Local potentiation of excitatory synapses by serotonin and its alteration in rodent models of depression

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1 Lol potentition of exittory synpses y serotonin nd its ltertion in rodent models of depression Xing Ci 1,8,9, Angy J Kllrkl 1,2,9, Mrk D Kvrt 1 3, Ssh Goluskin 4, Kitlin Gylor 4, Aileen M Biley 4, Hey-Kyoung Lee 5, Rihrd L Hugnir 6 & Sott M Thompson 1,2,7 npg 213 Nture Ameri, In. All rights reserved. The uses of mjor depression remin unknown. Antidepressnts elevte onentrtions of monomines, prtiulrly serotonin, ut it remins unertin whih downstrem events re ritil to their therpeuti effets. We found tht endogenous serotonin seletively potentited exittory synpses formed y the temporommoni pthwy with CA1 pyrmidl ells vi tivtion of serotonin reeptors (5-HT 1B Rs), without ffeting nery Shffer ollterl synpses. This potentition ws expressed postsynptilly y AMPA-type glutmte reeptors nd required lmodulin-dependent protein kinse medited phosphoryltion of GluA1 suunits. Beuse they shre ommon expression mehnisms, long-term potentition nd serotonin-indued potentition oluded eh other. Long-term onsolidtion of sptil lerning, funtion of temporommoni-ca1 synpses, ws enhned y 5-HT 1B R ntgonists. Serotonin-indued potentition ws quntittively nd qulittively ltered in rt model of depression, restored y hroni ntidepressnts, nd required for the ility of hroni ntidepressnts to reverse stress-indued nhedoni. Chnges in serotonin-medited potentition, nd its reovery y ntidepressnts, implite exittory synpses s lous of plstiity in depression. Depression is leding use of mortlity nd moridity worldwide 1 nd proly results from omintion of geneti nd environmentl risk ftors. Although the genes responsile for depression remin diffiult to identify, ommon risk ftor is stress 2. Current tretments for depression remin unstisftory, prtly euse the underlying iologil hnges in rin funtion in depression remin poorly understood. The disovery tht hnges in monomine levels lter the ffetive stte of ptients led to the hypothesis tht dysfuntion of monomine signling, prtiulrly serotonin, uses depression 3. Elevtion of serotonin levels with onventionl ntidepressnts, suh s seletive serotonin-reuptke inhiitors (SSRIs) nd triyli ntidepressnts, my modulte neuronl exitility nd plstiity y ltering the trnsription, trnsltion nd phosphoryltion stte of trget proteins 1,3. Neither the prinipl neurl iruits nor the key proteins tht re modulted y serotonin re known, however. Serotonin is ple of regulting the glutmtergi system, nd evidene of hnges in exittory synpti trnsmission in models of depression is umulting 4 6, ut it remins unler how these two neurotrnsmitter systems intert. Depression is likely used y dysfuntion in vriety of rin regions nd ell types, inluding the hippompus 7,8. Altered hippompl funtion my lso dversely influene the tivity of the ortex, mygdl nd other strutures ssoited with rewrd, motivtion nd emotionlity. For exmple, the hippompl formtion provides mjor soure of exittory input to the nuleus umens (NA) 9, where hroni stress wekens AMPA reeptor (AMPAR)-medited exittory synpti trnsmission 1. The highest onentrtion of serotoninergi fiers in the forerin is in strtum lunosum-moleulre (SLM) of hippompl res CA1 nd CA3 (ref. 11), where the xons of lyer III neurons in the entorhinl ortex form exittory synpses with the distl pil dendrites of pyrmidl ells. This temporommoni pthwy is required for some sptil reognition tsks nd for long-term onsolidtion of sptil memory 12. To understnd the funtion of serotonin etter, we studied its tions t temporommoni-ca1 (TA-CA1) synpses, nd oserved tht serotonin potentited these synpses postsynptilly nd tht this potentition ws ltered in rt model of depression. RESULTS Potentition of TA-CA1 synpses y serotonin We reorded field exittory postsynpti potentils (fepsps) in SLM of re CA1 of utely prepred rt hippompl rin slies in response to stimultion of the temporommoni pthwy (Supplementry Fig. 1). Bth pplition of the triyli ntidepressnt imiprmine (2 µm) or the SSRIs fluoxetine (2 µm) or itloprm (1 µm), to utely elevte extrellulr serotonin, ll inresed the slope of s (Fig. 1, nd Supplementry Fig. 1,). Potentition of synpti trnsmission y serotonin is notle, s most 1 Deprtment of Physiology, University of Mrylnd Shool of Mediine, Bltimore, Mrylnd, USA. 2 Progrms in Neurosiene nd Memrne Biology, University of Mrylnd Shool of Mediine, Bltimore, Mrylnd, USA. 3 Medil Sientist Trining Progrm, University of Mrylnd Shool of Mediine, Bltimore, Mrylnd, USA. 4 Deprtment of Psyhology, Sint Mry s College of Mrylnd, St. Mry s City, Mrylnd, USA. 5 The Znvyl Krieger Mind/Brin Institute, Johns Hopkins University, Bltimore, Mrylnd, USA. 6 Solomon H. Snyder Deprtment of Neurosiene, Howrd Hughes Medil Institute, Johns Hopkins University Shool of Mediine, Bltimore, Mrylnd, USA. 7 Deprtment of Psyhitry, University of Mrylnd Shool of Mediine, Bltimore, Mrylnd, USA. 8 Present ddress: Deprtment of Physiology, Southern Illinois University Shool of Mediine, Crondle, Illinois, USA. 9 These uthors ontriuted eqully to this work. Correspondene should e ddressed to S.M.T. (sthom3@umrylnd.edu). Reeived 4 Deemer 212; epted 14 Ferury 213; pulished online 17 Mrh 213; doi:1.138/nn VOLUME 16 NUMBER 4 APRIL 213 nture NEUROSCIENCE

2 npg 213 Nture Ameri, In. All rights reserved. Figure 1 5-HT 1B R tivtion seletively potentites TA-CA1 ell exittory synpses. () Promoting umultion of endogenous serotonin y th pplition of the triyli ntidepressnt imiprmine (2 µm) potentited s in SLM of re CA1 of utely prepred hippompl slies (n = 8 slies) in ontrol sline (lk open irles), ut not in slies pretreted with the 5-HT 1B R ntgonist ismoltne (1 µm) (red tringles, n = 9 slies). Smple tres efore (lk) nd 6 min fter imiprmine pplition (red) in ontrol sline (top) or in the presene of ismoltne (ottom) re shown t right. TA, temporommoni. () Group dt showing the effet of ntidepressnts on s in ontrol slies (fluoxetine, n = 4 slies; itloprm, n = 3 slies) nd the effet of npirtoline ( µm) on s in ontrol slies (red, n = 11 slies; ANOVA, F 3,2 = 9.751, P <.1) or slies pretreted for 6 min with ntidepressnts (n = 5 slies pretreted with imiprmine, n = 5 slies with fluoxetine, n = 3 slies with itloprm, F 3,2 = 7.32, P =.2). Bonferroni post ho tests reveled tht ismoltne prevented the inrese in fepsp slope oserved with ntidepressnts lone (P <.5 versus imiprmine, fluoxetine or itloprm) nd tht npirtoline tretment differed from eh of these pretretment + npirtoline onditions (P <.5). P <.5, P <.1 ompred with efore npirtoline or ntidepressnt, pired t test. () The seletive 5HT 1B R gonist npirtoline reversily inresed the slope of s in SLM of re CA1 (open irles, n = 11 slies). Ismoltne (1 µm) loked the effet of npirtoline on s (red tringles, n = 5 slies). (d) seletively enhned TA-CA1 EPSCs reorded in whole-ell voltge-lmp mode (open irles), ut not simultneously evoked SC-CA1 EPSCs (filled irles) in two-pthwy experimentl design (n = 9 ells). Smple tres re shown t right. The seletive inrese in s y serotonin, ut not of SC-CA1 fepsps, is onsistent with the seletive loliztion of 5-HT 1B Rs in SLM. SC, Shffer ollterl. neuromodultors inhiit synpti trnsmission in the mmmlin CNS, lthough there is evidene tht serotonin n inrese glutmte relese in some systems 13,14. Serotonin reeptor type 1B (Hrt1) mrna is present in CA1 ells t high levels nd 5-HT 1B R inding sites re onentrted in SLM 15,16. We therefore sked whether 5-HT 1B Rs medite the effets of ute elevtion of serotonin on TA-CA1 synpti trnsmission. Imiprmine pplition did not potentite slope in slies pretreted with the ntgonist ismoltne (1 µm) (Fig. 1,), nor did imiprmine or fluoxetine ffet s in slies of hippompus tken from mie lking 5-HT 1B Rs 17 (Supplementry Fig. 2,). In ontrst, the 5-HT 1A R ntgonist NAN-19 (1 µm) hd no effet on potentition of s y imiprmine (Supplementry Fig. 2). Consistent with these findings, seletive gonist for 5-HT 1B Rs, npirtoline ( µm) 18, enhned the slope of s (Fig. 1). Unlike the ute potentiting effets of the ntidepressnts, the effets of npirtoline on fepsp slope were fully reversed fter 6 min of wshout. Applition of ismoltne during wshout of imiprmine deresed the potentition (Supplementry Fig. 1d), suggesting tht the mintined potentition indued y ntidepressnts ws result of persistent inrese in serotonin levels. lso produed douling of TA-CA1 EPSP slope in whole-ell voltgelmp (Fig. 1d) nd urrent-lmp (Supplementry Fig. 2d) reordings. hd no effet on fepsps in slies pretreted with ismoltne (Fig. 1). Another 5-HT 1B R seletive gonist, CP94253 (5 µm), lso potentited slope (Supplementry Fig. 2e). ntgonizes 5-HT 3 reeptors t higher onentrtions 19, ut the 5-HT 3 reeptor ntgonist Y-2513 (5 µm) hd no effet on slope (Supplementry Fig. 2e), inditing tht npirtoline s effet ws medited y 5-HT 1B Rs. indued potentition ws unffeted y depletion of serotonin with p-hlorophenyllnine 2 (Supplementry Fig. 1e), inditing tht npirtoline s effet ws medited y postsynpti heteroreeptors nd not the result of n tion on presynpti inhiitory utoreeptors slope (% ontrol) slope (% ontrol) 3 2 Imiprmine TA stim TA stim + Ismoltne + Ismoltne Before + Imiprmine + Ismoltne d EPSC slope (% ontrol) 2 ms.2 mv TA stim SC stim on serotonergi nerve terminls 21. Finlly, pplition of npirtoline hd no effet on slope in slies pretreted for 6 min with ntidepressnts (Fig. 1), presumly euse the endogenous serotonin hd lredy mximlly tivted 5-HT 1B Rs, oluding the potentition. Serotonin-medited potentition ws speifi for the TA-CA1 exittory synpses in SLM, where 5-HT 1B Rs re onentrted. Neither npirtoline (Fig. 1d nd Supplementry Fig. 2d) nor fluoxetine (Supplementry Fig. 2f) hd ny effet on Shffer ollterl CA1 (SC-CA1) ell responses to stimuli delivered in strtum rditum. Thus, tivtion of 5-HT 1B Rs y serotonin produes highly lolized potentition of exittory synpses. Temporommoni inputs re reltively ineffetive t induing tion potentil firing in CA1 pyrmidl ells, lthough modest Shffer ollterl ell tivtion filittes this proess 22. enhned the suprliner summtion of temporommoni Shffer ollterl EPSPs nd promoted temporommoni Shffer ollterl indued tion potentil firing (Supplementry Fig. 3,). Ativtion of 5-HT 1B Rs y endogenously relesed serotonin nd susequent potentition of the TA-CA1 pthwy therefore promotes tion potentil output from re CA1. Moleulr sis of 5-HT medited potentition Is 5-HT 1B R medited potentition of s medited preor postsynptilly? Neither the mplitude of the temporommoni fier volley (16 ± 3% of ontrol, n = 7 slies, P >.5) nor the piredpulse rtio of s (1.39 ± 1.3 efore, 1.34 ± 1. fter npirtoline, n = 11 slies, P >.5; eliited with pirs of temporommoni stimuli seprted y ms) were hnged signifintly y npirtoline (Fig. 2). Furthermore, npirtoline signifintly potentited the AMPAR-medited omponent of the exittory postsynpti urrent (EPSC), reorded under voltge lmp t 7 mv (161 ± 5% of ontrol, n = 7 slies, t 6 = 4.88, P =.3), wheres the NMDA reeptor medited omponent of the EPSC t +4 mv ws unffeted (83 ± 13% slope (% ontrol) Before + slies Imiprmine Imip + ismoltne Fluoxetine Citloprm TA stim TA SC Pretret with imiprmine Pretret with fluoxetine Pretret with itloprm SC stim pa 2 ms 2 ms pa nture NEUROSCIENCE VOLUME 16 NUMBER 4 APRIL

3 npg 213 Nture Ameri, In. All rights reserved. Figure 2 Expression of 5-HT 1B R indued potentition of TA-CA1 synpses is medited postsynptilly. () Time ourse of hnges of slope (lk irles), fier volley (open tringles) nd pired-pulse rtio (PPR, red irles) efore nd fter npirtoline pplition in typil experiment. Representtive tres re shown t right, with the fier volley t higher resolution ove. Neither fier volley (t 6 =.39, P =.97) nor PPR (t 1 = 1.17, P =.27) hnged with npirtoline tretment. () Whole-ell voltge-lmp reordings of EPSCs t 7 nd +4 mv in the sme ell efore (lk) nd 4 min fter pplition of npirtoline (red). () Left, imge of CA1 ell filled with Alex 594 from somti whole-ell pipette inditing sites of mirophotolysis of ged glutmte on distl pil dendrites in SLM or olique dendrites in strtum rditum. Sle r represents 2 µm. Right, th pplition of npirtoline inresed photolysis-evoked EPSPs eliited from dendrites in SLM in utely prepred hippompl rin slies (n = 5 ells), ut not from dendrites in strtum rditum (n = 6 ells in seprte experiments), inditing tht 5-HT 1B R tivtion seletively enhned postsynpti glutmte responses t sites of temporommoni synpse formtion. Smple EPSP-like photolysis responses eliited from dendrites in SLM nd strtum rditum efore (lk) nd fter npirtoline pplition (red) re shown t right. of ontrol, n = 5 ells, t 4 = 1.838, P =.14; Fig. 2). A similr seletive potentition of phrmologilly isolted AMPAR-medited fepsps ws lso oserved in extrellulr reordings (Supplementry Fig. 3). 5-HT 1B R indued potentition is therefore inonsistent with lssil presynpti expression mehnisms. Seletive 5-HT 1B R indued enhnement of AMPAR-medited responses t temporommoni synpses my result from postsynpti inrese in AMPAR funtion. We tested this hypothesis using mirophotolysis of ged glutmte 23. With photostimuli delivered in SLM, npirtoline pplition produed ner douling of the slope of AMPAR-medited photolysis-evoked EPSPs (phepsps; Fig. 2). Consistent with its lk of effet on SC-CA1 EPSPs, npirtoline did not inrese the slope of AMPAR-medited phepsps eliited y mirophotolysis of ged glutmte onto proximl olique dendrites in strtum rditum (Fig. 2). Potentition of phepsps ws not ompnied y hnges in either holding urrent or input resistne (dt not shown). Thus, tivtion of 5-HT 1B Rs speifilly nd seletively inreses the numer or effiy of postsynpti AMPARs in the distl pil dendrites of CA1 pyrmidl neurons in highly lolized mnner. Stimultion of 5-HT 1B Rs n tivte severl protein kinses 24,25, inluding lium/lmodulin-dependent protein kinse (CMK). We first nlyzed threonine 286 on CMKII, whih is utophosphorylted when CMK is tivted 26, in SLM tissue wedges sudisseted from hippompl slies. inresed CMK T286 phosphoryltion with time ourse omprle to the indution of the potentition (Fig. 3,). Inution of hippompl slies with KN62 (5 µm), speifi CMK inhiitor, loked the effet of npirtoline on slope (Fig. 3). Similrly, intrellulr dilysis with seletive CMK inhiitor peptide, utomtide-2-relted inhiitory peptide (AIP, 2 µm; Fig. 3) loked potentition of TA-CA1 EPSP slope y npirtoline. In ontrst, npirtoline-indued potentition of fepsps ws not ffeted y the protein kinse A (PKA) inhiitor PKI (1 µm; Fig. 3). We onlude tht CMK is tivted rpidly in response to 5-HT 1B R tivtion nd tht this tivtion is neessry for the potentition of TA-CA1 trnsmission. Multiple phosphoryltion sites in AMPARs, inluding two serines in the C terminus of the GluA1 suunit, re importnt for synpti long-term potentition (LTP) 27,28. produed timedependent inrese in the phosphoryltion of GluA1 t serine 831 (142 ± 11% of untreted, n = 11 slies, t 1 = 3.17, P =.1), the trget of Response (% efore npirtoline) Before Lser spot 1 SLM Str. rditum Str. pyrmidle After npirtoline Photolysis EPSP slope (% ontrol) 2 AMPA fepsp slope PPR Fier volley NMDA + Str. rditum protein kinse C nd CMK, ut not t the PKA site serine 845 (116 ± 8% of untreted, n = 4 slies, P >.5), s reveled in western lots of SLM tissue wedges (Fig. 3,,d). Consistent with the lk of potentition of SC-CA1 synpses, npirtoline hd no effet on CMK utophosphoryltion or GluA1 S831 phosphoryltion in isolted tissue from strtum rditum (Supplementry Fig. 4) or in SLM in tissue from Htr1 / mie (Supplementry Fig. 4). Consistent with the persistent potentition indued y ute pplition of ntidepressnts (Fig. 1), ute fluoxetine tretment resulted in tivted CMK nd phosphoryltion of GluA1 S831 tht persisted fter fluoxetine wshout, unlike npirtoline (Supplementry Fig. 4). Comprle time-dependent inreses in CMK tivtion nd GluA1 S831 phosphoryltion in response to npirtoline were otined with tissue from the NA ore (Fig. 3e) nd medil prefrontl ortex (Supplementry Fig. 4d), suggesting tht lol regultion of the strength of synpti exittion y serotonin my our in multiple rin regions tht re importnt for ognitive nd ffetive funtion. We next tested the hypothesis tht CMK-medited phosphoryltion of S831 is required for npirtoline-indued potentition using trnsgeni mie in whih GluA1 S831 ws mutted to lnine (GluA1 S831A mie), rendering it inple of eing phosphorylted y CMK 29. Neither npirtoline (Fig. 3f) nor fluoxetine (Supplementry Fig. 4e) hd ny effet on slope in slies prepred from these mie, lthough they potentited slope in slies from wild-type littermte mie. We onlude tht serotonin enhnes AMPAR funtion y tivting CMK nd inresing GluA1 phosphoryltion t serine 831. Serotonin tion influenes in memory onsolidtion Ativtion of CMK nd phosphoryltion of GluA1 re required for oth serotonin-indued potentition of TA-CA1 synpses nd onventionl LTP t SC-CA1 synpses 28,3. Indeed, indution of LTP t TA-CA1 synpses prevented susequent potentition y npirtoline (Fig. 4,) nd TA-CA1 LTP ould not e indued fter potentiting fepsps with npirtoline (Fig. 4). In ontrst, npirtoline did not potentite SC-CA1 fepsps or olude SC-CA1 LTP (Fig. 4d). We onlude 1 2 pa ms SLM Str. rditum.1 mv 2 ms SLM Fier volley 5 ms 1 mv ms 466 VOLUME 16 NUMBER 4 APRIL 213 nture neuroscience

