Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision NO pa. 10 s ETMD

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1 doi:1.138/nture1136 Neuronl iruitry mehnism regulting dult quiesent neurl stem-ell fte deision Jun Song 1,2, Chun Zhong 1,2, Mihel A. Bonguidi 1,2, Gerld J. Sun 1,3, Derek Hsu 1, Yn Gu 4, Konstntinos Meletis 5, Z. Josh Hung 6, Shoyu Ge 4, Grigori Enikolopov 6, Krl Deisseroth 7, Bernhrd Lusher 8, Kimerly M. Christin 1,2, Guo-li Ming 1,2,3 & Hongjun Song 1,2,3 Adult neurogenesis rises from neurl stem ells within speilized nihes 1 3. Neuronl tivity nd experiene, presumly ting on this lol nihe, regulte multiple stges of dult neurogenesis, from neurl progenitor prolifertion to new neuron mturtion, synpti integrtion nd survivl 1,3. It is unknown whether lol neuronl iruitry hs diret impt on dult neurl stem ells. Here we show tht, in the dult mouse hippompus, nestinexpressing rdil gli-like quiesent neurl stem ells 4 9 (RGLs) respond tonilly to the neurotrnsmitter -minoutyri id (GABA) y mens of 2 -suunit-ontining GABA A reeptors. Clonl nlysis 9 of individul RGLs reveled rpid exit from quiesene nd enhned symmetril self-renewl fter onditionl deletion of 2. RGLs re in lose proximity to terminls expressing 67-kD glutmi id deroxylse (GAD67) of prvlumin-expressing (PV 1 ) interneurons nd respond tonilly to GABA relesed from these neurons. Funtionlly, optogeneti ontrol of the tivity of dentte PV 1 interneurons, ut not tht of somtosttin-expressing or vsotive intestinl polypeptide (VIP)-expressing interneurons, n ditte the RGL hoie etween quiesene nd tivtion. Furthermore, PV 1 interneuron tivtion restores RGL quiesene fter soil isoltion, n experiene tht indues RGL tivtion nd symmetril division 8. Our study identifies nihe ell signl reeptor trio nd lol iruitry mehnism tht ontrol the tivtion nd self-renewl mode of quiesent dult neurl stem ells in response to neuronl tivity nd experiene. Reent geneti linege-tring studies hve identified nestinexpressing RGLs s quiesent neurl stem ells (qnscs) in the dult mouse hippompus 4 9. In dult nestin mie 1, ells expressing green fluoresent protein ( 1 ells) in the sugrnulr zone (SGZ) with rdil proesses expressed GFAP (glil firillry idi protein) ut rrely MCM2 (minihromosome mintenne type 2), inditing quiesene (Supplementry Fig. 1, ). To ssess whether lol interneurons regulte dult qnscs diretly y mens of neurotrnsmitter relese, we exmined RGL responses to GABA in slies utely prepred from dult nestin mie y eletrophysiology (see Methods). 1 RGLs reorded under whole-ell voltge-lmp showed prominent responses to GABA (2 mm) or the GABA A reeptor (GABA A R) gonist musimol (2 mm), whih were olished y the GABA A R ntgonist iuulline (; 5 mm; Supplementry Fig. 1, d). GABA responses were potentited y dizepm (1 mm), whih speifilly enhnes 2 -ontining GABA A R responses to GABA 11. Indeed, 1 RGLs showed immunoretivity to 2 (Supplementry Fig. 1e). 2 -ontining GABA A Rs re present in non-neuronl ells nd n e found oth outside nd inside synpses in mture neurons 11. No spontneous or evoked synpti urrents in response to field stimultion of the dentte grnule ell lyer were deteted in 1 RGLs (n 5 25 ells; Supplementry Fig. 1f, g). Insted, toni GABA responses were reorded (n 5 18 ells; Fig. 1 nd Supplementry Fig. 1g, h), suggesting GABA spill-over from nery synpses 11. To exlude the possiility of synpti inputs with low relese proilities, we pplied hypertoni solution to enhne presynpti relese 12. Inresed GABA toni responses, ut not synpti urrents, were oserved (Supplementry Fig. 1h). Inhiition of the GABA reuptke trnsporter GAT1 with NO-711 (1 mm) lso inresed toni responses (Fig. 1), further supporting the toni nture of GABAergi responses in RGLs. We next explored phrmologil properties of toni GABA responses in RGLs 13. Consistent with the 2 involvement, dizepm (1 mm) signifintly inresed toni responses, wheres the enzodizepine ntgonist flumzenil (1 mm) deresed them (Fig. 1). The 5 -seletive enzodizepine gonist midzolm (1 mm), or the 3 -seletive positive llosteri modultor etomidte (ETMD; 1 nm), inresed toni GABA responses, wheres the 5 -seletive inverse gonist L (5 mm) deresed this response (Fig. 1). Together, these results suggest tht GABA A Rs re present in dult dentte RGLs to medite toni responses to GABA. To exmine the funtionl role of GABA in regulting dult dentte RGLs in vivo, we ssessed 5-ethynyl-29-deoxyuridine (EdU) inorportion nd MCM2 expression y RGLs fter tretment with dizepm (Supplementry Fig. 2). We identified RGLs s SGZ ells with nestin 1 rdil proesses (Fig. 2). Stereologil quntifition showed tht tretment with dizepm led to 45% derese in the numer of EdU 1 RGLs ompred with vehile tretment (Fig. 2). The numer NO pa 1 s Flumzenil ETMD Dizepm Midzolm L Figure 1 Toni tivtion of dult quiesent neurl stem ells y GABA y mens of GABA A Rs., Smple tres of whole-ell voltge-lmp reording from 1 RGLs treted with dizepm (1 mm), flumzenil (1 mm), midzolm (1 mm), ETMD (1 nm) or L (5 mm), followed y (1 mm) to otin seline for normlizing toni responses for eh ell., Summry of normlized mplitude of toni response. Vlues re mens nd s.e.m. (n 5 4 or 5 ells; ll signifintly different from the sl ondition; P,.5; Student s t-test). Perentge of sl toni response NO-711 Dizepm Flumzenil Midzolm ETMD L Institute for Cell Engineering, Johns Hopkins University Shool of Mediine, Bltimore, Mrylnd 2125, USA. 2 Deprtment of Neurology, Johns Hopkins University Shool of Mediine, Bltimore, Mrylnd 2125, USA. 3 The Solomon H. Snyder Deprtment of Neurosiene, Johns Hopkins University Shool of Mediine, Bltimore, Mrylnd 2125, USA. 4 Deprtment of Neuroiology nd Behviour, Stte University of New York t Stony Brook, New York 11794, USA. 5 Deprtment of Neurosiene, Krolinsk Institutet, S Stokholm, Sweden. 6 Cold Spring Hror Lortory, Cold Spring Hror, New York 11724, USA. 7 Deprtment of Bioengineering, Stnford University, Stnford, Cliforni 9435, USA. 8 Deprtment of Biology, Pennsylvni Stte University, University Prk, Pennsylvni 1682, USA. 15 NATURE VOL SEPTEMBER 212 Mmilln Pulishers Limited. All rights reserved 212

2 RESEARCH Vehile Dizepm Numer of EdU + nestin + RGLs per mm 3 Quiesent Nestin/MCM2/EdU/ 1,5 75 Nestin/ Numer of MCM2 + nestin + RGLs per mm 3 Nestin/EdU Ativted 3, 1,5 GFAP/ Ativted lones mong ll + lones (%) of MCM2 1 nestin 1 RGLs nd the perentge of RGLs tht were MCM2 1 were lso signifintly deresed (Fig. 2). Thus, systemi enhnement of 2 -medited GABA signlling promotes dult dentte RGL quiesene t the popultion level. To exmine ell-utonomous role of 2 in RGLs, we generted nestin-creer T21/2 ;Z/EG f/2 f/f ; 2 (KO) mie nd nestin- CreER T21/2 ;Z/EG f/2 ; 1/1 2 (ontrol) mie nd used low dose of tmoxifen for sprse indution to perform lonl nlysis of dult RGLs 9 (Supplementry Fig. 2 d). Immunohistology nd eletrophysiology indited highly effiient, ut not omplete, 2 deletion in 1 RGLs (Supplementry Fig. 2e, f). In KO mie, the perentge of RGL lones tht were tivted inresed mrkedly ompred with ontrol mie t 2 nd 7 dys fter indution (Fig. 2, d). Tretment with dizepm deresed the perentge of tivted RGL lones in ontrol mie t 7 dys fter indution, ut hd no effet in KO mie (Fig. 2e nd Supplementry Fig. 2g). These results showed diret role of GABA in mintining dult NSC quiesene through 2 signlling. We next exmined the fte hoie of tivted RGLs. There ws mrked inrese in pirs of losely ssoited 1 RGLs t 2 dys fter indution in dult KO mie ompred with ontrols, inditing inresed RGL symmetril self-renewl (Fig. 3, ). Detiled nlysis t 7 dys fter indution showed inresed symmetril nd strogliogeni symmetril RGL division in KO mie (Fig. 3). Conversely, tretment with dizepm deresed RGL symmetril division nd strogliogeni symmetri division in ontrol mie, ut hd no effet in KO mie (Fig. 3d). In supporting short-term linege-tring results, nlysis of lonl omposition t 3 dys fter indution showed deresed perentges of quiesent lones nd n inresed perentge of lones with multiple RGLs in KO mie (Fig. 3e, f nd Supplementry Fig. 