippocampal neurons (E17) in one side, allowing axonal growth onto the other side, isolated from the cell body. A cultured (DIV 3) microglial cell line (N9) was placed in the axonal side and their impact on axons was analysed by immunocytochemistry, counting the number of synapsin puncta per axonal length. It was observed that microglia tends to increase both the number of synapsin puncta (151.9±31%, n=4, as compared with axons cultured in the absence of microglia) as well as axonal length (163.4±52%, n=4, as compared with axons cultured in the absence of microglia). The present results suggest that, besides their role in synaptic pruning, microglia may be involved in events leading to axon maturation. This prompts us to hypothesise that changes in microglia during early phases of brain development may result in synaptic abnormalities associated with neurodevelopmental disorders. Supported by DARPA and FCT. Microglia, neuron interaction, brain development [email protected] 160

161 11:35 FIIIB2 s Cell ageing effects on microglia response to Aβ Dora Brites Cláudia Caldeira1,2, André Frederico1, Ana Filipa Oliveira1, Gonçalo Lidónio1, Ana Vaz1, Adelaide Fernandes1,3, Dora Brites1,3 (1) Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculty of Pharmacy, University of Lisbon, Portugal; (2) Centro de Investigação Interdisciplinar Egas Moniz, Monte de Caparica, Portugal; (3) Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal. There is a controversy on the microglia role in Alzheimer s disease progression. Some claim an excessive microglia reactivity and production of inflammatory mediators; others a loss of the phagocytic ability and less responsiveness, contributing to Aβ dissemination. Currently there are no means to isolate degenerating microglia for experimentation. Here we aimed to create an in vitro model of microglia senescence and to assess age-dependent vulnerabilities to Aβ. Microglia were isolated (Silva et al. J Neurochem 2011) and left in culture for 2 and 16 days in vitro (DIV) before incubation with Aβ (Aβ1-42: 50 nm/1 µm) for 24 h. Senescent microglia (16 DIV) evidenced diminished NF-kappaB reactivity, more MMP2 but less MMP9 secretion, a bipolar morphology instead of amoeboid (at 2 DIV), and decreased secretion of glutamate, phagocytic ability and migration ability. When exposed to Aβ, 2 DIV cells showed reduced expression of CX3CR1 and increased glutamate release, while 16 DIV cells exhibited increased GLT1, GLAST and MMP2/9, mainly for 1 µm Aβ. In sister experiments (A) Aβ-treated conditioned media from E16 hippocampal neurons at 4 and 18 DIV was added to aged-match microglia or (B) neuron-microglia cocultures were similarly treated with Aβ (B). Increased MMP2 activation occurred in A assay with young cells, and the opposite in aged ones with 1 µm Aβ. Considering glutamate and ATP secretion no significant changes were produced in A or B experiments. Preliminary data has shown increased mir-155 at 2 DIV microglia and a decrease at 16 DIV, upon Aβ treatment. Data point to the in vitro microglia ageing as a valuable model to assess cell senescence effects in neurodegenerative-neuroinflammatory diseases and evidence aged microglia as less reactive to the impact of Aβ and to the soluble factors released by aged-matched hippocampal neurons exposed to Aβ. Funded by FEDER (COMPETE Programme), National funds (FCT-project PEst- OE/SAU/UI4013/2011/2012), GEECD and Amadeus Dias. Age-related microglia activation, Alzheimer s disease, Microglial-neuronal communication, microglia reactivity to Aβ. [email protected] 161

162 11:40 - FIIIB3 s Absence of IFN-γ leads to an enhanced cognitive phenotype Susana Monteiro Susana Monteiro, Filipa Ferreira, Vitor Pinto, Susana Roque, Daniela Calçada, Margarida Correia-Neves, João José Cerqueira Instituto de Ciências da Vida e da Saúde In the past years, the crosstalk between the central nervous system and the immune system has gained increasing attention from the scientific community. Importantly, recent studies have shown that T-lymphocytes, and the cytokines they produce, appear to be modulators of normal brain functions. And namely, pro-inflammatory cytokines have been related with impaired cognitive function. In order to dissect the mechanisms underlying this relation on learning and memory we used mice that lack IFN-γ, a wellknown pro-inflammatory cytokine and compared their behavioral performance on the Morris Water Maze (MWM) and Novel Object Recognition test (NORT) to WT littermates. For understanding these behavioral alterations we proceeded to study the hippocampal morphology and synaptic plasticity in these mice. We analyzed volumes and neuronal numbers of the different dorsal and ventral hippocampal sub-regions. In order to assess possible alterations on cell proliferation we have counted the number of Ki-67 positive cells in the subgranular zone of the dorsal dentate gyrus. We have performed electrophysiological studies on dorsal hippocampal slices of these animals by inducing LTP in CA1 region upon theta burst stimulation of Schaffer collaterals. IFN-γ KO mice exhibit enhanced spatial learning abilities and increased recognition memory, which correlates with increased volumes in dentate gyrus and CA1 region of the dorsal hippocampus and enhanced synaptic plasticity. IFN-γ, cognition, hippocampus [email protected] 162

163 11:45 - FIIIB4 s Exploring anti-inflammatory strategies on motor neuron degeneration in ALS Ana Rita Vaz Ana Rita Vaz1, Marta Barbosa1, Andreia Ferreira1, Carolina Cunha1, Dora Brites1,2 1Neuron Glia Biology in Health & Disease, imed.ul, Faculty of Pharmacy, University of Lisbon, Portugal; 2Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal Amyotrophic lateral sclerosis (ALS) onset and progression has been associated with microglia activation, and up-regulation of inflammatory pathways. Examining motorneuron (MN) alterations and modulating strategies may provide new approaches to ALS therapy. Here, we investigated the benefits of microglia and glycoursodeoxycholic acid (GUDCA) in preventing MN dysfunction in ALS. We used a MN-like cell line expressing human-sod1 with G93A mutation (NSC- 34/hSOD1G93A) and the microglial cell line N9. Cells expressing human-sod1wt were considered as controls. In this cell line accumulation of mutated SOD1 was shown to occur at 4 DIV, together with cell dysfunction. To produce a mixed culture, we added N9 cells to mutated MNs at 0 and 2 DIV of differentiation and allowed incubation till the 7 DIV. We observed a decrease in SOD1 accumulation, together with a reduction in MMP-2 activation, and release of ATP and NO, suggesting that microglia reduced the inflammatory features. Next, NSC-34 were differentiated for 4 days in vitro (DIV), being exposed to 50 µm GUDCA at the beginning (0 DIV) or at 2 DIV of cell differentiation. GUDCA prevented apoptosis, mitochondrial dysfunction, and was able to completely restore NO and MMP-9 to control levels. We show that both microglia and GUDCA are able to counteract neuroinflammation and prevent mutated NSC-34 cells from degeneration. Combined strategies may be useful in delaying disease progression or even restoring the healthy state of MNs and glial cells in ALS. Supported by FEDER (COMPETE Programme) and by National funds (FCT-projects PTDC/SAU-FAR/118787/2010 to DB and PEst-OE/SAU/UI4013/2011). Amyotrophic Lateral Sclerosis, microglia, motor neuron degeneration, inflammation, glycoursodeoxycholic acid [email protected] 163

164 11:50 - FIIIB5 s Lipocalin 2 is produced in response to amyloid beta Mesquita SD Mesquita SD, Falcão AM, Ferreira AC, Sousa JC, Sousa N, Marques F and Palha JA Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus Gualtar, Braga, Portugal; ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The choroid plexus (CP) epithelial cells form the blood-cerebrospinal fluid (CSF) barrier and secrete most of the components of the CSF that are essential for brain s detoxification and homeostasis. We have shown that lipocalin 2 (LCN2), a protein initially described as an acute-phase protein that binds iron-loaded siderophores (iron chelators secreted by pathogens) is produced both by the CP epithelial cells and astrocytes under inflammatory conditions. Given the evidence of iron deregulation in Alzheimer s disease (AD), we were prompt to study, in vitro, the effects of LCN2 on the toxicity mediated by amyloid beta (Aβ) peptides. We observed that incubation with human Aβ1-42 alters the expression of Lcn2 in primary cultures of mice or rat CP epithelial cells and astrocytes, and that Aβ1-42-mediated toxicity seems to depend on LCN2. Lipocalin 2, Amyloid beta, Choroid plexus, Astrocytes [email protected] 164

165 11:55 - FIIIB6 s Histamine modulates dopaminergic neuronal survival by boosting microglial activity Tatiana Saraiva Tatiana Saraiva1*, Sandra Rocha1*, Marta Esteves1, Ana Clara Cristóvão1, Graça Baltazar1, Gilberto Alves1, Liliana Bernardino1,2 1Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal; 2Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal * Both authors contributed equally Microglial cells are the main players involved in the innate inflammatory responses in the adult brain. The response of microglia to brain injury involves the phagocytosis of death or damaged neurons, release of trophic and/or inflammatory factors, and the production of reactive oxygen species (ROS). Histamine is an amine found in high amounts in mast cells, histaminergic neurons, and leukocytes. In the CNS, histamine is also released by microglial cells and exerts its functions through the activation of four subtypes of G-protein coupled receptors: H1, H2, H3 and H4. Previously, we showed that histamine modulates microglial motility and cytokines release. The main aims of this work were: i) to evaluate the role of histamine in microglial phagocytic activity and ROS production in vitro and ii) to explore the consequences of histamine-induced microglia inflammation in dopaminergic neuronal survival in vivo. Initially, we showed that histamine induced an increase of phagocytosis via H1R activation in a N9 murine microglial cell line, as compared to control. This effect was accompanied by the rearrangement of microglial cytoskeleton monitored through phalloidin and acetylated tubulin immunostaining. Histamine also induced an increase of ROS production via H1R and H4R activation. Apocynin, a NADPH oxidase inhibitor, was able to fully inhibit phagocytosis and ROS production mediated by histamine. On the other side, the sterotaxic injection of histamine in the substantia nigra of adult C57Bl6 mice for 7 days induced an increase of microglia and astrocyte reactivity and a robust decrease in dopaminergic neuronal survival. Both apocynin and annexin IV (used as inhibitor of phosphatidylserine-induced phagocytosis) fully abolished the histamine-induced neurotoxicity of dopaminergic neurons. Overall, our results suggest that histamine induce microglial reactivity both in vitro and in vivo and that this effect may modulate dopaminergic neuronal survival in vivo. Microglia, Histamine, Phagocytosis, Reactive Oxygen Species, Neurotoxicity [email protected] 165

166 12:00 - FIIIB7 s Neuropeptide Y receptor Y1 modulates microglia activation in the rat retina Ana Raquel Santiago F. Elvas1,3,4, M. Madeira1, T. Martins1,4, A.R. Santiago1,2,4, C. Cavadas2,3, A.F. Ambrósio1,2,4 1Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra; 2Center for Neuroscience and Cell Biology, University of Coimbra; 3Faculty of Pharmacy, University of Coimbra. 4AIBILI, Coimbra, Portugal. Increasing evidence has shown that Neuropeptide Y (NPY) is a regulator of inflammation. In the retina, microglia activation has been shown to be associated to a number of different pathological states including central retinal artery occlusion, diabetic retinopathy, and glaucoma. Microglia activation results in elevated levels of pro-inflammatory cytokines, as well as increased expression of inducible nitric oxide synthase (inos) and increased NO production. The aim of this work was to investigate whether NPY, particularly through Y1 receptor activation, modulates retinal proinflammatory responses. Immunoreactivity of NPY and NPY Y1, Y2 and Y5 receptors was detected in microglia in cultured retinal explants. Lipopolysaccharide (LPS) increased inos immunoreactivity in microglial cells. Pre-treatment with NPY or [Leu31, Pro34]-NPY (LP-NPY; Y1/Y5 receptor agonist) inhibited the increase in inos immunoreactivity. Incubation with BIBP 3226 (Y1 receptor antagonist) abolished the effect of LP-NPY. Production of TNF-α, IL-1β and IL-6 was assessed by ELISA. Exposure to LPS increased the concentration of the cytokines. Pre-treatment with NPY inhibited the increase in the concentration of IL-1β and IL-6, but not TNF-α. Moreover, incubation with LP-NPY significantly inhibited the increase in TNF-α, IL-1β and IL-6 concentration, and incubation with BIBP 3226 abrogated the effect of LP-NPY. Retinal ischemia-reperfusion (I/R) injury was induced for 60 min followed by 24h reperfusion. Administration of NPY intravitreally injected prior ischemia inhibited the increase in the expression of TNF-α, IL-1β and IL-6 induced by I/R injury. In conclusion, retinal microglia express NPY and NPY Y1, Y2 and Y5 receptors. Y1 receptor activation and NPY induce an anti-inflammatory effect in retinal microglia, inhibiting microglia activation triggered by LPS and I/R injury, respectively. FCT (Grant PTDC/SAU-NEU/099075/2008 and Strategic Project PEst- C/SAU/UI3282/2011), Portugal and COMPETE-FEDER ischemia-reperfusion, microglia, neuroinflammation, Neuropeptide Y, retina [email protected] 166

167 12:05 - FIIIB8 s Microglia phagocytic ability is preserved irregardless changes in ATP levels and depends upon adenosine A2AR tonic activation Catarina V. Gomes1 Gonçalo Cristóvão, Jimmy George, FRancisco Queirós, Lisa Ferreira, Teresa Gonçalves, José Roberto Meyer Fernandes, Michelle Viegas, Otília Vieira, Rodrigo Cunha, Catarina Gomes 1CNC-Centre for Neuroscience and Cell Biology, 2Faculty of Medicine, University of Coimbra, Portugal; 3 WCU Multiscale Mechanical Design, Seoul National University, Seoul, Korea, 4 WCU Multiscale Mechanical Design, Seoul National University, Seoul, Korea; School of Mechanical and Aerospace Engineering, Seoul National University, Korea Microglial cells orchestrate inflammatory responses in brain disease. While beneficial at initial stages, inflammation must be time-limited, otherwise it exacerbates disease progression. Thus, controllers of microglia physiology, such as ATP and adenosine are attractive targets for disease modifying drugs. We reported recently that adenosine A2A receptor (A2AR) activation bolsters microglial proliferation, an effect requiring ATP conversion into adenosine since preventing ATP metabolism (yielding high ATP levels) impairs proliferation. This agrees with the fact that microglia surrounding damaged neurons, where ATP levels are high, do not proliferate and other cells must be recruited to phagocyte damaged neurons. We now tested if A2AR modulate the phagocytic capacity of microglia in conditions where ATP levels are low (adding the bacterial antigen, lipopolisaccharide, LPS) or high (adding glutamate). A microglial cell line (N9) was exposed for 6 h to LPS 100 ng/ml or to glutamate 0.5 mm in the absence or presence of the A2AR antagonist SCH nm. Phagocytosis was then measured by measuring (confocal z-stack analysis) the number of internalyzed apoptotic cells (aged red blood cells) during 30 min. We found that neither LPS nor glutamate modified phagocytic capacity, whereas A2AR blockade inhibits phagocytosis by 20-30% (n=2). These preliminary results suggest that phagocytosis is controlled by A2AR and is independent of ATP levels. This contrasts with proliferation, which is affected by ATP levels and controlled by A2AR only in conditions of low ATP levels. Altogether, the data suggest that two key microglial responses, proliferation and phagocytosis, are differentially regulated by ATP and adenosine. Supported by DARPA and FCT. Microglia, adenosine A2A receptors, ATP, phagocytosis, proliferation [email protected] 167

168 12:10 - FIIIB9 Motor neuron signaling to microglia: a clue for ALS pathogenesis Cunha C. s Cunha C., Vaz A.R, Brites D. Neuron Glia Biology in Health & Disease, imed.ul, Faculty of Pharmacy, University of Lisbon, Portugal Microglia may become protective or neurotoxic in several neurodegenerative disorders involving neuroinflammation, and decision for one of the sides of the coin apparently depend on signals microglia receive from other cells and from the brain parenchyma. Here we explored the reactivity of a microglial cell line towards lipopolysaccharide (LPS) and soluble factors released by a motor-neuron model of amyotrophic lateral sclerosis (ALS). We used a MN-like cell line (NSC-34) expressing human SOD1 with G93A mutation (NSC-34/hSOD1G93A). Our previous results have shown that our mutated NSC-34 cells accumulate SOD1 after 4 days in vitro (DIV) and present MN dysfunction. Thus, we have differentiated these cells for 1 and 4 DIV, and collected the supernatants at both periods. NSC-34 cells expressing human SOD1wt followed the same conditions and were used as controls. Next, microglial cell line N9 was incubated at 37ºC with either both MNs supernatants for 4 and 24 h, or 300 ng/ml LPS for 24 h. We observed that N9, while ramified in control conditions, changed to an amoeboid morphology (anti-iba1 staining) after LPS treatment, which was accompanied by increased phagocytosis (fluorescent latex beads) and decreased migratory ability (chemotaxis to ATP in a Boyden Chamber), indicating that LPS induces N9 activation. In addition, media from mutated MNs collected at 4 DIV and incubated for 24 h induced apoptosis (nuclei morphology), while changing microglia morphology into an active amoeboid state that evidenced reduced phagocytic ability. Curiously, no chemotactic activity was exerted by the released factors from degenerating MNs. Together, our results indicate features of microglia activation in response to LPS and to the MN model of ALS reinforcing the role that neuroinflammation may have in the ALS onset and progression. Supported by FEDER (COMPETE Programme) and by National funds (FCT - projects PTDC/SAU-FAR/118787/2010 to DB and PEst-OE/SAU/UI4013/2011). Microglia, motor neuron, neuroinflammation, ALS [email protected] 168

169 12:15 FIIIB10 s Adenosine A2A receptor modulates LPS-induced neuroinflammation in the retina M.H. Madeira M.H. Madeira1, F. Elvas1,2, A.F. Ambrósio1,2, A.R. Santiago1,2 1Center of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2AIBILI, Coimbra Neuroinflammation is a feature of neurodegenerative disorders involving activation of microglial cells, which leads to exacerbated expression of pro-inflammatory mediators that can be potent inducers of cell death. Adenosine is a neuromodulator in CNS, being involved in inflammatory responses and neuroprotection. Increasing evidence has demonstrated that blocking adenosine A2A receptor (A2AR) may prevent neurodegeneration in the brain by modulating the release of noxious factors by activated microglia. Little is known about the role of microglial cells in the control of the neuroinflammatory response in the retina. The aim of this work was to evaluate the effect of A2AR blockade in the control of retinal neuroinflammation. Purified retinal microglial cell cultures and cultured retinal explants were pretreated with A2AR agonist or antagonist, and challenged with LPS to mimic an inflammatory stimulus. Expression of A2AR increased in cultures of retinal microglial cells after exposure to LPS. Activation of A2AR potentiated the production of NO induced by LPS in microglial cells, while A2AR blockade inhibited LPS-induced NO increase and decreased the percentage of microglial cells incorporating fluorescent latex beads, as compared to LPS condition, indicating that blockade of A2AR prevents the increase in phagocytic activity. In cultured retinal explants, LPS increased the expression of A2AR in microglial cells in the retinal ganglion cell layer. A2AR blockade decreased the LPSinduced expression of the pro-inflammatory markers, such as TNF-α, IL-1β, IL6 and inos and reduced the release of pro-inflammatory cytokines, as IL-1β. Our results indicate that A2AR may have a role in the inflammatory responses in the retina, opening the possibility of the use of A2AR antagonists in retinal degenerative diseases involving inflammation. Support: FCT Fellowship SFRH/BD/75839/2011, FCT Project PTDC/BIM- MEC/0913/2012 and FCT Strategic Project (PEst-C/SAU/UI3282/2011), Portugal Retina, Microglia, Adenosine, Neuroinflammation [email protected] 169

170 12:20 FIIIB11 s Gaba enhances pain facilitation from the brain during inflammatory pain Isaura Tavares I. Martins1, P. Carvalho1, B.H. Westerink2,3, S.P. Wilson4, I. Tavares1 1 Department of Experimental Biology, Faculty of Medicine of Oporto and IBMC, Porto, Portugal 2 Dept. of Biomonitoring and Sensoring, University of Groningen, The Netherlands 3 Brains On-Line BV, Groningen, The Netherlands 4 Dept. of Physiology, Pharmacology and Neurosciences, University of South Carolina, School of Medicine, USA Gamma-aminobutyric acid (GABA) plays a key role in the pain control from the brain. Its the role in pain facilitatory areas is unknown. We studied the role of GABA at the dorsal reticular nucleus (DRt), an unique pain facilitatory area of the medulla oblongata, rich in GABAB-expressing neurons. We used in vivo microdialysis to evaluate GABA release at the DRt in the formalin model. This behavioral test has two phases, the second of which is more intense and mimics inflammatory pain. The effect of GABAB knock down at the DRt was evaluated using lentiviral-mediated gene transfer. Male Wistar rats were anesthetized and placed on a stereotaxic frame for cannula implantation or injection of lentiviral vectors into the left DRt. Microdialysis and behavioral studies were performed one week later. During microdialysis, the rats received a subcutaneous injection of formalin or saline into the left hindpaw (n=6/group) and 15 min fractions were collected before and for 2 hours after formalin or saline injections. The behavioral evaluation was performed in animals injected with a vector decreasing the GABAB1a subunit (LV-GBA; n=6) or a control vector expressing EFGP (LV-EGFP; n=5) by recording the number of paw jerks during 5-min periods for 60 min after formalin injection. The animals were perfused and the medulla oblongata was serially cut. A set of sections was counterstained with formol-thionin to confirm the position of the injections at the DRt. Viral transduction was determined by EFGP expression at control rats sections, showing EFGP-positive neurons limited to the DRt. During the second phase of the formalin test, GABA release increased and the number of paw jerks was significantly attenuated in LV-GBA-injected rats. The data suggest that increased GABA release at the DRt in response to the arrival of nociceptive input contributes to descending pain facilitation. This may account to chronic pain installation. Support: FCT/COMPTE project PTDC/SAU-NSC/110954/2009 Chronic pain, Descendind modulation, Gne therapy [email protected] 170

171 12:25 FIIIB12 s Can distinct cognitive performances among aged individuals be related with distinct immune system profiles? Cláudia Miranda Cláudia Miranda, Nadine Correia Santos, Nuno Sousa, Margarida Correia-Neves, Susana Roque Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal ; ICVS/3B's, PT Government Associate Laboratory, Braga, Guimarães, Portugal Aging has been an important hallmark of the world s population for the last decades. Some of the frequent alterations observed in older individuals are the progressive decline in immune function (immunosenescence) and in cognitive performance. In fact, in the last few years an increased body evidence has linked the immune system and cognitive functions, highlighting the strong interaction between these two systems. Particularly, the low-grade inflammation, frequently observed in older individuals, has been associated with cognitive decline. Moreover immunocompromised individuals present cognitive dysfunction that is restored when the levels of immune cells return to normal. The findings follow animal data evidencing that mice devoid of adaptive immune system present cognitive deficits, which are reverted by CD4+ T cell restoration. In this project we are using a novel strategy to investigate possible correlations between immunesenescence phenotype with cognitive phenotypes in a well characterized cohort of healthy agers with distinct cognitive performances. To achieve this objective we are assessing the most relevant immune cell populations by flow cytometry, belonging either to adaptive immune system (B and T cells compartments, regulatory T cells), or to innate immune system (monocytes, granulocytes, natural killer cells, dendritic cells), and correlating them with several cognitive dimensions. This integrative approach will allow us to investigate in great detail the interplay between different cognitive dimensions and the major cell populations of the immune system during aging. Aging, Cognition, Immunosenescence [email protected] 171

172 Neurodegeneration and neuroprotection III June 1 Auditorium 172

173 11:30 FIIIC1 s Retinoic acid-loaded polymeric nanoparticles induce neuroprotection in a mouse model of Parkinson s disease Marta Esteves1 Marta Esteves1, Tatiana Saraiva1, Sandra Rocha1, Graça Baltazar1, Lino Ferreira2, Liliana Bernardino1,2 1Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal; 2Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Retinoic acid (RA) has been highlighted as a therapeutic option for some neurodegenerative diseases due to its neuroprotective, anti-inflammatory and proneurogenic properties. However, RA presents undesirable properties like poor water solubility and short half-life. Therefore, nanoparticles (NPs) are an excellent alternative to control the undesired side effects and to ensure intracellular transport and controlled release of RA. The aim of this work was to evaluate the effects of RA-loaded NPs (NP+RA) in an in vivo mouse model of Parkinson s disease (PD) and to compare with effects of soluble RA. Adult C57BL6 mice were injected with 100ng/ml NP+RA or soluble RA (4nM and 10µM) in the right striatum, for 3 days, followed by intraperitoneal injection with the dopaminergic neurotoxin MPTP (60mg/kg), and then mice recovered for further 7 days. As expected, MPTP triggered a 40% reduction of tyrosine hydroxylase (TH) neurons in the substantia nigra (SN) and a decrease of TH terminals in the striatum, as compared to saline mice. Interestingly, NP+RA significantly reduced the MPTP lesion by increasing the percentage of TH+neurons in the SN to levels similar to saline mice as well as increasing the intensity and area occupied by TH+fibers in the striatum. This protective effect mediated by NP+RA was more robust than when soluble RA was injected using the same experimental conditions. In the SN of mice exposed to both NP+RA and MPTP there was also an increase of Pitx3 and Nurr1 mrna expression, transcription factors responsible for dopaminergic neuronal specification and survival, as compared with MPTP only-exposed cells. The same patterns of Pitx3 and Nurr1 mrna expression was obtained in old mice (24- month-old) treated with NP+RA plus MPTP versus MPTP alone. In conclusion, NP+RA show a higher protective effect against dopaminergic injury when compared to soluble RA, suggesting that NP+RA could be a good strategy to boost brain repair in PD. Retinoic-acid, nanoparticles, neuroprotection, dopaminergic neurons, MPTP [email protected] 173

174 11:35 FIIIC2 s Necroptotic neuronal death of hippocampal neurons submitted to an ischemic stimulus Marta Vieira M Vieira1,2, J Fernandes1,2, L Carreto3, M Santos3, CB Duarte1,4, AL Carvalho1,4 and AE Santos1,2 1 Center for Neuroscience and Cell Biology, 2 Faculty of Pharmacy, University of Coimbra, 3 RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, 4, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal Brain ischemia triggers a plethora of mechanisms that contribute to cell death. Despite recent advances, a complete understanding of these mechanisms remains elusive. Thus, in this work we aimed to study neuronal death mechanisms underlying in vitro ischemic insults using hippocampal neuronal cultures subjected to oxygen-glucose deprivation (OGD). We performed a microarray analysis to identify changes in gene expression induced by OGD and selected two altered genes, receptor interacting protein kinase 3 (RIP3) and caspase-8 (Casp8) due to their role in necroptosis, a recently described type of cell death. This form of cell demise has a necrotic phenotype; yet, like apoptosis, it is a programmed death process, requiring the activation of death receptors and in their vicinity, the assembly of protein complexes. RIP3 associates with RIP1 to induce necroptosis, in a mechanism dependent on RIP1/3 kinase activity, and negatively regulated by Casp8. Given the altered expression of RIP3 and Casp8 in neurons subjected to OGD, we hypothesized that necroptosis may be involved in hippocampal neuronal death under ischemic conditions. Indeed, the OGD-induced increase in RIP3 mrna and the decrease in Casp8 mrna levels observed in the microarrays were confirmed by RT-PCR and were found to be concomitant with increased RIP3 protein levels. Moreover, a necroptotic inhibitor, necrostatin-1, protected hippocampal neurons subjected to OGD, suggesting a role for necroptosis in ischemia-induced death. To investigate if the increase in RIP3 is determinant in triggering necroptosis, we overexpressed RIP3 in hippocampal neurons and observed a significant enhancement in cell death following OGD. On the other hand, RIP3 knockdown protected neurons against OGD. Overall, our results point towards a role of necroptosis to the cell death mechanisms induced by OGD, which may have relevant clinical implications. (Supported by FCT grants: PTDC/SAU-NEU/099440/2008 and PTDC/BIA- BCM/113738/2009) Global cerebral ischemia, Necroptosis, Oxygen-glucose deprivation, Programmed neuronal death, RIP3 [email protected] 174

