SUPPLEMENTARY MATERIAL

Transcription

1 SUPPLEMENTARY MATERIAL Lyer-speifi exittory iruits differentilly ontrol reurrent network dynmis in the neoortex Rirdo Beltrmo, Giuli D Urso, Mro Dl Mshio, Psqulin Frisello, Seren Bovetti, Yonne Clovis, Glend Lssi, Vlter Tui, Dvide De Pietri Tonelli & Tommso Fellin Supplementry figure 1 tdtomto ChR2-1 ChR2-eYFP NeuN mcherry 2 µm 8 Numer of ells ChR2+ (% of totl) f 1 µm tdtomto+ GABA+ tdtomto+ GABA+ f1 1 5 µm 1 µm tdtomto GABA 1 µm Supplementry figure 1. ChR2 expression in ortil neurons of infrgrnulr lyers. -1) Confol imge showing ortil setion of doule trnsgeni mouse (Rp4-Cre x tdtomto). Cells expressing tdtomto re shown t n expnded sle in 1. ) Confol imge of ortil setion from Rp4-Cre mouse injeted with AAVs trnsduing ChR2-mCherry. -1) ChR2-eYFP fluoresene in Rp4-Cre mouse injeted with AAV viruses. Arrows point to ChR2 expressing ells. Immunostining ginst the neuronl mrker NeuN is shown in (1). d) Histogrm showing the perentge of ChR2-expressing neurons ross ortil lyers. Dt were pooled from 3 nimls (3 5 setions per niml). e) Totl numer of tdtomto-positive, GABA-positive, tdtomto nd GABA-positive neurons loted in lyer V in Rp4-Cre x tdtomto reporter doule trnsgeni mie whih were immunostined for GABA. Dt were pooled from 4 nimls (3 setions per niml). f-f1) Confol imges showing GABA-immunostined ells (green) in setions from Rp4-Cre x tdtomto mouse. Nture Neurosiene: doi:1.138/nn.336

2 Supplementry figure 2 1 ms 2 mv 2 ms 2 ms 1 mv 2 mv d 1 µ m Supplementry figure 2. Funtionl hrteriztion of ChR2-expressing neurons in injeted Rp4-Cre mie. ) Representtive tres showing the memrne response to urrent injetions in ChR2-expressing neuron reorded in ortil slie from Rp4-Cre mouse injeted with AAV trnsduing ChR2. ) Currentlmp reording showing representtive memrne potentil response to light stimultion in neuron expressing ChR2. ) Histogrm showing the verge integrl (Are) of memrne depolriztion s funtion of light intensity in 6 reorded ChR2-expressing ells. d) Morphologil reonstrution of four ioytin-filled, ChR2-positive neurons from Rp4-Cre mie injeted with AAVs. Nture Neurosiene: doi:1.138/nn.336

3 Supplementry figure 3 1 mcherry ChR2-Venus mcherry 1 ChR2-Venus 2 merge 2 µ m # of eletroported ells (% of totl) d d 1 ChR2-Venus NeuN 2 µ m e mcherry e 1 GABA Numer of ells µ m 1 µ m 2 µ m mcherry+ GABA+ mcherry+ GABA+ Supplementry figure 3. ChR2 expression in ortil neurons of suprgrnulr lyers. - 1 ) Confol imge of neoortil setion from WT mouse eletroported in utero with the red fluoresent reporter mcherry (left) nd ChR2-Venus (right). - 2 ) Mgnified view showing eletroported ells in suprgrnulr lyers. ) Histogrm displying the perentge of trnsgene-expressing ells ross lyers in eletroported orties. Dt were pooled from 3 nimls (3 4 setions per niml). d-d 1 ) Left: ChR2-Venus fluoresene in n eletroported ortex. Right: Immunostining ginst the neuronl mrker NeuN. Arrows indite ChR2- expressing neurons. e-e 1 ) Confol imges showing GABA immunostined ells (green) in eletroported orties (mcherry fluoresene is shown in red). A mgnified view is displyed in e 1. f) Totl numer of mcherry-positive, GABA-positive, mcherry nd GABA-positive neurons in suprgrnulr ortil lyers ounted in setions of eletroported orties whih were immunostined ginst GABA. Dt were pooled from 4 nimls (4 setions per niml). Nture Neurosiene: doi:1.138/nn.336