3). Consistent with role of GABA signlling in promoting new neuron survivl 14, perentges of neurogeni lones d 1 5 MCM2 + nestin + /nestin + RGLs (%) Nestin/MCM2 1 5 e Ativted lones mong ll + lones (%) 2 7 Dys fter indution 1 5 RGL Vehile Dizepm Vehile Dizepm Cntl Figure 2 Cell-utonomous role of 2 -ontining GABA A Rs in mintining dult neurl stem ell quiesene.,, Dizepm promotes quiesene of nestin 1 RGLs in the dult dentte gyrus., Smple onfol imges of immunostining of nestin, MCM2, EdU nd 49,6-dimidino-2-phenylindole (). Arrows indite nestin 1 MCM2 1 or nestin 1 EdU 1 RGLs. Sle rs, 5 mm (left) nd 1 mm (lst olumn)., Summries of stereologil quntifition of RGL EdU inorportion nd MCM2 expression. Vlues re mens nd s.e.m. (n 5 4 nimls; sterisk, P,.1; Student s t-test). e, 2 deletion in individul RGLs leds to their tivtion., Smple onfol imges of immunostining. Sle rs, 1 mm. d, e, Summries of perentges of RGL lones tht were tivted (d) nd those treted with vehile or dizepm t 7 dys fter indution (e) for ontrol (ntl) nd KO mie. Vlues re mens nd s.e.m. (n nimls; sterisk, P,.1;, P..1; Student s t-test). e Among ll RGLontining + lones (%) f Among ll + lones (%) 7 dpi R R+R R+IPC R+A GFAP/ d Among ll RGLontining + lones (%) GFAP/ Single R R+R R+N R+A R+A+N no R 8 4 Figure 3 Clonl nlysis of RGL fte hoie fter onditionl 2 deletion in individul RGLs in the dult dentte gyrus. d, Short-term effet of 2 deletion on the tivtion nd fte hoie of dult dentte RGLs., Smple onfol imges of immunostining for 1 lone inditing symmetril division t 7 dys fter indution. Sle rs, 1 mm. d, Summries of perentges of lones inditing symmetril divisions t 2 nd 7 dys fter indution (), nd perentges of different types of RGL lones () nd those treted with vehile or dizepm (d) t 7 dys fter indution: R 1 R (two RGLs), R 1 intermedite progenitor ell (IPC; one RGL nd one GFAP 2 IPC) nd R 1 A (one RGL nd one GFAP 1 ushy stroyte). Vlues re mens nd s.e.m. (n nimls; sterisk, P,.5;, P..1; Student s t-test). e, f, Long-term effet (t 3 dys fter indution) of 2 deletion on the omposition of 1 lones in the dult dentte gyrus. e, Smple onfol imges of immunostining for lone onsisting of two GFAP 1 ells with rdil proesses. Sle rs, 1 mm. f, Summry of perentges of different lone types mong ll 1 lones: R, RGL; N, IPC or neuron; A, stroyte. Vlues re mens nd s.e.m. (n nimls; sterisk, P,.5; two sterisks, P,.1; Student s t-test). nd multilinege lones were deresed signifintly (Fig. 3f nd Supplementry Fig. 3e). In ontrst, lones without ny RGLs were inresed in KO mie (Fig. 3f), suggesting inresed RGL depletion fter 2 deletion. Together, these gin-of-funtion nd loss-of-funtion nlyses identified GABA s nihe signl to mintin dult NSC quiesene nd inhiit symmetril self-renewl nd stroyte fte hoie through 2 -ontining GABA A Rs under sl physiologil onditions. We next sought to identify GABA-relesing nihe ells mong multiple interneuron sutypes in the dult dentte gyrus 15,16. Immunohistologil nlysis of dult nestin mie showed lose ssoition etween 1 RGLs nd GAD67 1 terminls from PV 1 Symmetril division mong ll + RGL lones (%) 6 3 single R R+R R+IPC R+A 2 7 Dys fter indution +vehile +dizepm +vehile +dizepm 6 SEPTEMBER 212 VOL 489 NATURE Mmilln Pulishers Limited. All rights reserved

3 RESEARCH LETTER interneurons (Fig. 4 nd Supplementry Movie 1). To determine whether PV 1 interneurons intert funtionlly with RGLs, we took n optogeneti pproh nd used doule-floxed (DIO) denossoited virus (AAV) to express hnnelrhodopsin-2 (ChR2) or hlorhodopsin (enphr3.) speifilly in PV 1 interneurons, using d Numer of EdU + nestin + Numer of MCM2 + nestin + MCM2 + nestin + /nestin + RGLs (%) PV/nestin GAD67/PV/nestin AAV-ChR2 mcherry/ nestin mgc 1,5 75 3, 1, pa PV-Cre mie 2 s Cntl ChR2 NpHR RGL e VGA 1,2 6 2,6 1,3 8 4 Blue light Blue light Blue light SST-Cre mie Blue light PV neuron ChR2 f 1, 5 2, 1, Reording eletrode RGL mgc 2 pa 2 s Cntl ChR2 NpHR Cntl ChR2 NpHR 6 3 VIP-Cre mie Figure 4 Regultion of quiesene nd tivtion stte of neurl stem ells y PV 