175 11:40 FIIIC3 s Retinal ganglion cells express neuropeptide y and NPY receptors Dan Brudzewsky D. Brudzewsky1,2, F. Elvas1,2, J. Martins1, A.R. Santiago1,2,3, D.P.D. Woldbye4, A.F. Ambrósio1,2,3 1Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2AIBILI, Coimbra, Portugal; 3Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 4Department of Neuroscience and Pharmacology, University of Copenhagen, 2200 Copenhagen, Denmark Neuropeptide Y (NPY) is a 36 amino acid peptide widely distributed in the nervous systems. NPY has been implicated in several biological processes including vasoconstriction, and protection of neurons from apoptosis. In the rat, these effects are mediated through activation of several G-coupled receptors: NPY Y1, Y2, Y4 and Y5. Despite evidence of NPY and NPY receptor expression in the retina, it is unknown whether they are expressed in retinal ganglion cells (RGCs). The aim of this work was to identify the NPY receptors expressed in RGCs and to evaluate their functional activity. Identification of NPY receptors in RGCs was investigated in both albino and pigmented rats using reverse-transcription PCR, immunohistochemistry, and immunocytochemistry. NPY mrna was detected in freshly isolated RGCs from Long-Evans and Wistar rat pups (P4-P6). Similarly, in RGCs isolated from young adults (8-11 weeks) NPY mrna was detected in both rat strains. Also, mrna of tested NPY receptors (NPY Y1, Y2, Y4 and Y5) was detected in RGCs from pups and young adults from both strains. Immunoreactivity in RGCs (Brn3a-positive cells) of both NPY and tested NPY receptors was shown with immunohistochemistry performed on transverse sections of the retina of young adults, from both strains. Similar results were obtained in isolated RGCs. Receptor activity evaluation was performed using [35S]GTPγS functional binding assay. In retinal sections, a dose-response dependent increase of receptor activity was observed in RGCs upon either NPY or Pancreatic Polypeptide (PP) stimulation. NPY stimulated receptor activity is mediated through NPY receptors Y1, Y2 and Y5. Contrary to these receptors, Y4 receptor mediates the PP stimulated activity. The work presented here shows for the first time that NPY and NPY receptors Y1, Y2, Y4 and Y5 are expressed and functional active in RGCs. Support: FCT (PTDC/SAU-NEU/099075/2008 & Strategic Project PEst- C/SAU/UI3282/2011 & SFRH/BPD/69123/2010), PT, and COMPETE-FEDER. Neuropeptide Y, NPY receptors, Retinal Ganglion Cells, [email protected] 175

176 11:45 FIIIC4 s Diabetes alters the activity of rostroventromedial medulla (RVM) ON-like and OFF-like cells: a role in diabetic neuropathic pain? Carla Morgado Carla Morgado1, Marta Silva1, Diana Amorim2, Armando Almeida2, Filipa Pinto- Ribeiro2, Isaura Tavares1 1 Department of Experimental Biology, Faculty of Medicine of Porto and IBMC, University of Porto, Portugal. 2 Life and Health Sciences Research Institute, University of Minho, Portugal. Neuropathic pain is one of the most frequent complications of diabetes. The increased neuronal activity of primary afferents and spinal cord neurons in streptozotocin (STZ)- diabetic rats increases the recruitment of the nociceptive ascending pathways, which may affect the activity of pain control circuits in the brain. This study used in STZdiabetic rats to characterize the electrophysiological responses of neurons of the rostroventromedial medulla (RVM), a key brainstem area involved in descending modulation of nociceptive neurotransmission at the spinal cord. Spontaneous and noxious-evoked activity of ON-like cells (pain facilitatory cells) and OFF-like cells (pain inhibitory cells) in the RVM were analysed by single cell extracellular electrophysiological recordings in STZ-diabetic rats and in age-matched non-diabetic controls (CTRL). The electrophysiological analysis revealed an increase in the spontaneous activity of RVM pronociceptive ON-like cells in STZ-diabetic rats when compared to CTRL. On the contrary, the number of active antinociceptive OFF-like cells was significantly lower in the STZ-diabetic rats and their spontaneous activity was decreased when compared with CTRL. The responses of ON-like and OFF-like cells to the application of a peripheral noxious mechanical stimulus, defined as the evoked activity - spontaneous activity, were significantly decreased in the STZ-diabetic rats, despite similar discharge rates after stimulation in both experimental groups. The changes in the spontaneous activity of RVM pain modulatory cells in STZ-diabetic rats point to enhancement of descending pain facilitation, which may account for exacerbated pain-like behaviors in diabetic neuropathy. The low noxious-evoked responses of those cells in STZ-diabetic rats are due to the increased baseline activity, which is likely to alter electrophysiological properties of the cells. Support: FCT/COMPTE project PTDC/SAU-NSC/110954/2009. diabetic neuropathy, RVM, descending facilitation, pain [email protected] 176

177 11:50 FIIIC5 Mitochondrial dysfunction-dependent microtubule depolarization drives autophagic-lysosomal pathway impairment in MCI and AD cybrids s Diana F Silva Diana F Silva, A Raquel Esteves, Sandra Morais Cardoso Center for Neuroscience and Cell Biology, Faculdade de Medicina Universidade de Coimbra Mitochondrial abnormalities have been widely described in Alzheimer s disease (AD) and mild cognitive impairment (MCI), an intermediate state before declared dementia. Moreover, the accumulation of autophagosomes (AVs) in AD brains suggests that autophagy may be impaired, but the causes for this accumulation remain elusive. In this study we evaluated the role of AD and MCI mitochondrial by transferring mitochondria from MCI, AD, and control subject platelets to mtdna-depleted SH- SY5Y cells. Further, we differentiated cybrid cell lines by exposition to retinoic acid (RA). In differentiated cybrids we evaluated mitochondrial network/dynamics and found that mitochondria from MCI and AD cybrids are fragmented, in accordance with other observed mitochondrial abnormalities. We hypothesize that mitochondrial dysfunction causes a microtubule network disruption and exacerbates tau phosphorylation. This event leads to the decreased efficiency of autophagic-lysosomal pathway, resulting in decreased autophagic flux and accumulation of AVs in MCI and AD cybrids. Our results support the view that a mitochondrial dysfunction is a key event that leads to microtubule network defects and culminates with autophagic dysfunction. mitochondrial dysfunction, Alzheimer s disease, mild cognitive impairment, autophagy [email protected] 177

178 11:55 FIIIC6 s A Powerful Yeast Model to Investigate the Synergistic Interaction of a-synuclein and Tau in Neurodegeneration Gianmario Ciaccioli Gianmario Ciaccioli, Ana Martins, Catia Rodrigues, Helena Vieira, Patricia Calado Bioalvo S.A. Tec Labs, Campus FCUL Lisboa Several reports have proven a consistent overlap between synucleinopathies and tauopathies, α-synuclein (ASYN) was found in neurofibrillary tangles (NTFs) mainly composed by tau protein and tau was also found in Lewy bodies mainly composed by ASYN protein. Direct binding between these two proteins has been described and it was also confirmed that ASYN can act as a pathological initiator of tau phosphorylation and aggregation. We designed a yeast model to investigate the synergistic cytotoxic effect mediated by the co-expression and interaction of ASYN and tau proteins. Our goal is to perform genome wide screening assays to identify target genes that modulate this interaction, in order to shed light into the pathological mechanism of cell dysfunction and to provide new targets for future therapeutic intervention. We started by validating the synergistic toxicity of ASYN and tau co-expression by developing episomal and integrative yeast strains. In the episomal strains no growth delay was observed upon coexpression of tau and ASYN isoforms but the presence of ASYN led to increased tau insolubility and correlated with increased tau phosphorylation in S396/404, which is mainly mediated by RIM11, the human orthologue of GSK3β in yeast. The integrative strains showed a strong synergistic toxic effect upon co-expression of ASYN and tau, related to high levels of ASYN inclusions and increased tau phosphorylation and aggregation. Tau phosphorylation level in S396/404 plays a crucial role in this model as depletion of RIM11 completely abolishes the synergistic cytotoxic effect mediated by ASYN and tau. The integrative model was used to perform a genome wide screening assay using a Yeast Genomic Tiling Collection Assay Ready Pooled DNA to identify new target genes for potential therapeutic intervention. By preliminary screening we have identified about ten different putative genes of interest that are currently being further investigated. α-synuclein, tau, yeast, genome wide screening [email protected] 178

179 12:00 FIIIC7 s LRRK2 as a player on mitochondrial and microtubule dynamics impairment in sporadic Parkinson s disease Ana Raquel Esteves A. Raquel Esteves1, Sandra M. Cardoso1,2 1Centro de Neurociências e Biologia Celular, 2Faculdade de Medicina, Universidade de Coimbra, Portugal LRRK2 mutations are an important cause of late-onset Parkinson s disease (PD). Moreover common variants in the LRRK2 gene may act as a risk factor for sporadic PD (spd). A growing body of evidence have been implicating LRRK2 on remarkably diverse pathways, including quality control mechanisms (autophagy), and regulation of microtubule network and mitochondrial dynamics. Interestingly these pathways have been shown to be impaired in spd. However current data do not explain much of LRRK2 underlying cellular mechanisms in spd. Therefore we set out to elucidate the exact cellular mechanisms of LRRK2 in spd. To do so we began by evaluating LRRK2 contribution to mitochondrial deficits and microtubule network alterations in CT and spd cybrids exposed to an inhibitor of LRRK2 kinase activity, LRRK2-IN-1. We have previously demonstrated that PD cybrids harbour inherent mitochondrial deficits leading to microtubule-trafficking alterations relatively to CT cybrids. Herein, we observed that PD cybrids show an increase in LRRK2 phosphorylation at ser935 indicating an enhanced LRRK2 kinase activity, which decreases after LRRK2-IN-1 treatment. Furthermore, LRRK2-IN-1 promoted the destabilization of microtubule dynamics as visualized by an increase in free/polymerized α-tubulin ratio and a decrease in acetylated alpha-tubulin in CT cybrids. Interestingly, no alteration was observed in PD cybrids indicating that these cells already show microtubule disruption. Accordingly, we also observed an impairment of mitochondrial trafficking in CT cybrids treated with LRRK2-IN-1. As a result we observed an increase in mitochondrial elongation and a perturbation in mitophagy in CT cybrids treated with LRRK2-IN-1. Overall, we shed light to LRRK2 role on mitochondrial-mediated microtubule trafficking and more importantly in placing LRRK2 in the molecular cascade events that sustain the mitochondrial cascade hypothesis for spd. LRRK2, microtubule dynamics, mitochondria, Parkinson s disease [email protected] 179

180 12:05 FIIIC8 Functional Gellan Gum Hydrogels and Cell based Therapies A Novel Therapeutic Approach for Spinal Cord Injury Regeneration Eduardo Gomes s E. Gomes, S. Samy, M.M. Carvalho, F.G. Teixeira, H. Leite-Almeida, R.L. Reis, N. Sousa, N.A. Silva, A.J. Salgado Life and Health Sciences Research Institute (ICVS) Spinal Cord Injury (SCI) remains one of the most devastating diseases of the Central Nervous System. The lack of an effective therapy to treat this condition is still a major concern nowadays. Knowing that single therapies usually fail when translated to the clinics, combining more than one approach could lead to novel improvements in the field. Multidisciplinary approaches such those presented by tissue engineering concepts hold great promise for SCI treatment. In this sense we previously functionalized the gellan gum hydrogel with a fibronectin derived peptide - GRGDS. Moreover, it was already demonstrated that Adipose Mesenchymal Stem Cells (ASCs) secrete factors that promote neuronal proliferation; in addition, Olfactory Ensheathing Cells (OECs) are well known for promoting neuronal regeneration and guidance. Therefore, we propose to conjugate the hydrogel with ASCs and OECs in order to promote SCI repair. To study possible synergistic or antagonistic effects we co-cultured both ASCs (5.000 cells/cm2) and OECs ( cells/cm2) for 7 days either in 2D or in 3D. Results demonstrated that the number and morphology of both cell types was not affected when in direct cocultures. Despite of the absence of synergistic effects, our results showed that these cells can be cultured together without having a harmful effect on the growth and morphology of each other. This indicates that these cells may be successfully transplanted together. Having this in mind we have performed an in vivo experiment, in which adult rats were subjected to a hemisection lesion. The animals were divided into different groups according to the treatment: transplant of ASCs and OECs, implantation of the Gellan Gum-GRGDS hydrogel scaffold or a combination of both (cells encapsulated in the hydrogel). The effectiveness of our proposed therapy is currently being evaluated through locomotor improvements of the paralyzed left hindlimb. Spinal Cord Injury, Tissue Engineering, Adipose Stem Cells, Olfactory Ensheathing Cells, Hydrogels [email protected] 180

181 12:10 FIIIC9 s A2AR blockade prevents ltp impairment induced by alpha-synuclein Diana G. Ferreira1,2 Diana G. Ferreira1,2, Joana E. Coelho1, Vânia L Batalha1, António Albino-Teixeira3,4, Tiago F. Outeiro1,2,5 and Luísa V. Lopes1 1Instituto de Medicina Molecular, Faculty of Medicine Lisbon, 2Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen Germany, 3Instituto de Farmacologia e Terapêutica, Faculdade de Medicina, Universidade do Porto, Portugal, 4IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal, 5Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal Alpha-synuclein (asyn) deposits are a hallmark of Parkinson s Disease (PD) and correlate with progressive neuronal dysfunction and, eventually, death of affected neuronal populations. Cognitive deficits are concomitant with these pathological changes, since these deposits are not restricted to the substantia nigra, but extend to several brain areas including those related to memory, such as the hippocampus and cortex. While the precise mechanisms underlying asyn neurotoxicity are unclear, recent studies suggested that the blockade of the A2A receptor (A2AR), whose expression becomes aberrant during aging and in PD patients, is beneficial against asyn mediated-synaptic impairments. We performed immunoblot analyses in transgenic rats overexpressing the human A2AR (CAMKII-hA2A) in the forebrain. We observed an alteration of the endogenous levels of asyn in the hippocampus and striatum, as well as an enrichment of asyn in the presynaptic terminal, where the A2ARs are known to be preferentially located. Based on our previous data showing that asyn impairs long-term potentiation (LTP), we investigated the ability of A2AR blockade to prevent this synaptic damage. Hippocampal slices pre-incubated with an A2AR antagonist, SCH58260 (110min; 50 nm), maintained the ability to respond to LTP when incubated with asyn oligomers (500 nm; 90 min) and resulted in LTP levels similar to those obtained in the control (LTPcontrol 50.9 ± 5.0%, n=9; LTPaSyn 9.1 ± 3.3%, n=6; LTPSCH+aSyn 50.6 ± 13.0%, n=5). Overall, our results suggest that A2AR may influence the levels and subsynaptic distribution of endogenous asyn which in turn may lead to synaptic deterioration. Importantly, if we block these receptors we are able to prevent asyn-impairment of neuronal function. These results open new avenues for the study of A2ARs as potential therapeutic targets in PD-related cognitive deficits. Funding: FCT, Fritz Thyssen Stiftung, and DFG Research Center (CNMPB) Alpha-synuclein, Adenosine A2A receptors, memory, LTP, hippocampus [email protected] 181

182 12:15 FIIIC10 s In vitro ischemia in hippocampal neurons triggers a transcriptional response to down-regulate synaptic proteins Joana Fernandes J. Fernandes1,2, M. Vieira1,2, L. Carreto3, M. Santos3, C. B. Duarte1,4, A.L. Carvalho1,4 & A. E. Santos1,2 1-CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2-Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal 3-RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal 4-Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal Excitotoxicity, caused by overactivation of glutamate receptors, is implicated in the neuronal death observed in global cerebral ischemia, which leads to the selective and delayed death of hippocampal neurons. However, excitotoxicity-induced changes in gene expression under ischemic conditions remain to clarify. Thus, we aimed to investigate the contribution of ionotropic glutamate receptors to the excitotoxic cell death in hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. Primary cultures were subjected to 2h OGD, in the absence or presence of selective antagonists of the AMPA and NMDA subtype of glutamate receptors. Cell viability was assessed 24h later by the LDH release and analysis of the nuclear morphology. The results show that both antagonists were neuroprotective, confirming the essential role of these receptors to the excitotoxic component of this insult. Additionally, we investigated the changes in the transcriptome in cultures of hippocampal neurons submitted to OGD. For that purpose, total RNA was extracted 7h and 24h after OGD and used in a whole-genome RNA microarray to identify genes related to an early and a delayed ischemic response. Analysis of the gene ontology showed that genes related with synaptic activity, such as those encoding subunits of the ionotropic glutamate receptors and other synaptic proteins, such as PICK1, calsyntenins, SAPAP2 and SNAP-25, were differently expressed after OGD. These results were confirmed by qpcr and western blot analysis. The changes in the expression levels of these genes indicate that OGD activates a transcriptional program leading to a repression of proteins of excitatory synapses, suggesting that changes in the synaptic organization take place after the ischemic event. (Supported by PTDC/SAU-NEU/099440/2008 and PTDC/SAU-NEU/104297/2008) Oxygen and glucose deprivation, hippocampal neurons, cell death, synaptic proteins [email protected] 182

183 12:20 FIIIC11 s Luteolin and 3-alkyl-luteolin derivatives: potential neuroprotective agents in Huntington s disease striatal cells Ana M. Oliveira Ana M. Oliveira1,3; Susana M. Cardoso2; Márcio Ribeiro3; Raquel S.G.R. Seixas1; Artur M.S. Silva1; A. Cristina Rego3,4 1Chemistry Department & QOPNA, University of Aveiro, Aveiro, Portugal; 2CERNAS - School of Agriculture, Polytechnic Institute of Coimbra, Coimbra, Portugal; 3CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 4Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of CAG repeats in the exon 1 of the HD gene encoding for huntingtin (Htt). This mutation results in alteration of multiple intracellular mechanisms leading to oxidative stress and cell death by apoptosis. Flavones are compounds with an important protective role in several diseases, including neurodegenerative disorders. Therefore, in this study, we analyzed the protective effects of luteolin (Lut, 3-alkyl-3,4,5,7-tetrahydroxyflavone) and four Lut derivatives bearing alkyl chains of n = 1, 4, 6 and 10 carbons (Lut-C1, 4, 6, 10) in striatal cells derived from HD knock-in mice, expressing normal (STHdh7/7) or mutant (STHdh111/111) Htt. Mutant cells showed increased caspase-3 activity and intracellular ROS, which were significantly rescued following treatment with Lut-C4 and Lut-C6 under concentrations that did not affect cell viability. Interestingly, mutant cells exhibited an increase in phospho-nrf2 (nuclear erythroid 2-related factor 2) in total extracts of mutant cells, which was largely decreased following treatment with Lut-C6. No significant changes in SOD1 (superoxide dismutase 1, a Nrf2 target), were observed. However, decreased GCLc (glutamate-cysteine ligase catalytic subunit, another target of Nrf2) was largely ameliorated in mutant cells treated with Lut-C6, suggesting modified synthesis of reduced glutathione. Data suggest that luteolin derivatives bearing alkyl chains of n = 6 carbons may be relevant for the development of novel antioxidant strategies in HD. Work supported by FCT Portugal: PEst-OE/AGR/UI0681/2011, PEst- C/QUI/UI0062/2011, PTDC/SAU-FCF/108056/2008, and M.R. PhD fellowship SFRH/BD/41285/2007. Huntington disease, Luteolin, Luteolin derivatives, Reactive oxygen species, Apoptosis [email protected] 183

184 12:25 FIIIC12 Adult female rat brain is less prone to Alzheimer disease hallmarks than male brain: the role of insulin/igf-1 signaling E. Candeias s E. Candeias, A. I. Duarte, C. Carvalho, S. Correia, M. A. Fernandes, R. Seiça, M. S. Santos, C. R. Oliveira, P. I. Moreira Center for Neuroscience and Cell Biology, University of Coimbra For a long time, insulin (INS) was thought to be unable to cross the blood-brain barrier, and type 2 diabetes (T2D) was considered only a peripheral disease. However, this idea is long past and studies proved that not only insulin and insulin receptors (IR) are expressed in the brain, but also that brain INS signaling dysfunction is associated with cognitive decline, being INS the key feature in T2D-increased risk for neurodegenerative diseases, including Alzheimer disease (AD). More strikingly, some studies reported variability in the prevalence of AD, T2D and cognitive dysfunction, between women and men, the distinct hormone signaling profiles being pointed as one of the main causes for the gender-dependent brain differences. In this regard, we hypothesized that gender affects IR/insulin growth factor (IGF-1R)-mediated intracellular signaling pathways and AD-like pathological hallmarks in adult rat brains. Herewith, we analyzed brain cortical homogenates from adult (8-months-old) male and female Wistar rats. We observed a significant decrease in steroid hormone metabolism (as given by cholesterol, DHEA, testosterone and estrogen levels) in adult Wistar female brain compared to age-matched males. In addition, the protein expression levels of estrogen receptor were decreased, while a higher density of IR and IGF-1R were observed in female brain, thereby suggesting a compensatory mechanism, despite the decrement in both INS and IGF-1 levels. The increased phosphorylated Akt expression in female brains may play a pivotal role in the decrement in the amyloid beta peptide levels (AD hallmark), as well as in TBARS levels, as the alternative signaling pathway the phosphorylated ERK1/2 proteins was decreased in females. In conclusion, our results suggest that increased INS/IGF-1-mediated intracellular signalling in adult Wistar female cerebral cortex may render it less prone to the development of T2D and AD-related hallmarks than male cerebral cortex. insulin, type 2 diabetes, brain, Alzheimer disease, gender [email protected] 184

185 Posters 185

186 Neurodegeneration and neuroprotection 186

187 P1 s Effects of Chronic Ethanol Treatment and Withdrawal on the Neuropeptide Y Content and Cholinergic Innervation of the Rat Cerebral Cortex P.A. Pereira P.A. Pereira, M. Vilela, S. Sousa, A. Cardoso and M.D. Madeira Department of Anatomy, Faculty of Medicine, University of Porto, Portugal Chronic ethanol treatment (CET) and withdrawal (W) have profound effects on the brain neuropeptide Y (NPY)-ergic and the cholinergic systems. NPY is expressed in various brain regions, including the cerebral cortex, where its content is strongly influenced by cholinergic afferents originating in the basal forebrain. Forebrain cholinergic neurons depend on nerve growth factor (NGF) for phenotype maintenance and there is evidence that exposure to ethanol and W alters the NGF neurotrophic support. Thus, we examined the effects of CET and W on the density of NPY-immunoreactive (NPY-ir) neurons and cholinergic varicosities in the primary somatosensory cortex (SI) and in the hippocampal dentate hilus and investigated if NGF administration would interfere with the content of NPY and the cholinergic innervation of those regions. Twenty Wistar male rats were assigned to control, ethanol-treated, withdrawn and NGFtreated withdrawn groups. At the end of the experiments, rats were perfused and brain sections were processed for NPY and vesicular acetylcholine transporter immunostaining and for Nissl staining. Our estimates show that the density of NPY-ir neurons and cholinergic varicosities in the SI are altered neither by CET nor by W. In the hilus, CET did not change, but W increased the number of NPY-ir neurons. Conversely, CET reduced the density of cholinergic varicosities in the hilus and W aggravated it. In all analyzed areas, the density of cholinergic varicosities is higher in NGF-infused than in the remaining rats. Our results point towards the existence of region-specificity in the effects of CET, W and NGF treatment on the cortical NPY-ergic and cholinergic systems, and might be of importance for understanding the role of these systems in various alterations associated with CET and W as well as the potential therapeutic role of NGF. Cerebral cortex, chronic ethanol treatment and withdrawal, neuropeptide Y, cholinergic system, nerve growth factor [email protected] 187

188 P2 s Structural dynamics of synapse: the role of TAU and its vulnerability to stress hormones Sara Pinheiro Sara Pinheiro, Joana Silva, Vítor Pinto, Nuno Sousa, Ioannis Sotiropoulos ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The dynamic nature of the structure of synapses is essential for neuronal connectivity and brain function while synaptic malfunction/atrophy has a key role in stress-related pathologies (e.g. depression and Alzheimer s disease). Important clues of synaptic disruption mechanism(s) previously implicated in pathophysiology of AD have been recently suggested to also contribute in brain aging; e.g. we have previously demonstrated that chronic stress and elevated glucocorticoid (GC) levels trigger hyperphosphorylation of the cytoskeletal protein Tau resulting to cognitive deficit. Since plethora of evidence supports the importance of synaptic failure and loss in stress pathology and Tau is recently shown to have a synaptic role binding to NMDA receptors, this study aims to monitor TAU subcellular distribution and its relationship with Tau phosphorylation underling the stress/gc-induced neuronal atrophy and malfunction based on both electron microscopy and WB analysis. Using 3-4 months old Wistar rats, we found that exposure to high GC levels for 15 days induced accumulation of total and various phosphorylated species of Tau in dendritic compartment of hippocampal neurons followed by a reduction of synaptic Tau levels. These findings reveal novel insights about the underlying mechanisms of GC-evoked synaptic remodeling. Alzheimer's Disease, Tau Protein, Synapses, Stress hormones [email protected] 188

189 P3 s Neurobehavioral deficits in C. elegans mutants of neurodevelopmental disorders Carlos Bessa Carlos Bessa, Filipa Pereira, Bruno Vasconcelos, Filipe Marques, Adriana Miranda, Patrícia Maciel*, Ana João Rodrigues* Life and Health Sciences Research Institute (ICVS), School of Health Science, University of Minho. Neurodevelopmental disorders such as epilepsy, intellectual disability (ID) and autism spectrum disorders (ASD) occur in over 2% of the population. In the last years, the use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. However, this brings the need to validate genetic associations from a functional perspective in model systems in fast and effective manner. We are currently using Caenorhabditis elegans (C. elegans) to ascertain the function of several genes associated with the above-mentioned disorders. Of 60 mutant strains analyzed, (25 strains associated with ID 18 genes, 13 strains with ASD 9 genes, and 22 strains with epilepsy 16 genes), we have found several strains with developmental and neurological defects. Developmental defects are variable and range from mild larval arrest to severe embryonic lethality. No gross anatomical defects were found with the exception of one strain. 8 strains presented a chemotaxis defect for NaCl and 18 for isoamylic alcohol. 48% of mutants displayed altered motility, indicative of neurological (or muscular) deficits. We are now analyzing the most promising candidate mutants regarding their neuronal architecture and synaptic processes. In addition, we are performing additional behavioral tests to assess memory and learning. Neurodevelopment, C. elegans, Screening [email protected] 189