4 npg 213 Nture Ameri, In. All rights reserved. Figure 3 tivtes CMK nd inreses phosphoryltion of GluA1 t serine 831 in SLM tissue. (,) Representtive western lots () nd quntifition () showing tht npirtoline ( µm, 2 h) time dependently inresed phosphoryltion of lph nd et CMK t T286 (moleulr weights 52 nd 6 kd; F 5,18 = 4.651, P =.7) nd GluA1 t S831 (F 5,24 = 3.76, P =.13), ut did not ffet totl GluA1 expression (F 5,18 =.863, P =.524) in isolted CA1 SLM tissue setions, sudisseted from whole hippompl slies. P <.5, P <.1 ompred with efore npirtoline. () The effet of npirtoline on s ws loked y th pplition of the speifi CMK inhiitor KN62 (5 µm) (n = 6 slies), ut not y th pplition of the ell-permele PKA inhiitor PKI (PKI, 1 µm, n = 3 slies). Intrellulr dilysis of the CMK inhiitor utomtide-2 relted inhiitory peptide (AIP, 2 µm) vi pth-pipettes (n = 5 ells) loked potentition of TA-CA1 EPSPs reorded in whole-ell mode. (d) inresed phosphoryltion of GluA1 t S831 in isolted CA1 SLM tissue setions, ut not t S845. (e) (Anp, µm for 6 min) inresed phosphoryltion of CMK T286 (left, Mnn-Whitney U test, z = 2.9, P =.4) nd GluA1 S831 (right, Mnn-Whitney U test, z = 2.9, P =.4) in tissue punhes tken from the ore of the NA (n = 6 slies eh). Con, ontrol. (f) did not potentite s in slies from GluA1 S831A mie (n = 7 slies from 4 nimls), ut produed strong potentition in slies from wild-type littermte mie (n = 4 slies from 4 nimls). Full-length lots re presented in Supplementry Figure 7. tht serotonin-indued potentition nd onventionl synpti LTP olude eh other t TA-CA1 exittory synpses, proly euse they shre ommon signling nd expression mehnisms. TA-CA1 synpses re strongly tivted during ognitive tsks, inluding oth sptil nd nonsptil tsks tht involve working nd ssoitive memory in primtes 31. In rodents, TA-CA1 synpses support lerning in the Morris wter mze 32 nd re required for long-term memory onsolidtion 12. To determine whether serotonin tivtes 5-HT 1B Rs t TA-CA1 synpses in intt rts, we sked whether 5-HT 1B R ntgonist would hve ny influene on onsolidtion of sptilly ued memory using the Morris wter mze (Fig. 5). slope (% efore HFS) slope (% efore npir) 2 HFS Adjust 12 HFS slope (% efore HFS) 2 e pcmk CMK pglua1 S831 Totl GluA1 slope (% ontrol) 5 min 1 min 3 min 1 h 2 h 4 NA pcmk Totl CMK pglua1 S831 Totl GluA1 Adjust HFS 4 8 LTP Adjusted + d SC-CA1 fepsp slope (% efore HFS) HFS PKI KN62 AIP 8 12 Phosphoprotein (% ontrol) 2 1 CMK Con S831 Anp Con Anp f slope (% ontrol) Phosphoprotein level (% ontrol) 3 2 d min 3 1 min 4 3 min 5 1 h 6 2 h pglua1 S831 Totl GluA1 pcmkll pglua1 S831 Totl GluA1 pglua1 S845 Totl GluA1 3 min Wild-type mie GluA1 S831A The time required for rts to lote hidden pltform eme shorter fter 6 d of trining. Suessful memory of the lotion ws verified in proe tril 24 h fter the finl trining session. Rts were then divided into two groups: one group reeived the 5-HT 1B R ntgonist SB (4 mg kg of ody weight, intrperitonel, dily for 2 weeks strting immeditely fter the proe tril) 33 nd the others did not (ontrols, sline, intrperitonel, dily). The rts underwent seond proe tril to test onsolidtion 28 d fter the end of trining. Both groups performed etter in this tril thn they hd efore trining, inditing tht they hd onsolidted some memory of the pltform lotion. Rts treted with SB216641, however, displyed shorter lteny to find the pltform lotion thn the untreted ontrols. Furthermore, the lteny of the SB treted rts to the pltform lotion ws not sustntilly different 28 d fter trining thn it ws in the 24 h proe tril. Treted rts lso rossed through the pltform re signifintly more times thn the ontrol rts (P <.5), Figure 4 5-HT 1B R medited potentition oludes LTP t TA-CA1 synpses nd influenes memory onsolidtion. () After indution of LTP with high-frequeny stimultion (HFS, four trins, 1 s per trin t Hz, 5-min intervl), th pplition of npirtoline ( µm) filed to indue further potentition of slope (n = 8 slies). () The effet of npirtoline on slope remined oluded fter indution of LTP, even when the stimultion intensity ws deresed to return the fepsp to the seline level (n = 5 slies). Representtive tres re shown elow. Sle rs represent.1 mv nd 2 ms. () -indued potentition of slope oluded tetnus-indued LTP t TA-CA1 synpses (n = 4 slies). (d) neither enhned SC-CA1 fepsps nor oluded LTP of SC-CA1 synpses (red, n = 7 slies). LTP of SC-CA1 fepsps in ontrol slies shown in lk (n = 5 slies). Only every other dt point is plotted to llow the two dt sets to e distinguished. nture NEUROSCIENCE VOLUME 16 NUMBER 4 APRIL

5 npg 213 Nture Ameri, In. All rights reserved. Figure 5 Sptil memory onsolidtion is ffeted y endogenous tivtion of 5-HT 1B Rs. () Lteny to find the trget pltform in the Morris wter mze deresed signifintly etween the first (nive) nd lst trining tril (trined), inditing tht the rts lerned the lotion of the pltform. Lteny remined short during proe tril dministered 24 h fter the lst trining tril, inditing tht the rts rememered the pltform lotion. Finlly, lteny remined signifintly shorter thn nive when tested 28 d fter the finl trining tril, demonstrting memory onsolidtion. Rts dministered 5-HT 1B R ntgonist (SB216641, 4 mg per kg, intrperitonel) dily for 14 d strting fter the 24-h proe tril displyed signifintly shorter espe lteny in the onsolidtion tril thn the ontrol group (.9% NCl, intrperitonel). Representtive exmples of the swim pth in the mze from the strt (white) to stop (lk) positions re shown t left for ontrol nd SB treted individuls. (,) SB treted rts lso spent more time in the trget qudrnt during the 28-d onsolidtion proe tril thn ontrols () nd swm signifintly shorter distne thn ontrols (). Both groups hd identil swim speeds (), however, inditing tht the differene in performne ws not result of ltered motor funtion. Full swim pths for two different individuls in the proe trils re shown t left. Dshed line indites rndom performne. P <.5, P =.6, n = 8 rts per group, post ho t test. Further sttistil informtion n e found in the Online Methods. nd spent more time in the trget qudrnt, inditing more urte serh pttern. Consistent with previous reports tht Htr1 / mie hve improved short-term lerning in the Morris wter mze 34, we onlude tht tivtion of 5-HT 1B Rs y endogenous serotonin interts with the memory onsolidtion proess, perhps through intertion with LTP t TA-CA1 synpses. Potentition is ltered in depression model The serotonin hypothesis of depression postultes tht hnges in the tions of serotonin ontriute to its pthology. Depression is Figure 6 Chroni stress enhnes, wheres hroni ntidepressnts eliminte, the effet of npirtoline on TA-CA1 synpti trnsmission. () Chnges in TA-CA1 EPSP slope in slies from CUS rts (red, n = 11 ells) nd ontrol littermtes (lk, n = 1 ells) showing the lrger mplitude nd persistene of potentition in CUS rts ompred with ontrols. Representtive tres efore nd fter npirtoline pplition nd fter 6 min of wshout re shown t right. Sle rs represent 2 mv nd ms. () Chnges in TA-CA1 EPSP slope indued y npirtoline in slies from CUS rts (red) were lrger nd more persistent thn the potentition seen in slies from ontrols (lk) (ontrols, pek hnge in fepsp slope = 162 ± 3%, fepsp slope fter 6-min wsh = 16 ± 3%, n = 1 slies; CUS, pek hnge = 226 ± 23%, 218 ± 21% fter 6-min wsh, n = 11 slies). () produed no signifint effet on fepsp slope in slies from rts treted for d with 24-h proe tril Consolidtion tril 24-h proe tril Consolidtion tril Vehile +5-HT 1B R ntgonist 3 Stressed rts TA-CA1 EPSP slope (% ontrol) slope (% ontrol) Vehile +5-HT 1B R ntgonist Vehile +5-HT 1B R ntgonist Time in trget qudrnt (%) Lteny to pltform lotion (s) Nive Vehile +5-HT 1B R ntgonist 24-h proe tril Trined purely humn ondition, ut depressive-like ehviorl stte n e indued in rodents. We therefore investigted serotonin-indued potentition in rts sujeted to the hroni unpreditle stress (CUS) model of depression, whih hs fe, onstrut nd preditive vlidity 35. After 3 weeks of CUS, rts displyed oth derese in surose preferene nd inresed lteny in novelty suppressed feeding (Supplementry Fig. 5,), s reported previously for rodent models of depression 35,36. We oserved tht the npirtoline-indued potentition of TA-CA1 EPSPs ws muh greter in slies from CUS rts thn in unstressed littermtes, nd tht EPSP slope remined littermtes Chroni imiprmine 2 4 Before + Wsh Stressed rt littermte littermtes 6 8 Chroni fluoxetine d slope (% ontrol) 3 Consolidtion tril (28 d) TA-CA1 EPSP slope (% ontrol) h proe tril Perent vehile-treted Consolidtion tril (28 d) Stressed rts Pth length Vehile +ntg Consolidtion tril (28 d) s Swim speed Vehile +ntg + Wshout s Stressed rts Stress + fluoxetine the ntidepressnts imiprmine ( mg l 1, red) or fluoxetine (8 mg l 1, lue), wheres it douled fepsp slope in slies from ontrols in interleved experiments (lk) (imiprmine, n = 9 slies; fluoxetine, n = 5 slies; ontrols, n = 7 slies). (d) Administrtion of fluoxetine for the finl 3 weeks in rts sujeted to 6 weeks of CUS (lue, n = 12 slies) resulted in restortion of trnsient npirtoline-indued potentition (lk, n = 11 slies), whih ws lerly different from the irreversile potentition in CUS rts (red, n = 5 slies). 468 VOLUME 16 NUMBER 4 APRIL 213 nture neuroscience