1, ut not SST 1 or VIP 1 interneuron tivity, in the dult dentte gyrus., Smple onfol imges of nd immunostining of PV nd GAD67 (See Supplementry Movie 1). Sle rs, 5 mm., Smple onfol imge nd shemti digrm of eletrophysiologil reording. Sle r, 1 mm., Smple whole-ell voltge-lmp reording tres of responses fter light stimultion of ChR2 1 PV 1 interneurons from mture grnule ell (mgc; 1 Hz) nd 1 RGL (8 Hz) in ute slies, nd fter tretment with (5 mm) or vigtrin (VGA; 1 mm). d f, Regultion of RGL tivtion in the dult dentte gyrus y lol interneuron tivity. Shown re summries of stereologil quntifition of RGL EdU inorportion nd MCM2 expression fter in vivo tivtion (ChR2) or suppression (NpHR) of PV 1 (d), SST 1 (e)or VIP 1 (f) interneurons or shm tretment (ntl; see Supplementry Figs 5 nd 6e for experimentl proedures). Vlues re mens nd s.e.m. (n 5 3or4 nimls; sterisk, P,.1; Student s t-test). dult PV-Cre mie 17 (Supplementry Fig. 4). Immunostining nd eletrophysiology onfirmed the speifiity nd effiy of AAVmedited opsin expression in ontrolling the firing of dentte PV 1 interneurons (Supplementry Fig. 4 e). In ute slies from PV- Cre 1/2 ;nestin 1/2 mie, phototivtion of PV 1 interneurons indued synpti responses in mture dentte grnule ells nd toni responses in 1 RGLs to GABA (Fig. 4, ). Furthermore, derese in GABA turnover with the GABA trnsminse inhiitor vigtrin (1 mm) drstilly inresed 1 RGL responses to PV 1 interneuron tivtion (Fig. 4). Together, these results indite tht dult RGLs respond tonilly to GABA relesed from lol PV 1 interneurons. To ssess the funtionl impt of PV 1 interneuron tivity on RGL ehviour, we phototivted or suppressed PV 1 interneurons in the dentte gyrus of dult PV-Cre mie for 5 dys (Supplementry Fig. 5). In omprison with shm tretment without light stimultion, EdU inorportion nd MCM2 expression y RGLs were signifintly deresed fter tivtion of PV 1 interneurons expressing ChR2 tgged with yellow fluoresent protein (ChR2 YFP), resulting in 53% derese in RGL tivtion t the popultion level (Fig. 4d nd Supplementry Fig. 5). Conversely, suppression of PV 1 interneurons expressing enphr YFP led to 95% inrese in RGL tivtion (Fig. 4d). These results identified PV 1 interneurons s ritil nihe omponent nd showed tht PV 1 interneuron tivity n ditte the RGL hoie etween quiesene nd tivtion in the dult dentte gyrus. Do other sutypes of lol interneurons lso regulte RGL ehviour in vivo? We developed similr optogeneti strtegies to mnipulte somtosttin-expressing (SST 1 ) or vsotive intestinl polypeptideexpressing (VIP 1 ) interneurons 16 (Supplementry Fig. 6). Both SST 1 nd VIP 1 interneurons showed elorted proesses in the SGZ nd hilus region (Supplementry Fig. 6, d nd Supplementry Movie 2), nd our proedure lelled greter numers of SST 1 nd VIP 1 interneurons thn PV 1 interneurons in the dult dentte gyrus (Supplementry Fig. 6). Eletrophysiologil reoding of 1 RGLs did not detet ny toni or synpti responses fter light-indued tivtion of SST 1 or VIP 1 interneurons in ute slies (Supplementry Fig. 6, d). Funtionlly, phototivted or suppressed dentte SST 1 or VIP 1 interneurons hd no effet on EdU inorportion nd MCM2 expression y RGLs (Fig. 4e, f nd Supplementry Fig. 6e). Thus, oupling of neuronl iruit tivity to RGL ehviour seems to e distintive of PV 1 interneurons rther thn ourring rodly ross different lol interneuron sutypes. Finlly, we ssessed whether GABA lso serves s nihe signl to medite experiene-dependent regultion of RGLs. We sujeted mie to soil isoltion regime, whih dereses neuronl tivity in the dult dentte gyrus 18 nd ws reently shown to promote RGL expnsion 8. Clonl nlysis t 7 dys fter indution showed tht, in ontrst with group housing, soil isoltion led to signifint inrese in 1 RGL tivtion nd symmetril nd strogeni division, in similr mnner to 2 deletion in RGLs (Fig. 5, nd Supplementry Fig. 7). 