190 P4 s Mitoplasticity during hypoxic preconditioning: the brain battles to survive! Sónia C. Correia Sónia C. Correia, Maria S. Santos, Paula I. Moreira CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Brief episodes of sublethal hypoxia reprogram brain response to face subsequent lethal stimuli by triggering adaptive and pro-survival mechanisms - a phenomenon denominated by hypoxic preconditioning (HP). Notably, we previously demonstrated the effectiveness of HP in preventing Alzheimer s disease-related pathological features including mitochondrial dysfunction. Given the importance of mitochondria in determining cells fate, the present study was devoted to monitor the structural and functional alterations of brain mitochondria in response to a well-established protocol of HP induced by the cyclic exposure to moderate hypoxia (10% O2 during 2 hours) with intervening 24 hours reoxygenation periods, during 3 consecutive days. Several parameters related with mitochondrial bioenergetic function, biogenesis, and fusion and fission machinery were evaluated in the cortex and hippocampus of adult rats immediately, 6 and 24 hours after the last hypoxic session. HP induced a decrease in respiratory state 2 and an increase in ADP/O ratio in brain cortical and hippocampal mitochondria. Immediately after the last hypoxic episode, a significant increase in the protein levels of nuclear respiratory factor-1, and mitochondrial transcription factor A was observed. Twenty-four hours after the last hypoxic episode, a shift in the mitochondrial fusion-fission balance towards fusion occurred, as evidenced by the significant increase in the protein levels of optic atrophy protein 1 and a decrease in the protein levels of dynamin-like protein 1 in the brain cortex, and the significant reduction in the fission protein 1 levels in the hippocampus. Consistently, the electron microscopy analysis revealed that HP generated mitochondria with an elongated phenotype. Overall, these results indicate that HP enhances mitochondrial bioenergetic function, probably due to a coordinated interplay between mitochondrial biogenesis and fusion/fission events, increasing brain tolerance. Brain tolerance, mitochondrial biogenesis, mitchondrial fission and fusion, preconditioning [email protected] 190

191 P5 s Effects of the cholinesterase inhibitors galantamine and tacrine on in vivo release of dopamine from rat striatum Hanna Tak H. Tak, R. Durán, M. Alfonso, L.R. F. Faro Department of Functional Biology and Health Sciences. Faculty of Biology. University of Vigo (Spain) The inhibition of the acetylcholinesterase (AChE) by AChE inhibitors increase the levels of acetylcholine (ACh) in the synaptic cleft. Ach modulates the synthesis, release and reuptake of several neurotransmitters such as dopamine (DA) in the striatum. Acknowledging the fact that the synthesis and release of DA can be modified by the increase of extracellular ACh levels, in this work we evaluated the effects of two AChE inhibitors: galantamine, which is currently used for the treatment of Alzheimer s Disease, and tacrine on in vivo striatal release of DA and its metabolites (DOPAC and HVA). To this, Sprague-Dwley female rats were used the experiments. Tacrine (0.1, 0.5 and 2mM) and galantamine (2 and 5mM) were infused directly in the striatum through a microdialysis probe. DA, DOPAC and HVA levels, sampled by microdialysis, were measured by HPLC-EC. ANOVA/Student-Newman-Keuls test was used for the statistical analysis. Considered differences: P<0.05, P<0.01 and P< Intrastriatal administration of tacrine at concentrations of 0,5 and 2mM during 1 hour significantly increased DA levels (1189±101% and 6232±886% respectively, compared to basal levels). Regarding to metabolites, only the infusion of tacrine at the concentration of 2 mm significantly increased DOPAC levels (351 ± 39%, compared to basal levels). The administration of 5 mm of galantamine also produced significant increases on in the levels of DA and DOPAC (155±21% and 343±64%, compared to basal levels, respectively). On the other hand, the infusion of galantamine 2 mm decreased the levels of DA (5±1%, compared to basal levels). These results suggest that instrastriatal administration of tacrine increments the release of DA in a concentration-dependent manner, and that this increase is more significant than the one produced by galantamine under the same experimental conditions. Galantamine, Tacrine, Microdialysis, HPLC, anticholinesterase [email protected] 191

192 P6 s The β-amyloid (1-42) peptide-induced impairment of hippocampal LTP is eliminated in adenosine A2A receptor knock-out mice João Pedro Lopes João Pedro Lopes(1), Daniel Rial(1), Paula M. Canas(1), Rodrigo A. Cunha(1,2) (1) Center for Neuroscience and Cell Biology, Univ. Coimbra, Portugal; (2) Faculty of Medicine, Univ. Coimbra, Portugal Alzheimer's disease (AD) is characterized by an initial synaptic dysfunction and damage causing progressive cognitive impairment. Amyloid-beta peptides (Aβ) have been proposed as culprits of AD, since their brain levels are increased in this pathology and can trigger synaptic and memory dysfunction. Indeed, Aβ affect synaptic plasticity, namely impairing long-term potentiation. Epidemiological and animal studies have converged to show that caffeine, an adenosine receptor antagonist, prevents the synaptotoxicity and memory deficits caused by Aβ, an effect mimicked by blockade of adenosine A2A receptors (A2AR). Albeit A2AR control synaptic plasticity, it has not been tested if A2AR can abrogate Aβ-induced impairment of synaptic plasticity. This was tackled by comparing electrophysiological responses in Schaffer fibers/ca1 pyramid synapses of hippocampal slices collected from wild type (WT) and A2AR knockout (KO) mice upon induction of long-term potentiation (LTP) with a train of 100 Hz for 1 s. Our preliminary results show that exposure of slices from WT mice to oligomeric Aβ1-42 (50 nm for 30 min) decreased the amplitude of LTP, with a reduction of 20-41% when compared to LTP magnitude in the absence of Aβ. In contrast, in slices from A2AR-KO mice, the amplitude of LTP was similar in the absence or in the presence of Aβ. However, it should be noted that, in accordance with the known ability of A2AR blockade to depress hippocampal LTP, the amplitude of LTP in the absence of Aβ was 25-29% lower in A2AR-KO than in WT mice. These results suggest the need of A2AR in order for Aβ to impair LTP, which supports our working hypothesis that A2AR blockade prevents the impact of Aβ on synaptic function and viability. However, the qualitative similarity on LTP between the effects of Aβ and A2AR genetic elimination casts doubts on the relation between hippocampal LTP and memory performance, which ought to be experimentally resolved. Supported by FCT grant PTDC/SAU-NMC/114810/2009 Alzheimer s disease, adenosine A2A receptors, LTP, β-amyloid peptides, hippocampus [email protected] 192

193 P7 s Activation of IR,IGF-1R signaling pathways promote mitochondrial function and energy metabolism in Huntington s disease human lymphoblasts Luana Naia Naia L.1,2, Ferreira I.L.1, Cunha-Oliveira T.1, Ribeiro M.1, Duarte A.I.1, Ribeiro M.J.3, Oliveira C.R.1,2, Saudou F.4, Humbert S.4, A. Cristina Rego1,2 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 3CEDOC- Chronic Diseases Research Center, FCM NOVA, Lisbon, Portugal; 4Institut Curie, Orsay, F-91405, France. Huntington s disease (HD) is a neurodegenerative disorder caused by a CAG expansion in HD gene. This trinucleotide expansion encodes for a polyglutamine stretch in the huntingtin protein (Htt), resulting in progressive death of striatal neurons with clinical symptoms of chorea and dementia. Mutant Htt interacts with mitochondria affecting their function and leading to energy failure. In the present work we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin on intracellular signaling pathways, and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways, linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt and ERK was largely prevented by IGF-1 and, at to a less extent, by insulin in HD lymphoblasts. Importantly, both compounds stimulated Htt phosphorylation at Ser421 in HD cells. IGF-1 and insulin also prevented energy depletion in HD peripheral cells, as evaluated through a decrement in ATP and phosphocreatine levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential in HD lymphoblasts, which was correlated with increased cytochrome c protein levels. HD lymphoblasts further exhibited increased intracellular Ca2+ before and following exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca2+ accumulation. The later was recovered by both IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, data support an important role for IR,IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts. This work was supported by FCT, projects reference PTDC/SAU-FCF/66421/2006, PTDC/SAU- FCF/108056/2008 and PEst-C/SAU/LA0001/2011, COMPETEQREN and FCT PhD fellowship SFRH/BD/86655/2012. Huntington's disease, mitochondrial dysfunction, energy metabolism, insulin, IGF-1 [email protected] 193

194 P8 s Unravelling the effect of different Abeta species on BDNF effects upon hippocampal synaptic plasticity Caetano AP Caetano AP, Jerónimo-Santos A, Vicente H, Outeiro TF, Sebastião AM, Diógenes MJ Institute of Pharmacology and Neurosciences, Cell and Molecular Neuroscience Unit, Physiology Unit, Faculty of Medicine of Lisbon and Unit of Neurosciences, Instituto de Medicina Molecular, University of Lisbon, Portugal Department of Neurodegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Gottingen, Germany The brain-derived neurotrophic factor (BDNF) acts as regulator of neuronal survival, differentiation, synaptic plasticity and memory. BDNF signaling is impaired in Alzheimer s disease (AD), a neurodegenerative disorder where the accumulation of amyloid-β (Aβ) peptide is involved. Long-term potentiation (LTP) is the most studied cellular mechanism for learning and memory. Our recent data shows that heterogenous Aβ peptides impair the facilitatory effects of BDNF upon LTP, but the effect of specific Aβ species upon LTP enhancement induced by BDNF has not been established yet. Therefore the present work was designed to evaluate the effect of different species of Aβ on BDNF effects upon hippocampal LTP. The different forms of Aβ 42 (oligomers, fibrils, monomers) were prepared and its structural analysis was confirmed by atomic force microscopy. Hippocampal slices from adult male Wister rats were incubated for 3h with the different Aβ species (200nM). FEPSP were recorded through extracellular microelectrodes placed in the stratum radiatum of the CA1 area. LTP was induced by θ-burst protocol. In the hippocampal slices not exposed to Aβ, BDNF (20ng/mL) significantly increased LTP magnitude (LTPCtrl:12.9±7.7, n=5; LTPCtrl+BDNF:34.6± 4.75, n=5, p<0.01), whereas LTP enhancement induced by BDNF was strongly impaired in the hippocampal slices incubated with Aβ oligomers (LTPoli:6.4±7.4, n=4; LTPoli+BDNF:16.7± 5.6, n=4, p>0.05) and fibrils (LTPfib:10.3±3.7, n=3; LTPfib+BDNF:20.8±13.2, n=3, p>0.05). In the presence of monomers the impairment of BDNF effects was not so severe (LTPmon:11.9±10.1, n=4; LTPmon+BDNF: 29.5±3.6, n=4, p=0.127). Moreover, LTP magnitude was not affected by the different forms of Aβ when compared to the control. These results suggest there is no significant difference regarding the effect of specific Aβ assemblies upon LTP enhancement induced by BDNF, although there is a tendency for Aβ monomers to be less effective in provoking impairment of BDNF action BDNF, LTP, Aβ, Hippocampus [email protected] 194

195 P9 s Circadian oscillation of memory performance, hippocampal long-term potentiation and cortical mitochondrial function in a mouse model of Alzheimer s disease Antonio Manuel Carvalho da Silva António Carvalho da Silva1, John Jones1, Ana Cristina Rego1,2, Rodrigo A. Cunha1,2 1Center for Neuroscience and Cell Biology, 2Faculty of Medicine, University of Coimbra, Portugal There is increasing evidence that modifications of the circadian rhythm are associated with neurorological disorders, including Alzheimer s disease (AD). In this study we evaluated the impact of the circadian profile on cognitive performance in an animal model of AD, the 3xTg-AD mice, and explored how this correlated with hippocampal long-term potentiation (a proposed neurophysiological correlate of learning and memory) and modified mitochondrial function (an indicator of brain metabolism critically affected in AD). Additionally, we also tested the chrono-pharmacological effect of A2A receptor (A2AR) blockade, a validated strategy to control AD-related symptoms in animal models. The cognitive function of 3xTG-AD mice was evaluated using the Morris water maze (MWM) at different times of the day, namely Zeitgeber (ZT) 04h and 16h. Preliminary data suggest that the MWM performance is worst at 04h than at 16h. Regarding, the amplitude of hippocampal LTP, recorded extracellular at Schaffer fibers/ca1 pyramid synapses, not different at 04h (55,75% over baseline) than at 16h (51,44%, n=2). Finally, the basal rate of oxygen consumption in nerve terminals isolated from the cerebral cortex of 3xTg-AD mice was similar at ZT04 & ZT16, but when challenged with FCCP maximal oxygen consumption was observed at ZT04, compared to ZT16. In preliminary experiments, we also found that the administration of the A2AR antagonist, SCH58261 (50 nm) decreased LTP amplitude markedly at 04h than at 16h (26,43% vs 37,26%;n=2). These results set up the basis to evaluate, by comparison with control mice of the same age, if there is a different impact of the circadian rhythm on some key traits of AD pathogenesis and if there is an optimal time window for pharmacological intervention through the blockade of adenosine A2AR. Supported by FCT SFRH/BD/51675/2011 and DARPA. Chronobiology, A2a, LTP, Alzheimer's, Bioenergetics [email protected] 195

196 P10 Neuropsychological and Saccadic Measures in Premanifest and Manifest Huntington s Disease Filipa Júlio s Filipa Júlio 1, Gina Caetano 1, Cristina Januário 1, 2, Miguel Castelo-Branco 1 1 Instituto Biomédico de Investigação da Luz e Imagem, Faculdade de Medicina, Universidade de Coimbra, Portugal; 2 Centro Hospitalar e Universitário de Coimbra, Portugal Huntington s disease (HD) is a genetic neurodegenerative disorder that primarily affects the basal ganglia. Oculomotor abnormalities have been described as one of the earliest symptoms presented by HD patients and have also been reported in pre-manifest HD gene carriers. This study aims to understand the role of saccades as a potential biomarker of disease progression and severity. A comprehensive battery of neuropsychological tests was used to assess the overall cognitive functioning of the participants: 10 early HD patients, 9 premanifest HD subjects (prehd) and 14 healthy controls. An experimental paradigm was designed to assess oculomotor function. Subjects had to complete four different horizontal saccadic tasks: prosaccade, antisaccade, n-back memory prosaccade and n-back memory antisaccade. Data was recorded using an iviewx high-speed eye tracker. Error rate (saccades that did not meet ROI area criteria), latency, directional and timing errors were calculated for each task. Early HD patients exhibited deficits in several of the neuropsychological tests applied, while no significant differences were found in memory, attention, executive or visual perception tests between prehd and control subjects. Preliminary oculomotor results indicate that the early HD group was significantly different from controls in the number of valid saccades (error rate), and number of directional and timing errors made across the different conditions, except prosaccade. Significant differences were also found between prehd and control subjects for error rate in anti-saccade conditions. The saccadic impairments shown by early HD patients seem to increase with task difficulty, having a higher prevalence in the n-back conditions. Likewise, the oculomotor performance of the prehd group seems to deteriorate when an executive or/and memory load is added to the task. These preliminary data suggest that saccades can potentially mark the presence of early neurodegeneration in HD. Huntington's Disease, Eye Movements, Cognition [email protected] 196

197 P11 s Spinal redox status in peripheral neuropathic pain Raquel M. Sousa Raquel M. Sousa, Liliana S. Carvalho, Marina Morais, Dora Pinho, Isaura Tavares, António Albino-Teixeira IBMC and FMUP Spinal redox dysfunction has been described as playing an important role in the pathophysiology in several animal models of neuropathic pain. In this work we aimed at evaluating redox dysfunction in spinal cord at early stages of peripheral neuropathic pain induced by spared nerve injury (SNI) in Rat, and correlating that dysfunction to the animals pain behavior. Three experimental groups were constituted: SNI, sham-operated and naïve Wistar Han rats. Paw responses to mechanical (von Frey and pinprick tests) and cold stimuli (acetone test) were evaluated at 1, 3, 7 and 14 days after surgery. At these timepoints, rats were sacrificed and their spinal cords were immunoreacted for the DNA oxidative damage marker 8-OHdG and for the peroxynitrite marker nitrotyrosine. Results were analyzed with 2-way ANOVA followed by SNK test. SNI animals showed mechanical allodynia and hyperalgesia during the study and cold allodynia on day 14. Although the behavior of sham animals was mostly identical to naïves, they showed mechanical allodynia on days 3 and 7 after surgery. Despite the behavioral differences, both SNI and sham animals exhibited similar levels of oxidative and nitrosative damage in both lumbar and cervical spinal segments. Furthermore, no differences could be found between ipsi- and contralateral sides of either SNI or sham animals. Our results suggest that spinal redox dysfunction may not, per se, be causally involved in the development of neuropathic pain in SNI animals, at least up to 14 days after surgical induction. The differences between SNI and other models of neuropathic pain might be explained by different nerve injury location or inflammation levels. Funded by FCT and COMPETE, project PTDC/SAU-NEU/101090/2008 neuropathic pain, redox dysfuntion, allodynia, hyperalgesia, spinal cord [email protected] 197

198 P12 s Heterogeneity in cognitive aging: looking for structural correlates C. Mota C. Mota, S. Neves, S. Martins, JC. Sousa, N. Sousa and JJ. Cerqueira Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal and ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Human aging is associated with memory impairments; however, there are some individuals who retain their cognitive abilities until late in life. Understanding these diversities can help us promote healthy aging. We recently showed that in humans, the profile of cognitive deficits in old-age is broad and the severity of cognitive decline highly variable. To try to understand the underpinnings of this individual and functional heterogeneity, we decided to use rats as models of cognitive aging. In this study, we behaviorally characterized a cohort of 150 naïve old-age (18 to 24 months) male Wistar- Han rats in a standardized behavioral assessment. This was composed of well-described swimming-based tests to assess spatial working memory, spatial reference memory, behavioral flexibility and egocentric versus allocentric spatial navigation skills. Our results showed that all cognitive tests were impaired in some individuals and that almost all individuals were affected in some tests, without a clear pattern of impaired cognitive functions affecting a large proportion of subjects. Given this heterogeneity, we were able to cluster aged animals by their cognitive performance as 1) cognitively intact (good performers) and 2) cognitively impaired (bad performers). We then hypothesized that such differences could be due to structural alterations within the different groups. In order to uncover the structural correlates of such behavioral differences, ongoing stereological estimates of specific brain regions and 3-dimensional morphometric dendritic analysis are being performed. In summary, this study shows that, as in humans, cognitive function in aged rats is heterogeneous. Our ongoing stereological assessments will also provide valuable insights on the mechanisms of aging. Cognitive aging, Heterogeneity, Rats, Behavioral assessment, Structural Correlates [email protected] 198

199 P13 s Establishing a fast-throughput system for the identification of therapeutic compounds in a C. elegans model of Machado-Josep disease (MJD) Mafalda Martins-Araujo Mafalda Martins-Araujo, Andreia Teixeira-Castro, Ana Jalles, Liliana Santos, Patrícia Maciel ICVS/ECS High-throughput screening strategies in C.elegans models have been hampered by classical labour-intensive assays. We have evaluated the potential of a new automated phenotyping system, Wmicrotracker (trademark of CONICET, Argentina), in which animals freely moving in liquid culture are detected by light refraction (1). This system is composed by an infrared emitter positioned bellow the microtiter plates containing animals, and a phototransistor (receiver) localized above. When animals move across the light beam, a transient fluctuation in the signal received by the phototransistor is generated and movement is detected by digital analysis of this signal and presented as activity counts. Here we show that despite being an indirect measure of locomotion this system was able to detect significant differences in activities of strains with previously characterized locomotion deficiencies as well as to validate the therapeutic potential of compounds previously found to affect locomotion of our C.elegans MJD model in crawling assays. In addition, frequencies of animal lateral swimming ( thrashing ) measured by direct tracking systems parallel the activity signals obtained with the Wmicrotracker. Altogether these results support this new automated system as a good strategy for screening compounds in our C. elegans model of Machado-Joseph disease and in other C.elegans locomotion-defective models. (1) Simonetta SH and Golombek DA. (2007). An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application. J. Neurosci. Methods 161, C. elegans; screening; high-throuput; automation; Machado-Joseph [email protected] 199

200 P14 s Oculomotor and Executive Function in Huntington s Disease: an fmri study Gina Caetano Gina Caetano1, Filipa Júlio1, Cristina Januário2, Miguel Castelo-Branco1 1-IBILI, Faculty of Medicine, University of Coimbra, Portugal, 2-Centro Hospitalar e Universitário de Coimbra, Portugal BACKGROUND: Huntington Disease (HD) is an autosomal dominant, inherited neurodegenerative disorder, characterized by motor symptoms, cognitive decline, and psychiatric disturbances. Genetic testing for HD provides reliable information on disease status but not on disease stage. Basal ganglia are among the first areas to be affected, and have also been shown to play a crucial role in oculomotor function. This study focused on unravelling HD neuronal alterations in oculomotor and executive function. METHODS: Functional brain images were acquired during prosaccades (PS), antisaccades (AS), and n-back memory antisaccades (MAS), in separate runs, using an fmri block-design. Eye movements were measured during scanning. The pool of participants include a premanifest HD (prehd) group (N=8), a manifest HD group (n=7), and a healthy age- and education-matched control group (N=13). A comprehensive battery of neuropsychological tests was used to assess the overall cognitive functioning of the participants. RESULTS: Neuropsychological tests differences were identified between manifest HD and pre HD/control groups (Mann-Whitney, p<0.05). Contrast maps Task vs. Baseline (FDR, q = 0.01), for each of the groups, show that the saccadic paradigms activated a network of regions commonly reported in oculomotor studies. Contrast maps between Controls and prehd groups (FDR, q = 0.01) reveal that cortical areas and basal ganglia are differentially recruited. The anterior part of caudate nuclei or ventral-striatum, bilaterally, is comparatively less recruited in prehd with task difficulty. Additionally, HD show increased recruitment of visual, parietal and frontal areas, more pronounced for executive load, compared with prehd. REMARKS: These preliminary results have shown differential recruitment of cortical and basal-ganglia regions, in prehd and HD patients, during saccadic tasks involving inhibitory processes with/without executive load. Huntington, fmri, oculomotor, executive, inhibition [email protected] 200

201 P15 s Underpinning alterations in Cortical thickness and Basal Ganglia Volumetric measures in Huntington s Disease Gina Caetano Gina Caetano1, Rui Leitão1, Filipa Júlio1, Gil Cunha2,Cristina Januário2, Miguel Castelo-Branco1 1-IBILI, Faculty of Medicine, University of Coimbra, Portugal, 2-Centro Hospitalar e Universitário de Coimbra, Portugal Neuroimaging has shown that in manifest HD patients (early-hd) the striatum is most severely affected, whereas also other brain areas show signs of neurodegenerative processes, such as the cortex. Nevertheless, a linear relationship between neuronal alteration in premanifest HD (prehd) and early-hd is still lacking. Thus, this project aims at a three years follow of HD participants, and here, we present findings from the first year measurements. Participants underwent structural MRI scans on a 3T Siemens scanner, using a multiecho MPRAGE sequence. Two anatomical scans were acquired per participant, and processed with FreeSurfer5.1. The results presented here correspond to a sample of twenty-eight participants: 13 controls, 8 premanifest HD (prehd), and seven manifest HD (early-hd). Estimates of volumes of interest were extracted (bilateral Caudate, Putamen, and Pallidum) and normalized to account for total intracranial volume. We applied nonparametric tests at a significance level of 0.05, Bonferroni corrected. Surface-based cortical thickness analysis was performed with QDEC, fitting a general linear model at each surface vertex. Correction for multiple comparisons was performed using a False Discovery Rate with q=0.01. We have unveiled, bilaterally, smaller Caudate, Putamen, and Globus Pallidum volumes when comparing early-hd to controls. Similarly, with striatum volumes, when comparing early-hd with pre-hd. Cortical thinning in early-hd, derived from comparison with the control group, is observed throughout an extensive region of the cortex, compound primarily of occipital, inferior parietal, temporal and frontal regions. Cortical thinning in pre-hd, again derived from comparison with the control group, is mostly observable in the left hemisphere parietal and temporal regions. Finally, differences between prehd and controls were not identified. These preliminary results already enable to pinpoint neurodegeneration at different stages of HD. Huntington, MRI, Cortical Thickness, Basal Ganglia [email protected] 201

202 P16 The role of glutathione and dithiothreitol in proctecting against clothianidin dopamine induced release in striatum of freely moving rats. Alfonso M, s Alfonso M, Oliveira IM, Faro LRF, Durán R. University of Vigo Introduction. Clothianidin (CLO) is an insecticide which belongs to the neonicotinoids group. This insecticide is known to presenting an agonist action on nicotinic acetylcholine receptors (nachrs), leading to an acute dopamine (DA) release by dopaminergic neurons. In previous studies (Oliveira et al. Tox Lett. 192, 2010) an increased striatal dopamine release induced by CLO, in a concentration dependent way, was observed. The objective of this study was to evaluate the role of glutathione (GSH) and dithiothreitol (DTT) on protecting against acute dopamine release in freely moving rats. Methods. Female Sprague-Dawley adult rats (250 g, 5/group) were used in the experiments. CLO (3.5 mm) and GSH and DTT (0.4 mm and mm respectively) were administered directly into the striatum through a membrane probe for brain microdialysis for 60 minutes. There were two different experimental groups, one group using CLO and GSH and another group using CLO and DTT, and a control group. Levels of DA were measured using HPLC-EC. Statistical analysis was made by means of ANOVA and Student-Newmans-Keuls test. Significant differences were P< Results. Intrastriatal administration of 3.5 mm CLO has increased extracellular DA levels to 2356 ± 701% respect to basal levels. Intrastriatal infusion of CLO in GSH pre-treated animals reduced the DA levels a 81%, with respect to the administration of CLO only. The perfusion of DTT also inhibited the release of DA stimulated by CLO, 72% of decrease, respect to the infusion of CLO only. Conclusions. Both antioxidant substances used could significantly inhibit the effects on DA release observed with CLO administration only. However, the specific mechanism of action, the direct action of GSH and DTT on nachrs and consequently to hamper the union of CLO nachrs or the combination of GSH and DTT to CLO and the reduction of its capacity on fixing to the nachrs, was not the onjective of the study. Financial Suport. Contract-program of Universit pesticide, clothianidin, dopamine, microdialysis [email protected] 202