4 Supplementry figure 4 1 ms 2 mv 2 ms 2 ms 1 mv 2 mv d 1 µ m Supplementry figure 4. Funtionl hrteriztion of ChR2-expressing neurons in eletroported ortex. ) Representtive tres showing the memrne response to urrent injetions in one ChR2-expressing neuron reorded in slie preprtion from n eletroported ortex. ) Memrne potentil response to light stimultion. ) Histogrm showing the verge integrl (Are) of memrne depolriztion s funtion of light intensity in 4 ChR2-expressing ells. d) Morphologil reonstrution of 6 ioytin-filled ChR2-positive neurons from eletroported orties. Nture Neurosiene: doi:1.138/nn.336

5 Supplementry figure mv s.5 mv 1 µ V 2 Power mp (mv /Hz) Frequeny (Hz) s 2 mv s 1 µ V 1 s 2 mv Supplementry fig. 5. Extrellulr nd intrellulr reordings of spontneous reurrent tivity in the neoortex. ) Ninety seonds of ontinuous LFP nd MU reording displying spontneous network dynmis in wild type mouse in vivo. Three 5-seond time windows re shown t n expnded time sle in the ottom pnels. ) Power mplitude spetrum of the LFP tre shown in. ) Sixty seonds of ontinuous pth-lmp reording from neoortil neuron in vivo. The insets on the right show two 5-seond windows t n expnded time sle. Nture Neurosiene: doi:1.138/nn.336

6 Supplementry figure 6 Hz.5 mv µ V 5 Spontneous Spontneous LFP norm log 1 power MU Light-evoked Light-evoked.5 s norm log 1 power 3 6 Hz power 6 9 Hz power 1 9E-4 6E-4 3E-4 6E-4 4E-4 2E-4 Numer of spikes 8 4 Spontneous Up Up Up 3 6 Hz power 6 9 Hz power 1 4E-4 3E-4 2E-4 1E-4 4E-4 2E-4 Numer of spikes 4 2 Light-evoked Supplementry fig. 6. Light stimultion in Thy1-ChR2 mie genertes network up stte-like trnsitions. ) LFP tres showing spontneous up-stte (left) nd light-evoked (right) event in Thy1-ChR2 mouse. The orresponding spetrogrms re displyed on the ottom. 1 ) MU signls orresponding to the tres shown in () for one spontneous up-stte (left) nd one light-evoked response (right). Corresponding PSTHs re displyed in the ottom pnel. ) Averge power in the 3 6 (top) nd 6 9 (ottom) Hz frequeny nd efore (pre), during (up) nd fter (post) spontneous up-sttes. Power vlues re normlized to the totl power in the pre period. One-wy, repeted mesures ANOVA for omprison etween pre, up nd post periods during spontneous events ws used, N = Hz, p = 4.E 4; 6 9 Hz, p = 3.2E 6. 1 ) Averge numer of spikes reorded in the MU signl efore (pre), during (up) nd fter (post) spontneous up-sttes. Vlues re normlized to the verge numer of spikes in the pre period. One-wy, repeted mesures ANOVA, N = 6, p = 3.2E ) Sme s in (- 1 ) for light-evoked responses. One-wy, repeted mesures ANOVA, N = 6. ) 3 6 Hz, p = 1.2E 4; 6 9 Hz, p = 2.2E 4. 1 ) p = 3.9E 5. Nture Neurosiene: doi:1.138/nn.336

7 Supplementry figure 7 lyer V ChR2 lyer II/III ChR2.5 s 1 mv.5 s 1 mv Supplementry fig. 7. Initil tivtion of ChR2-positive ells either in lyer V or II/III spreds to ChR2- negtive neurons. -) In vivo representtive pth-lmp reording from lyer V ChR2-negtive ell while stimulting sugroup of lyer V neurons () nd from ChR2-negtive neuron loted in lyer II/III while stimulting supopultion of ChR2-positive neurons in the sme lyer (). Nture Neurosiene: doi:1.138/nn.336