2 -defiient RGLs showed no dditionl tivtion or fte lterntion fter soil isoltion (Fig. 5). At the popultion level, EdU inorportion nd MCM2 expression y RGLs were inresed signifintly fter soil isoltion (Fig. 5 nd Supplementry Fig. 7, ). PV 1 interneuron tivtion olished the inrese in RGL tivtion indued y soil isoltion (Fig. 5). Thus, dentte PV 1 interneurons lso medite experiene-dependent regultion of dult qnscs through GABA- 2 signlling. Preise ontrol of somti stem ell tivity is essentil for the longterm mintenne of tissue homeostsis nd needs to e losely linked to tissue demnds t ny given time. Our study of dult RGLs t oth lonl nd popultion levels identified previously unknown nihe mehnism tht regultes oth dult qnsc tivtion nd self-renewl mode in response to neuronl tivity nd experiene (Supplementry Fig. 8). GABA hs een shown to derese the prolifertion of other stem ells nd progenitors in vitro, inluding mouse emryoni stem 152 NATURE VOL SEPTEMBER 212 Mmilln Pulishers Limited. All rights reserved 212

4 RESEARCH RGL+RGL Soil isoltion GFAP/ when lol iruitry tivity levels re low, while keeping NSCs in quiesene when tivity levels re high (Supplementry Fig. 8). Given tht oth the numer nd properties of hippompl PV 1 interneurons re regulted y physiologil nd pthologil onditions, suh s geing, Alzheimer s diseses, epilepsy, hroni stress, shizophreni nd other severe psyhitri illness 21 26, our findings hve rod implitions. Among ll RGL-ontining + lones (%) Numer of EdU + nestin + 1 1,2 6 5 Isoltion + isoltion Single R R+R R+IPC R+A Isoltion Hz + + Numer of MCM2 + nestin , 1, MCM2 + nestin + / nestin + RGLs (%) ells, y mens of GABA A Rs, the phosphtidylinositol-3-oh kinse (PI(3)K)-relted kinse fmily nd the histone vrint H2AX 19,2. PTEN deletion in individul RGLs lso leds to tivtion nd symmetril self-renewl in the dult dentte gyrus 9, suggesting onserved mehnism regulting the prolifertion of vrious stem ells through the GABA A R nd PI(3)K/PTEN pthwy. Our optogeneti pproh identified PV 1 interneurons s ritil nd unique nihe omponent mong different interneuron sutypes tht ouples neuronl iruit tivity to qnsc regultion in vivo under oth physiologil onditions nd in response to speifi experiene. PV 1 interneurons re undnt in the hippompus nd hve een implited in higher rin funtion nd ognitive dysfuntion 15. In the dult dentte gyrus, PV 1 interneurons reeive exittory inputs from dentte grnule ells nd, to smller extent, from entorhinl ortil inputs (Supplementry Fig. 8). We reonstruted one PV 1 interneuron in the dult PV-Cre 1/2 ;nestin 1/2 mie nd estimted tht it overed more thn 2 1 RGLs in the dentte gyrus (Supplementry Movie 3). A hrteristi feture of PV 1 interneurons is the formtion of ensemles oupled y oth eletril (through gp juntions) nd hemil onnetions (through reiprol innervtions) 15. Thus, PV 1 interneurons re well suited to ouple lol iruit tivity to the regultion of lrge numer of dult NSCs in the hippompus s n dptive mehnism inresing qnsc tivtion Figure 5 Contriution of GABA signlling from PV 1 interneurons to experiene-dependent regultion of dult quiesent neurl stem ells.,, Clonl nlysis of RGL fte hoie fter soil isoltion., Smple onfol imges of immunostining for n tivted lone with two RGLs t 7 dys fter indution fter soil isoltion (see Supplementry Fig. 7 for experimentl proedure). Sle rs, 1 mm., Summry of different types of lone t 7 dys fter indution. Vlues re mens nd s.e.m. (n nimls; sterisk, P,.5; Student s t-test)., Summries of stereologil quntifition of RGL EdU inorportion nd MCM2 expression. Vlues re mens nd s.e.m. (n 5 4 nimls; sterisk, P,.5;, P..1; Student s t-test). METHODS SUMMARY Wild-type (C57BL/6), nestin 1, PV-Cre 17, SST-Cre 16, VIP-Cre 16, nestin- CreER T21/2 ;Z/EG f/2 ; 2 f/f (ref. 27) were used in the present study. Cre-dependent reominnt AAV 17 ws used for interneuron sutype-speifi expression of opsins in the dult dentte gyrus. Eletrophysiologil reordings nd nlysis were performed s desried previously 28. Immunohistohemistry, onfol imging nd proessing were performed s desried previously 9. Stereologil quntifition ws ssessed s desried previously 29. All nlyses were performed y investigtors lind to experimentl onditions. All niml proedures were performed in ordne with institutionl guidelines. Full Methods nd ny ssoited referenes re ville in the online version of the pper. Reeived 1 Novemer 211; epted 11 June 212. Pulished online 29 July Zho, C., Deng, W. & Gge, F. H. Mehnisms nd funtionl implitions of dult neurogenesis. Cell 132, (28). 