203 P17 s Effects of isatin (an endogenous MAO inhibitor) on release of dopamine and its metabolites in experimental Parkinsonian rats induced by reserpine. An in vivo study by brain microdialysis. Lorenzo Antonio Justo Cousiño Lorenzo Antonio Justo Cousiño, Daniel Fajardo Blanco, Hanna Tak Kim, Rafael Durán Barbosa. University of Vigo Introduction Isatin (indole-2,3-dione) is an endogenous monoamine oxidase (MAO) inhibitor that increases dopamine (DA) levels in the brain. It has been determined that levels of isatin in urine increases in relation to severity of Parkinson's disease (PD). However, there is little evidence of isatin on experimental models of PD. Objective The objective of this study was to determine, in awake and free moving rats, the effects of isatin on in vivo release of striatal DA and its metabolites (DOPAC and HVA) in Parkinsonian rats induced by reserpine. Material and Methodology Isatin (10 mm) has been administrated during a period of 1 hour, directly in striatum of female Sprague-Dawley adult rats (4-6/group) through a microdialysis probe. Reserpine (10 mg/kg) has been administrated intraperitonally one hour before recollected microdialysis samples. DA and its metabolites levels, sampled by microdialysis, have been measured by HPCL-EC. The statistical analysis was made by ANOVA and Student-Newman-Kleus multiple range test. Considered different P< 0,05; P<0,01 and P<0,001. Results and Discussion Reserpine injection caused catatonia in all experiments and significantly decreased striatal DA levels to 68.7 ± 7.2%, DOPAC levels to 61.9 ± 4.4% and HVA levels to 62.5 ± 6.9%. Administration of isatin increased striatal DA levels to ± 145.9%, and significantly decreased DOPAC levels to 72.2 ± 5.9% and HVA levels to 81.3 ± 7.8%. Intrastriatal perfusion of isatin in Parkinsonian rats increased DA levels to ± 135.2%, and decreases DOPAC and HVA levels to 56.5 ± 4.1% and 40.5 ± 3.9% respectively. Conclusions These results suggest that intrastriatal infusion of isatin induce increases extracellular DA levels and decreases its metabolites in rat striatum in a parkinsonism model induced by reserpine. Finally, these results support the hypothesis that isatin may be useful in the Parkinson s disease treatment, but more investigation is necessary. Isatin, reserpine, microdialysis, Parkinsonian rats. [email protected] 203

204 P18 s Mechanisms underlying paraoxon-induced dopamine release from rat striatum: an in vivo microdialysis study Daniel Fajardo Blanco Daniel Fajardo Blanco; Lorenzo Antonio Justo Cousiño; Hanna Tak Kim; Miguel Alfonso Pallares University of Vigo INTRODUCTION: Paraoxon (POX) is the active metabolite of Parathion, a highly neurotoxic organophosphate insecticide that acts by inhibition of the enzyme acetylcholinesterase, resulting in cholinergic toxicity. In previous studies, we have observed that POX also increased striatal dopamine (DA) levels in rat striatum. However it had not been elucidated all the mechanism of action for this pesticide. OBJECTIVE: The aim of this study was to determine the mechanisms of action of POX on in vivo DA release from rat striatum. To do so, we used tetrodotoxin (TTX), a voltage-sensitive Na+ channel blocker; reserpine (RES), a vesicular DA depletor agent; and nomifensine (NOM), a DA uptake inhibitor. METHODOLOGY: POX, NOM and TTX have been administrated directly in striatum of female Sprague- Dawley adult rats ( g, 4-6/group) through a microdialysis probe. RES was administrated by intraperitoneal injection. DA levels, sampled by microdialysis, have been measured by High-Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC). The statistical analysis was made by ANOVA/Student-Newman- Keuls test. Significant differences: *P< 0.05; **P<0.01 and ***P< RESULTS AND DISCUSSION: Intrastriatal administration of 5 mm POX through the microdialysis probe increased striatal DA levels to 1066 ± 119%, with respect to basal levels. Infusion of 5 mm POX in TTX (20 µm) or RES pretreated animals, increased striatal DA levels to 291±51% and 205±78%, with respect to basal levels, respectively, being these increases 73% and 81% smaller than those induced by POX in non-pretreated animals. Coinfusion of POX and 50 µm NOM evoked an additive effect on striatal DA levels (2429 ± 417%). CONCLUSIONS: Our results suggest that the effects of POX are mainly mediated by an exocytotic mechanism, vesicular and voltage-dependent, and not by a mechanism mediated by the DA transporter (trough a decreased DA uptake). Paraoxon, dopamine, striatum, microdialysis, mechanism. [email protected] 204

205 P19 s Angiotensin II overexpression in the hippocampus may be responsible for the death occurred during pilocarpine induced status epilepticus Gouveia TLF Gouveia TLF, Almeida SS, Araujo R, Iha HA, Silva Jr, JA, Casarini DE, Pesquero JB, Pesquero J, Cavalheiro EA, Naffah Mazzacoratti MG Neurology and Neurosurgery Department - Universidade Federal de São Paulo (UNIFESP) Purpose: Previous data of our group showed alterations in renin-angiotensin system in rat s hippocampus after pilocarpine-induced epilepsy. Tonin is an enzyme responsible for the cleavage of angiotensinogen releasing angiotensin II (AngII). In addition, angiotensin converting enzyme (ACE) releases AngII after cleavage of AngI. Thus, we analyzed if transgenic mice, expressing rat tonin and as consequence high amount of AngII in the brain, were more susceptible to status epilepticus (SE). Methods: Transgenic mice, expressing high rat tonin in the brain (TGM-rTon) and wild type mice (wt-c57black/6) were submitted to pilocarpine-induced SE (320mg/kg) or saline-treated. Control groups (saline-treated), Acute groups (3h of SE), tonic-clonic groups (death after a tonic-clonic seizure) of both lineage were employed. ACE was analyzed in the hippocampi of these mice by real time PCR (mrna) and by enzymatic activity using fluorescent peptides as substrates. Statistical evaluations were performed using Student s t-test and variance analysis was done followed by Student Newman- Keuls as post-hoc test and p< 0.05 was accepted. Results: Death related to tonic-clonic seizure was very increased in TGM-rTon group, when compared to wt-c57black/6 group, after SE induction. Few transgenic mice survived after SE, showing increased vulnerability of this lineage. Comparing mice from both lineages we found increased activity of ACE in saline-treated, acute and tonicclonic TGM-rTon mice, when compared to wt-c57blacl/6 groups, suggesting an increased release of AngII in the hippocampus of these animals. Conclusion: Taken together, these data show that the synchronic action of both converting enzymes (tonin and ACE) may release a large amount of AngII in the hippocampus during SE. This fact may modify all synaptic and vascular hippocampal environments in these animals and promoting their death. Supported by: CInAPCe, FAPESP, MCT-Instituto Nacional de Neurociência Translacional-INNT, CAPES, epilepsy, pilocarpine, angiotensin, [email protected] 205

206 P20 Omega-3 fatty acid supplementation reduces resting heart rate of rats with epilepsy Fulvio Alexandre Scorza s Fulvio Alexandre Scorza, Márcio D. Lopes, Diego B. Colugnati, Antônio C. Lopes, Carla A. Scorza, Esper A. Cavalheiro, Roberta M. Cysneiros Escola Paulista de Medicina/Universidade Federal de São Paulo (EPM/UNIFESP) and Universidade Presbiteriana Mackenzie Since cardiovascular dysfunction may contribute to sudden unexpected death in epilepsy (SUDEP), the consumption of omega-3 fatty acids (omega-3 FAs) might be beneficial as an adjunctive therapy for SUDEP prevention. It is well recognized that omega-3 FAs exert positive effects on the cardiovascular system including heart rate (HR) reduction, a major risk factor to sudden death. Thus, we evaluated the effects of chronic supplementation of omega-3 FAs on the HR of rats with epilepsy. In agreement with our previous investigations, this study also showed that the HR of animals with epilepsy is higher than that of the control group. Quite interesting, chronic supplementation with omega-3 FAs restored the HR of rats with epilepsy toward control values. In conclusion, although further investigations are still required, our preliminary results showed a potential preventive effect of omega-3 FA supplementation against SUDEP. epilepsy, sudden death, omega-3, brain, herat [email protected] 206

207 P21 s Role of glutamate receptors and nitric oxide on the effects of glufosinate ammonium, an organophosphate pesticide, on in vivo dopamine release from rat striatum Lilian R. Ferreira Faro Lilian R. Ferreira Faro, Miguel Alfonso, Lorenzo A. Justo Cousiño, Rafael Durán University of Vigo Glufosinate ammonium (GLA) is an organophosphate herbicide that induces convulsions in humans and rodents. It has been shown that treatment with NMDA receptor antagonists and nitric oxide synthase (NOS) inhibitors blocks the convulsions induced by GLA. Thus, we may presuppose that GLA might act stimulating NMDA receptors or inducing imbalances in the glutamate (GLU) levels to induce that excitatory effects. So, in the present work we investigate the involvement of ionotropic glutamatergic receptors and NOS activation on the effects of GLA on striatal DA release in freely moving rats. The possible implication of NMDA receptor antagonist [2R]- amino-5-phosphonovaleric acid (AP5) and NOS inhibitor 7-nitro-indazol (7-NI) on the GLA-induced DA release from rat striatum was investigated using in vivo microdialysis. Female Sprague-Dawley adult rats ( g, 5/group) were used in the experiments. GLA (10 mm), AP5 (650 µm) and 7-NI (100 µm) were administered directly into the striatum through a membrane probe for brain microdialysis during 60 minutes. Levels of DA and GLU, obtained from dialysates, were measured using HPLC- EC or FLU. Statistical analysis was made by means of ANOVA and Student-Newman- Keuls test. Significant differences were as follow: P<0.05 and P<0.01. Intrastriatal infusion of GLA significantly increased DA levels (1035±140%, respect to basal) and produced no significant changes in GLU levels. Administration of GLA to AP5 or 7-NI pretreated animals produced increases in DA levels that were ~90% and ~75% smaller, respectively, than those observed in not pretreated animals. In summary, GLA appears to act, at least in part, through an overstimulation of NMDA receptors with possible NO production to induce in vivo DA release. Administration of NMDA receptor antagonist and NOS inhibitor partially blocks the release of DA from rat striatum. Glufosinate ammonium, dopamine, striatum, microdialysis, HPLC-EC [email protected] 207

208 P22 Ataxin-3 deubiquitylase contributes to neuronal differentiation through the modulation of alpha-5 integrin subunit levels Andreia Neves-Carvalho s Neves-Carvalho A, Martins AM, Freitas A, Logarinho E, Duarte-Silva S, Heutink P, Relvas J and Maciel P Life and Health Sciences Research Institute (ICVS); ICVS/3B's - PT Government Associate Laboratory Machado-Joseph disease (MJD) is a late onset neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). This protein is known to bind ubiquitylated substrates, act as a deubiquitylase in vitro, and might be involved in transcription regulation. Although mouse and C. elegans KOs for this protein do not show overt phenotypes, functional analyses in different models support its relevance for protein quality control pathways, cellular stress responses, and cytoskeleton and cell adhesion regulation. However, the physiological role(s) of ATXN3 in the nervous system and its contribution to the disease process remain mostly unknown. Here we show that stable depletion of ATXN3 using shrna leads to an abnormal differentiation in SH-SY5Y cells. Upon differentiation, shatxn3-silenced cells present reduced mrna levels for many neuronal markers and maintain their proliferating activity. Also, these cells exhibit marked morphological changes associated with decreased adhesion, and an increased number of filopodia, in contrast with a reduced number of longer processes. This cellular phenotype is accompanied by an altered structure of the actin filaments and a deregulation of several cytoskeletal regulators. Cell death is also increased in ATXN3 depleted cultures. Interestingly, very similar morphological and cytoskeletal alterations are observed in SH-SY5Y cells overexpressing ATXN3 with a mutation in its catalytic site (C14A) that abolishes deubiquitylase activity. We show that the absence of ATXN3 leads to an abnormal differentiation and an overt cytoskeletal phenotype in neuronal cells. Since neurons are highly dependent on a wellstructured cytoskeletal network, depletion of ATXN3 may have several downstream consequences in terms of neuronal function. Our results suggest that ataxin-3 plays a role in the organization of the cytoskeleton during neuronal differentiation, that is likely related to specific targets of its DUB activity. ataxin-3, deubiquitylation, differentiation, neurons, cytoskeleton [email protected] 208

209 P23 s P2X7 receptor blockage promotes neuroprotection in different subregions of the hippocampus of rats subjected to pilocarpine-induced status epilepticus. Araújo, M.G.L. Araújo, M.G.L.; Amorim, R.P.; Fernandes, M.J.S Federal University of São Paulo - Departamento de Neurologia e Neurocirurgia - Disciplina de Neurologia Experimental Objectives: Previous studies have shown increased expression of P2X7 receptors in the hippocampus of rats subjected to pilocarpine (Pilo) model. The expression was located in excitatory terminals and in glial cells of CA1, CA3 and dentate gyrus. The activation of this purinoreceptor promotes entry of calcium in the cell, release of glutamate and proinflammatory cytokines which could contribute to cell death processes. The aim of this study was to evaluate the effect of the antagonist of P2X7 receptor in the cell death induced by pilocarpine. Methods: The Fluoro Jade-B (FJB) method was used to evaluate cell death. Male Wistar rats ( g) were used for the study. The P2X7 receptor antagonist AZ was administrated intracerebroventricularly in the following doses: 1µg, 2µg and 3µg, 30 minutes after Pilo injection. The control group was treated with saline instead of Pilo. Results: The saline group treated with AZ had no hippocampal cell staining by FJB. A dose dependant neuroprotection was observed in the Pilo group treated with AZ in the layer of pyramidal cells of CA1 and CA3, although the pattern of cell death in the entorhinal cortex and in the hillus of the dentate gyrus did not changed in comparison with the Pilo group. Conclusion: This is a pilot study to determine if pharmacological blockade of P2X7 is able to promote neuroprotection and which dose of AZ is necessary to obtain this effect. We have demonstrated that AZ is neuroprotective in the Pilo model and the mechanisms needs to be further elucidated. Financial support: Fapesp-Cinapce, CNPq, CAPES, INNT/MCT, FAP-Unifesp. temporal lobo epilepsy, pilocarpine model, P2X7 receptor, antagonism, neuroprotection [email protected] 209

210 P24 s Neuropeptide Y receptors activation protects rat retinal cells against glutamate excitotoxicity Ana Santos-Carvalho Ana Santos-Carvalho1,2; Filipe Elvas2,3,4; Ana Rita Álvaro5; António Francisco Ambrósio1,3,4; Cláudia Cavadas1,2 (1) CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; (2) Faculty of Pharmacy, University of Coimbra, Portugal; (3) Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Portugal; (4) Association for Innovation and Biomedical Research on Light and Image (AIBILI), Coimbra, Portugal; (5) Department of Biology and Environment, University of Trás-os- Montes and Alto Douro. Glutamate excitotoxicity might play a role in the pathogenesis of several retinal degenerative diseases. Neuropeptide Y (NPY) is neuroprotective against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. Thus, the main aim of this study was to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500µM) for 24h. Necrotic and apoptotic cell death were evaluated by propidium iodide (PI) and TUNEL and caspase-3 assays, respectively. NPY receptors selective agonists and antagonists were used to assess their involvement in NPY neuroprotection. Pretreatment of cells with NPY (100nM) inhibited both necrotic and apoptotic cell death triggered by glutamate. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38k. In the animal model, NPY (2.35nmol) was intravitreally injected 2h before glutamate (500nmol) injection into the vitreous. The potential protective role of NPY was assessed 24h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy. This work was supported by the FCT, FEDER and COMPETE (SFRH/BD/45311/2008, PTDC/SAU-NEU/73119/2006; PTDC/SAU-NEU/099075/2008; PTDC/NEU- OSD/1113/2012; PEst-/SAU/LA0001/2011; PEst-C/SAU/UI3282/2011). Retinal cells, neuropeptide Y, NPY receptors, neuroprotection, glutamate [email protected] 210

211 P25 s Imaging axonal transport of nanoparticles functionalized with the tetanus toxin fragment HC Cátia Lopes Cátia Lopes, Estrela Neto, Paula Sampaio, Ana Paula Pêgo INEB - Instituto de Engenharia Biomédica Gene therapy can contribute to the development of treatments of peripheral neuropathies by mediating the delivery of therapeutic genes to neurons. We have previously reported the efficient transfection of sensorial neurons by using non-viral vectors based on poly(ethylene imine) [1]. As a strategy to specifically target the delivery to peripheral neurons, the surface of these vectors was functionalized with the non-toxic carboxylic fragment of the tetanus toxin (HC). The developed nanoparticles were tested in vivo (rat model) being observed the transfection of dorsal root ganglion (DRG) neurons when the particles were injected in the footpad of the animal. Remains to be dissected if the nanoparticles process of transport in vivo is the one described for the HC fragment. Using compartmented neuronal cultures, treatments can be applied separately to cell bodies and axonal ends, and their effect or mode of action can be individually analyzed. The aim of this project is to explore the mechanism of transport in vitro, under circumstances that more directly model the in vivo situation than the conventional neuronal cultures. In order to track the nanoparticles by microscopy methods we labelled the HC fragment with quantum dots (QD) via an amidation reaction. To test if the tethering of the QDs to the HC fragment would alter its internalization and retrograde transport we conducted compartmented cultures of adult mouse DRG neurons where the HC-QD conjugates were administered in the distal axonal compartment. Our first results showed that QD-conjugation did not influenced the biological activity of HC protein, which could be observed in proximal axonal part in the cell body compartment of the cultures after 5 hours of incubation. Presently, we are testing the behaviour of functionalized nanoparticles conjugated with QDs. With this work it is highlighted that nanotechnology can give an important contribution in the field of neurosciences. [1] J Control Release, 2010,143,350 Gene therapy, nanomedicine, non-viral vectors, peripheral nervous system. [email protected] 211

212 P26 s In situ mitochondrial dysfunction evoked by immediate exposure to amyloid-beta 1-42 and NMDA receptor activation involving calcium release from endoplasmic reticulum I. Luísa Ferreira I. Luísa Ferreira*1,2, Elisabete Ferreiro*1,2, Sandra I. Mota1,2, João M. Cardoso3, Cláudia M. F. Pereira1,3, Catarina R. Oliveira1,2,4, A. Cristina Rego1,2,4 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 2Institute for Interdisciplinary Research (IIIUC), 3Instrumentation Center, Physics Department and 4Faculty of Medicine, University of Coimbra, Coimbra, Portugal * The authors contributed equally for this study. Early cognitive deficits in Alzheimer s disease (AD) are thought to be related to glutamate receptor dysregulation evoked by amyloid-beta peptide (Aβ). Aβ appears to interfere with N-methyl-D-aspartate receptors (NMDARs) activity, which may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. In order to evaluate the role of mitochondria in NMDARs activation mediated by Aβ, we followed in situ single cell simultaneous measurement of cytosolic free Ca2+ (Ca2+i) and mitochondrial membrane potential in rat brain primary cortical neurons. Our results show that direct exposure to Aβ increases Ca2+i, although in a lower extent when compared to NMDA or Aβ+NMDA. Aβ induced immediate mitochondrial depolarization and an increase in Ca2+i in cortical neurons, being the latter partially mediated by GluN2B-containing NMDARs. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Ca2+i evoked by Aβ or NMDA, indicating that mitochondria control Ca2+ entry. However, incubation with rotenone did not preclude mitochondrial Ca2+ retention in cells treated with Aβ. Both pre- and simultaneous exposure to Aβ affected the response to NMDARs activation, largely causing mitochondrial depolarization, when compared to Aβ or NMDA alone. Aβ+NMDA further induced higher mitochondrial Ca2+ retention involving the release of Ca2+ from the endoplasmic reticulum (ER) through the inositol-1,4,5-triphosphate receptors. Altogether, data highlight the role of NMDARs and mitochondrial capacity to retain Ca2+ as a mechanism implicit in Aβ-mediated early neuronal dysfunction in AD. Supported by QREN DoIT, FCT projects PTDC/SAU-NEU/71675/2006, PEst- C/SAU/LA0001/2011 and FCT fellowships SFRH/BPD/43536/2008 and SFRH/BD/43430/2008; Lundbeck Foundation. Alzheimer's disease; amyloid beta oligomers; mitochondria; N-methyl-D-aspartate receptor, GluN2B subunit. [email protected] 212

213 P27 STEP expression in the nigrostriatal pathway: effect of the dopaminergic lesion Rita Videira (1) s Rita Videira (1), Ana Saavedra (2) and Graça Baltazar (1) (1) Centro de Investigação em Ciências da Saúde, Covilhã, Portugal (2) Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in neuronal signal transduction. STEP is present at high levels in striatal medium spiny neurons where it is regulated by dopamine receptors. Surprisingly, there is no information on the expression of STEP in Parkinson s disease (PD), a pathology characterized by the progressive degeneration of dopaminergic neurons from the substantia nigra (SN) that project to the striatum, resulting in reduced striatal levels of dopamine. With this work we intended to determine if changes in dopaminergic signalling, resultant from a selective dopaminergic lesion, can influence the expression of STEP in the nigrostriatal pathway. For the determination of this effect, neuron-astrocyte midbrain co-cultures were stimulated with MPP+ and, in parallel, C57BL/6 mice were submitted to an intraperitoneal injection of MPTP. Our results showed that exposure to the dopaminergic toxins MPP+ and MPTP significantly increased STEP61 levels in the substantia nigra. We observed that, besides neurons, STEP is also expressed by nigral astrocytes, and that the augmented STEP levels parallels with increased astrocyte reactivity characteristic of the MPTP model. Moreover, inducing astrocytes reactivity through exposure of primary cultures to lipopolysaccharide significantly increased STEP expression. Taken together these results suggest that STEP expression is regulated by dopaminergic lesion through a process involving both changes in the dopamine signalling and astrocytic reactivity. STEP, dopaminergic neurons, Parkinson s disease. [email protected] 213

214 P28 Neuroprotective role of Neuropeptide Y in a lentiviral mouse model of Machado- Joseph Disease. Joana Neves s Neves, Joana; Gonçalves, N.; Cavadas, C & Pereira de Almeida, L. CNC - Centro de Neurociências e Biologia Celular, Universidade de Coimbra; Faculdade de Farmácia da Universidade de Coimbra Machado-Joseph disease (MJD), the most common dominantly-inherited ataxia worldwide, is a polyglutamine disorder: more than 55 repetitions of the CAG trinucleotide in the MJD1 gene translate into a mutant ataxin-3 protein, causing progressive impairment of motor coordination, which is associated to the neurodegeneration of cerebellum, pons, brain stem, substantia nigra and striatum. There is no effective therapy for this disorder. Neuropeptide Y (NPY) is one of the most abundant and widely distributed neuropeptides in the mammalian CNS and is involved in various physiological functions, such as feeding behavior, memory processing and cognition. Evidences of NPY neuroprotective potential in neurodegenerative disorders have been reported. The aim of this study was to assess the potential of NPY overexpression to prevent or attenuate MJD neuropathology. For this purpose, a mouse model of MJD was generated by stereotaxically injecting in striatum lentiviral vectors encoding mutant ataxin-3 (72Q). Co-injection of lentiviral vectors encoding NPY or EGFP, as control, allowed evaluation of the protective effects of NPY. Four and eight weeks after vectors injection, the animals were sacrificed, the brain tissue was collected and analyzed by immunohistochemistry. NPY expression mediated significant decrease in the number of cells exhibiting mutant ataxin-3 inclusions (19480 ± 3080 inclusions versus ± inclusions, n=4, student s t-test, p<0.01), compared to the control group. Importantly, mutant ataxin-3 expresssion induced a striatal lesion characterized by depletion of DARPP-32 immunoreactivity, which was reduced by 55% upon NPY overexpression: DARPP-32 staining volume depletion ( ± /mm3 versus ± /mm3, n=4, student s t-test, p<0.01). These results suggest that the overexpression of NPY may be promising in the reduction of MJD neuropathology, which might contribute to the development of a new therapeutic strategy for MJD. Machado-Joseph Disease, Neuropeptide Y, neuroprotection [email protected] 214

215 P29 s Citalopram as a candidate therapeutic agent for Machado-Joseph Disease Andreia Teixeira-Castro Sofia Esteves, Andreia Teixeira-Castro, Anabela Silva-Fernandes, Patricia Maciel Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, and ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal. Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract (polyq) in the C-terminus of the ATXN3 gene product, ataxin-3. We have generated a new transgenic mouse model expressing human ataxin-3 with an expanded CAG tract under the control of the CMV promoter: CMVMJD135, carrying 135 glutamines. CMVMJD135 mice develop a severe and progressive neurologic phenotype, with intranuclear inclusions in neurons and brain pathology consistent with human disease. Antidepressants have been shown to prolong lifespan, improve motor phenotypes and neuropathology and to enhance neurogenesis in mouse models of polyglutamine diseases. In an unbiased screening of 1200 small molecules Citalopram was identified as markedly reducing motor impairment in our C. elegans model of MJD. To validate these findings in a mammalian model, two Citalopram dosages are being tested chronically in the CMVMJD135 mouse. Citalopram was able to rescue the loss of limb strength given by the hanging wire test, at 10 and 14 weeks of age in a dose-dependent manner. In the vertical pole test there was also a trend towards amelioration, although it was not statistically significant. Body weight loss was rescued at 14 weeks of age with both dosages. Motor coordination tests, including Beam Walk, Rotarod and Motor Swimming test, Footdragging analysis and also SHIRPA protocol are being conducted. In these tests, no amelioration of the phenotype was observed at 14 weeks of age. Further neuropathologic, neurochemical and molecular studies are being performed to understand the effects of the compound in the CMVMJD135 mouse model. Machado-Joseph Disease, Therapy, Serotonine, SSRI [email protected] 215

216 P30 s Effect of caffeine administration in the electrophysiological response of the retina in a model of retinal ischemia-reperfusion Pedro Tralhão P. Tralhão1, R. Bóia1, D. Brudzewsky1,2, A.F. Ambrósio1,2, A.R. Santiago1,2 1Center of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, 2AIBILI, Coimbra, Portugal Caffeine is one of the main biologically active compounds of coffee and the most widely consumed psychoactive substance in the world. Caffeine at low concentrations can block all four subtypes of adenosine receptors (A1, A2A, A2B and A3), most actions being mediated through inhibition of the high-affinity A1 and A2A receptors. Ocular ischemia plays an important role in the pathophysiology of various ocular diseases, which may lead to neuronal death. Retinal neuroprotection is often quantified as molecular and histological changes. However, these changes provide no retinal functional information unlike retinal electrophysiology, a non-invasive in vivo approach. Here the aim was to investigate if long-term caffeine administration prevents changes in electroretinography (ERG) induced by retinal ischemia-reperfusion (I-R) injury. Caffeine (1g/L) was administered in drinking water from 2 weeks prior retinal I-R injury. Intraocular pressure (IOP) was measured regularly. Caffeine did not affect IOP values when compared to control animals (water only). ERGs were recorded after dark adaptation at 4 time points: before caffeine administration; after 2wks of caffeine; 24h after I-R injury and 7d after I-R. The ERG consisted of a 4-step exam: scotopic; light adaptation; photopic and flicker. ERG analysis of all ischemic eyes revealed a similar pattern. Scotopic condition showed severe amplitude reduction of a- and b-waves 24h and 7d post-i-r, while OP amplitudes were barely measurable. Photopic and flicker conditions also showed serious defects in amplitude of b-waves and 1st harmonic, respectively. None of these ERG components showed a significant recovery at 7d after I-R injury. This data suggests that 1g/L caffeine, given orally, did not prevent ERG alterations induced by I-R, indicating that caffeine does not protect retinal function after I-R injury. Support: FCT (Proj. PTDC/BIM-MEC/0913/2012 & FCT Strat. Proj. PEst- C/SAU/UI3282/2011), Portugal, FEDER-COMPETE Adenosine, caffeine, electroretinography, ischemia-reperfusion, retina [email protected] 216