6 npg 213 Nture Ameri, In. All rights reserved. Surose preferene (%) Bseline SDS Fluoxetine ± SB Time (weeks) enhned even fter min of wshout in rts sujeted to CUS, ut not ontrol rts, in oth whole-ell (Fig. 6,) nd extrellulr (Supplementry Fig. 5,d) reordings. The persistene of the potentition ws independent of its mgnitude (Fig. 6) nd ws resistnt to ismoltne tretment (Supplementry Fig. 5e). Despite the inresed mgnitude of the response in CUS rts, the potentition remined speifi to the TA-CA1 synpse (Supplementry Fig. 5d). Elevtion of extrellulr serotonin with ute pplition of imiprmine lso produed lrge enhnement of slope in slies from rts sujeted to CUS (nive, 23 ± 46%, n = 4 slies; CUS, 37 ± 88%, n = 3 slies; 2 2 ANOVA min effet of imiprmine F 1,5 = 18.22, P =.8; min effet of group nive versus CUS, F 1,5 = 2.264, P =.2). We onlude tht endogenous serotonin n still e relesed from dorsl rphe fferents in the hippompus in rts sujeted to CUS nd tht the resting level of extrellulr serotonin is still regulted y the serotonin trnsporter, ut tht serotonin s downstrem signling t exittory synpses is inresed in rts sujeted to CUS. Mny ntidepressnts elevte extrellulr serotonin levels, nd this hroni elevtion of serotonin inreses AMPAR phosphoryltion in the hippompus 37. We sked wht effet hroni ntidepressnt tretment of nive rts might hve on 5-HT 1B R medited potentition. In hippompl slies from rts given imiprmine or fluoxetine vi their drinking wter for d, npirtoline-indued potentition of slope ws sent, ut ws present in slies from untreted littermte ontrols (Fig. 6). In ontrst, npirtoline produed norml (no SDS) SB (no SDS) SDS SDS + fluoxetine SDS + fluoxetine + SB Figure 7 5-HT 1B R dependent potentition of GluA1 reeptors is neessry for the therpeuti tion of ntidepressnts. () Surose preferene ws high in vehile-treted ontrol C57BL6j mie (lk) nd remined high. SDS eliminted surose preferene fter 1 week (light lue). Fluoxetine (8 mg l 1 ) restored surose preferene in SDS mie in whih it hd een lost (red). The 5-HT 1B R ntgonist SB (4 mg l 1 in drinking wter) hd no effet on surose preferene in non-sds mie (gry), ut prevented restortion of surose preferene in SDS mie treted with fluoxetine (drk lue). Asterisk indites signifintly less thn orresponding seline. () Surose preferene ws high in nive Htr1 / mie nd wild-type (Sv129Imj) mie, nd ws deresed in oth strins fter CUS. Fluoxetine restored surose preferene in wild-type, ut not in Htr1 /, mie. P <.5, different thn seline in sme genotype (Bonferroni orreted post ho). P <.5, different thn fter CUS (Bonferroni orreted post ho). Thus, 5-HT 1B R tivtion is required for fluoxetine to exert its therpeuti tion. () CUS deresed surose preferene in ontrol C57BL6j mie (Bonferroni orreted post ho, P <.2), Surose preferene (%) Sv129lmj Htr1 / 3 Bseline CUS CUS + fluox d Time immolie (s) 2 potentition in slies from rts treted with imiprmine for only 36 h (dt not shown), insuffiient time for most therpeutilly relevnt ehviorl effets in rodents 38 nd depressed humns. Chroni ntidepressnt tretment therefore prevents 5-HT 1B R tivtion from potentiting TA-CA1 synpses, n effet tht is diretly opposite to the enhnement of 5-HT 1B R indued potentition seen fter CUS. Finlly, we oserved tht restortion of norml surose preferene nd novelty suppressed feeding ehviors in rts sujeted to CUS for 3 weeks, followed y n dditionl 3 weeks of CUS nd fluoxetine dministrtion (Supplementry Fig. 5,), ws ompnied y restortion of norml mgnitude, reversile potentition of TA-CA1 synpses in response to npirtoline (Fig. 6d). A rief period of fluoxetine dministrtion (36 h), in ontrst, filed to restore either surose preferene or the reversile npirtoline response (Supplementry Fig. 6). Thus, the response of TA-CA1 synpses to 5-HT 1B R tivtion orreltes with the ffetive stte of the rts under severl onditions. Potentition required for ntidepressnt tion Is the 5-HT 1B R dependent potentition of exittory synpti trnsmission oserved in rin slies in response to ute elevtion of serotonin involved in the tion of hronilly dministered SSRIs in ssys of ntidepressnt response? The surose preferene test ws hosen s ehviorl endpoint euse of its preditive vlidity. To nswer this question, we first used phrmologil pproh in C57BL6j mie sujeted to the soil defet stress (SDS) model of Surose preferene (%) Til suspension test C57BL6j GluA1 S831A + Imipr + Imipr C57BL6j GluA1 S831A Bseline CUS CUS + fluox e Perent wild type GluA1 S831A Novelty Open suppressed field feeding whih ws restored y fluoxetine (seline versus fluoxetine, P >.5). Surose preferene ws lower in GluA1 S831A mie thn in C57BL6j mie (Student s t test, t 19 = 3.37, P <.5). Surose preferene in GluA1 S831A mie ws not ffeted y fluoxetine (t 14 =.335, P >.7 with pired t test), nd ntidepressnt-treted GluA1 S831A mie hd lower surose preferene thn ontrols (Student s t test, t 25 = 2.42, P <.5 with Bonferroni djustment for multiple omprisons). indites signifintly less thn C57BL6j seline, indites signifintly different thn C57BL6j seline, ut not GluA1 S831A seline. (d) In the til-suspension test, C57BL6J littermtes treted with imiprmine spent less time immoile thn those treted with sline (t 1 = 4.1, P <.5). GluA1 S831A mie treted with imiprmine spent less time immoile thn GluA1 S831A mie treted with sline (t 13 = 2.35, P <.5). indites signifintly different thn ontrol in sme genotype. A ehviorl response to ute dministrtion of ntidepressnts ws preserved in this ssy, unlike the response to hroni ntidepressnts in the surose preferene test. (e) Lteny to feeding in the novelty-suppressed feeding test ws longer in GluA1 S831A mie (n = 14) thn in wild-type C57BL6j mie (n = 13) (t 27 = 2.1, P <.5). GluA1 S831A mie did not differ from wild-type littermtes with regrd to time spent in the enter in the open field test, ut ompleted fewer line rossings thn littermtes, onsistent with hypo-loomotor phenotype, ut not inresed nxiety (t 13 = 2.29, P <.5). indites signifintly different thn wild type. Tken together with lower surose preferene, GluA1 S831A mie displyed depressive-like ehviorl phenotype. Further sttistil informtion n e found in the Online Methods. Lteny Center time Line rosses nture NEUROSCIENCE VOLUME 16 NUMBER 4 APRIL

7 npg 213 Nture Ameri, In. All rights reserved. depression 39 (Fig. 7). Surose preferene ws high in ontrol mie, nd remined high in the sene of SDS, ut eme deresed fter 1 week of SDS nd remined low during sueeding weeks of SDS. Administrtion of fluoxetine vi the drinking wter restored high level of surose preferene to SDS mie fter 1 week. Administrtion of the 5-HT 1B R ntgonist SB (4 mg l 1 ) 4 hd no effet on the high surose preferene of nive mie, ut prevented the inrese in surose preferene produed y hroni fluoxetine dministrtion in mie tht hd lost their surose preferene fter SDS. Similrly, nive Htr1 / mie hd high surose preferene 41 tht ws lost fter 3 5 weeks of CUS (Fig. 7). Surose preferene ws restored y 3 weeks of fluoxetine dministrtion in wild-type Sv129Imj mie sujeted to CUS, ut not in Htr1 / mie. We onlude tht 5-HT 1B R tivtion is neessry for the effiy of SSRIs in this ehviorl test, onsistent with previous evidene tht ute tivtion of 5-HT 1B Rs is suffiient for n ntidepressnt-like effet of SSRIs in the fored swim test 42. Is 5-HT 1B R medited potentition of AMPARs lso required? GluA1 S831A mie displyed low preferene for 1% surose during the seline period (Fig. 7). Administrtion of fluoxetine for 3 weeks filed to indue n inrese in preferene for 1% surose in GluA1 S831A mie, ut did restore surose preferene in wildtype C57BL6j mie tht hd lost their surose preferene fter CUS. In ontrst, GluA1 S831A mie did respond to ute imiprmine dministrtion in the til suspension test (Fig. 7d), inditing tht serotonin uptke remins sensitive to inhiition y triylis in these mie nd highlighting the importne of serotonin-indued plstiity of glutmtergi trnsmission in the ility of ntidepressnts to reverse nhedoni. The lk of preferene for 1% surose in nive GluA1 S831A mie suggested tht they might hve depressive-like phenotype. We therefore ompred novelty suppressed feeding in these mie with their wild-type ontrols (Fig. 7e). The GluA1 S831A mie required lmost twie s long s wild-type C57BL6j mie to egin eting food in the rightly lit, novel ren, onsistent with ehviorl hnges indued in severl niml models of depression 36. They lso displyed hypo-loomotor phenotype in the open field test, without evidene of nxiety (Fig. 7e). Tken together, our dt suggest tht 5-HT 1B R tivtion nd