2. Kriegstein, A. & Alvrez-Buyll, A. The glil nture of emryoni nd dult neurl stem ells. Annu. Rev. Neurosi. 32, (29). 3. Ming, G. L. & Song, H. Adult neurogenesis in the mmmlin rin: signifint nswers nd signifint questions. Neuron 7, (211). 4. 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Soil isoltion rering-indued impirment of the hippompl neurogenesis is ssoited with defiits in sptil memory nd emotion-relted ehviors in juvenile mie. J. Neurohem. 15, (28). 19. Andng, M. et l. Histone H2AX-dependent GABA A reeptor regultion of stem ell prolifertion. Nture 451, (28). 2. Fernndo, R. N. et l. Cell yle restrition y histone H2AX limits prolifertion of dult neurl stem ells. Pro. Ntl Ad. Si. USA 18, (211). 21. Lolov, I. & Dvidoff, M. Age-relted morphologil nd morphometril hnges in prvlumin- nd lindin-immunoretive neurons in the rt hippompl formtion. Meh. Ageing Dev. 66, (1992). 22. Stoh, J., Tir, T., Sno, M., Nkym, H. & Tteishi, J. Prvluminimmunoretive neurons in the humn entrl nervous system re deresed in Alzheimer s disese. At Neuropthol. 81, (1991). 23. Msiulis, I., Yun, S. & Eish, A. J. Theinterestinginterplyetweeninterneurons nd dult hippompl neurogenesis. Mol. Neuroiol. 44, (211). 24. Knle, M. B., Bri, B. M., Wester, M. J., Medor-Woodruff, J. & Torrey, E. F. Moleulr normlities of the hippompus in severe psyhitri illness: 6 SEPTEMBER 212 VOL 489 NATURE Mmilln Pulishers Limited. All rights reserved

5 RESEARCH LETTER postmortem findings from the Stnley Neuropthology Consortium. Mol. Psyhitry 9, (24). 25. Gonzlez-Burgos, G., Fish, K. N. & Lewis, D. A. GABA neuron ltertions, ortil iruit dysfuntion nd ognitive defiits in shizophreni. Neurl Plst. 211, (211). 26. Andre, V., Mresux, C., Nehlig, A. & Fritshy, J. M. Altertions of hippompl GABAergi system ontriute to development of spontneous reurrent seizures in the rt lithium-pilorpine model of temporl loe epilepsy. Hippompus 11, (21). 27. Shweizer, C. et l. The 2 suunit of GABA A reeptors is required for mintenne of reeptors t mture synpses. Mol. Cell. Neurosi. 24, (23). 28. Ge, S. et l. GABA regultes synpti integrtion of newly generted neurons in the dult rin. Nture 439, (26). 29. Kim, J. Y. et l. DISC1 regultes new neuron development in the dult rin vi modultion of AKT-mTOR signling through KIAA1212. Neuron 63, (29). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk L. H. Tsi for initil help in the study; memers of the Song nd Ming lortories for disussion; H. Dvoudi for help; nd Q. Hussini, Y. Ci nd L. Liu for tehnil support. This work ws supported y grnts from the Ntionl Institutes of Helth (NIH) (NS47344) to H.S., the NIH (NS48271, HD69184), the Ntionl Alline for Reserh on Shizophreni nd Depression nd the Adelson Medil Reserh Foundtion to G.L.M., the NIH (MH89111) to B.L., the NIH (AG429) ndnewyork StteStem Cell SienendtheEllisonMedilFoundtion to G.E., nd y postdotorl fellowships from the Life Sienes Reserh Foundtion to J.S. nd from the Mrylnd Stem Cell Reserh Fund to J.S., C.Z. nd K.C. Author Contriutions J.S. led the projet nd ontriuted to ll spets. C.Z., M.A.B., G.J.S., D.H. nd K.C. helped with some experiments. Y.G. nd S.G. ontriuted regents. J.H. provided SST-Cre mie. G.E. provided nestin mie. K.D. nd K.M. provided initil help on optogeneti tools. B.L. provided f/f 2 mie. J.S., G-l.M. nd H.S. designed experiments nd wrote the pper. Author Informtion Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of this rtile t Correspondene nd requests for mterils should e ddressed to H.S. (shongju1@jhmi.edu) or G.M. (gming1@jhmi.edu). 154 NATURE VOL SEPTEMBER 212 Mmilln Pulishers Limited. All rights reserved 212