217 P31 s Abeta disaggregating proteins counteract Abeta induced effects on intracellular sapp accumulation by remodeling actin network Ana Gabriela Henriques Henriques A.G., de Oliveira J.M., Gomes B., Ruivo R., da Cruz e Silva E.F.* and da Cruz e Silva O.A.B. Laboratório de Neurociências, Centro de Biologia Celular, Universidade de Aveiro Abeta aggregation and deposition into senile plaques is a key feature in Alzheimer s disease pathology. Aggregated Abeta was shown to induce intracellular retention of secreted APP (isapp). Potentially, isapp accumulation of non-amyloidigenic origin (isappalpha), which has extracellular neurotrophic and neuroprotective properties, may be a mechanism by which Abeta can contribute to increased neurotoxicity and neurodegeneration. Further, our group also showed that this isapp retention was associated with cytoskeletal network abnormalities induced by the Abeta peptide. Hence, therapeutic strategies aimed at reversing Abeta polymerization provide attractive avenues for intervention. Laminin and gelsolin provide such a model, given that both prevent Abeta fibril formation and decrease Abeta toxicity, giving rise to Abeta-laminin and Abeta-gelsolin complexes. In the present work, both Abeta disaggregating proteins, were able to counteract the Abeta effects, at the neurotoxic and isapp accumulation levels. The data presented validate the potential of both proteins as therapeutic targets to prevent Abeta-mediated effects. Acknowledgements: This work was supported by the Fundação para a Ciência e Tecnologia of the Portuguese Ministry of Science and Technology: PTDC/BEX- BCM/0493/2012, PTDC/QUI-BIQ/101317/2008 and JPND BIOMARKAPD - Biomarkers for Alzheimer s disease and Parkinson s disease, and also the Center for Cell Biology UA - REEQ/1023/BIO/2005. Laminin, Gelsolin, AD therapeutics, fibril inhibition, aggregation [email protected] 217

218 P32 s The Role of N-terminal Phosphorylation on Mutant Huntingtin Oligomerization, Aggregation and Toxicity Joana Branco dos Santos Joana Branco dos Santos, Federico Herrera, Tiago Fleming Outeiro Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa Background: Aggregation of mutant huntingtin (Htt) is a dynamic process that starts with the association of a few misfolded Htt monomers in small, soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic species of mutant Htt and that the largest aggregates are rather neuroprotective. In order to prevent Htt aggregation and toxicity it is essential to understand the molecular mechanisms of oligomerization. Aims: Elucidate the role of Htt N-terminal region on its oligomerization, aggregation and toxicity, with particular emphasis on the phosphorylatable residues (T3, S13 and S16). Methods/Techniques: We have recently developed a cellular model for the visualization of Htt oligomeric species in living cells, based on the bimolecular fluorescence complementation (BiFC) assay. In this model, Htt exon-1 is fused to two non-fluorescent halves of the Venus protein (V1 and V2). When Htt dimerizes, the two halves get together and reconstitute the functional fluorophore. We used these BiFC constructs to create a series of phosphoresistant (T3A, S13A, S16A) and phosphomimic (T3D, S13D, S16D) Htt mutants. Results/Outcome: When phosphomimic mutations were present in both 103QHtt-V1 and 103QHtt-V2 BiFC constructs, the generation of inclusion bodies was completely abolished. However, the levels of oligomeric species were similar to the non-mutated and the phosphoresistant BiFC pairs. The combinations of a non-mutated construct with a phosphomimic construct produced intermediate phenotypes in terms of aggregation. Phosphoresistant BiFC pairs did not produce overt phenotypes. Mutations in N-terminal residues had varied effects on Htt toxicity, which apparently were not associated with the levels of oligomeric species or inclusion bodies. Conclusions: The phosphorylation state of Htt N- terminal region is a key player in the formation of inclusion bodies and the toxicity of the protein. huntingtin, oligomerization, bimolecular fluorescence complementation, phosphorylation [email protected] 218

219 P33 s 17-DMAG induces autophagy and delays disease progression in a mouse model of Machado-Joseph disease Sara Duarte-Silva Duarte-Silva S., Silva-Fernandes A., Neves-Carvalho A, Amorim M., Soares-Cunha C., Oliveira P., Teixeira-Castro A., Maciel P. Life and Health Sciences Research Institute (ICVS); ICVS/3B s - PT Government Associate Laboratory Objective: Machado-Joseph disease (MJD) is a late-onset inherited neurodegenerative disease currently lacking effective treatment. Our goal was to generate a new mouse model of MJD (CMVMJD135) with a robust behavioral phenotype and specific neuropathology, providing a framework for pre-clinical trials, and to assess the therapeutic effect of 17- DMAG, a brain-permeable HSP90 inhibitor not tested before in MJD. Methods: CMVMJD135 mice express human ataxin-3 (ATXN3) carrying 135 glutamines under the regulation of the ubiquitous CMV promoter, resulting in ubiquitous expression of ATXN3 at near-endogenous levels. The model was characterized using a battery of motor behavioral tests and immunohistochemistry and stereological tools for neuropathologic evaluation. 17-DMAG (25mg/kg) was administered from 6 to 30 weeks of age and behavioral and neuropathologic studies were performed to assess therapeutic efficacy. Results: CMVMJD135 mice manifest MJD-like motor symptoms since 6 weeks of age that progress slowly over time, show ATXN3 intranuclear inclusions (INIs) in several brain regions and cell loss in the pontine nuclei. 17-DMAG-treatment led to later onset and slower progression of the motor coordination deficits, decreased neuropathologic involvement, including less INI formation, and a reduction in ATXN3 protein levels, in parallel with a marked induction of autophagy. Interpretation: CMVMJD135 mice mimic several features of the human disease, making this model a valuable tool for the study of MJD pathogenesis and for pre-clinical studies. 17-DMAG treatment markedly delayed disease progression, decreased ATXN3 aggregation and levels of the mutant protein, possibly through autophagy induction. This suggests that Hsp90 inhibition is a promising therapeutic strategy for MJD. polyglutamine diseases, autophagy, therapy [email protected] 219

220 P34 Synaptic Localization of the Secretases Involved in the Metabolism of Amyloid Precursor Protein Anna Pliássova s Anna Pliássova 1, Catarina R. Oliveira 1,2, Rodrigo A. Cunha 1,2, Paula Agostinho 1,2 1 - Center for Neuroscience and Cell Biology, 2 - Faculty of Medicine, University of Coimbra, Portugal The early stages of Alzheimer s disease (AD) are associated with alterations in synaptic efficiency, which are thought to be caused by oligomeric assemblies of soluble -amyloid peptides (A ). A are derived from the proteolytic cleavage of the amyloid precursor protein (APP) through the action of secretases (, or. Although it is widely accepted that synaptic A metabolism is crucial for AD initial damage, the synaptic metabolism of secretase-mediated APP-derived production of A in different terminals remains to be clarified. Thus, we aim to define: i) the synaptic, sub-synaptic and astrocytic distribution of APP and secretases and ii) their co-localization in different nerve terminals. We compared by Western-blot analysis the levels of APP, BACE1 ( -secretase), ADAM- 10 ( -secretase) and PS-1 ( -secretase) in synaptosomes (nerve terminals), gliosomes (membranes of astrocytes) and total membranes of the hippocampus of adult mice (C57/Bl6), as well as in subsynaptic (pre- pos- and extra-synaptic) fractions. We found that APP, BACE1 and ADAM-10 were present in nerve terminals, although they were not enriched in these fractions as compared with their densities in total membranes. Considerable levels of APP were also found in gliosomes. Preliminary immunoblot results suggested that ADAM10 was more abundant in the postsynaptic density than in pre- and extra-synaptic fractions. Immunocytochemical analysis of purified nerve terminals indicated that APP was more frequently associated with glutamatergic (37.5±0.8%, n=2) rather than with GABAergic terminals (23.9±1.3%, n=3). BACE was also more abundant in glutamatergic (60.6±5.1%, n=3) rather than in GABAergic terminals (22.0±13.4%, n=2). The data obtained with this work will be important to define the synaptic localization of different proteins involved in A formation, which is considered the major culprit in AD. (Supported by FCT grant PTDC/SAU-NMC/114810/2009 and CAPES-FCT) Synapse, Alzheimer's Disease, BACE, ADAM10, PS1 [email protected] 220

221 P35 Effects of type 1 diabetes and insulin therapy on brain mitochondrial dynamics and autophagy Renato X. Santos s Renato X. Santos 1, Sónia C. Correia 1, Maria S. Santos 1,2,Paula I. Moreira 1,3 1 Center for Neuroscience and Cell Biology, University of Coimbra, 2 Dept. of Life Sciences Faculty of Sciences and Technology A healthy pool of mitochondria depends on a balanced mitochondrial dynamics. and autophagic degradation of dysfunctional organelles. It has been shown that type 1 diabetes can affect several organs including the brain and these effects can be halted or attenuated by insulin therapy. This study aimed to evaluate the effects of type 1 diabetes and insulin treatment on rat brain mitochondrial dynamics and autophagy. For this purpose we induced type 1 diabetes to 2-month-old rats by i.p. injection of streptozotocin (STZ, 50mg/kg body weight). 2 months after the induction of diabetes, a group of animals were maintained for 1 month with a daily s.c. injection of insulin using a protocol intended to normalize the levels of glucose in the blood. Animals were sacrificed 3 months after the induction of diabetes. No significant alterations were observed in the protein levels of active mtor (phosphorylated in Ser2448), an autophagy repressor and Beclin 1, an autophagy stimulator. LC3-II, a recognized marker of autophagosomes, remained unchanged. These observations and p62, an autophagic substrate, are in agreement. Regarding mitochondrial dynamics, a significant increase in the levels of active DRP1 (phosphorylated in Ser616 residue), a fission-related protein, was observed in STZ animals. Insulin treatment promoted a slight decrease of active DRP1. Interestingly, an increase in the number of mtdna copies is observed in STZ animals whereas insulin reverted that effect. Altogether our results show that type 1 diabetes affects, mitochondrial dynamics favoring mitochondrial fission, which may predispose to neurodegenerative events. Renato X. Santos was a recipient of a PhD fellowship from the Fundação para a Ciência e a Tecnologia (SFRH/BD/43972/2008). Autophagy, insulin, mitochondrial dynamics, type 1 diabetes [email protected] 221

222 P36 Protective effects of antioxidants on the glufosinate ammonium-induced striatal dopamine release in vivo. Rafael Durán s Rafael Durán. Lilian R. Ferreira Faro. Brenda V. Ferreira Nunes. Miguel Alfonso Universidad de Vigo (España) Glufosinate ammonium (GLA) is a phosphinic acid derivative of glutamate (GLU), being a structural analogue of this molecule. GLA is neurotoxic and causes various effects including convulsions in humans and animals, being this effect possibly due to an action of GLA on extracellular GLU levels or GLU receptors. GLA also induces in vivo dopamine (DA) release from rat striatum (Nunes et al. Arch. Toxicol. 84, 777, 2010). In the present work, we have investigated the possible protective effects of three antioxidants: glutathione (GSH), trolox (TRX) and alpha-lipoic acid (ALA) on the GLAinduced DA release from rat striatum. Female Sprague-Dawley adult rats ( g, 5/group) were used in the experiments. GLA (10 mm), GSH (400 µm), TRX (1 mm) and ALA (400 µm) were administered directly into the striatum through a membrane probe for brain microdialysis. Levels of DA, obtained from dialysates, were measured using HPLC-EC. Statistical analysis was made by means of ANOVA and Student-Newman- Keuls test. Significant differences were as follow: P<0.05 and P<0.01. Intrastriatal infusion of 10 mm GLA significantly increased DA levels (1035±140%, respect to basal levels). Administration of GLA to GSH or TRX pretreated animals increased DA overflow to 382±143% and 149±14%, respectively, respect to basal levels. These increases were 63% and 86% smaller than that observed in not pretreated animals. Conversely, the infusion of GLA in ALA pretreated rats increased DA levels to 671±163% respect of basal levels, an increase not statistically different from that produced by GLA alone. In summary, the administration of antioxidants GSH and TRX produced significant decreases in the GLA-induced DA release. On the other hand, the infusion of ALA not decreased the DA release in the same experimental conditions. ALA is a co-factor that acts protecting against lipid peroxidation and GLA probably do not act by this mechanism to induce DA release from rat striatum. Glufosinate, Dopamine, In vivo, Antioxidants [email protected] 222

223 P37 s Ataxin-3 phosphorylation and its effects on protease activity and Machado-Joseph disease mechanisms Carlos Matos Carlos A. Matos, Clévio Nóbrega, Marilisa Vigorita, Luísa Cortes, Luís Pereira de Almeida, Sandra de Macedo-Ribeiro and Ana Luísa Carvalho Center for Neuroscience and Cell Biology Machado-Joseph disease (MJD) is the most common form of dominantly inherited ataxia in the world. It develops when the polyglutamine tract of ataxin-3 (atx3), a deubiquitinase (DUB) involved in protein homeostasis and transcription regulation, is expanded beyond a critical threshold. Though atx3 displays ubiquitous expression throughout the human body, neuronal death associated with MJD occurs only in discrete regions of the nervous system, suggesting that the cellular context is a crucial factor mediating expanded atx3-induced degeneration. Post-translational modifications (PTMs) are frequently described as toxicity modulators in many neurodegenerative diseases and may be responsible for differential regulation of atx3 properties between different cell types, being important for the selective disease mechanisms. We set out to identify novel atx3 PTMs with possible functional relevance. Through a mass spectrometric analysis of atx3 purified from transfected mammalian cell lines we identified two novel phosphorylation sites, one in the catalytic DUB domain and the other in the second ubiquitin interacting motif of atx3. We then focused our study on the phosphorylation of the catalytic domain of atx3, because the identified phosphorylation site serine 12 is in close proximity to the catalytic triad, making it a good candidate for an activity regulator. Phosphorylation of serine 12 in cells was further confirmed by immunoblot with a phospho-specific antibody raised against the modified residue. Using phosphomimetic and phosphodead mutants of atx3 we performed activity assays in vitro using DUB model substrates and observed that modification at position 12 modulates atx3 DUB activity. The effect of atx3 phosphorylation on its aggregation, a key event in MJD pathogenesis, is being assessed in cells cultures and in an in vivo lentiviral rat model with striatal pathology. Machado-Joseph disease, ataxin-3, neurodegenerative diseases, phosphorylation, deubiquitinases [email protected] 223

224 Cell signaling 224

225 P38 s Potential role for mglur5 in pain inhibition from the infralimbic cortex in rodents Ana David-Pereira Ana David-Pereira, Sónia Puga, Armando Almeida, Filipa Pinto-Ribeiro ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The prefrontal cortex (PFC), an area primarily involved in cognition, has also been demonstrated to participate in the descending modulation of pain. Concomitantly, glutamate (GLU) has been show to partly mediate the output activity of this area. In this work, we aimed at assessing the effects of the transient activation (with GLU) of the Prelimbic (PrL) and Infralimbic (IL) cortices upon acute nociception, using the paw withdrawal test (PW) (Hargreaves model). In control animals, GLU injection in the PrL cortex resulted in an increased PW latency (antinociceptive effect) as soon as 30s after its administration. Interestingly, the administration of GLU to the IL cortex promoted a long lasting pronociceptive effect (decreased PW latency) that peaked only 20 minutes after its administration. Further studies, on the IL, using specific ligands showed that this facilitatory effect of GLU was mimicked by the administration of metabotrobic GLU receptors type 5 agonists (CHPG). Moreover, preliminary data show that these mglur5 also facilitate nociception in an animal model of experimental monoarthritis, demonstrating that this pathway is not disrupted and might thus contribute to the aberrant pain syndromes so common in clinical conditions. Our data show that mglur5 is a potential player in the facilitation of nociception in both control and arthritic animals. PFC, nociception, metabotropic glutamate receptors, chronic inflammatory pain [email protected] 225

226 P39 s BACE-ics in thymocyte differentiation: implications for Alzheimer s disease therapeutics Ana Ribeiro Ana Ribeiro, Susana Roque, Margarida Correia Neves, Nuno Sousa and Ioannis Sotiropoulos ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal APP misprocessing and generation of the neurotoxic amyloid beta (A ) peptide represent a key pathological mechanism in Alzheimer s disease (AD) highlighting the therapeutic potential of enzymes involved in this pathway such as BACE-1 and presenilin 1. While presenilin inhibitors are previously shown to have detrimental role e.g. damaging thymocytes development, there is no evidence about side effects of BACE-1 inhibition despite the fact that BACE-1 inhibitors has been suggested as alternative therapeutic approach for AD. Using both in vivo and in vitro approaches, this study demonstrated that lack of BACE-1 enzyme result in increase of thymus weight and total cell numbers followed by enlarged thymic cortex and elevation of CD4+CD8+ percentages. Moreover, BACE-KO thymocytes were less vulnerable to GC-induced cell death suggesting the involvement of BACE-1 on thymocytes differentiation. All together, these findings suggest that BACE-1 has an essential role in thymocytes differentiation and thymus structure providing evidence about the underlying cellular pathways involved. thymus, BACE, amyloid-beta, Alzheimer's disease [email protected] 226

227 P40 s A whole organism screen identifies serotonin signaling as modulator of Machado- Joseph disease pathogenesis Andreia Teixeira-Castro Andreia Teixeira-Castro, Ana Jalles, Rick Morimoto and Patrícia Maciel Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, and ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal. Despite the many efforts that are under way to develop therapeutic strategies, no preventive treatment is yet available for any of the polyglutamine (polyq) diseases. Machado-Joseph disease (MJD) is one of the polyq disorders caused by the expansion of a polyq tract within the C-terminal of the ataxin-3 (ATXN3) protein. Mutant ATXN3 acquires the ability to self-associate and enter an aggregation process, which is associated with several pathophysiological consequences for neurons. The lack of therapeutic strategies that effectively prevent neurodegeneration in MJD patients prompted us to search for compounds that modulate mutant ATXN3-related pathogenesis. Recent data from our lab have shown that many aspects of MJD can be properly modeled in the round worm Caenorhabditis elegans. This study is based on the idea that our C. elegans MJD model is amenable for large-scale drug screenings, in which the identification of effective drugs can be accomplished by looking simultaneously at protein aggregation in the live neuronal cells, and on its impact on neuron-regulated behavior of the whole-animal. Our goal was to screen a library of ~1200 mainly FDA-approved out-of-patent compounds for their ability to prevent or delay mutant ATXN3-mediated neuronal dysfunction. Ten percent of the non-toxic compounds significantly reduced the locomotion deficits of the animals. The majority of our hits are FDA-approved compounds or are currently in clinical trials for other neurological disorders. We found that one group of the newly identified compounds exert their function in C. elegans by increasing serotonin signaling. Rescue of MJD pathogenesis seems to be dependent on at least one of the G-protein-coupled serotonin receptors. We should be able to identify efficacious compounds that can be tested in higher organisms, including our transgenic mouse model, and eventually enter clinical development. Machado-Joseph disease, Therapy, C. elegans, Serotonin [email protected] 227

228 P41 A new understanding of the interplay insulin-glucose in neurons from the rat hippocampus Bastos A.E.P. s Bastos A.E.P.(1), Mondragão M.(1), Costa G.(1), Costa P.F.(2), Lima P.A.(1, 2) (1)Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Ed. C8, Lisboa, Portugal (2) Faculdade de Ciências Médicas da Universidade de Lisboa Insulin is recognized by its role in promoting cellular glucose-uptake. Recently, new functions of insulin and its receptor (InsR) in the nervous system have been revealed, such as facilitating long-term-potentiation and memory. However, there is limited information about the underlying neuronal mechanisms. The present work investigates the influence of glucose concentration on the insulin effects on neuroexcitability. In our laboratory, we found that insulin increases neuroexcitability by reducing activity of slow-voltage-activated K+ channels in hippocampal neurons acutely isolated from fed but not fasted animals. Here we show that this effect is balanced by glucose concentration. Firstly, we used whole-cell voltage-clamp to record voltage activated K+ currents in differentiated neuroblastoma cells N1E-115. During the records, these cells were superfused with different glucose concentration (3mM and 25mM). The results disclose that the insulin inhibitory effect on slow voltage-activated K+ currents (Islow) is larger in the presence of high glucose levels (17,2±2,5%; n=4 for 25mM glucose and 7,8±2,7%; n=5 for 3mM glucose). In order to test whether such phenomenon is related to glucose influence in the expression levels of membrane InsRs, western blots were performed with anti-beta-insr antibody in differentiated N1E-115 cells incubated for 24h in media with different glucose concentration (0mM; 5mM and 25mM glucose). The results show that the expression levels of the InsRs are as higher as the glucose concentration used. Such variations might explain the insulin-response in the presence of different glucose levels. Overall, the present study gives new insights into the understanding of the mechanisms underlying the neuronal insulin effect during the feeding cycle. Furthermore, this work contradicts the general current theory that the brain is isolated from metabolic systemic changes ( the selfish brain theory). Insulin; Glucose; Insulin Receptor; N1E-115 cells [email protected] 228

229 P42 s Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion Castro Neto EF Castro Neto EF, Cunha RH, Silveira DX, Yonamine M, Lima CAS, Gouveia TLF, Cavalheiro EA, Amado D, Naffah-Mazzacoratti MG UNIFESP Psychotropic effects have been described during ingestion of the infusion of the Banisteriopsis caapi roots known as Ayahuasca and it is used for religious purposes. Physical, psychological and spiritual cures have been attributed to Ayahuasca and some studies on medical applications of this infusion show inhibition of MAO activity. The aim of the present work was to evaluate changes in neurotransmission induced by ayahuasca in the hippocampus and amygdala of naïve rats. The level of monoamines, their metabolites and amino acid concentrations were quantified using chromatography (HPLC). Four groups of rats were employed: saline-treated, and rats receiving 250 mg/kg, 500 mg/kg and 800 mg/kg of ayahuasca infusion (gavage). Animals were killed 40 min after drug ingestion and the structures stored at -80ºC until HPLC assay. The results showed decreased concentrations of glycine (GLY) and gamma-aminobutyric acid (GABA) in the amygdala of rats that received 500 and 800 mg/kg of ayahuasca. In the hippocampus increased level of GABA was found. Increased concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) and a reduction of their main metabolites were detected in the amygdala suggesting increase in the utilization rate of monoamines and the release of inhibitory amino acids. ayahuasca, amino acids, monoamines, hippocampus, amygdala [email protected] 229

230 P43 s Differential expression of the Kv1.3 channel/current throughout the rat hippocampus Clara Patrício Clara Patrício, Joana E. Coelho, Ana M. Sebastião, Luísa V. Lopes, Pedro A. Lima Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa; Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa; Departamento de Fisiologia, Faculdade de Ciências Médicas The hippocampus has a major function in memory and learning. Anatomically, a transversal slice of the hippocampus is divided in the Ammon s horn (CA1 to CA4) and the dentate gyrus. Along its longitudinal axis, the hippocampus is often regarded a homogeneous, structure. However, there is increasing evidence for differences throughout its dorsal-ventral axis not only at the anatomic but also at the molecular and functional levels. This study aims to investigate the dorsal-ventral distribution of the voltage-activated potassium (K+) channel Kv1.3 as well as the resulting K+ currents. Three different approaches have been applied, using preparations obtained wistar rats (p21-29). Firstly, immunohistochemistry assays with an anti-kv1.3 antibody were carried out using 30 m thick coronal slices from anterior and posterior sections of the hippocampus. The resulting images show that, in the CA1 region, the Kv1.3 expression is mainly somatic. Interestingly, in CA1 s pyramidal layer, the dorsal Kv1.3 signal is higher than in the ventral region. Secondly, to further quantify such trend, western-blot analysis using the same antibody has been employed to samples from dorsal, intermediate and ventral hippocampus. Finally, the functional implications of these findings have been investigated using whole cell voltage clamp recordings, from pyramidal neurons acutely isolated from the CA1 region of the dorsal and ventral portions of the hippocampus. Margotoxin (MgTx, 100 pm), a specific antagonist for Kv1.3 was applied in the superfusing medium. The MgTx sensitive voltage-dependent current component from neurons of the dorsal and ventral regions were compared. As a whole, these findings suggest that similar CA1 pyramidal neurons have different neuroexcitability profiles depending on their location (dorsal/ventral) in the hippocampus. Indeed Kv1.3 has a clear role in neuronal activity, as MgTx(100pM) increases the firing frequency during current clamp recordings from brain slices. hippocampus, Kv1.3, dorsal-ventral, rat, immunohistochemistry [email protected] 230

231 P44 The chronic consumption of caffeine leads to enhanced plasma levels of caffeine and the appearance of both demethylated and deaminated metabolites in mouse tissues Eszter C. Szabó s Eszter C. Szabó 1,2, Angelo R. Tomé 1,3, Rodrigo A. Cunha 1,4 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal, 2Department of Physiology and Neurobiology, Eötv The regular consumption of moderate doses of caffeine alleviates the incidence of different neuropsychiatric conditions, such as depression, Alzheimer s or Parkinson s diseases. Although it is well established that caffeine acts through the antagonism of adenosine receptors, it remains to be explored if caffeine metabolites might also be involved in neuroprotection. Thus, caffeine (a tri-methylated xantine, 1,3,7-TMX) is metabolized by the liver, mainly to form demethylated products such as theophylline (1,3-DMX), theobromine (3,7-DMX) and paraxanthine (1,7-DMX), whereas the formation of deaminated products such as 1,3-dimethyluric acid (DMU), 1,7-DMU and 3,7-DMU has largely been over-looked. We now optimized an HPLC separation method to probe the profile of caffeine metabolites found in different tissues. Our preliminary results show that the theobromine (3,7-DMX) is the main metabolite present in the plasma, liver and brain of adult mice treated for 16 days with caffeine (2 mg/kg); notably, we also identified the presence of a deaminated metabolite of caffeine, 1,3-DMU, in these same samples (n=4). Also, the plasma levels of caffeine increased with time, being 1.44±0.41 M after 2 days and 2.39±0.83 M after 16 days of caffeine administration. We also evaluated the brain of 1 day pups from dams consuming caffeine (0.3 g/l) through the drinking water: together with caffeine (estimated concentration of 1.18±0.21 M), we also found both demethylated (3,7-DMX: 0.75±0.02 M) and deaminated metabolites (1,3-DMU: 0.23±0.04 M) (n=5). The present results show that caffeine metabolism and accumulation might be a factor impacting on neuroprotection, with chronic caffeine consumption leading to higher plasma levels of caffeine and with the appearance of different, hitherto unrecognized, metabolites in different tissues, which biological activity needs to be explored. Supported by DARPA (09-68-ESR-FP-010) and FCT (PTDC/SAU-NSC/122254/2010). caffeine, neuroprotection, dimethyluric acid, theobromine, metabolism [email protected] 231