8 Supplementry figure 8 Norm 1 Hz power mp d ON OFF.5 s.2 mv 1s Norm power mp 1 µ V 5 e Spike # (% of totl) Frequeny (Hz) ON OFF Supplementry figure 8. Rhythmi light stimultion entrins spontneous network tivity in trnsgeni Thy1-ChR2 mie. ) Representtive LFP tres efore (), during () nd fter () light stimultion t 1 Hz (light ON durtion, 5 ms; light OFF durtion, 5 ms) in the neoortex of trnsgeni Thy1-ChR2 nimls in vivo. ) Power mplitude spetr of the LFP signl efore (, lk line), during (, red line) nd fter (, lue line) light stimultion. ) Averge vlues of LFP power mplitude in the 1 Hz frequeny in in the three different experimentl onditions (one wy, repeted mesures ANOVA, N = 8, p =.7E 3). d) Representtive MU signls reorded efore (), during () nd fter () lue light stimultion. Bottom: orresponding PSTHs re shown for eh tre. e) Men vlues of the numer of spikes in the MU signl ourring during the light on (ON) nd during the light off (OFF) yles of the stimultion. Vlues re normlized to the totl numer of spikes reorded (pired Student s t-test, N = 9 nimls, p = 1.1E 9). Nture Neurosiene: doi:1.138/nn.336

9 Supplementry figure 9.5 Hz Hz 2.1 mv 1s 1 4 Hz 2 d d 1 8 Hz d2 e e 1 1 Hz e2 f Numer of spikes (% of totl) *** *** ON OFF ** ** **.5 Hz 2 Hz 4 Hz 8 Hz 1 Hz Supplementry figure 9. Boosting ortil osilltions in the.5 1 Hz frequeny rnge. -e) Representtive MU tres efore (), during () nd fter () light stimultion t.5 ( 1 ), 2 ( 1 ), 4 ( 1 ), 8 (d 1 ) nd 1 (e 1 ) Hz in Thy1-ChR2 mie. f) Averge numer of spikes in the ON nd OFF yles during stimultion t vrious frequenies (N = 3 nimls,.5 Hz, p = 2.7E 4; 2 Hz, p = 3.2E 4; 4 Hz, p = 2.8E 3; 8 Hz, p = 3.4E 3; 1 Hz, p = 1.6E 3, pired Student t-test). Nture Neurosiene: doi:1.138/nn.336

10 Supplementry figure 1 Norm 1 Hz power mp NS NS d WT WT.5 s.2 mv Norm power mp 1 s 5 5 µ V e Frequeny (Hz) 1 Numer of spikes (% of totl) 5 ON NS OFF Supplementry figure 1. Light hs no effet on the LFP nd MU signls in wild type mie. ) Cortil LFP reorded in WT mie efore (), during () nd fter () light stimultion t 1 Hz. ) Corresponding power mplitude spetr under the different experimentl onditions. ) Averge power mplitude of the 1 Hz in efore, during nd fter stimultion t 1 Hz (one-wy ANOVA, N = 5 nimls, p =.24). d) MU tres nd orresponding PSTH efore (), during () nd fter () light stimultion of the neoortex in WT mouse. e) Averge numer of spikes during the ON nd OFF yles (pired Student s t- test, N = 6 nimls, p =.46). Nture Neurosiene: doi:1.138/nn.336

11 Supplementry figure 11 Thy1-ChR2 non-rem.25 mv 2 Power (mv /Hz).5 s 1E-2 1E-3 1E-4 Thy1-ChR2 non-rem 1 Hz stim Norm 1 Hz in power * * 1E Frequeny (Hz) Supplementry figure 11. Potentition of low frequeny rhythms in nturlly sleeping mie. ) Representtive EEG tres during non-rem sleep efore (top tre, ), during (middle tre, ) nd fter (ottom tre, ) light stimultion (1-Hz repetition frequeny, light ON durtion, 5 ms; light OFF durtion, 5 ms) in ehving Thy1-ChR2 mie. -) Power spetr () nd verge power of the 1 Hz in () under the different experimentl onditions. In, one wy ANOVA, N = 5, p = 9.E 3. Vlues were normlized to the totl power in the period. Nture Neurosiene: doi:1.138/nn.336