susequent phosphoryltion of GluA1 reeptors y CMK is required for the ility of hronilly dministered SSRIs nd triyli ntidepressnts to reverse nhedoni in these ssys. Beuse the surose preferene test does not depend on the hippompus, these results support the onlusion tht serotonin-indued potentition of exittory synpti trnsmission ours in multiple rin regions involved in hedoni ehviors. DISCUSSION We oserved tht SSRIs, triyli ntidepressnts, gonists of 5-HT 1B Rs nd onventionl LTP ll potentite exittory synpti trnsmission t TA-CA1 synpses nd olude eh other, suggesting tht the moleulr events triggered y serotonin nd onventionl ntidepressnts overlp with those underlying tivity-dependent synpti plstiity to notle degree 5. Ativtion of 5-HT 1B Rs enhned AMPAR-medited synpti exittion y tivting CMK nd using phosphoryltion of GluA1 t serine 831. We lso oserved tht long-term memory onsolidtion, whih depends on intt TA-CA1 synpti iruits 12,32, ws enhned y inhiiting 5-HT 1B Rs. We suggest tht relese of endogenous serotonin nd tivtion of 5-HT 1B Rs ours during memory onsolidtion, fter lerning hs tken ple, nd influenes the ility of TA-CA1 synpses to prtiipte in the onsolidtion proess. Unlike LTP, 5-HT 1B R medited potentition ws reversed rpidly fter withdrwl of gonist. Given the similrity of the iohemil pthwys, this differene is surprising nd nnot e redily explined. Indution of LTP requires CMK tivity, wheres mintenne of LTP does not 43. It therefore ppers tht LTP indution is ompnied y reruitment of some dditionl signling proess(es) tht re not reruited y 5-HT 1B R tivtion. A etter understnding of the seond-messenger pthwys downstrem of this form of potentition my revel key iohemil steps distinguishing the indution of potentition, whih seems to our vi ommon mehnism in oth forms of potentition, from the mintenne of potentition, whih only ours fter indution of LTP. TA-CA1 synpses were potentited y serotonin, ut nery SC-CA1 synpses were not. The signling proesses underlying this phenomenon must therefore e highly lolized in postsynpti CA1 ell dendrites. Serotonin relese n e predited to hnge the flow of informtion through the hippompl iruitry, fvoring the so-lled diret pthwy t the expense of the indiret pthwy. Regulting informtion flow through neurl iruit is unique mehnism of tion for neuromodultory trnsmitters nd my e generlly pplile in other rin regions. Conversely, dysregultion of informtion flow s result of ltered serotonin reeptor signling or ltered regultion of exittory synpses my ontriute to the ognitive dysfuntion of depression. Animls sujeted to hroni stress exhiit elevted gluoortioid levels, nd elevted gluoortioid levels dmge neuronl struture nd funtion in the hippompus 44, inluding impiring Shffer ollterl LTP 45. We oserved tht serotonin-indued potentition of TA-CA1 synpses ws ltered in two distint wys in hronilly stressed rts: the potentition ws inresed in mgnitude nd eme irreversile. We do not yet know the explntion for these hnges. A derese in initil synpti strength 6 ould potentilly ount for the inrese in the mgnitude of the potentition, ut the persistene of the potentition in CUS mie ws independent of its mgnitude (Fig. 6). Chroni ntidepressnt dministrtion in nive mie deresed the potentition nd restortion of surose preferene in CUS mie treted hronilly with ntidepressnts ws ompnied y restortion of the norml trnsient potentition. Short-term dministrtion of ntidepressnts hd no effet on serotonin-indued potentition in either nive or CUS mie. The response of TA-CA1 synpses to 5-HT 1B R tivtion orreltes well with the ffetive stte of the nimls under five experimentl onditions, nd therefore represents unique endophenotype of this model of depression. There is inresing evidene tht exittory synpses re ltered in models of depression 4,5. Depression of AMPAR-medited exittion of neurons in the rewrd iruitry of the NA is required for the genesis of some depressive-like ehviors fter hroni restrint stress, inluding nhedoni in the surose preferene test 1. The soure of the inputs tht eome depressed ws not identified, ut the NA reeives prominent projetions from the hippompus nd frontl ortex. The serotonin-medited plstiity tht we oserved my t to ountert this depression of NA exittory synpses in two wys. First, serotonin-indued potentition of TA-CA1 synpses inresed hippompl tion potentil dishrge (Supplementry Fig. 3), nd should inrese the exittory drive onto NA ells nd promote de-depression of NA inputs, therey ting to reverse nhedoni nd restore the rewrding vlues of peripherl stimuli, suh s surose. Consistent with this suggestion, tivtion of hippompl 5-HT 1B Rs hs een shown to inrese dopmine levels in the NA 46, nd inresed dopmine llevites depressive-like ehviors 47. The enhned nd persistent tion of serotonin in the CUS mie (Fig. 6) my e interpreted not only s unique stress-indued form of 47 VOLUME 16 NUMBER 4 APRIL 213 nture neuroscience

8 npg 213 Nture Ameri, In. All rights reserved. plstiity of exittory synpses, ut lso s potentil ompenstory response, ting to promote the de-depression of hippompl- NA inputs. Seond, medium spiny ells in the NA express high levels of Hrt1 mrna. If the plstiity tht we oserved lso ours in NA D1-expressing neurons, then diret potentition of exittory synpses should ountert stress-indued depression of exittory synpses 1. Consistent with this possiility, we oserved 5-HT 1B R indued tivtion of CMK nd phosphoryltion of S831 of GluA1 reeptors in the NA. Our results link ntidepressnt-indued elevtion of serotonin to funtionl potentition of exittory synpses in vivo for the first time, to the est of our knowledge. We found tht phrmologil inhiition or geneti deletion of 5-HT 1B Rs prevented the ility of hronilly dministered fluoxetine to restore surose preferene in two hroni stress models. Furthermore, surose preferene ws low in GluA1 S831A mie nd ws not inresed y 3 weeks of fluoxetine dministrtion. We therefore suggest tht onventionl ntidepressnts exert t lest some of their therpeuti tions y promoting on-going serotonin-indued, 5-HT 1B R medited potentition of exittory synpti trnsmission. It is noteworthy tht ute pplition of SSRIs produed rpid potentition of exittory synpti trnsmission in rin slies, wheres the therpeuti tion of SSRIs in depressed humns nd in niml models of depression requires severl weeks. We do not yet hve n explntion, ut suggest tht indution of potentition is ritil first step tht improves ffetive stte y promoting tion potentil firing, whih susequently triggers other slower, synergisti, tivity-dependent proesses 1, suh s growth ftor signling, neurogenesis nd hnges in gene expression. Repeted outs of potentition my e required to produe lsting hnges, s hs een oserved with deep rin stimultion nd eletroonvulsive therpy. Given the plethor of ehviorl hnges tht define depression, it is unlikely tht there is one speifi rin region tht would e ritil to the ility of ntidepressnts to restore these funtions. The plstiity tht we oserved for TA-CA1 synpses is ttrtive s potentil explntion for some of serotonin s therpeuti tions euse it is generlizle: similr to LTP for lerning nd memory, this form of plstiity my, in priniple, tke ple t ny exittory synpse t whih 5-HT 1B Rs re expressed nd therey re-normlize tivity in lmost ny region in the depressed rin. This is in ontrst with the well-doumented ility of serotonin to promote dentte gyrus neurogenesis, whih does not our in ny other rin region. The TA-CA1 synpse serves s useful rhetype to study the tions of serotonin on exittory synpti trnsmission nd the orreltes of depression-like stte. Elevting monomine levels improves the ffetive stte of depressed ptients, whih led to the serotonin hypothesis of depression 3, ut the evidene of defiieny in serotonin synthesis or relese in depression is modest. Insted, our results suggest n exittory synpse hypothesis of depression. We postulte tht hnges in exittory synpses re fundmentl to the genesis of depression nd tht their restortion is ritil to the relief of depression. We found tht indution of serotonin-medited potentition ws normlly enhned nd prolonged fter CUS nd tht GluA1 S831A mie, similr to Gri1 / mie 48, displyed depressive-like phenotype. Similrly, glutmte reeptor expression is deresed 6 nd exittory synpti trnsmission is depressed 1 in other systems fter repeted stress. Serotonin-medited potentition would e predited to restore the norml strength of synpses wekened y hroni stress. Indeed, we oserved tht 5-HT 1B R tivtion nd GluA1 phosphoryltion t S831 were neessry for fluoxetine to restore surose preferene in hronilly stressed nimls, suggesting tht this tion my e ritil to the therpeuti effiy of SSRIs. It is noteworthy tht these effets of onventionl, slow-ting ntidepressnts, suh s SSRIs nd triylis, re shred y newly identified, fst-ting ntidepressnts. For exmple, ketmine potentites exittory synpses in hippompl slies nd its immeditely effetive therpeuti tions hve een ttriuted to enhned tivtion of glutmtergi iruits 49. Potentition of exittory synpti trnsmission y serotonin in multiple rin regions involved in ognitive funtion, nd its idiretionl ltertion y stress nd hroni ntidepressnts, provides link etween the pthology of depression nd its tretment, nd represents new perspetive on depression nd ntidepressnt tion. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Note: Supplementry informtion is ville in the online version of the pper. Aknowledgments We thnk S. Ahmri nd R. Hen (Columi University) for providing Hrt1 / mie, B. Alger, T. Blnpied, T. Gould, J. Kim nd M. MCrthy for their omments, S. Aungst for dvie on immunohistohemistry, nd H. Zimmermn, L. Mok nd M. Tylor for tehnil ssistne. This work ws funded y Mr. nd Mrs. Roert nd Lee Peterson Southwest Florid Ntionl Alline for Reserh on Shizophreni nd Depression Young Investigtor Awrd (X.C.), the Howrd Hughes Medil Institute (R.L.H.) nd the US Ntionl Institutes of Helth (R.L.H., H.-K.L., X.C. nd S.M.T.). AUTHOR CONTRIBUTIONS X.C., A.J.K., M.D.K., A.M.B. nd S.M.T. designed the study. X.C., A.J.K., M.D.K., S.G., K.G. nd A.M.B. performed the experiments nd nlyzed the dt. H.-K.L. nd R.L.H. provided the GluA1 S831A mie. X.C., A.J.K. nd S.M.T. prepred the mnusript. All of the uthors disussed the results nd ommented on the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Krishnn, V. & Nestler, E.J. The moleulr neuroiology of depression. Nture 455, (8). 2. Billings, A.G., Cronkite, R.C. & Moos, R.H. Soil-environmentl ftors in unipolr depression: omprisons of depressed ptients nd nondepressed ontrols. J. Anorm. Psyhol. 92, (1983). 3. Heninger, G.R., Delgdo, P.L. & Chrney, D.S. The revised monomine theory of depression: modultory role for monomines, sed on new findings from monomine depletion experiments in humns. Phrmopsyhitry 29, 2 11 (1996). 4. Dumn, R.S. & Aghjnin, G.K. Synpti dysfuntion in depression: potentil therpeuti trgets. Siene 338, (212). 5. Snor, G., Zrte, C.A., Krystl, J.H. & Mnji, H.K. Trgeting the glutmtergi system to develop novel, improved therpeutis for mood disorders. Nt. Rev. Drug Disov. 7, (8). 6. Yuen, E.Y. et l. Repeted stress uses ognitive impirment y suppressing glutmte reeptor expression nd funtion in prefrontl ortex. Neuron 73, (212). 7. Cmpell, S. & Mqueen, G. The role of the hippompus in the pthophysiology of mjor depression. J. Psyhitry Neurosi. 29, (4). 8. Hikie, I. et l. Redued hippompl volumes nd memory loss in ptients with erly- nd lte-onset depression. Br. J. Psyhitry 186, (5). 9. Phillipson, O.T. & Griffiths, A.C. The topogrphi order of inputs to nuleus umens in the rt. Neurosiene 16, (1985). 1. Lim, B.K., Hung, K.W., Grueter, B.A., Rothwell, P.E. & Mlenk, R.C. Anhedoni requires MC4R-medited synpti dpttions in nuleus umens. Nture 487, (212). 11. Ihr, N., Ued, S., Kwt, M. & Sno, Y. Immunohistohemil demonstrtion of serotonin-ontining nerve fiers in the mmmlin hippompl formtion. At Ant. 132, (1988). 12. Remondes, M. & Shumn, E.M. Role for ortil input to hippompl re CA1 in the onsolidtion of long-term memory. Nture 431, (4). 13. Lme, E.K., Goldmn-Rki, P.S. & Aghjnin, G.K. Serotonin indues EPSCs preferentilly in lyer V pyrmidl neurons of the frontl ortex in the rt. Cere. Cortex 1, (). nture NEUROSCIENCE VOLUME 16 NUMBER 4 APRIL

9 npg 213 Nture Ameri, In. All rights reserved. 14. Koyshi, K., Iked, Y., Hned, E. & Suzuki, H. Chroni fluoxetine idiretionlly modultes potentiting effets of serotonin on the hippompl mossy fier synpti trnsmission. J. Neurosi. 28, (8). 15. Aït Amr, D., Segu, L., Nili, S. & Buhot, M.C. Serotonin 1B reeptor regultion fter dorsl suiulum defferenttion. Brin Res. Bull. 38, (1995). 16. Sri, Y. et l. Cellulr nd suellulr loliztion of 5-hydroxytryptmine1B reeptors in the rt entrl nervous system: immunoytohemil, utordiogrphi nd lesion studies. Neurosiene 88, (1999). 17. Sudou, F. et l. Enhned ggressive ehvior in mie lking 5-HT 1B reeptor. Siene 265, (1994). 18. Svenningsson, P. et l. Altertions in 5-HT 1B reeptor funtion y p11 in depressivelike sttes. Siene 311, 77 8 (6). 19. Göthert, M. et l. 5-HT 3 reeptor ntgonism y npirtoline, mixed 5-HT 1 reeptor gonist/5-ht 3 reeptor ntgonist. Br. J. Phrmol. 114, (1995). 2. Dewr, K.M., Grondin, L., Crli, M., Lim, L. & Reder, T.A. [ 3 H]proxetine inding nd serotonin ontent of rt ortil res, hippompus, neostritum, ventrl mesenephli tegmentum, nd midrin rphe nulei region following p-hlorophenyllnine nd p-hloromphetmine tretment. J. Neurohem. 58, (1992). 21. Shrp, T., Brmwell, S.R. & Grhme-Smith, D.G. 5-HT 1 gonists redue 5-hydroxytryptmine relese in rt hippompus in vivo s determined y rin mirodilysis. Br. J. Phrmol. 96, (1989). 22. Jrsky, T., Roxin, A., Kth, W.L. & Spruston, N. Conditionl dendriti spike propgtion following distl synpti tivtion of hippompl CA1 pyrmidl neurons. Nt. Neurosi. 8, (5). 23. Ci, X. et l. Unique roles of SK nd Kv4.2 potssium hnnels in dendriti integrtion. Neuron 44, (4). 24. Hsu, E.H., Lohn, A.C. & Cowen, D.S. Ativtion of Akt1 y humn 5-hydroxytryptmine (serotonin)1b reeptors is sensitive to inhiitors of MEK. J. Phrmol. Exp. Ther. 298, (1). 25. Leone, A.M., Errio, M., Lin, S.L. & Cowen, D.S. Ativtion of extrellulr signlregulted kinse (ERK) nd Akt y humn serotonin 5-HT(1B) reeptors in trnsfeted BE(2)-C neurolstom ells is inhiited y RGS4. J. Neurohem. 75, (). 26. Giese, K.P., Fedorov, N.B., Filipkowski, R.K. & Silv, A.J. Autophosphoryltion t Thr286 of the lph lium-lmodulin kinse II in LTP nd lerning. Siene 279, (1998). 27. Rohe, K.W., O Brien, R.J., Mmmen, A.L., Bernhrdt, J. & Hugnir, R.L. Chrteriztion of multiple phosphoryltion sites on the AMPA reeptor GluA1 suunit. Neuron 16, (1996). 28. Lee, H.K., Brrosie, M., Kmeym, K., Ber, M.F. & Hugnir, R.L. Regultion of distint AMPA reeptor phosphoryltion sites during idiretionl synpti plstiity. Nture 45, (). 29. Lee, H.-K., Tkmiy, K., Hen, K., Song, L. & Hugnir, R.L. Speifi roles of AMPA reeptor suunit GluR1(GluA1) phosphoryltion sites in regultion synpti plstiity in the CA1 region of the hippompus. J. Neurophysiol. 13, (21). 3. Mlinow, R. & Mlenk, R.C. AMPA reeptor trffiking nd synpti plstiity. Annu. Rev. Neurosi. 25, (2). 31. Syirsk, E., Dvhi, L. & Goldmn-Rki, P.S. Prominene of diret entorhinl- CA1 pthwy tivtion in sensorimotor nd ognitive tsks reveled y 2-DG funtionl mpping in nonhumn primte. J. Neurosi. 2, (). 32. Nkshi, T., Young, J.Z., MHugh, T.J., Buhl, D.L. & Tonegw, S. Trnsgeni inhiition of synpti trnsmission revels role of CA3 output in hippompl lerning. Siene 319, (8). 33. Ttrzyńsk, E., Kłodzińsk, A., Sthowiz, K. & Chojnk-Wójik, E. Effets of seletive 5 HT1B reeptor gonist nd ntgonists in niml models of nxiety nd depression. Behv. Phrmol. 15, (4). 34. Buhot, M.C. et l. Sptil lerning in the 5 HT1B reeptor knokout mouse: seletive filittion/impirment depending on the ognitive demnd. Lern. Mem. 1, (3). 35. Willner, P., Towell, A., Smpson, D., Sophokleous, S. & Must, R. Redution of surose preferene y hroni unpreditle mild stress, nd its restortion y triyli ntidepressnt. Psyhophrmology (Berl.) 93, (1987). 36. Dvid, D.J. et l. Neurogenesis-dependent nd -independent effets of fluoxetine in n niml model of nxiety/depression. Neuron 62, (9). 37. Svenningsson, P. et l. Involvement of stritl nd extrstritl DARPP-32 in iohemil nd ehviorl effets of fluoxetine (Proz). Pro. Ntl. Ad. Si. USA 99, (2). 38. Bondi, C.O., Rodriguez, G., Gould, G.G., Frzer, A. & Morilk, D.A. Chroni unpreditle stress indues ognitive defiit nd nxiety-like ehvior in rts tht is prevented y hroni ntidepressnt drug tretment. Neuropsyhophrmology 33, (8). 39. Mltynsk, E. & Knpp, R. Dominnt-sumissive ehvior s models of mni nd depression. Neurosi. Bioehv. Rev. 29, (5). 4. Gster, L.M. et l. The seletive 5 HT1B reeptor inverse gonist 1 -methyl-5-[[2 - methyl-4 -(5-methyl-1,2,4-oxdizol-3-yl)iphenyl-4-yl]ronyl]-2,3,6, 7-tetrhydro-spiro[furo[2,3-f]indole-3,4 -piperidine](sb ) potently loks terminl 5-HT utoreeptor funtion oth in vitro nd in vivo. J. Med. Chem. 41, (1998). 41. Behtholt, A.J., Smith, K., Gughn, S. & Luki, I. Surose intke nd fsting gluose levels in 5-HT(1A) nd 5-HT(1B) reeptor mutnt mie. Physiol. Behv. 93, (8). 42. O Neill, M.F. & Conwy, M.W. Role of 5-HT 1A nd 5-HT 1B reeptors in the medition of ehvior in the fored swim test in mie. Neuropsyhophrmology 24, (1). 43. Burd, I. et l. CMKII utonomy is required for inititing ut not for mintining neuronl long-term informtion storge. J. Neurosi. 3, (21). 44. Krugers, H.J., Lussen, P.J., Krst, H. & Joëls, M. Chroni stress effets on hippompl struture nd synpti funtion: relevne for depression nd normliztion y nti-gluoortioid tretment. Front. Synpti Neurosi. 2, 1 1 (21). 45. Pvlides, C., Nivon, L.G. & MEwen, B.S. Effets of hroni stress on hippompl long-term potentition. Hippompus 12, (2). 46. Boulenguez, P. et l. Modultion of dopmine relese in the nuleus umens y 5HT lb gonists: Involvement of the hippompo-umens pthwy. Neurophrmology 35, (1996). 47. Chudhury, D. et l. Rpid regultion of depression-relted ehviours y ontrol of midrin dopmine neurons. Nture 493, (213). 48. Chourji, S. et l. AMPA reeptor suunit 1 (GluR-A) knokout mie model the glutmte hypothesis of depression. FASEB J. 22, (8). 49. Autry, A.E. et l. NMDA reeptor lokde t rest triggers rpid ehviourl ntidepressnt responses. Nture 475, (211). 472 VOLUME 16 NUMBER 4 APRIL 213 nture neuroscience