232 P45 s Adenosine A2A receptors in the prefrontal cortex regulates impulsive behavior in rats Fernanda da Rosa Douglas Leffa 1,2, Nuno J. Machado 1, Nélio Gonçalves 1, Carolina M. Souza 1, Fernanda da Rosa 1,3, Diogo O. Souza 2, Luisa V. Lopes 4, Pablo Pandolfo 2,5, Rodrigo A. Cunha 1,6 1 CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2 Federal University of Rio Grande do Sul, Brazil; 3 Federal University of Santa Maria, Brazil; 4 Institute of Molecular Medicine; Faculty of Medicine, University of Lisbon, Portugal; 5 Federal Fluminense University, Brazil; 6Faculty of Medicine, University of Coimbra, Portugal Attention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed psychiatric disorder in children, yet its primary symptoms of hyperactivity, inattention and impulsivity can persist into adolescence and adulthood. Although the cause of ADHD is unknown, prefrontal cortex (PFC) dysfunction stems as prime candidate. We have previously reported that adenosine A2A receptors (A2AR) are up-regulated in the PFC and chronic treatment with caffeine, an adenosine receptor antagonist, improved memory and attention deficits in an animal model of ADHD (Pandolfo et al., 2012, Eur Neuropsychopharmacol, in press). However, the impact of PFC A2AR on impulsivity has not been tested. We evaluated rats injected bilaterally in PFC with lentivirus expressing shrna either to neutralize A2AR (sha2ar) or a control (shctr) as well as rats over-expressing A2AR (oea2ar) vs controls in an impulsivity test of intolerance to delayed reward paradigm (IDRP), consisting of 3 phases: habituation to the apparatus (T-maze), pre-training (learning the side of the greater reward) and training (delaying the access to the greater reward) phases. No differences were observed between groups either in the pre-training phase of IRDP or in locomotor activity (open field). During the training phase, sha2ar rats made significantly less choices of the greater reward (34.5 ± 3.3, n=8; p<0.05) compared to shctr animals (64.0 ± 6.7, n=8) while there were no differences between oea2ar (n=7) and control (n=6) rats. The present study shows that A2AR silencing in the PFC increased impulsivity, strongly heralding the potential of manipulating PFC A2AR to control abnormal impulsivity in ADHD. Supported by FCT (PTDC/SAU-NSC/122254/2010), DARPA (09-68-ESR-FP-010) and CNPq. Attention deficit hyperactivity disorder (ADHD), adenosine A2A receptors (A2AR), impulsivity, prefrontal cortex (PFC). [email protected] 232

233 P46 Adenosine A2A receptor activation promotes microtubule dynamics Filipa F. Ribeiro s Filipa F. Ribeiro (1,2), Telma Santos (3), Mónica Sousa (3), Ana M. Sebastião (1,2) (1) Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Portugal; (2) Unidade de Neurociências, Instituto de Medicina Molecular, Lisboa, Portugal; (3) Nerve Regeneration Group, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. We have previously observed that the neuromodulator adenosine, through A2A receptor (A2AR) activation, induces axon elongation during neuronal maturation (Ribeiro FF et al. VII FENS Forum of Neuroscience, Barcelona, Spain, 2012). In addition, microtubules (MTs) are the major components of the axon and their dynamics is associated with axonal growth. Therefore, the objective of the study now reported was to evaluate how A2ARs activation affects microtubule (MT) dynamics, by studying MT stability and MT growth speed at the growth cones. E18 forebrain neurons from Sprague-Dawley rats were seeded at 4x10^4cells/mL, and treated, at DIC 3, with the A2AR agonist, CGS (30nM), in the presence/absence of the A2AR antagonist, ZM (50nM), for 5h until fixation. Neuronal growth cones were analyzed in terms of the ratio of fluorescence intensities of acetylated/tyrosinated alpha-tubulin. Regarding MT growth speed, neurons were transfected with pegfp-eb3 (end-binding protein 3). Time-lapse recordings (100 frames every 2 seconds) at DIC 3 were taken using an Andor Revolution XD Spinning Disk. All data were expressed as mean ± SEM, where 100 growth cones were counted for the MT stability assay and 100 MTs were analyzed in at least 10 growth cones in the plus-end tracking assay, from each condition from each independent culture. Data on MT stability show that A2AR activation by the A2AR agonist, CGS 21680, induce a decrease in the ratio acetylated/tyrosinated alpha-tubulin (22%, 100 growth cones, p<0.05), which was prevented by the presence of A2AR antagonist, ZM (100 growth cones, p>0.05). Results from live imaging of pegfp-eb3 transfected neurons show that A2AR activation induced an increase in MT growth speed (69%, 200 MTs, p<0.05). Results suggest that activation of A2AR promotes a decrease in MT stability and an increase in MT growth speed in axonal growth cones, which reflects an increase in MT dynamics. Work and FF Ribeiro (BD/74662/2010) supported by FCT. adenosine A2A receptor, axon elongation, microtubule dynamics [email protected] 233

234 P47 s Characterization of the role of phospholipase D in Caenorhabditis elegans. Francisca Vaz Bravo Francisca Vaz Bravo, Andreia Castro, Robin B. Chan, Gilbert Di Paolo, Nuno Sousa, Tiago Gil Oliveira. Life and Health Sciences Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal Lipids are the major constituent of the brain. Growing evidence, suggests that a group of enzymes called phospholipases, that modulated signaling lipids, have an important impact in neuronal physiology. In this study we are mainly interested in a specific enzyme, phospholipase D (PLD), which hydrolyses phosphatidylcholine to phosphatidic acid. Phosphatidic acid plays an important role in multiple aspects of cell physiology, including signaling and membrane trafficking processes. In order to study the role of this enzyme, a battery of behavioral, imaging and biochemical tests were performed using PLD knock-out (KO) C. elegans animals. Through mass spectrometry analysis it was confirmed that PLD KO worms had no PLD activity. In our behavioral analysis, PLD KO worms had no locomotor deficits, no defective attraction to isoamilic acid as well as to NaCl and were not susceptible to proconvulsivant drugs (Pentylenetetrazol and Aldicarb). Currently, we are continuing the characterization of these PLD KO animals with established behavioral tests. Our preliminary data indicates that although PLD is a key signaling modulator, it is not essential for the survival or for a myriad of behavioral tests in a C. elegans KO model. Lipids, Phosphatidic acid, Phospholipase D, Behavior, C.Elegans [email protected] 234

235 P48 s Modulation of NMDA receptor activity through adenosine A2A receptors in the hippocampus Francisco Melo Albuquerque Saraiva Mouro Francisco Mouro, Raquel Baptista Dias, Diogo Rombo, Ana Maria Sebastião Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa - Instituto de Farmacologia e Neurociências, Unidade de Neurociências Hippocampal excitatory synaptic plasticity is often considered the synaptic basis for memory formation. NMDAR activity is deeply involved in long-lasting changes in synaptic plasticity. Adenosine modulatory actions upon excitatory glutamatergic transmission are well described. However, the modulatory actions of adenosine A2ARs upon NMDARs activity in CA1 pyramidal cells have never been reported. Thus, the effect of A2AR activation on NMDARs-mediated postsynaptic currents (PSCs) was examined in CA1 pyramidal neurons of young (3-10 weeks) rat hippocampal slices, by using the whole-cell patch-clamp technique (Vh = -60mV). NMDARs-mediated currents were evoked through pressure application of NMDA (150 M) (NMDAR agonist) directly onto the cell soma. Bath application of A2AR agonist CGS (30nM) induced significant increases on NMDA-evoked PSCs (23%±4,7% n=6, p<0.005). To further address if this effect was caused by an A2AR-mediated modulation of NMDAevoked PSCs, CGS (30nM) was superfused in the presence of SCH (100nM), an A2AR antagonist, which was added to the perfusion 10 minutes before the agonist. NMDA-evoked PSCs were not significantly altered by the presence of CGS (30nM) when A2ARs were previously blocked by SCH (100nM) (2%±9,0% n=5, p>0.05). To assure that the measured currents were elicited by NMDARs activity, CNQX (10 M) was used to block the activity of AMPA/kainate receptors. The results show no significant changes in NMDA-evoked PSCs in the presence of CNQX (99%±2.9% n=3, p>0.05), suggesting that these currents were mediated by NMDARs. In the presence of DL-APV (50 M), a NMDARs antagonist, NMDA-evoked PSCs significantly decreased (76%±4.9% n=3, p<0.005). Together these results allow to conclude that A2ARs exert a modulatory effect over NMDARs activity at the CA1 neurons of the hippocampus, resulting in potentiation of NMDA-evoked PSCs. Adenosine, Hippocampus, NMDA receptor, Neuromodulation [email protected] 235

236 P49 Adenosine, acting on A1 receptors, negatively modulates acetylcholine and ATP release from cholinergic nerves of the bladder of patients with benign prostatic obstruction Isabel Silva(1) s I. Silva(1) J. Correia(1), F. Ferreirinha(1), M. Silva-Ramos(2), J. Sévigny(3) & P. Correia-de-Sá(1) (1)Instituto de Ciências Biomédicas Abel Salazar/UMIB - UP, Portugal; (2)Ser. Urologia, CHP-Porto, Portugal; (3)Centre Recherche en Rhumatol. et Immunol., CHU and Départ. Microbiol.-Infectiol. et d Immunol., Fac. Médecine, Univ. Laval, Québec, QC, Canada. Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function, and in the pathophysiology of detrusor overactivity. In vitro studies from our lab showed that stimulated cholinergic nerves from patients with benign prostatic obstruction (BPH) release 3-times more ATP than controls. We and others demonstrated that increased ATP bioavailability, due to slow inactivation kinetics of the nucleotide, contributes to hyperexcitability of bladder nerves (via P2X3 receptors) and detrusor reactivity (via P2X1 receptors) in obstructed BPH patients. The role of ATP may, however, be counteracted by compartmentalization of purinoceptors together with ecto-ntpdases at cell surface microdomains, which may have profound implications on the activity of co-localized inhibitory P1 receptors by endogenously formed adenosine (ADO) from the catabolism of ATP. Using selective receptor agonists and enzymatic inhibitors our data showed that ADO A1 receptors that negatively modulate cholinergic nerve activity are up-regulated in the bladder of BPH patients; this situation was confirmed by immunofluorescence confocal microscopy using an antibody against the A1 receptor. That is, inhibition of electrically-evoked [3H]ACh release by the A1 receptor agonist, R-PIA (300 nm), was of higher magnitude in BPH patients (45±1%, n=4) than in control individuals (30±8%, n=6). In spite of this, endogenous formation of ADO was insufficient to tonically activate up-regulated inhibitory A1 receptors in BPH patients, since inactivation of the nucleoside with adenosine deaminase (0.5 U/ml, n=6) was devoid of effect on evoked [3H]ACh release. In BPH patients, restoration of the A1 inhibitory tonus was observed upon increasing endogenous ADO by inhibiting the nucleoside uptake (with 0.5 µm dipyridamole) and/or extracellular deamination (with 50 µm EHNA). Interestingly, EHNA (50 µm) also significantly decreased ATP release from stimulate Adenosine, ATP release, human bladder, ACh release, benign prostatic patients [email protected] 236

237 P50 Opioidergic modulation of a pain facilitatory area of the brain using gene transfer Isaura Tavares s P. Carvalho 1, I. Martins 1, S.P. Wilson 2, I. Tavares 1 1 Department of Experimental Biology, Faculty of Medicine of Oporto and IBMC, Porto, Portugal 2 Department of Physiology, Pharmacology and Neurosciences, University of South Carolina, School of Medicine, USA Opioids are most commonly used drugs in chronic pain management. Their mechanism of action in brain areas involved in pain facilitation remains understudied. The dorsal reticular nucleus (DRt) is a medullary area which plays a unique role in descending pain facilitation. In the DRt, µ-opioid receptors (MOR) are expressed in spinally-projecting and presumptively interneurons. Here we studied the effects MOR expression at the DRt by gene transfer in an inflammatory pain model (the formalin test), characterized by having several phases of response. Male Wistar rats were stereotaxically injected into the left DRt with lentiviral vectors expressing EGFP (LV-EGFP) and decreasing (MOR- R vector) or increasing (MOR-F vector) MOR expression. Seven days later, the animals were divided in 2 sets. One set of animals (n=6/vector) was injected with formalin into the left hindpaw and the number of jerks was recorded for 60 min. Rats were transcardially perfused, the brains and the L4 spinal segment were removed and serially cut. The expression of MOR and c-fos were immunohistochemically evaluated in medullary and spinal sections, respectively. The second set of animals (n=4/vector) was sacrificed and variations in the expression of MOR were evaluated in DRt micropunches by western blotting. Knock down and overexpression of MOR at the DRt was confirmed by immunohistochemistry and western blot. The number of paw jerks was significantly attenuated in the formalin test during the early second in the case of MOR-F and during the late second phase by MOR-R. Both vectors reduced the numbers of spinal neurons expressing c-fos. The detected antinociception likely results from opioid-mediated inhibition of descending facilitation from the DRt. We are currently evaluating the neuronal circuits involved and preliminary data suggest that GABAergic MORexpressing interneurons which block descending facilitation from the DRt are recruited. Support: FCT/COMPTE project PTDC/SAU-NSC/110954/2009. Morphine, Descending pain control, gene therapy [email protected] 237

238 P51 The role of dopaminergic system in the modulation of the Indo-pacific bluestreak cleaner wrasse Labroides dimidiatus cooperative behavior João P. Messias s João P. Messias 1 ; José R. Paula 1 ; Alexandra Grutter 3 ; Rui Oliveira 1,4 ; Redouan Bshary 2 ; Marta C. Soares 1,2 1- Unidade de Investigação em Eco-Etologia, ISPA Instituto Universitário, Lisboa, Portugal; 2- Université de Neuchâtel, Institut de Zoologie, Neuchâtel, Switzerland; 3- The University of Queensland, School of Biological Sciences, St. Lucia, Australia; 4- Champalimaud Neuroscience Programme, Instituto Gulbenkian de Ciência, Oeiras, Portugal The cleaner wrasse Labroides dimidiatus is one of the best studied examples of cleaning mutualism, an interspecific interaction with positive payoffs for both sides of the involving parties. Although its behavioral basis is well-known, its physiological counterpart is less comprehended. The cleaner wrasse Labroides dimidiatus is a fish species highly specialized in this type of interaction, and given the extremely complex social network these animals are exposed to, we assume neuropeptide systems must be involved in the modulation of this animal s behavior. Dopamine is a neurotransmitter involved in a variety of behavioral modulating actions, usually being tied with aggressive behavior and reward-related learning systems. Here we tested if the administration of exogenous agonists (SKF38393, Quinpirole) and antagonists (SCH23390, Metoclopramide) would produce any changes in their motivation for interand intra-specific interactions. To our knowledge, our study is the first to link the effects of dopamine actions and mechanisms to cooperative behaviour, to which we expect have tremendous effects on the output behaviour of these highly social fish. Cleaning mutualism, Dopamine, Labroides dimidiatus, behavior, modulation [email protected] 238

239 P52 s Serotonin Neuromodulation of Cooperative Behaviour in a Cleaner Fish José Paula José Paula, João Messias, Alexandra Grutter; Rui Oliveira; Redouan Bshary; Marta Soares Unidade de Investigação em Eco-Etologia, ISPA - Instituto Universitário The cleaning behavior has been used as a classic example of mutualism, with benefits to cleaners and clients. Although much has been studied on the behavioural aspects of these mutualistic interactions, little is known about physiological processes that underlie these interactions. The neurotransmitter serotonin or 5-hydroxytryptamine (5-HT) is involved in the regulation of vertebrate social behaviour while its activity is usually related with social status and aggressive behaviour. Here we tested if the serotoninergic system is responsible for the modulation of cooperative behaviour, in the best studied cleaning mutualism, between the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus and their visiting clientele. We ve found that exogenous administration of 5- HT agonist 8-Hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) and selective serotonin reuptake inhibitor (SSRI) fluoxetine caused a substantial increase of cleaners motivation to inspect clients without affecting their intraspecific interactions, which suggests a particular effect of 5-HT on interspecific behaviour but not of an overall effect on social behaviour. Additionally we ve discovered that 5-HT antagonists WAY and p-chlorophenylalanine lead to a substantial decrease of cleaning inspections. To our knowledge, our study is the first to link the effects of neurotransmitter action (serotonin) to cooperative behaviour, beyond the usual focus of its influence on conspecific social behaviour. Our findings are consistent with those observed with arginine vasotocin influence on cleaning behaviour, which might indicate that there is an important role of serotonin-related mechanisms in modulating neuropeptide and steroid responses as demonstrated in bluehead wrasse Thalassoma bifasciatum where fluoxetine-treated males presented lower AVT mrna expression in preoptic area of hypothalamus. cooperative behaviour, neurotransmitters, serotonin, Labroides dimidiatus [email protected] 239

240 P53 s Differential localization and enzymatic activity of ecto-ntpdases and ecto-5 - nucleotidase in the rat ileum Margarida Duarte Araújo M. Duarte-Araújo(1), S. Monteiro(1), C. Tavares(1), H. Lima(1), F. Ferreirinha(1), M.T. Magalhães-Cardoso(1), J. Sévigny(2) & P. Correia-de-Sá(1) (1) Laboratório de Farmacologia e Neurobiologia, UMIB, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Portugal. (2) Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec, QC, Canada Previous findings demonstrated that ATP (acting via P2X2 receptors) exerts a transient facilitatory effect on spontaneous acetylcholine (ACh) release from myenteric motoneurons. As a consequence of its rapid metabolism in the extracellular milieu, nerve-evoked ATP responses may be controlled by adenosine through the activation of inhibitory A1 and facilitatory A2A receptors located on cell bodies and axon terminals of myenteric nerves, respectively. In addition, activation of P2Y1 purinoceptors by ADP may be functionally relevant to restrain stimulation-induced ACh release. Considering the relevance of the purinergic cascade, we decided to investigate the regional distribution and activity of ectonucleotidase at the rat ileum. Confocal microscopy studies showed that the ileum lacks immunoreactivity against NTPDase1, but PGP 9.5- positive neuronal cell bodies of the myenteric plexus exhibit high immunoreactivity against NTPDase2 and NTPDase3. NTPDase3 immunoreactivity was also evident in myenteric nerve terminals at the muscle layer. The catabolism of ATP was prevented by POM-1 (100 µm), visualized by both Wachstein-Meisel histochemical reaction and HPLC analysis of ATP (30 µm) metabolism. Ecto-5 -nucleotidase immunolabeling was predominant in the muscular layers of the ileum and its inhibitor, α,β-methyleneadp (200 µm), blocked muscular deposition of phosphates. Data suggest that ATP may be sequentially hydrolyzed into ADP by NTPDase 2 allowing the activation of inhibitory P2Y1 receptors on myenteric nerve cell bodies. Localization of NTPDase 3 at the myenteric neuromuscular junction yields AMP formation from both ATP and ADP, which may be subsequently dephosphorylated into adenosine by ecto-5 -nucleotidase bound to smooth muscle fibers. This might explain why formation of adenosine from the extracellular catabolism of adenine nucleotides activates preferentially facilitatory A2A receptors on myenteric nerve terminals. (FEDER funding, PEst-OE/SAU/UI0215/2011). Ectonucleotidases, Myenteric plexus, ATP, ADP, Adenosine [email protected] 240

241 P54 s Protein Phosphatase 1C Isoform Associated Interactions in Brain Margarida Fardilha Sara L.C. Esteves, Luís Korrodi-Gregório, Odete A.B. da Cruz e Silva, Edgar F. da Cruz e Silva and Margarida Fardilha Centre for Cell Biology, University of Aveiro Reversible protein phosphorylation is the most important regulatory mechanism involved in cellular signaling transduction pathways. Thousands of human proteins are phosphorylated and the tight regulation of phosphorylation states is crucial for cell health and vitality. Protein phosphorylation occurs primarily on serine, threonine, and tyrosine residues, through the balanced actions of protein kinases and phosphatases. The catalytic subunit of PhosphoProtein Phosphatase 1 (PPP1C), a major Ser/Thrphosphatase, associates with a huge variety of regulatory subunits that specify its substrates, regulate its activity and target it to specific subcellular locations. PPP1C has been shown to bind to different proteins in brain in order to execute key and differential functions. This work reports the identification of proteins expressed in human brain that interact with PPP1CC1 and/or PPP1CC2 isoforms by the Yeast Two Hybrid method. An extensive search of PPP1C binding motifs was performed for the proteins identified, revealing already known PPP1C regulators but also novel interactors. Moreover, our results were integrated with data of PPPCC1 interacting proteins from several public web databases, which allowed for the development of physical maps of the novel interactions. Thus we obtained the PPP1CC interactome and identified novel PPP1C interacting proteins, supporting novel functions for PPP1CC isoforms in human brain. Acknowledgments: This work was supported by the Centre for Cell Biology of the University of Aveiro, by grants from Fundação para a Ciência e Tecnologia of the Portuguese Ministry of Science and Higher Education to OBCS (POCTI/BIA- BCM/58469/2004) and from the European project to EFCS (APOPIS - PL503330). PhosphoProtein Phosphatase 1, Yeast Two Hybrid, Interactome, Brain [email protected] 241

242 P55 s Uncovering novel micrornas involved in homeostatic plasticity Mariline M. Silva Mariline M Silva; Joana Fernandes; Sandra D. Santos; Ana Luísa Carvalho CNC-Center for Neuroscience and Cell Biology, University of Coimbra Synaptic scaling is a homeostatic mechanism responsible for the adjustment of the overall synaptic strength in a neuron s synapses to a set point, in order to stabilize neuronal firing. Dendritic protein synthesis is crucial for synaptic scaling but the mechanisms that regulate the derepression of mrnas are still unclear. MicroRNAs, well known posttranscriptional regulators, are modulated upon changes in neuronal activity, which indicates that mirnas may have a role in the regulation of homeostatic plasticity. Our main goal is to unveil novel mirna players during synaptic scaling and to further investigate their role in this form of plasticity. We performed a gene expression microarray analysis of rat hippocampal neurons under chronic blockade of activity and submitted the results to biological interpretation using the GoMiner tool. Several gene categories, relevant for synaptic events, were selected and mirna target sites were predicted for those genes using the following algorithms: miranda, TargetScanS and MirTarget2. Focusing on a restricted group of altered genes with a crucial role in synaptic scaling and/or in synaptic function, we identified a group of 18 predicted mirnas, and performed a screening panel for their expression levels in primary cultures of rat hippocampal neurons subjected to synaptic scaling up conditions. This analysis revealed that several mirnas present altered expression in scaling up conditions and therefore suggests an important role for these mirnas in synaptic scaling mechanisms. Scaling up; mirnas [email protected] 242

243 P56 Subcelular localization of adenosine a2a receptors in hippocampal synapses Sara Carvalho (1) s Sara Carvalho (1), Joana E. Coelho (1), Diana G. Ferreira (1,2) and Luísa V. Lopes (1) (1) Instituto de Medicina Molecular, Faculty of Medicine Lisbon, Portugal (2) Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Germany A2ARs are constitutively activated G-protein coupled-receptors, preferentially expressed by the striatopallidal medium spiny striatal neurons. They exhibit however a very distinct pattern of expression in the hippocampus and cortex where their expression is very low in physiological conditions and mainly in the nerve terminal. Our team and others have found compelling evidence of cortical and hippocampal upsurge of A2AR expression/function associated to cognitive deficits. This is accompanied by clear behavioural deficits in hippocampal-dependent tasks, such as spatial memory in rats. However, the mechanisms by which A2AR dysregulation drives synaptic, cognitive and AD-related pathological hallmarks is unknown; and whether these deficits, are generated by A2ARs located pre- or postsynaptically remains to be uncovered. We now used transgenic rats overexpressing human A2AR in forebrain, under CAMKII promoter (CAMKII-hA2A), which display learning and memory deficits. In order to verify if these deficits are related to pre- or post-synaptic A2AR overexpression, we performed a sequential cellular fractionation protocol, using hippocampi derived from wildtype (WT) and transgenic (Tg) male rats. We were able to efficiently separate the pre- and pos- synaptic fractions, as assessed by obtaining exclusive positive SNAP25 or PSD95, respectively. The total level of A2AR was increased by 3 fold in Tg versus WT rats (n=4). In both WT and Tg, A2AR were found preferentially located in SNAP25 positive fractions and absent from PSD95 positive fraction (n=4). This shows that A2AR overexpression in the hippocampus occurs preferentially in the nerve terminal, rather than in the postsynaptic density. The pre-synaptic location of A2AR in these animals is in agreement with electrophysiology and behavior results, and follows the age progressive physiological overexpression in the hippocampus. Funding: FCT. A2A Tg rats generated by Michael Bader, MDC, Berlin, Germany. hippocampus; adenosine A2A receptor; synaptic localization; transgenic [email protected] 243

244 P57 s Ecological relevance determines arginine vasotocin influence on cleanerfish learning abilities Sónia C. Cardoso Sónia C. Cardoso, José R. Paitio, Renata Mazzei, Redouan Bshary, Rui F. Oliveira and Marta C. Soares ISPA - Instituto Universitário According to an ecological approach to cognition, a species ability to solve any task depends on its evolutionary history and on its specific ecological selective pressures. Proximate mechanisms are required to establish individual social competence, which optimizes social behaviour in relation to ecological significance. Here we show that arginine-vasotocin (AVT) is directly linked to the learning performance of the cleaner wrasse (Labroides dimidiatus). We tested the influence of this neuro-hormone upon these cleaners ability to solve two problems that varied in ecological relevance (a social and an asocial task). Cleaners learned to solve the social task more rapidly than the asocial one; however the influence of our tested neuropeptides differed considerably between problems. While blocking the effects of AVT increase these cleaners learning abilities, these effects only stand for a task that reflects their specific learning rules under natural conditions. In the absence of social relevance, AVT was only responsible for a significant decrease of their learning aptitude. Our results show that neuropeptides (AVT and Isotocin) pathways should be directly implicated on the building of these animals social cognitive competence and should also have a prominent role in the learning of key behavioural decision rules associated to their ecological context. Cognitive abilities.ecological relevance.neuropeptides.cleanning Mutualism [email protected] 244

245 P58 s Interaction between ecto-5 -nucleotidase and adenosine A2A receptors in nerve terminals of the mouse prefrontal cortex Tiago Silva Tiago Silva, Rodrigo A. Cunha, Paula Agostinho CNC-Center for Neuroscience and Cell Biology The activation of adenosine A2A receptors (A2AR) is mediated by adenosine selectively originated from the extracellular catabolism of released ATP. Ecto-5 - nucleotidase (e-5 N) plays a key role in the formation of ATP-derived adenosine and in the subsequent activation of A2AR to control synaptic plasticity. Upon brain injury, ATP is released as a stress signal and both e-5 N and A2AR are up-regulated in parallel. This prompts the hypothesis that e-5 N and A2AR could be co-localized and coregulated. The present study aims to define: i) the synaptic and sub-synaptic localization of e-5 N in cortical regions of adult C57/Bl6 mice, ii) the co-localization of e-5 N with A2AR in cortical nerve terminals, iii) if the genetic deletion of A2AR affects the density of synaptic e-5 N in cortical regions. The comparison by Western blot analysis of the density of e-5 N in hippocampal total membranes and synaptosomes revealed that e- 5 N, albeit present in significant amount in nerve terminals (79±15%, n=3), was not enriched in hippocampal synapses. The fractionation of hippocampal synaptosomes unveiled the presence of two different isoforms of e-5 N, one being more present at the pre-synaptic and extra-synaptic fractions (~50 kda) and the other (~70 kda) at the postsynaptic fraction. The pull-down of A2AR revealed the co-immunoprecipitation of A2AR with e-5 -N in the prefrontal cortex. However, the synaptic density of e-5 N was not affected in the hippocampus or prefrontal cortex of global A2AR knockout mice. The results show that e-5 N is co-localized with A2AR in nerve terminals, thus supporting their functional interplay, but also indicate that e-5 N and A2AR are independently regulated, paving the way to consider e-5 N an independent target to manage neurodegeneration. Ecto-5'-nucleotidase, purines, A2A receptor, neurodegeneration, prefrontal cortex [email protected] 245