6 RESEARCH METHODS Animls, housing, dministrtion of tmoxifen, EdU nd AAV, nd optogeneti mnipultions. The following genetilly modified mie nd rosses etween them were used for eletrophysiologil nlysis: nestin 1 (CB57BL/6 kground), PV-Cre 17 (JAX lortory; stok numer 869; stok nme B6;129P2-Pvl tm1(re)arr /J), SST-Cre 16 (JAX lortory; stok numer 1344; stok nme Sst tm2.1(re)zjh /J), VIP-Cre 16 (JAX lortory, stok numer 198; stok nme Vip tm1(re)zjh /J). The following mie were used for neurogenesis nlysis: wild-type (C57BL/6), nestin-creer T21/2 ;Z/EG 1/2 ; floxed/floxed 2 (ref. 27; C57BL/6) nd nestin-creer T21/2 ;Z/EG 1/2 (C57BL/6), PV-Cre (B6;129), SST- Cre (B6;129), nd VIP-Cre (B6;129). Animls were housed in stndrd 14 h light/1 h drk yle. Soilly isolted nimls were individully housed immeditely fter wening for t lest 6 weeks efore injetion with tmoxifen or EdU, nd hd free ess to food nd wter 8. A single dose of tmoxifen (62 mg kg 21 )ws injeted intrperitonelly into 6 1-week-old mie s desried previously 9. For optogeneti mnipultions, Cre-dependent reominnt AAV vetors were used sed on DNA ssette rrying two pirs of inomptile loxp sites with the opsin genes (ChR2-H134 mcherry, ChR2-H134 YFP or enphr3. YFP) inserted etween lox sites in the reverse orienttion s desried previously 17 (Supplementry Fig. 4). The reominnt AAV vetors were serotyped with AAV2/9 for ChR2 (pkged t the UPenn Vetor Core) nd with AAV9 for enphr3. (pkged t University of North Crolin Vetor Core). The following finl virl onentrtions were used for AAV viruses ( prtiles ml 21 ): 7.4 (ChR2 YFP), 36 (ChR2 mcherry) nd 8 (enphr3. YFP), respetively. AAV ws delivered stereottilly into the dentte gyrus with the following oordintes (in mm): nterioposterior 522 from regm; lterl 561.5; ventrl Fire opti nnule (Dori Lenses, In.) were implnted t the sme injetion sites immeditely fter AAV injetion with dorsl ventrl depth of 1.6 mm from the skull. Animls were then llowed to reover for t lest 4 weeks fter surgery. For nlysis of RGL tivtion t the popultion level fter optogeneti mnipultions, littermtes of nimls were used nd n in vivo light regime ws dministered 8 h per dy for five onseutive dys (Supplementry Figs 5, 6e nd 7). For ChR2 YFP stimultion, flshes of lue light (472 nm; 5 ms t 8 Hz) through the DPSSL lser system (Lser Century Co. Ltd) were delivered in vivo every 5 min for 3 s per tril. For enphr YFP stimultion, ontinuous yellow light (593 nm) ws delivered in vivo. On the fifth dy, nimls were injeted with EdU (41.1 mg per kg ody weight) six times with n intervl of 2 h. Animls were killed 2 h fter the lst EdU injetion nd were proessed for immunostining s desried previously 9. All niml proedures were performed in ordne with institutionl guidelines. Eletrophysiology. Mie were nesthetized nd proessed for slie preprtion s desried previously 28. In rief, rins were quikly removed into the ie-old solution (in mm: 11 holine hloride, 2.5 KCl, 1.3 KH 2 PO 4, 25. NHCO 3,.5 CCl 2, 7 MgSO 4, 2 dextrose, 1.3 sodium L-sorte,.6 sodium pyruvte, 5. kynureni id). Slies 3 mm thik were setioned with virtome (Lei VT1S) nd trnsferred to hmer ontining the externl solution (in mm: 125. NCl, 2.5 KCl, 1.3 KH 2 PO 4, 1.3 MgSO 4, 25. NHCO 3, 2 CCl 2, 1.3 sodium L-sorte,.6 sodium pyruvte, 1 dextrose, ph 7.4, 32 mosm), uled with 95% O 2 /5% CO 2. Eletrophysiologil reordings were otined t uc. 1 RGLs loted within the SGZ in dult nestin 1/2 mie were reveled y differentil interferene ontrst nd fluoresene mirosopy. A whole-ell pth-lmp onfigurtion ws employed in the voltge-lmp mode (V m 5265 mv) or urrent-lmp mode. Miroeletrodes (4 6 MV) were pulled from orosilite glss pillries nd filled with the internl solution ontining (in mm) CsCl gluonte, 15 KCl, 4 MgCl 2,.1 EGTA, 1. HEPES, 4 ATP (mgnesium slt),.3 GTP (sodium slt) nd 7 phosphoretine (ph 7.4, 3 mosm). All RGL reordings were performed in the presene of kynureni id (5 mm). Dt were olleted with n Axon 2B mplifier nd quired with DigiDt 1322A (Axon Instruments) t 1 khz. For mesuring GABA-indued responses from 1 RGLs, fol