10 npg 213 Nture Ameri, In. All rights reserved. ONLINE METHODS All protools were pproved y the University of Mrylnd Shool of Mediine nd St. Mry s College of Mrylnd Institutionl Animl Cre nd Use Committees. Aute slie eletrophysiology. Hippompl slies were prepred from 3 6-week-old mle Sprgue-Dwley rts or mie (C57BL6j or Sv129Imj, s desried). Dissetion ws done in ie-old rtifiil ererospinl fluid (ACSF; 124 mm NCl, 3 mm KCl, 1.25 mm NH 2 PO 4, 1.5 mm MgCl 2, 2.5 mm CCl 2, 26 mm NHCO 3 nd 1 mm gluose) uled with 95% O 2 /5% CO 2. Brin slies (4 µm) were ut on virtome nd kept in holding hmer t 2 22 C t the interfe of ACSF nd humidified 95% O 2 /5% CO 2 for >1 h. The slies were then trnsferred to sumersion-type reording hmer nd perfused t 2 22 C with ACSF (flow rte = 1 2 ml min 1 ). Pirotoxin (.1 mm) nd CGP52432 (2 µm) were inluded to lok GABA A nd GABA B reeptors. Are CA3 nd the dentte gyrus were ut from the slie to prevent spontneous epileptiform dishrge (Supplementry Fig. 1). Conentri ipolr tungsten eletrodes were pled either in SLM to stimulte temporommoni fferents or in strtum rditum to stimulte Shffer ollterl fferents (Supplementry Fig. 1). Extrellulr reording pipettes were filled with ACSF (3 5 MΩ) nd pled µm from the stimulting eletrodes. Stimuli (-µs durtion) were delivered t.5 Hz. The stimulus intensity ws set t % of threshold intensity, resulting in fepsp of.1.2 mv. All ompounds were pplied y perfusion. fepsps were reorded using Axolmp 2B (Moleulr Devies) or n.p.i. (NPI) mplifiers, filtered t 1 khz, nd mplified efore digitiztion. Whole-ell intrellulr reordings were otined with pth pipettes (tip resistnes = 3 6 MΩ) filled with 135 mm esium or potssium methnesulphte (for voltge- or urrent-lmp reording, respetively), 1 mm HEPES, 1 mm NCl, 1 mm MgCl 2,.1 mm K 4 BAPTA, 2 mm Mg 2+ -ATP nd 1 mm phosphoretine (djusted to ph 7.3 with KOH). All experiments were performed t 2 22 C. Whole-ell reordings were mde from ell odies nd were mde lind. Photolysis. As desried previously 23, n rgon ion lser fitted with ultrviolet optis ws used to produe ontinuous 3-mW em of ultrviolet light t the 355- nd 361-nm lines. The light ws foused into 25-µm multimode qurtz fier nd delivered to the preprtion vi dihroi mirror, so s to permit simultneous wide-field exittion with n HBO lmp. Lser flsh durtion ws ontrolled y high-speed eletromehnil shutter. The proximl end of the fier ws foused vi rely lens ssemly in onjugte imge plne with respet to the ell nd positioned with miromnipultors ner the enter of the field of view through the 6 (1. N.A.) wter-immersion ojetive of n upright mirosope (Nikon). The lotion nd fous of the ultrviolet spot in the tissue ws determined from exittion of dye-filled dendriti shft y n ttenuted em. Intrellulr Alex 568 fluoresene ws imged with CCD mer (Hmmtsu Or ER II, effetive pixel size =.18 µm 2 ) ontrolled y Simple PCI softwre (Compix). Cged glutmte (.5 mm N-Nm-Glu) nd the ntioxidnt Trolox (.1 mm) were dded vi perfusion. Western lotting. Protein expression ws quntified using western lotting. Are SLM or strtum rditum tissue wedges were disseted from hippompl slies (3 4) under ie-old sline, pooled, nd homogenized in ie-old lysis solution ontining phosphtse nd protese inhiitors (PPI, Sigm) nd smple uffer (Lemmli, Sigm), oiled, nd loded into 4 12% Bis-Tris gel (Invitrogen). After running in 1 NuPge MOPS SDS uffer, the gel ws trnsferred onto polyvinylidene difluororide memrnes in 1 Nupge trnsfer uffer (in 1% methnol, wt/vol). The memrne ws loked with 5% nonft dry milk (wt/vol) in uffer ontining 1 M Tris-uffered sline nd.5% Tween (wt/vol), nd proed with ntiodies to Ser845-phosphorylted GluA1 (1:1,, Millipore AB5849), Ser831-phosphorylted GluA1 (1:1,, Sigm A4352) nd Thr286-phosphorylted CMKII (1:5,, Cell Signling Tehnology #3361) t 4 C overnight. After rinses in TBS-Tween, the memrne ws inuted for 1 h t 2 22 C in horserdish peroxidse onjugted got ntiody to rit IgG (1:1,, Cell Signling Tehnology #774). The immunolot ws developed with enhned hemiluminesene (Amershm). Memrnes were then stripped, loked nd reproed with ntiodies to GluA1 (.5 µg ml 1, Thermo Sientifi PA ), CMKII (1:2,, Cell Signling Tehnology #3362) or β-tin (1:2,, Cell Signling Tehnology #4967). Levels of phosphoryltion, expressed s the rtio of phospho-speifi intensity divided y totl protein intensity nd omputed with ImgeJ, were used for sttistil nlysis. For disply purposes, lots were ropped (see Supplementry Fig. 7) nd rightness nd ontrst were djusted glolly using Photoshop. Serotonin depletion. Rts were given two injetions of the tryptophn hydroxylse inhiitor p-hlorophenyllnine (3 mg per kg, intrperitonel, 24 h prt) 2 efore prepring slies for eletrophysiologil reording. Tissue from the sme nimls ws fixed, mounted in geltin, setioned t µm, nd stined with polylonl ntiody to serotonin (1:1,, Millipore AB125), followed y leling with seondry ntiody onjugted with the fluorophore Cy2 nd epifluoresene mirosopy. CUS proedure. Adult mle Sprgue-Dwley rts (3 4 weeks old) were rndomly divided into ontrol nd CUS groups. CUS rts were treted s follows: dy 1, ge rottion (3 h), fored swim (5 min), food deprivtion (16 h); dy 2, stroe light (3 min), restrint (3 min), food nd wter deprivtion (16 h); dy 3, stroe light (3 min), soil isoltion (16 h); dy 4, stroe light (3 min), restrint (3 min); dy 5, ge rottion (3 h), wter deprivtion (16 h); dy 6, restrint (3 h), soil isoltion (16 h); dy 7, ge rottion (3 h), restrint (3 min). The yle ws repeted over 3 5 weeks. Eletrophysiologil experiments were then performed nd nlyzed with the experimenter linded to the experimentl ondition of the rts. SDS proedure. Individul mle test mie (>4 weeks old) were introdued into the home ge of n unfmilir resident mouse for 5 min nd oserved to ensure tht they were physilly ttked nd defeted, s indited y freezing ehvior nd sumissive posture. Resident mie were CD1 reeders seleted for short ttk ltenies. After 5 min of physil intertion, the test mouse ws kept for 1 h in the resident s ge while eing seprted y perforted sreen tht proteted the test mouse from physil ontt, ut permitted olftory, visul nd uditory ontt. The SDS protool ws mintined for 5 weeks. To void individul differenes in intensity of defet, the test nimls were onfronted with different resident eh dy. Surose preferene. After 3 h of wter deprivtion, rts were given hoie etween two ottles for 3 4 h, one with 1% surose solution (wt/vol) nd nother with norml drinking wter. To prevent the possile effets of side preferene in drinking, the position of the ottles ws reversed fter 2 h. Mie were tested over 12 h (6 h light/6 h drk) with ottle positions swithed fter 6 h. The onsumption of wter nd surose were mesured y weighing the ottles. Preferene for surose ws lulted s perentge of onsumed suose-ontining solution reltive to the totl mount of liquid intke. % mens tht they drnk eqully from oth ottles, tht is, they hd no preferene for surose. Nive rts were tested repetedly in group housing until the ge hd onsistent preferene for 1% surose, ut were lwys tested individully therefter. For experiments with only wild-type rts, ny individul tht did not demonstrte surose preferene >65% ws disrded. In some experiments, rts were first trined to the tsk using 2% surose. Tests were repeted one per week during the ourse of CUS or ntidepressnt dministrtion. Novelty supressed feeding. Novelty suppressed feeding tests were performed s previously. The test pprtus ws rightly lit ren ( m for rts, 4 25 m for mie) with solid floor pled in dimly lit room. The floor of the ox ws overed with lyer of edding. Two lortory how pellets were pled on white pper irle pltform positioned in the enter of the ox. Rts tht hd een food deprived for 24 h, nd mie tht were food deprived for 3 h, were gently pled in orner of the ren. The lteny to egin eting, defined s tive hewing of the pellet, ws reorded. A mximum time llowne ws set t 6 s. Immeditely fter the test, nimls were returned to their home ge nd llowed to feed for 5 min. Food pellets were weighed efore nd fter the 5 min, nd the mount of food onsumed ws lulted. Rts tht te less thn.3 g of food in this 5-min period were removed from ll nlyses to ensure tht only suffiiently hungry nimls were inluded. Similrly, mie tht filed to tively hew on pellet were not inluded. doi:1.138/nn.3355 nture NEUROSCIENCE