246 P59 s The redox interplay between nitrite, ascorbate and nitric oxide in the brain Nuno R. Ferreira Nuno R. Ferreira, Lourenço CF, Barbosa RM, Laranjinha J Center for Neurosciences and Cell Biology and Faculty of Pharmacy, University of Coimbra, Portugal In the brain, stimulation of NMDA glutamate receptors activates nitric oxide synthase with production of nitric oxide ( NO), a free radical transmitter implicated in a number of processes, such as neurovascular coupling and neurodegeneration. The brain has circulating levels of nitrite ranging from 50 nm up to c.a. 1 µm. Under hypoxic conditions, nitrite has shown to increase blood flow. Given the critical role of NO in the brain and the high concentration of ascorbate (AA) in neurons we hypothesized an AA dependent redox conversion of nitrite to NO. This was assessed in vivo in the rat hippocampus by means of stereotaxic insertion of selective microelectrodes for NO and AA. Results support a temporal and functional connection between extracellular AA and NO upon glutamatergic stimulation. NO induced the release of AA in a concentrationdependent way and AA release was significantly diminished by decreasing NO production with pharmacological probes. Local injection of nitrite also increased NO signal, supporting the notion that nitrite can be converted back to NO by AA released from neurons during increased neuronal activity. Supported by FCT, Grant PTDC/SAU-NEU/103538/2008 Nitric Oxide, nitrite, ascorbate, microelctrodes [email protected] 246

247 Neurogenesis and neural cell differentiation 247

248 P60 s Mesenchymal Stem Cells Secretome: a Step Towards Improving Neuronal Survival and Recovery A.O.Pires A.O. Pires, A. Neves-Carvalho, Graça Baltazar, N. Sousa, A. J. Salgado Life and Health Sciences Research Institute (ICVS), School of HealthSciences, University of Minho, Mesenchymal Stem Cells (MSCs) capability to secrete neuroregulatory and immunomodulatory molecules, have prompted these cells as powerful tools for CNS regenerative medicine. MSCs are commonly isolated from bone marrow, adipose tissue, umbilical cord blood and the Wharton s jelly of umbilical cord. In the last decade, MSCs have arisen as a potential therapeutic tool for CNS regenerative medicine, particularly, in what concerns to neuronal protection or recovery in neurodegenerative diseases. However, the potential therapeutic effects that the MSCs secretome can hold towards CNS pathologies remains to be unveiled. Therefore, in the present work we aimed to evaluate the effects of the MSCs secretome in neuronal survival, differentiation and neuro-protection/recovery. A SH-SY5Y cell line was used to determine the effect of media conditioned from MSCs derived from bone marrow (hbmscs) in cell survival and differentiation of these cells into neuron-like cells. The media conditioned of hbmscs was able to maintain SH-SY5Y cultures viable but, most importantly, were capable of inducing differentiation of SH-SY5Y cells in neuron-like cells. We also evaluated the effect of hbmscs media conditioned in promoting the neuroprotection of ventral midbrain dopaminergic cells exposed to the toxin 6-hydroxydopamine. Our preliminary results suggest that hbmscs conditioned media are capable to promote the protection/recovery of dopaminergic cells previously exposed to the dopaminergic toxin. Following our in vitro results, further work focused on understanding the neuroprotection/recovery role of hbmscs secretome in a 6-OHDA unilateral injected animal. So far, our recent results indicate that treatment with hbmscs CM (24H) seems to promote animal motor recovery and protection of injured DA neurons. MSCs, secretome, 6-OHDA,CNS, Regenerative Medicine [email protected] 248

249 P61 s Modulatory role of Adenosine A2A receptors on brain-derived neurotrophic factor (BDNF)-induced neurogenesis in murine dentate gyrus stem/progenitor cell cultures Mendes-Pedro D Mendes-Pedro D, Duarte-Samartinho M, Mendes-Pedro D, Ribeiro FF, Ferreira FF, Sebastião AM, Xapelli S Instituto de Farmacologia e Neurociências, Faculdade de Medicina da Universidade de Lisboa Constitutive neurogenesis takes place in both adult mammalian subventricular zone and, in less extent, in the subgranular zone of the dentate gyrus (DG). Since BDNF has been implicated in the regulation of neurogenesis and a functional interaction between Adenosine A2A Receptor (A2AR) and BDNF has been suggested, our study aims to evaluate the role of A2AR on BDNF-induced neurogenesis. DG neurospheres were prepared from early postnatal (P1-3) Sprague-Dawley rats in serum-free medium supplemented with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Neurospheres with 6 days were then grown in differentiation conditions (withdrawal of EGF and FGF-2) and were seeded onto poly-d-lysine coated coverslips. One day after plating, the medium was renewed in the presence or absence (control) of BDNF (1-100 ng/ml) and/or ZM (A2AR anatagonist, 50nM). We observed that both TrkB and A2AR were detected in neurospheres, plated differentiated cells and in adult rat DG. We evaluated cell viability by propidium iodide assay after incubating the cultures for 48h with the drugs and we observed that none of them altered cell viability. Importantly, BDNF treatment increased proliferation (increase in BrdUpositive cells) at 48h and neuronal differentiation (increase in NeuN-positive cells) after 7 days in DG cell cultures in a concentration-dependent manner. Cell proliferation was not affected by A2AR activation, although ZM prevented the effect mediated by BDNF. Moreover, it was observed that A2AR activation increased neuronal differentiation similarly to BDNF, without synergism. Both effects were attenuated in the presence of ZM Finally, BDNF and A2AR activation promoted an increase in βiii tubulin expression without affecting astrogliogenesis. Our data suggest that BDNF promotes cell proliferation and neuronal differentiation, while A2AR only promotes neuronal differentiation. Moreover, the role of BDNF on neurogenesis seems to depend on A2AR BDNF, A2A receptors, Neurogenesis [email protected] 249

250 P62 s The role of the transcription factor AP2γ on the modulation of adult glutamatergic neurogenesis in stress-related disorders Nuno Dinis Alves N. D. Alves, A. Mateus-Pinheiro, P. Patrício, J. Ninkovic, H. Schorle, M. Goetz, N. Sousa and L. Pinto Life and Health Sciences Research Institute (ICVS), School of Health Sciences (ICVS), University of Minho, Braga, Portugal; ICVS/3B s, PT Government Associate Laboratory, Braga/Guimarães, Portugal. Several studies suggest a crucial role of adult hippocampal neurogenesis in the pathogenesis and treatment of stress-related disorders, such as depression. However, the molecular signature behind these events is still poorly characterized. The understanding of important players in processes such as proliferation, maturation and specialization of neuronal precursors may reveal promising adult neurogenesis modulators with therapeutical potential. Recent studies highlighted neuronal transcription factors including AP2γ, which regulates proliferation of cortical neurons precursors and the specification of glutamatergic neurons during early development. Interestingly, the embryonic cortical transcriptional network is conserved postnatally. Thus, it became relevant to understand the potential of AP2γ as an effective modulator of the adult glutamatergic neurogenesis process in the healthy brain and in the context of depression. For that, wild-type (WT) and AP2 +/- mice were exposed to a chronic mild stress (CMS) protocol, which triggers depression-like features. We accessed the molecular and behavioral impact of AP2γ heterozygous deletion in healthy and depressive-like animals. Furthermore, we measured markers of neuronal proliferation, morphology and genesis in adult neurogenic niches and evaluated behavioral domains typically affected by depression. Preliminary results revealed that, AP2γ +/- mice presented a mild neuronal atrophy and decreased number of bromodeoxyuridine (BrdU+) and doublecortin (DCX+) cells in the hippocampal dentate gyrus. Hence, AP2γ is essential for neuronal differentiation and maturation in the adult hippocampal niche. Additionally, AP2γ is important for cognition, both hippocampal and prefrontal cortex-dependent functions, anxiety and explorative behaviors. On the other hand, under stress conditions, AP2γ deletion induces an increase of adult neurogenic progenitors consistent with a resilient effect to the CMS-induced deficits in cognitive tasks. AP2γ, neurogenesis, stress, depression, cognition [email protected] 250

251 P63 s Development of cones in the retina of adult turbots. An electron microscopy study. E. de Miguel Villegas A. Peteira-Martínez: R. Álvarez-Otero; E. de Miguel Villegas. University of Vigo Throughout the fish life, the retina grows continuously producing new cells from a proliferative peripheral area named ciliary marginal zone (CMZ). In this location, cells proliferate and differentiate in a similar way to that observed during retinal ontogeny1. The present study describes the ultrastructure of developing cones in the CMZ of adult turbots. Cell differentiation in the CMZ progress from the periphery to the center of the retina. We observed that the undifferentiated neural retina is closely attached to the retinal pigment epithelium at the CMZ contiguous with the iris. Toward the proximal limit of the CGZ, cytodifferentiation of cones is underway. In this retinal location, broad cytoplasmic process extends into the optic ventricle. A few scattered mitochondria (ellipsoid primordium) lye in the apical cytoplasm, while endoplasmic reticulum and Golgi cisternae, which characterize the myoid of the photoreceptor, were observed more basally. Although no outer segments were yet developed, occasional connecting cilia were noted. Close to the differentiated retina, cone outer segments begin to differentiate from vitreal to scleral. The most conspicuous inner disks were surrounded by calycal process, while the apical ones were tapered by cytoplasm. In the outer plexiform layer, differentiating cones have dilated synaptic terminals which contain numerous synaptic vesicles and scarce small electron-dense lamellae (ribbon) associated to processes that penetrate superficially in the receptor cell terminal. Double cones were characterized in the newly retina closest to the CMZ. They raise from single cones which differentiate prominent subsurface cisternae along the inner segment. References: [1] Easter Jr SS, Hitchcock PF. Stem cells and regeneration in the retina: What fish have taught us about neurogenesis. Neuroscientist 2000;6: Retina, flatfish, development, photoreceptors, electron-microscopy. [email protected] 251

252 P64 Amyloid β peptides modulate differentiation of mouse neural stem cells by an autophagy-dependent manner Maria B. Fonseca1 s Maria B. Fonseca1, Susana Solá1,2, Joana M. Xavier1, Pedro A. Dionísio1, Cecília M. P. Rodrigues1,2 1Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; 2 2Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal Although regarded as neurotoxic, amyloid β (Aβ) peptides may also mediate a wide range of non-pathogenic processes. Autophagy has been implicated in Aβ-mediated effects, although its precise function in neural differentiation remains unknown. Here, we addressed the role of different Aβ fragments in neural stem cell (NSC) proliferation and differentiation, and investigated whether autophagy is involved in Aβ-induced alterations of neural fate. Our results demonstrate that Aβ1-40 enhances neurogenesis in NSCs, as determined by βiii-tubulin, NeuN and MAP2 neuronal marker immunoreactivity. In contrast, Aβ1-42 appears to favor gliogenesis, while Aβ25-35 does not influence NSC fate. The effect of Aβ1-40 on neurogenesis is partially dependent on its role in NSC self-renewal as both S-phase of the cell cycle and BrdU labeling were markedly increased. Nevertheless, Aβ1-40 resulted also in increased Tuj1 promoter activity. Autophagy, assessed by conversion of endogenous LC3-I/-II, fluorescence of pgfp-lc3 transfected cells, and Atg9 protein levels was evident in both Aβ1-40- and Aβ1-42-treated NSCs, independently of reactive oxygen species production and apoptosis. Finally, inhibition of autophagy by pharmacologic means abrogated Aβinduced lineage-specific protein markers. These results support distinct roles for different Aβ peptides in NSC fate decision and underline the importance of autophagy control of this process. Alzheimer s disease, Gliogenesis, Neural stem cell fate, Neurogenesis, Proliferation [email protected] 252

253 P65 Optimization of cell culture parameters for the 3-D culture of single embryonic stem cell-derived neural stem cells in fibrin. Ana Rita Bento s Ana Rita Bento 1, A.P. Pêgo 1,2,3, I.F. Amaral 1,2 1 INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; 2 Faculdade de Engenharia da Universidade do Porto, Porto, Portugal; 3 Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. Spinal cord injury is a devastating condition leading to permanent neurological deficits. So far there is no consensual clinical treatment or cure. This work aims to develop a fibrin-based hydrogel for the delivery of embryonic stem (ES)-derived neural stem cells (NSCs) into the injured spinal cord. Fibrin (Fb) will be functionalized with ligands for α6β1 integrin receptors to enhance NSC migration following transplantation into the spinal cord. Being the optimization of the cell culture conditions a fundamental step in the in vitro assessment of the biological performance of the functionalized Fb gels, we report the 3- D culture of single NSCs in Fb, using different cell seeding densities and concentrations of aprotinin in the gel. For that purpose, mouse ES-46C cells were expanded in serumfree conditions and committed to the neural phenotype. The percentage of GFP/SOX-1+ cells was assessed. NSCs were cultured in Fb gels prepared by mixing equal volumes of a plasminogen-free human fibrinogen solution containing factor XIIIa and a thrombin solution containing CaCl 2 and aprotinin (final concentration: 6 mg/ml fibrinogen; 2 NIH U/mL thrombin; 2.5 mm CaCl 2 ). NSCs were added to the fibrinogen solution prior to Fb polymerization. The cell-matrix constructs were cultured for periods up to 14 days, following a protocol for neuronal differentiation. The use of a cell seeding density of 10^6 cells/ml and 25 µg/ml of aprotinin led to the formation of small-sized neurospheres (50-75 µm of diameter) and a dense neuronal network, allowing the retention of cell viability (91.4% of viable cells), as shown by CLSM analysis and flow cytometry. We established the culture conditions for 3-D culture of single ES-NSCs in fibrin, where cells proliferate and differentiate into neural phenotypes. Acknowledgments Portuguese FCT through project PTDC/SAU-BMA/118869/2010 and FEDER through COMPETE (FCOMP FEDER ). AR Bento is supported by a FCT fellowship (SFRH/BD/86200/2012). neural stem cells, hydrogels, spinal cord injury [email protected] 253

254 Neuroinflammation 254

255 P66 s The role of TNF-α in METH-induced barrier dysfunction Vanessa Coelho-Santos1,2 Vanessa Coelho-Santos1,2*, Ricardo Alexandre Leitão1,2*, Filipa Lourenço Cardoso3, Carlos Fontes-Ribeiro1,2, Maria Alexandra Brito3, Ana Paula Silva1,2 1Lab. Pharmacology and Exp. Therapeutics, and 2IBILI, Fac. Medicine, Univ. Coimbra, Coimbra; 3iMed, Univ. Lisbon, Lisbon Methamphetamine (METH) is a powerful psychostimulant drug of abuse that causes severe alterations in the central nervous system (CNS). Recently, some studies have suggested that METH can also compromise the blood-brain barrier (BBB) function. However, the mechanisms underlying the effect of this drug on brain endothelial cells (ECs) remain unclear. Furthermore, astrocytes play a crucial role in modulating the structure and function of the cerebral endothelium, but the effect of METH on the crosstalk between ECs and astrocytes has never been addressed before. Thus, we aimed to clarify how METH influences the crosstalk between astrocytes and ECs. Our results demonstrate that 1 or 50 µm METH did not induce astrocytes or endothelial cell death. Nevertheless, it decreased transendothelial electric resistance (TEER) and increased permeability to sodium fluorescein. Additionally, METH triggered the release of tumor necrosis factor-alpha (TNF-α) by both astrocytes and ECs, and increased the expression of intercellular adhesion molecule 1 (ICAM-1). We further investigated the effect of METH on the crosstalk between ECs and astrocytes by exposing ECs to astrocyte-conditioned medium (ACM). The use of control ACM had, by itself, a protective effect on ECs with an increase of TEER and decreased in permeability. However, the ACM obtained from astrocytes previously exposed to METH (50 µm) impaired the barrier functions of ECs, which was due to the TNF-α released by astrocytes. In conclusion, our results show that METH can directly interfere with endothelial barrier properties or indirectly via astrocytes. Moreover, TNF-α seems to be a key mediator of BBB dysfunction in response to METH. (*These authors contributed equally to this work. This work was supported by Project PTDC/SAU-FCF/098685/2008, COMPETE and FEDER funds, FCT, Portugal, Pest- C/SAU/UI3282) Astrocytes, blood-brain barrier, endothelial cells, methamphetamine, TNF-α. [email protected] 255

256 P67 s Mycobacterium avium chronic infection induces alterations in the hippocampal cytokine profile Daniela Calçada Susana Roque, Daniela Calçada, Carlos Branco, Bruno Rodrigues, Susana Monteiro, Nuno Sousa, Joana A. Palha, Margarida Correia-Neves Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal; ICVS/3B's, PT Government Associate Laboratory, Braga, Guimarães, Portugal The etiology of depression is a matter of intense debate. In the last decades, a disruption in the interplay between the immune and central nervous systems has been suggested as a potential cause of depression. In fact, the cytokine theory of depression suggests that enhanced production of pro-inflammatory cytokines is associated with the pathophysiology of this mood disorder. Increased production of pro-inflammatory cytokines occurs naturally as part of the immune response to infection. In animal models, acute infection or administration of LPS, which are both associated with high levels of pro-inflammatory cytokines during a short period of time, cause what is called sickness behavior. This syndrome is characterized by a pattern of behavioral alterations that share similarities with those of depressed individuals (e.g. reduction in motor activity, reduced appetite and weight, anhedonia). However depression is a chronic disease. Therefore, it is more likely that a chronic process is associated with the mechanics underlying this disorder. Chronic infections are very frequent and information regarding their possible role in depression is still scarce. To investigate the role of chronic infection in mood disorders we choose the mouse model of Mycobacterium avium infection since it is a very well characterized model, and also because mycobacterial infections are among the major health threats worldwide. Here we show that this infection induces distinct alterations in the cytokine profile in the hippocampus of three different strains of mice. Since we have not found an association between these alterations in cytokine milieu and alterations in mood behavioral phenotype, we are currently exploring their impact on hippocampal neurogenesis and neuronal plasticity. Depression, cytokine theory, chronic infection, mood behavior [email protected] 256

257 P68 s An engineered alginate-based platform to mimic a glial scar Daniela N. Rocha Daniela N. Rocha, Cristina C. Barrias, João B. Relvas*, Ana P. Pêgo INEB- instituto de engenharia Biomécica; * IBMC- Instituto de Engenharia Biomédica Astrogliosis is a hallmark of central nervous system lesions. Therefore, the recreation of a glial scar environment in vitro can lead to the development of an important tool for the screening of strategies to treat a number of conditions in which the neuronal function is impaired by the glial scar. Mechanical forces can influence nearly every tissue and organ of the human body. As such, mechanotransduction has become a topic of increasing scientific interest in many fields of research. For this we have prepared 3 dimensional (3D) alginate gels with different rigidities, by varying the alginate mass content (0.3-2% in wt/vol). Primary astrocytes could be successfully maintained in 3D culture and their phenotype was found to be dependent on the mechanical properties of the hydrogels. Two-dimensional studies have shown the ability of primary meningeal fibroblast conditioned medium (MFCM) to activate astrocytes by increasing glialfibrillary acidic protein (GFAP) production. Furthermore, axonal outgrowth of cortical neurons cultured on top of these astrocytes was significantly inhibited. By culturing astrocytes in low alginate content (soft) 3D hydrogels in the presence of MFCM we were able to induce astroglyosis. The classical, up-regulation of GFAP, collagen IV and vimentin was observed, as well, as the augmentation of glycosaminoglycan production, suggesting that astrogliosis can be achieved in these 3D culture conditions. Hydrogel mechanical properties and culture medium conditions were also found to have an impact on matrix metalloproteinase (MMP) production, with higher MMP 2 and 9 levels being detected in culture conditions where astrocytes are activated. The modulation of the matrix mechanical properties as a function of the culture time is currently under study. hydrogels, glial scar, astrogliosis, 3D platform [email protected] 257

258 P69 s Caffeine inhibits neuroinflammation and cell death in the retina induced by ischemia-reperfusion injury Bóia, R Bóia, R.1, Tralhão, P.1, Madeira, M.H.1, Elvas,F.1,2,Tomé A. R.3, Ambrósio, A.F.1,2, Santiago, A.R. 1,2 1Center of Ophthalmology and Vision Sciences - IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2AIBILI, Coimbra; 3Center for Neuroscience of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal Caffeine is the most commonly used psychostimulant in the world and its effects are partly mediated by antagonizing adenosine receptors. Evidence suggests that caffeine attenuates inflammatory responses and affords protection upon CNS injury, by modulating adenosine A2A receptor (A2AR). Glaucoma is a neurodegenerative retinal disease and the second cause of blindness worldwide, characterized by optic nerve damage and retinal ganglion cell (RGC) death. It is known that microglia plays a key role in glaucoma and their overactivation leads to the production of proinflammatory mediators. Retinal ischemia-reperfusion injury (I-R) model mimicks clinical situations such as acute glaucoma, and has been used to investigate retinal neuronal cell damage. The aim of this work was to investigate whether long-term caffeine intake prevents retinal neuroinflammation induced by ocular ischemia-reperfusion injury in rats. Caffeine was administrated in the drinking water (1 g/l) two weeks before ischemia and until the end of the experiment. Retinal ischemia was induced in one eye by elevating the intraocular pressure for 60 min. Retinal reperfusion was reestablished, and the animals were euthanized at 7 days after ischemia. Caffeine intake was 132±4 mg/kg/day and concentration of caffeine in the serum was 97 µm at 7 days of reperfusion. Caffeine consumption did not affect the intraocular pressure. The number of activated microglia and the levels of the proinflammatory cytokines TNF-α, and IL-1β were decreased. In addition, caffeine decreased the number of TUNEL-positive cells induced by I-R. These results suggest that caffeine can modulate retinal neuroinflammation and protect neurons against damage induced by I-R injury. Support: FCT (Project PTDC/BIM-MEC/0913/2012 and FCT Strategic Project PEst- C/SAU/UI3282/2011), Portugal, FEDER-COMPETE., adenosine A2A receptors, ischemia-reperfusion, retina, microglial cells [email protected] 258

259 Neural systems 259

260 P70 s The sources of gamma-band oscillatory activity during a simultaneous EEG/fMRI ambiguous perception task João Castelhano João Castelhano; Catarina Duarte; Eugénio Rodriguez; Miguel Castelo-Branco IBILI- Faculty of Medicine, University of Coimbra EEG studies suggested increased Gamma-band activity during object perception but the neural sources of this oscillatory response to ambiguous perception remain elusive. Here we have recorded simultaneous EEG/fMRI signals during a visual perception task using ambiguous stimuli. Data were acquired from 10 healthy subjects that performed a forced choice discrimination task between Mooney categories (prototypical upright and inverted faces, prototypical guitars and scrambled versions) stimuli. Stimuli were delivered using Presentation software (Neurobehavioral Systems). Stimulus duration and inter-stimulus-interval were 150ms and 6s, respectively. Functional MR data were acquired on a 3T MR scanner. EEG was recorded using an MR compatible EEG system (NeuroScan, USA) with a cap providing 64Ag/AgCl electrodes. EEG and fmri recordings were acquired in a continuous way and were synchronized by means of a Syncbox (NordicNeuroLab). EEG MR gradient and pulse artifacts were corrected offline using Independent Component Analysis. All channels were re-referenced to average reference and data were analyzed in the 1-70Hz range. Epochs were obtained locked to the stimuli and event-related potential (ERP) measures, time-frequency analysis and fmri informed source localization were performed. Behavioural data show that subjects discriminate between categories with high performance levels (>75%). We replicated the typical face N170 peak and found oscillatory activity was enhanced within the high beta/low gamma range (20-40Hz) for the perception moments. The latencies of oscillatory activity peaks were used as general linear model (GLM) predictors for fmri source localization. We found that different gamma sources are related to perception, spanning from temporo-parietal areas to frontal regions. Gamma-band Oscillations, Ambiguous Perception, EEG/fMRI [email protected] 260

261 P71 s Characterization of the sleep-wake cycle of the neotropical rodent proechimys. José C da Silva Silva JC, Matos G, Andersen ML, Schoorlemmer GHM, Tufik S, Cavalheiro EA. Escola Paulista de Medicina / Universidade Federal de São Paulo - Brasil Introduction: The comprehension of the sleep-wake cycle characteristics of the neotropical rodent Proechimys is important in processes related to comparative neurobiology. Objective: The aim of this study was to characterize the sleep pattern in this wild rodent. Methods: Adult female Wistar rats and Proechimys (n=8) were housed under controlled conditions (light/dark cycle with lights on from 07:00 to 19:00 hours, C), with food and water ad libitum. All procedures were approved by the ethic committee of UNIFESP (Nº 2132/11). Using stereotaxic surgery, animals from both groups were implanted with 2 cortical electrodes (1 at right and 1 at left) and 1 bipolar electrode was aimed to the right hippocampus (CA1), which were used for electrographic recordings (EGRs). A reference electrode was implanted in the nasal bone. Electrodes for electromyography were surgically implanted into the neck muscle in both species. After surgical recovery and adaptation period, EGRs were realized for periods as long as 48 hours. Results: In both the light and dark phases of the cycle, significant differences in several electrographic patterns could be observed between Proechimys and Wistar animals. For instance, in the Proechimys the duration of the wakefulness, the latency to paradoxical sleep, the duration of slow wave sleep and the number of awakenings were higher than those observed in the Wistar rat. However, the latency and duration of slow-wave and paradoxical sleep as well as the number of episodes of paradoxical sleep were similar between the species. Conclusions: Although Proechimys has nocturnal habits and polyphasic sleep pattern similar to Wistar rats, its sleep-wake cycle presents distinct characteristics. Together with previous findings of different neuroanatomical, neurophysiologic and behavioral characteristics, the present study allow us to better understand adaptive changes of the neotropical rodent Proechimys. Sleep, Proechimys, Wistar, EGRs. [email protected] 261

262 P72 Configural processing of faces in high-functioning adults with autism: an EEG/ERP study. Paula Tavares s Tavares, P.; Mouga, S.; Guiomar, O.; Castelo-Branco, M. Instituto Biomédico de Investigação da Luz e Imagem (Faculdade de Medicina, Universidade de Coimbra) People with autism spectrum disorders (ASD) have marked deficits in the social domain, most notably in face perception, which manifests since early on in a decreased attention or interest to faces. Healthy adults expertise in recognizing faces has been attributed to configural processing. According to current models, there are at least three levels of face processing: first-order (two eyes, above a nose, which is above a mouth), second-order (the relative distance between features) and holistic (ability to recognize as faces images that lack distinctive facial features). We used event-related potentials (ERPs) in 9 high-functioning adults with ASD and 14 healthy controls, during a face decision task, using photographic, schematic and Mooney upright and inverted faces, and control scrambled images, to determine whether people with ASD are generically impaired in facial configural processing or whether this impairment is selective to specific levels of configural processing. Behaviorally, there were no differences in performance between ASD and healthy controls. At the electrophysiological level subjects with ASD displayed a normal N170 inversion effect (being significant bilaterally). Processing differences were supported by the fact that ASD subjects had lower N170 amplitude to Photographic, Schematic and Mooney faces than healthy controls (right hemisphere). Moreover, whereas healthy controls had delayed N170 to Mooney faces compared to photographic and schematic faces, ASD showed instead faster N170 latencies to Mooney faces than controls (right hemisphere). These differences could all be explained away by using IQ measures as covariates. Moreover these cognitive measures showed all significant correlations with amplitude measures. We conclude that the ASD group shows sparing of first-order configural and holistic face processing when cognitive levels are taken into account. Autism ERP Face Holistic Cognitive [email protected] 262