12 Supplementry figure 12 2 µ m 2 mv 2 AP # 1.2 s 1 µ m Supplementry figure 12. Expression of inhiitory opsins in ortil neurons of infrgrnulr lyers. -) Confol imges showing expression of inhiitory opsins in Rp4-Cre mie injeted with AAV trnsduing Arh nd enphr. Cells re shown t n expnded sle in. Arrows point to opsin-expressing ells. ) Top: slie reording displying memrne potentil response to light (yellow line) in representtive opsin-positive neuron. Ation potentil dishrge ws indued in the neuron y urrent injetion nd ws inhiited y light. Bottom: PSTH orresponding to the tres shown in the top pnel. d) Histogrm displying the instntneous frequeny (IF) of tion potentils efore (pre), during (stim) nd fter (post) light stimultion. One-wy, repeted mesures ANOVA, N = 5, p = 3E 4. Eletrophysiologil properties of reorded neurons were: resting potentil, 65 ± 3 mv; input resistne, 189 ± 24 MΩ; tion potentil frequeny t 2 pa urrent injetion, 28 ± 3 Hz; tion potentil threshold, 4 ± 2 mv, N = 5 ells. Nture Neurosiene: doi:1.138/nn.336

13 Supplementry figure 13 mcherry Arh/ArhT-GFP mcherry Arh/ArhT-GFP 2 µ m 2 µ m 2 mv.2 s 2 AP # 1.2 s 1 µ m 1 µ m Supplementry figure 13. Expression of inhiitory opsins in ortil neurons of suprgrnulr lyers. - ) Confol imges showing mcherry (left) nd Arh-GFP nd ArhT-GFP (right) expression in eletroported mie. Cells re shown t n expnded sle in. Arrows indite opsin-expressing ells. ) Top: slie reording showing memrne potentil response to light (yellow line) in representtive opsin-positive neuron loted in suprgrnulr lyers. Ation potentil dishrge ws indued in the neuron y urrent injetion nd ws inhiited y yellow light. Bottom: PSTH orresponding to the tres shown in the top pnel. d) Averge instntneous frequeny (IF) of tion potentils efore (pre), during (stim) nd fter (post) light stimultion. One-wy, repeted mesures ANOVA, N = 4, p = 3.6E 3. Eletrophysiologil properties of reorded ells were: resting potentil, 78 ± 3 mv; input resistne, 111 ± 13 MΩ; tion potentil frequeny t 2 pa urrent injetion, 13 ± 3 Hz; tion potentil threshold, 37 ± 1 mv, N = 4 ells. Nture Neurosiene: doi:1.138/nn.336

14 Supplementry figure 14 Lyer V ChR2 12 Lyer V ChR2.2 s 1 mv 1 mv 4 ms Amplitude (mv) 8 4 Lyer II/III ChR2.2 s 1 mv 1 mv 4 ms d Amplitude (mv) Lyer II/III ChR2 Supplementry fig. 14. Synpti relese of exittory neurotrnsmitter medites the propgtion of exittion. ) Representtive urrent-lmp reording from lyer V ChR2-negtive neuron in ortil slies from Rp4-re mouse injeted with AAVs trnsduing ChR2 under ontrol onditions (top), nd in the presene of DNQX (1 µm) nd D-AP5 (5 µm, ottom). ) Averge mplitude of light-evoked EPSPs in ChR2-negtive ells under the two different experimentl onditions. Pired Student s t-test, N = 4, p =.27. -d) Sme s in - for reordings from lyer II/III ChR2-negtive neurons while stimulting supopultion of ells in the sme lyer in slies from eletroported orties. Pired Student s t-test, N = 4, p =.3. Nture Neurosiene: doi:1.138/nn.336