11 npg 213 Nture Ameri, In. All rights reserved. Open field test. Mie were pled in m plexiglss ox for 5 min. The ox ws divided into 12 squres using tpe on the ottom of the ox. Mie were video reorded nd linded experimenter lulted the time spent in enter two squres nd the numer of line rossings for eh niml. Til suspension test. Mie were intrperitonelly injeted with sline or 3 mg per kg of imiprmine 3 min efore testing. Eh mouse ws tped to wooden horizontl r 2 inhes from the se of its til. A linded experimenter reorded the mount of time spent immoile for 6-min period. Antidepressnt tretment. Animls were given ntidepressnts vi their drinking wter to minimize stress (imiprmine, mg l 1 ; fluoxetine, 8 mg l 1 ). Animls were housed singly nd drinking wter ws hnged every 3 d. Animls were given ntidepressnts ontinully for 3 4 weeks. nimls reeived wter only. Experiments were then performed nd nlyzed with the experimenter linded to the experimentl ondition of the nimls. Dt nlysis. Experiments were performed with the investigtors linded to the genotype of the nimls or the identity of the sustne eing pplied whenever possile. The lind ws not roken until dt nlysis ws omplete. For quntifition of npirtoline nd ntidepressnt tions in eletrophysiologil experiments, fepsp or EPSC slope vlues were verged nd quntified over 3-min period preeding pplition of the sustne (ontrol) nd 3-min period t the end of the sustne pplition (effet). Memory onsolidtion. 16 mle 7 8-d-old Sprgue Dwley rts were housed two per ge in temperture-ontrolled room. Rts were kept on 12 h:12 h light-drk shedule nd ll testing ws done during the light phse. All rts were hndled for pproximtely 5 min d 1 for the 4 d efore testing. The rts were given unlimited ess to food nd wter throughout the testing periods. The wter mze (139.7 m in dimeter) ws filled with lk-olored wter reted with non-toxi lk pint. During trining, ler Plexigls pltform (1.16 m in dimeter) ws sumerged.5 m elow the wter surfe. Three lk nd white geometri sptil ues were pled round the mze nd remined in fixed lotion throughout trining nd testing. Eh rt ws rndomly ssigned qudrnt (NW, NE, SE, SW) in whih the pltform would lwys e loted. As desried previously 12, rts were then given ten trining loks in the wter mze ross 6 d. Dys 1 nd 6 ontined one trining lok. Dys 2 5 ontined two trining loks seprted y minimum of 2 h. Eh trining lok onsisted of four trining trils in whih the niml ws pled in rndom strting lotion in the pool (NW, NE, SE, SW) nd given 12 s to swim to the pltform. The niml ws llowed to remin on the pltform for 3 s. If the rt did not reh the pltform in 12 s, it ws guided to the pltform nd left to sit on the pltform for 3 s. Animls were given 2-min intertril intervl. Two proes tests were onduted in whih the pltform ws removed, nimls were pled in rndom strting lotion nd were then llowed to swim in the mze for 6 s. One proe test ws given 24 h fter the ompletion of the finl trining lok. The seond proe ws given 28 d fter the originl proe tril. Lteny to the trget, distne to trget, time spent in the trget qudrnt nd swim speed were reorded y HVS Imge softwre. Following ompletion of the first proe test, nimls were treted with either SB (Toris) or.9% physiologil sline (wt/vol) for 14 onseutive dys. Intrperitonel injetions of SB (4 mg per kg, 4 mg ml 1 ) or sline were given t the sme time eh dy immeditely fter light onset in the housing room. The nimls did not differ in performne during the originl trining loks. A 2 2 mixed ANOVA (Blok Group) ws used to exmine performne on the originl nd finl trining lok. The espe lteny deresed signifintly from lok 1 (men = 44.34, s.e.m. = 3.1) to lok 1 (men = 8.1, s.e.m. =.9) (F 1,13 = 17.4, P <.1). There ws no differene etween untreted (men = 26.4, s.e.m. = 2.1) nd future SB treted (men = 26., s.e.m. = 2.1) nimls (F 1,13 =.2, P >.5). There ws no intertion (F 1,13 =.36, P >.5). Exmintion of performne on the proe tril given 24 h fter the finl trining lok lso showed no group differenes. There ws no differene in the perentge of time spent in the trget qudrnt (t 14 =.91, P >.5). There ws no differene in pth tken to the trget re (t 14 =.46, P >.5). There ws no differene in lteny to the trget re (t 14 =.3, P >.5) nd no differene in swim speed etween the nimls (t 14 =.51, P >.5). The nimls were tested for onsolidtion of originl trining 28 d fter the end of trining in the wter mze. Animls treted with SB demonstrted signifintly improved retention of sptil informtion. During the proe tril, nimls treted with SB (men = 42.66, s.e.m. = 3.4) spent n inresed perentge of time in the trget qudrnt ompred with the untreted nimls (men = 32.41, s.e.m. = 3.69) (t 14 = 2.4, P =.6). Animls treted with SB (men = 1.77, s.e.m. =.42) used shorter pth to the trget re thn the untreted nimls (men = 4.5, s.e.m. =.82) (t 14 = 2.48, P =.27). The SB treted nimls (men = 11.11, s.e.m. = 2.6) lso demonstrted signifintly shorter lteny to the trget re thn the untreted nimls (men = 26.26, s.e.m. = 5.89) (t 14 = 2.41, P =.3). There were no differenes in swim speed etween the two groups (t 14 =.5, P >.5). Therpeuti tions of 5-HT 1B Rs. SB ws used s the 5HT 1B R ntgonist nd ws given to mie vi drinking wter. SB ws dministered t 4 mg l 1. Wter levels were heked dily to ensure eh mouse ws reeiving the intended SB dose throughout the llotted period. Preferene for surose ws nlyzed in the experiments shown in Figure 7 y 5 5 mixed ANOVA (time group). There ws signifint effet of time (F 4, = 9.33, P <.1) s the overll surose preferene deresed ross weeks. There ws signifint effet of group (F 4,25 = 47.3, P <.1) nd there ws signifint intertion etween time nd group (F 16, = 2.99, P <.1). Eh time point ws then nlyzed y etween-sujets one-wy ANOVA. There were no differenes etween the groups in surose preferene t week 1 (seline) (F 4,32 = 2.26, P >.5). The groups were signifintly different in surose preferene levels t week 2 (F 4,32 = 9.2, P <.1). At week 2, ll soilly defeted nimls (SDS, SDS + fluoxetine, fluoxetine + SB224289) hd signifintly lower surose preferene thn oth ontrol groups s mesured y independent t tests (ll P s <.3). This pttern remined t week 3, s the overll groups were signifintly different (F 4,26 = 36.2, P <.1) nd ll soilly defeted nimls hd signifintly lower surose preferenes thn the ontrols (ll P s <.1). At week 4, the groups were signifintly different (F 4,26 = 6.78, P =.1). Independent t tests indited tht the ontrol nimls hd signifintly higher surose preferene thn the SDS group (t 8 = 9.95, P <.1) nd signifintly higher surose preferene thn the SB fluoxetine group (t 12 = 2.5, P =.3). The SB group hd signifintly higher surose preferene thn the SDS group (t 8 = 4.57, P =.2). However, the SDS + fluoxetine group hd signifintly higher surose preferene thn the SDS group (t 9 = 5.6, P <.1), ut ws not different thn the SB fluoxetine group (t 13 = 1.889, P =.81). Groups differed in overll surose preferene t week 5 (F 4,25 = 6.9, P =.1). The ontrol nimls hd signifintly higher surose preferene thn the SDS group (t 8 = 5.2, P =.1) nd the SB fluoxetine group (t 11 = 4.19, P =.2). The SB group hd signifintly higher surose preferene thn SB fluoxetine group (t 11 = 2.77, P =.18), ut ws not different from the SDS group (t 8 = 2.9, P =.7). The SDS + fluoxetine group hd signifintly higher surose preferene thn the SDS group (t 9 = 3.34, P =.9) nd the SB fluoxetine group (t 12 = 3.28, P =.7). Finlly, dependent t tests indited tht surose preferene signifintly inresed in the SDS + fluoxetine group from week 3 to week 4 (t 6 = 4.28, P =.5) nd remined higher t week 5 ompred to week 3 (t 6 = 3.11, P =.2). However, no inrese in surose preferene ws seen in the SB + fluoxetine nimls etween week 3 nd 4 (t 7 = 1.77, P =.12) or etween week 3 nd week 5 (t 6 = 1.7, P =.15). In the experiment with Htr1 / mie (Fig. 7), two-ftor ANOVA reveled signifint effet of genotype (F 1,12 = 2.965, P =.111) nd ondition (F 2,24 = , P <.1), nd n intertion etween genotype nd ondition (F 2,24 = 8.9, P =.2). In the experiment with GluA1 S831A mie (Fig. 7), there ws signifint effet of ondition in the ontrol mie y repeted-mesures ANOVA (F 2,1 = 8.67, P <.1). Finlly, one-wy ANOVA reveled signifint group effet in the til suspension test (F 3,23 = 7.7, P =.1; Fig. 7d).. Sntrelli, L. et l. Requirement of hippompl neurogenesis for the ehviorl effets of ntidepressnts. Siene 31, (3). nture NEUROSCIENCE doi:1.138/nn.3355