263 P73 s Cortical structural alterations due to peripheral visual loss in Retinitis Pigmentosa Andreia C. Pereira Andreia C. Pereira1, Sónia Ferreira1 Bruno Quendera1, Catarina Mateus1, Maria do Rosário Almeida3, Eduardo Silva2, Miguel Castelo-Branco1 1 Visual Neuroscience Laboratory, IBILI - Faculty of Medicine - University of Coimbra, Coimbra, Portugal; 2 Ophthalmology, University Hospital of Coimbra, Coimbra, Portugal; 3 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Introduction and Purpose: Retinitis Pigmentosa (RP) is a retinal disease characterized by photoreceptor degeneration. Symptoms are early onset night blindness followed by progressive loss of peripheral vision, and eventually leading to complete blindness after several decades. Using MRI we studied the impact of peripheral vision loss on cerebral cortex anatomy. Subjects and Methods: Six patients (two females, 42.8±4.1 yrs) and six age- and gendermatched controls were scanned in a 3T Siemens scanner. Brain cortical thickness (CT) and surface area (SA) of Brodmann areas (BA) were obtained using Freesurfer and exported for statistical analysis with SPSS. Patients and controls hemispheres (n=12 per group) were compared. Patients visual field and disease duration were assessed. Results: Patients visual capacity ranged from peripheral visual loss (22º of maximum visual field) to blindness, with loss of central acuity. Disease duration ranged from 20 to 50 years. Visual cortical CT was preserved in patients although BA 18 (V2 - secondary visual cortex) showed a tendency for smaller SA (p=0.058). Importantly, patients BA4p (primary motor cortex) showed significantly increased CT (p=0.015). Discussion and Conclusion: Our results suggest a surprising link between peripheral visual loss and motor cortical alterations in RP. These results suggest compensatory motor cortical reorganization triggered by peripheral visual function loss. These results are consistent with the notion that peripheral visual sensitivity is relevant in vision for action dorsal stream loops. cortical reorganization, peripheral vision loss, Retinitis Pigmentosa, MRI [email protected] 263

264 P74 s Peripheral Vision Loss and Visual Cortical Organization in Retinitis Pigmentosa Sónia Ferreira Sónia Ferreira; Andreia C. Pereira; Bruno Quendera; Catarina Mateus; Maria do Rosário Almeida; Eduardo Silva; Miguel Castelo-Branco Visual Neuroscience Laboratory - IBILI - Faculty of Medicine-University of Coimbra Retinitis Pigmentosa (RP) is an inherited retinal disease characterized by progressive degeneration of photoreceptors cells and consecutive loss of peripheral vision and, in later stages, of central vision. This study aims to determine the influence of rod-cone dystrophy on visual cortical function, by using functional magnetic resonance imaging. Anatomical and functional brain images of two RP subjects (one female; 43.50±9.19 years old) and four age- and gender-matched controls were acquired with a 3T Siemens magnetic resonance scanner and were analyzed with BrainVoyager software. BOLD responses resulted from the monocular presentation of a sequence of two checkerboard rings (central and paracentral, with a maximum diameter of 1.90 and 9.52 degrees, respectively), during a passive viewing condition and a one-back task condition. Visual field diameter was lower than 23 degrees and corrected visual acuity was higher than 4/10 for RP subjects. RP subjects showed a significant peripheral retinotopic activation in striate and extrastriate visual areas for paracentral rings, between task and passive viewing conditions (p<0.05, uncorrected). Given that rings sequences were equal in both conditions, this difference in activation arose from task demands and not from passive visual stimulation. Results show a functional reorganization of visual cortex in RP subjects, as suggested by previous studies [1] [2] [3]. We propose that visual attention boosts activity in peripheral representations under active task demands in RP. References: [1] Nakadomari et al, 2005, IOVS, 46(5), 5681; [2] Poggel et al, 2007, IOVS, 48(5), 935; [3] Masuda et al, 2010, IOVS, 51(10), Cortical Reorganization, Visual Cortex, Retinal Degeneration, Retinitis Pigmentosa, fmri [email protected] 264

265 P75 Neuronal activation of the nucleus tractus solitarii (NTS) in spontaneously hypertensive rats with concurrent diabetic neuropathy Liliana S. Carvalho s Carvalho LS (1,2), Sousa RM (1,2), Patinha D (1,2), Pinho D (1,2), Tavares I (2,3), Albino-Teixeira A (1,2) (1) Departamento de Farmacologia e Terapêutica, Faculdade de Medicina, Universidade do Porto; (2) IBMC Instituto de Biologia Molecular e Celular, Universidade do Porto; (3) Departamento de Biologia Experimental, Faculdade de Medicina, Universidade do Porto Neuropathic pain and hypertension are frequent complications of diabetes. The NTS is a key area in cardiovascular and pain control and both cardiovascular and noxious stimuli can promote neuronal activation in this nucleus. We aimed to evaluate the development of diabetic neuropathy in spontaneously hypertensive rats (SHR) and the activation of NTS under these conditions. Diabetes was induced by streptozotocin (STZ, 60 mg/kg, i.p.) in 12-week old male SHR rats and their normotensive controls, Wistar-Kyoto (WKY). Body mass, glycemia, blood pressure (BP)-values and responses to Hargreaves, acetone and von Frey tests were evaluated pre- and weekly post-stz, up to week 4. On week 5, rats were subjected to formalin test and sacrificed 2h later. Brainstem cryosections were immunohistochemically analysed for Fos expression. All STZ rats developed hyperglycemia and presented lower body mass than controls. Diabetic SHR BP-values decreased over time, reaching normotensive values on the 4th week. Behavioral tests showed that, although both diabetic groups developed thermal hypoalgesia, only SHR-STZ animals exhibited mechanical, cold and chemical allodynia. Accordingly, SHR-STZ rats showed higher Fos-expression than WKY-STZs, whereas no diferences could be found between non-diabetic SHR and WKYs. NTS neuronal activation upon formalin stimulus was higher in diabetic animals than in their respective controls. Under basal conditions (no formalin stimulus), SHR-STZs presented higher neuronal activation than WKY-STZs, while no differences were found between SHR and WKY control groups. Unlike WKY rats, SHRs develop a severe form of painful diabetic neuropathy within a few weeks after STZ administration. This hypersensitive state is accompanied by basal neuronal activation of the NTS and an even higher activation upon formalin stimulus. These results highlight the importance of NTS in pain modulation in diabetic neuropathy. Funded by FCT and COMPETE, project PTDC/SAU-NEU/101090/2008. hypertension, NTS, STZ-induced diabetic neuropathy, neuropathic pain, Fos expression. [email protected] 265

266 P76 s Differential impact of right-/left-sided neuropathy in reversal learning: a c-fos expression study in the prefrontal cortex Marco Rafael Guimarães Marco Rafael Guimarães, Helena Anjos-Martins, João José Cerqueira, Nuno Sousa, Armando Almeida and Hugo Leite-Almeida Life and Health Sciences Research Institute (ICVS), Braga We demonstrated recently that left-sided pain was more anxiogenic than right-sided which in turn was more detrimental to executive function. We hypothesized that the prefrontal cortex (PFC) could be involved in the phenomenon: i. it is functionally lateralized; ii. it is morphofunctionally affected in chronic pain conditions; iii. it is a core area in emotional behaviour and executive function. To test this hypothesis, we have analysed c-fos expression in the PFC after an attentional set-shifting task (ASST) in animals with left- and right-sided neuropathic pain. The spared nerve injury (SNI) model of chronic neuropathic pain was induced in the left (SNI-L) or in the right (SNI-R) side. 1 month later animals were tested in the ASST. In this task animals are required to find a reward hidden in 1 of 2 bowls according to a series of rules involving 2 stimuli sets odors and textures. 90 minutes after completion, animals were sacrificed and brains removed. 50 µm brain slices were obtained for c-fos immunohistochemistry. c-fos positive cells density was assessed in the medial (mpfc) and orbital (OFC) PFC regions. SNI-R presented a significantly higher number of trials to completion in the reversal steps (REV; relevant cue changed to previously irrelevant cue of the same dimension) when compared to both SNI-L and controls. No differences were observed in other ASST steps. Concerning c-fos expression a task-related increase was observed but no side specific differences were observed in mpfc or OFC. The behavioural observations were in accordance with our previous studies showing an impairment of executive function in SNI-R but not in SNI-L animals. PFC c-fos expression however did not confirm our initial hypothesis. Such could result from the functional PFC heterogeneity (e.g. in the rostral-caudal axis), which was not accounted in our approach or from the involvement of other areas Chronic Pain, Neuropathy, SNI Model, Emotional and Cognitive Behaviour [email protected] 266

267 P77 s Variable delay-to-signal: a new paradigm for decision and response impulsivity assessment Hugo Leite-Almeida Hugo Leite-Almeida, António Almeida Melo, José Miguel Pêgo, Sara Bernardo, Nuno Sousa, Armando Almeida and João José Cerqueira ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal Impulsivity is defined as a tendency to act prematurely without foresight. It is a nonunitary construct embracing different levels of response inhibition including impulsive response and impulsive choice. Testing impulsive behavior in rodents is challenging and labor-intensive. We have therefore developed a new behavioral paradigm the Variable Delay-to-Signal (VDS) to provide a rapid and simultaneous assessment of response and decision impulsivity in rodents. The VDS protocol is executed in a conventional 5-hole operant box and consists in the presentation of a light, at variable delays, signaling the action (nosepoke) for a reward. Two blocks of 25 trials at 3s delay flank a block of 70 trials in which the signal is presented with large and randomly presented, 6s/12s delays. Responses during the delay period are recorded as premature/impulsive responses. The VDS proper is preceded by a shaping protocol consisting of 8-10 sessions (100 trials each) at 3s delay. In this case, premature responses are punished, determining reward retention and trial interruption. We validated the VDS by employing two drugs with a well-established action on impulsive behavior, methamphetamine (0.5 mg/kg) and MK-801 (0.03 mg/kg), and by comparing the individual performance against two reference paradigms, the 5-choice serial reaction time task (5-csrtt) and the delay-discounting (DD). The exposure to large delays boosts the rate of premature responses when the delay drops to 3s and this parameter correlated with the delay-discounting paradigm but not with the 5-choice serial reaction time task. Acute methamphetamine challenge blunted this effect while increasing response impulsivity in the initial segments of the delays. The NMDA agonist MK-801 increased the rate of impulsivity. The VDS provides in a single session a multi-dimensional assess of impulsive behavior. variable delay-to-signal, impulsive response/choice, animal behavior, 5-choice serial reaction time task, delay-discounting [email protected] 267

268 P78 s Effects of bariatric surgery in gustatory function: exploring preliminary data Marta Camacho Marta Camacho, Ana B. Fernandes, Catarina Castro, Teresa Branco, Sandra Martins, Rui M. Costa, Albino-Oliveira Maia Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisboa, Portugal. The hedonic value of food is closely related to the perception of taste. However, experiments exploring the relationship between taste perception, eating behavior and body mass, have not reached consensual conclusions, possibly due to differing methodologies. Some have suggested that obese individuals taste sweet as being less intense but more pleasant than normal-weight individuals, others have shown that people with high body mass index (BMI) report lower pleasantness and others still have not detected differences in sweet taste perception in different BMI groups. Moreover, while it has been suggested that certain weight-loss surgery approaches could impact gustatory function, the impact of treatments for weight loss on taste perception have not been addressed in longitudinal studies. In an attempt to clarify these questions, we are testing changes in psychophysical and psychometric parameters in severely obese subjects before and after 2 different interventions: 1) intensive modification of lifestyle (diet + exercise), 2) bariatric surgery. Some of these measures were also collected in non-obese control individuals. In these three groups we applied an extensive battery of psychological assessment, determined electrogustometric threshold and assessed gustatory function through subjective ratings of intensity and pleasantness on labelled magnitude scales. In our exploratory analysis at baseline, we found significant differences between obese and non-obese subjects regarding intensity ratings of bitter and sweet taste. Additionally, we found a significant correlations between BMI and mean intensity ratings for sweet and bitter taste. Post-intervention measurements suggest no differences in intensity and pleasantness ratings after weight loss from lifestyle modification. However, testing for longitudinal analyses are ongoing and, based on existing literature, we expect to find differences in intensity and pleasantness ratings after bariatric surgery. Obesity; Taste Perception; Weight Loss; Lifestyle modification; Bariatric Surgery. [email protected] 268

269 P79 s The role of cannabinoids in the brain-gut axis Fátima Marques Ramalhosa Fátima Ramalhosa, Ana Franky Carvalho, Nuno Sousa Neuroscience Research Domain, School of Health Science, Life and Health Sciences Research Institute (ICVS) The coordination of gastrointestinal (GI) motility involves the enteric nervous system (ENS), gastric hormones, interstitial cells of Cajal (ICCs) and smooth muscle cells. In addition, GI function is also affected by central stimuli. Since endocannabinoids can impact both the central nervous system (CNS) and GI motility we believe that endocannabinoids may be a major player in the brain-gut axis. The impact of several diseases in the anatomical distribution of ICCs, ENS and cannabinoid receptor 1 (CB1r) are still unknown. In the present study, we show immunohistochemistry for CB1r and its relation with substrates of the GI tract. In addition, we explore the effects of a perinatal stress in animal behavior and expression of CB1r in both the GI tract and the CNS. Cannabinoid receptor 1 (CB1r), Interstitial cells of Cajal (ICC), Perinatal stress [email protected] 269

270 P80 s The Sweet Drive Test (SDT): a novel paradigm to measure anhedonic behavior in rodents Santos, A. Santos A.*, Mateus-Pinheiro A.*, Patrício P.*, Alves ND., Bessa J., Sousa N & Pinto L Life and Health Sciences Research Institute (ICVS), University of Minho; ICVS/3B s PT Government Associate Laboratory Anhedonic behavior is a pathological hallmark of depressive spectrum disorders. In rodent models of depression, Sucrose or Sacarine consumption tests are gold-standard methods to measure hedonic behavior, but often present inconsistent results between experimental sets and only moderate sensitivity to detect anhedonic phenotype. Recently, a cookie-test was developed and claimed to be a robust experimental paradigm; however, as it relies in a neophobia paradigm, animal performance in this test seems to be strongly related to anxiety behavior. We have developed a new test, in the attempt to overcome limitations of the existing methods, which combines food preference measurement in a non-aversive environment with 50 KHz ultrasonic vocalizations (USVs) measurement during the test execution. Results demonstrate that the newly designed Sweet Drive Test (SDT) efficiently detects anhedonic behavior associated with feeding behavior and accurately descriminates between naïve male rats and rats exposed to an unpredictable chronic mild stress protocol. Curiously, stressed animals exhibiting depressive-like behavior, besides having low preference for sugared-food, present a low number of 50 KHz USVs during the test. Additionally, these results were also reproducible in female rats. Overall, results demonstrate that SDT can robustly caractherize anedhonic behaviour both in male and female rat models of depression, and constitutes a promising tool to be used in the field. Animal models of depression, Anhedonia, Stress. [email protected] 270

271 P81 s Stress and cooperative levels in a cleaner fish mutualism Marta Soares Marta Soares, Sónia Cardoso, Alexandra Grutter, Rui Oliveira, Redouan Bshary ISPA-Instituto Universitário Both human and other animals alike need to be able to shift their behavioural output in order to optimize their decision-making process, along with a dynamic and sometimes stressful social environment. Stress plays a unique role in facilitating local adaptation and behavioural synchronization but also amplifies phenotypic and genetic variation, which should vital for the expression of behavioural flexibility. Here we tested a hypothesis that cooperative behavioural flexibility is mediated by stress-mediated mechanisms, in a cleaning mutualism involving the cleaner fish Labroides dimidiatus. When interacting with other fish, cleaner fish are faced with the decision to cooperate by removing other fish ectoparasites or to cheat by removing their mucus instead; and are known to use a highly diverse behaviour repertoire to persuade their clientele to visit, to increase the duration of inspection and to promote their return in the near future. We found that stressed cleaners responded to their visiting clientele by providing more tactile stimulation however, they were also cheating clients more often. On the contrary, the blocking of the glucocorticoids receptors led to an increase of cleaning inspection duration. Our results demonstrate that the role of stress and stress responses are key for producing changes on individuals levels of cooperation. This might be the primary hormonal mechanism affecting vertebrate interspecific cooperation through which cognitive social flexibility develops and further adjustments to ecological and social factors arise. Behaviour, Cortisol, Cooperative levels, Mutualism [email protected] 271

272 P82 s Lipocalin-2 is involved in cognition and emotional behaviors Ana Catarina Ferreira Ana C Ferreira1,2, Vítor Pinto1,2, Sandro D Mesquita1,2, Ashley Novais1,2, João C Sousa1,2, Margarida Correia-Neves1,2, Trude H Flo3, Cevayir Coban4, Nuno Sousa1,2, Joana A Palha1,2 and Fernanda Marques1,2 1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus Gualtar, Braga, Portugal. 2ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal. 3Department of Cancer Research and Molecular Medicine, Norges Teknisk-Naturvitenskapelige Universitet, and St. Olavs Hospital, Trondheim, Norway. 4Laboratory of Malaria Immunology, Immunology Frontier Research Center (IFReC) The mechanisms underlying the control and the interplay between emotional and cognitive behaviors are still largely unknown and an increasing body of evidence is the comorbidity shared between the immune system and the central nervous system with implications in such behaviors. Lipocalin-2, an iron-traffic protein, has a denotable function as part of the innate immune response for bacteria depletion but also in mediating cell proliferation, apoptosis and survival in basal conditions. In the central nervous system, lipocalin-2 acts as an acute-phase protein at the brain barriers, with a recent involvement in the promotion of stress-related changes in spine morphology and in regulating neuronal excitability and anxiety. In the present study, we assessed the behavioral profile of lipocalin-2-null mice and observed that these animals displayed anxious and depressivelike behaviors, cognitive impairment in spatial learning tasks and deficits in synaptic plasticity. The present observations bring into light the existence of another immunerelated protein as a novel player in the fine-tuning of behavior and neuronal excitability. Lipocalin-2, behavior, anxiety, depression, hippocampus [email protected] 272

273 P83 s Different distribution of 5-HT1B and 5-HT2C receptor in two different genetic mouse models of depression Carolina M de Souza Carolina M de Souza1, Paula M Canas1, Malika El Yacoubi2,3, Jean-Marie Vaugeois4, Rodrigo A Cunha1,5 1CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2Faculty of Pharmacy Pharmacie, University of Lyon 1, France; 3Lyon Neuroscience Research Center, France; 4Department Pharmacology, Rouen University, France; 5Faculty of Medicine, University of Coimbra, Portugal. Major depressive disorders are the leading burden of disease in Europe. We generated two mouse models of depression by selective breeding of mice with heightened responses either in the tail suspension test (TST) or in the forced-swim test (FST). Since the serotonin (5-HT) system is considered central to depression, we now evaluated if the density of 5-HT1A, 5-HT1B and 5-HT2C receptors was altered in different brain regions in these two models of depression. Mice were divided into 4 groups (n=4 per group): mice with a helpless phenotype in the TST (HTST) or in the FST (HFST) and their controls with a non-helpless phenotype (NHTST and NHFST). The quantification of 5-HT1A, 5-HT1B and 5-HT2C receptor (R) density in the prefrontal cortex (PFC), amygdala and hippocampus (CA1, CA3, SGZ) was performed by immunohistochemistry (optical density). There were no differences in the density of 5-HT1AR between HTST and NHTST mice. HTST mice had a greater 5-HT1BR density in the CA1 (NHTST: 107.5±6.6; HTST: 138.8±5.2), CA3 (NHTST: 125.0±5.7; HTST: 149.9±6.0) and SGZ (NHTST: 92.2±6.5; HTST: 113.6±3.1) but there was no difference in the PFC and amygdala. Finally, 5- HT2CR density in the PFC was lower in the HTST (115.7±2.2) than in NHTST (127.9±2.5). In the FST-selected mice, there was no difference of 5-HT1AR or 5- HT1BR densities, whereas 5-HT2CR density was lower only in CA3 (HFST: 97.5±1.3; NHFST: 107.7±2.1) and SGZ (HFST: 81.5±2.3; NHFST: ±2.8). In conclusion, in these 2 mouse models of depression, there was no change in 5-HT1AR density, 5-HT1BR density was selectively higher in the hippocampus of one strain but not of the other, whereas 5-HT2CR was lower in the PFC in one model and changed in the hippocampus of the other. This indicates that the density of these 5HT-R is not a reliable marker predicting learned helpless behavior characteristic of depressive conditions. Supported by DARPA (09-68-ESR-FP-010), FCT (PTDC/SAU-NSC/122254/2010), Égide-Pessoa and CAPES. Depression, 5-HT receptor, Helpless/Rouen mice, hippocampus, prefrontal cortex. [email protected] 273

274 P84 s The impact of antipsychotic drugs in depression: a role of adult neurogenesis? Mónica Morais Mónica Morais(1), António Pinheiro(1), Patrícia Patrício(1), Nuno Dinis Alves(1), Luísa Pinto(1), Nuno Sousa(1), João Bessa(1) (1)Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus Gualtar, , Braga, Portugal and ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal. Considering the crescent number of depressive patients and the increase of nonresponders/incomplete responders to antidepressant treatment, it is essential to explore new strategies in order to achieve a full remission and to prevent recurrent depressive episodes. Recently, antipsychotics received FDA approval for the treatment of antidepressant-resistant forms of major depression. However the mechanism triggered by these drugs remains widely undisclosed. Although adult neurogenesis has been involved in the actions of antidepressants, its importance concerning antipsychotics in depression remains unclear. To address this question, an unpredictable chronic mild stress (ucms) paradigm was implemented during 7 weeks to induce core symptoms of depressive-like behavior in rats. During the last 3 weeks of ucms, two different classes of antipsychotics, clozapine and haloperidol, were daily administered. At the end of treatment, learned helplessness was evaluated in the forced swimming test and anxietylike behavior was assessed through the elevated-plus maze. Cognitive function was assessed by different tasks in the Morris water maze. Anhedonia was assessed through the sucrose preference test. We found that clozapine reduced both measures of depressive-like behavior. Haloperidol only reverted anhedonic phenotype and aggravated learned helplessness. Haloperidol-treated animals displayed cognitive impairments in the working memory and reverse learning task. Our findings also suggest that haloperidol and clozapine lead to different neuroplastic adaptive responses. Clozapine promotes cell proliferation and neurogenesis in the hippocampus and in the subependymal zone while haloperidol leads to a decrease in neurogenesis in these brain regions. The present results suggest an association between the modulations of adult neurogenesis and the emotional/cognitive changes observed in response to antipsychotic treatment in an animal model of depression. Depression, Antipsychotics, Neurogenesis, Hippocampus, Subependimal [email protected] 274

275 P85 s Potential role for galanin in pain-depression comorbidity Diana Amorim Diana Amorim1,2, Ana Pereira1,2, Armando Almeida1,2 and Filipa Pinto-Ribeiro1,2 1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 2- ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, Chronic inflammatory pain and depression are enormous burdens on our society, with depression also being the major comorbidity of chronic pain disorders. While an acute inflammatory response triggers a complex biological cascade of molecular and cellular signals in order to protect areas affected by physical injury; chronic inflammation induces neuronal plasticity in brain wiring of central pathways mediating pain. Galanin (GAL) is an injury-responsive peptide whose expression during chronic inflammation is enhanced in the dorsomedial nucleus of the hypothalamus (DMH), a nucleus that has recently been proposed to promote hyperalgesia. Finally, since GAL and its receptors have been reported to be involved in the pathophysiology of depression, we hypothesize that GAL might be a common player in the comorbidity between chronic inflammation and depression. Four weeks after the induction of experimental monoarthritis (kaolin/carrageenan model), animals displayed an anxious- and depressive-like phenotype. Anxiety-like behaviour was associated with a decrease in the time spent in the open arms of the elevated plus maze. Depressive-like behaviour was associated to decreased (1) preference for sucrose and (2) latency to immobility at the expense of climbing and swimming in the forced swimming test. The decrease in paw-withdrawal latency after GAL in the DMH was mimicked by GalR1 agonist (M617) pointing to the possibility of a role for GalR1 in GAL-mediated pronociception in monoarthritis. Moreover, our results are in accordance with previous data where GalR1 agonists promote depressivelike behaviours in rodents, concomitant with decreased pain thresholds. Our data show that GAL is a potential player in the comorbidity between chronic inflammation and depression and that this pathway should be investigated as new therapeutic target for the treatment of pain, depression, and their comorbidity. Chronic inflammatory pain, depression, anxiety, galanin, galanin receptor [email protected] 275

276 P86 s Mapping the functional organization of striatal circuits Sevinc Mutlu Sevinc Mutlu, Catia Feliciano, Gabriela Martins, Fatuel Tachupetla, Rui M. Costa Fundacao Champalimaud, Champalimaud Neuroscience Programme Clonal relationship of cortical pyramidal cells has been shown to be an important factor defining their connectivity and their response characteristics in vivo. Recent data made a strong link between the ontogenic column hypothesis and the functional column hypothesis of cortical organization, and unified the rules of structural and functional organization in the cortex. However, it is not known if ontogenetic relationship of cells in other brain structures, like basal ganglia circuits, also contributes to their functional organization. We are first using adeno associated viruses to express different fluorescent proteins on genetically defined populations to map the organizational rules of the cortical input on the striatum, and to map the target structures of direct and indirect pathway medium spiny neuron projections in a medio-lateral gradient. We are also using fluorescent protein expressing retrovirus to label ontogenetically related, genetically defined cell populations to study the functional organization of striatal circuits. striatal circuits, mapping, functional organization, ontogenetic relationship [email protected] 276

277 P87 s Matching oxygen and glucose dynamics during neurovascular coupling in rat hippocampus Cátia F. Lourenço Lourenço CF, Ledo A, Cateano MA, Laranjinha J, Barbosa RM Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal The brain is a highly energy-demanding organ and lacks substantial substrate stores, which implies a continuous supply of blood flow-carried substrates to maintain its functional and structural integrity. The increase in neuronal activity is, under physiological conditions, tightly coupled to changes in local blood flow (neurovascular coupling) and glucose and O 2 utilization (neurometabolic coupling). Thus, both O 2 and glucose are permanently being balanced by opposite driving forces: decreased levels due to enhanced utilization and increased levels due to their delivery from blood supply. How this balance is affected during neuronal activation it is not, however, fully understood. In this work, by simultaneously measuring cerebral blood flow (CBF), O 2 and glucose in vivo, we studied the relationship between the CBF dynamics and the changes of O 2 and glucose concentration in the brain, as well as the interplay between them. The correlation between CBF and O 2 dynamics was also addressed in aging. Overall, this work provides clues for a better understanding of the processes governing the neurometabolism. CFL acknowledges the Post-Doctoral fellowship SFRH/BPD/82436/2011 from FCT. This work was supported by FCT grant PTDC/SAU-NEU/103538/2008 and PTDC/SAU-BEB/103228/2008. Cerebral blood flow, oxygen, glucose, sensors [email protected] 277