pressure ejetion of 2 mm GABA or musimol through puffer pipette ontrolled y Piospritze (2 s puff t 3 5 l in 22 )wsused to tivte GABA A Rs under the whole-ell voltge-lmp. A ipolr eletrode (World Preision Instruments) ws used to stimulte (.1 ms durtion) the dentte grnule ell lyer. Low-frequeny stimuli (.1 Hz) nd thet ursts (8 Hz with trin of 1 stimuli) were delivered. The stimulus intensity (5 ma) ws mintined for ll experiments. The following phrmologil gents were used: dizepm (1 mm), NO-711 (1 mm), flumzenil (1 mm), midzolm (1 mm), ETMD (1 mm), L (5 mm) nd vigtrin (1 mm). All drugs were purhsed from Sigm exept iuulline (5 or 1 mm; Toris). RGL reordings under optogeneti mnipultion in ute rin slies were performed t lest 4 weeks fter injetion with AAV. To stimulte ChR2 in lelled interneurons, light flshes (5 ms t 1, 8 or 1 Hz) generted y Lmd DG-4 plus high-speed optil swith with 3 W Xenon lmp nd 472 nm filter set (Chrom) were delivered to oronl setions through 34 ojetive lens (Crl Zeiss). To stimulte enphr in lelled interneurons, ontinuous yellow light generted y DG-4 plus system with 593 nm filter set were delivered to oronl setions ross full high-power (34) field. Immunohistohemistry, onfol imging, proessing nd quntifition. For immunostining with nti-nestin nd nti-mcm2, n ntigen retrievl protool ws performed y mirowving setions in oiled itri uffer for 7 min s desried previously 9. For 2 immunostining, wek fixtion protool using live tissues ws dopted s desried previously 29,3. For hrteriztion of different interneuron sutypes, the following ntiodies were used: nti-pv (Swnt; mouse or rit; 1:5 dilution), nti-gad-67 (Millipore; mouse or rit; 1:5 dilution), nti-sst (Millipore; rt; 1:2 dilution) nd nti-vip (Immunostr; rit; 1:2 dilution). For lonl nlysis, oronl rin setions (4 mm) through the entire dentte gyrus were olleted in seril order, nd immunostining ws performed with the following primry ntiodies s desried previously 9 : nti- (Roklnd; got; 1:5 dilution), nti-nestin (Aves; hik; 1:5 dilution), nti-mcm2 (BD; mouse; 1:5 dilution), nti-gfap (Millipore; mouse or rit; 1:1, dilution) nd nti-psa-ncam (Millipore, mouse lgm; 1:5 dilution). For quntifition of 1 lones t 2 nd 7 dys fter indution, single 1 RGL ws sored s quiesent lone. Two or more nulei in 1 RGL lone were sored s tivtion. Clonl nlysis t 3 dys fter indution ws onduted extly s desried previously 9. For experiments with dizepm (5 mg kg 21 ody weight; one dily for 5 dys), oronl rin setions (4 mm) through the entire dentte gyrus were olleted in seril order. For optogeneti mnipultions, setions within distne of 1. mm nterior nd 1. mm posterior to injetion sites were used for quntifition, given the estimted light spred in vivo. Immunostining ws performed on every sixth setion s desried previously 9. EdU lelling ws performed with Clik-iT EdU Alex Fluor imging kit (Invitrogen). Imges were quired on Zeiss LSM 71 onfol system (Crl Zeiss) with 34 ojetive lens using multitrk onfigurtion. Stereologil quntifition of ells positive for vrious moleulr mrkers ws ssessed in the dentte gyrus with modified optil frtiontor tehnique 29. For quntifition of EdU 1 or MCM2 1 RGLs, n inverted Y shpe from nti-nestin stining superimposed on EdU 1 or MCM2 1 nuleus ws sored doule positive for nestin nd EdU or MCM2. All nlyses were performed y investigtors lind to experimentl onditions. Sttistil nlysis ws performed with Student s t-test. For genertion of movie files, imges were serilly reonstruted in Reonstrut (J. C. Fil, NIH), normlized, nd deonvolved with Autoqunt X2 (Medi Cyernetis). Imges were then segmented in MATLAB (The Mthworks) using ustom ode nd imported into Imris (Bitplne). Surfe renderings nd movies were mde using the Surfe nd Animtion funtions, respetively, in Imris (Supplementry Movies 1 3). 3. Shneider Gsser, E. M. et l. Immunofluoresene in rin setions: simultneous detetion of presynpti nd postsynpti proteins in identified neurons. Nture Protools 1, (26). 212 Mmilln Pulishers Limited. All rights reserved