Sensory inputs control the integration of neurogliaform interneurons into cortical circuits

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1 Sensory inputs control the integrtion of neurogliform interneurons into corticl circuits Ntli De Mrco Grcí 1 4, Rshi Priy 1 3, Sebnem N Tuncdemir 1 3, Gord Fishell 1 3 & Theofnis Krynnis 1 3 Neuronl microcircuits in the superficil lyers of the mmmlin cortex provide the substrte for ssocitive corticl computtion. nhibitory interneurons constitute n essentil component of the circuitry nd re fundmentl to the integrtion of locl nd long-rnge informtion. Here we report tht, during erly development, superficilly positioned Reelin-expressing neurogliform interneurons in the mouse somtosensory cortex receive fferent innervtion from both corticl nd thlmic excittory sources. Attenution of scending sensory, but not intrcorticl, excittion leds to xo-dendritic morphologicl defects in these interneurons. Moreover, brogtion of the NMDA receptors through which the thlmic inputs signl results in similr phenotype, s well s in the selective loss of thlmic nd concomitnt increse in intrcorticl connectivity. These results suggest tht thlmic inputs re criticl in determining the blnce between locl nd long-rnge connectivity nd re fundmentl to the proper integrtion of Reelin-expressing interneurons into nscent corticl circuits. The bility of the neocortex to process sensory informtion nd trnsform it into meningful motor output depends on functionl neurl circuits involving diversity of cell types. At the microcircuit level, neurons fce the chllenge of integrting peripherl sensory informtion nd recurrent locl corticl ctivity. ndeed, the mintennce of proper blnce between corticl nd thlmic inputs is fundmentl to informtion processing becuse the former inputs regulte the gin of thlmic excittion 1. However, the mechnisms by which the formtion of these complex corticl circuits is regulted, including the estblishment nd mintennce of n pproprite blnce between thlmic nd intrcorticl inputs, re unknown. Recent work hs highlighted the importnce of interneurons in the superficil lyers of the cortex for corticl computtion. ndeed, these neurons re strtegiclly positioned to receive feedforwrd inputs from the thlmus nd feedbck inputs from the cortex. Superficil corticl interneurons, delineted by Reelin (Re) or vsointestinl peptide (P) expression, constitute pproximtely 30% of the totl number of interneurons in the mouse brin nd 50% in the humn brin 2,3. The increse in bundnce of this popultion cross species is speculted to reflect the lrger demnd for ssocitive functions in the humn brin 3. n the mouse, superficil corticl interneurons hve been shown both to prticipte in locl disinhibitory circuits 4,5 nd to control the trnsfer of long-rnge informtion from the motor cortex 6, contrlterl hemisphere 7 nd mygdl 8. Despite the centrl role of these interneurons in corticl function, the developmentl steps tht regulte the estblishment of their specific connectivity ptterns re not understood. n n effort to understnd the contribution of ctivity to interneuron development, we compred in preceding study how dmpening excittion of interneurons t prticulr developmentl time points ffected the positioning nd mturtion of specific interneuron subtypes 9. We observed mrked differences in the requirement for ctivity in P multipolr cells, which were pprently unffected by ttenuted levels of excittion, unlike Re + neurogliform cells, whose migrtion nd morphologicl development were mrkedly impired 9. Despite discovering requirement for ctivity for the development of Re + neurogliform cells, the presynptic source nd the neurotrnsmitter used, s well s receptor(s) required for this process, ll remined unknown. To begin to ssess whether spects of the developing circuit ply role in the proper integrtion of Re + interneurons, we here exmined the erly connectivity of this subtype using modified version of the rbies-bsed monosynptic trcing method. Although lyer hs been clssiclly regrded s the primry recipient of scending sensory informtion crried vi thlmic terminls 11, we found, unexpectedly, tht during the first postntl week, in ddition to inputs from intrcorticl sources, Re + interneurons in lyers lso receive direct thlmic inputs. Notbly, presynptic interference with thlmic but not intrcorticl inputs impired the morphologicl development of Re + interneurons. n exploring the mechnisms underlying this observtion, we found tht thlmocorticl synpses use NMDA receptors (NMDARs) contining the NR2B subunit nd tht cell-utonomous removl of these receptors (but not NR2A-contining receptors) led to severe morphologicl defects in Re + interneurons. Strikingly, in the bsence of NMDAR-medited currents, the rtio of thlmocorticl to intrcorticl innervtion ws drmticlly ltered such tht concomitnt increse in intrcorticl innervtion occurred t the expense of thlmic fferents (Supplementry Fig. 1). Our results indicte tht scending excittory thlmic ctivity controls the blnce of fferents impinging on select subtypes of superficil lyer interneurons. 1 NYU Neuroscience nstitute, New York Lngone Medicl Center, New York, New York, USA. 2 Deprtment of Neuroscience nd Physiology, New York Lngone Medicl Center, New York, New York, USA. 3 Deprtment of Neurl Science, New York University, New York, New York, USA. 4 Present ddress: Brin nd Mind Reserch nstitute, Weill Cornell Medicl College, New York, New York, USA. Correspondence should be ddressed to G.F. (fisheg01@nyumc.org). Received 14 October 14; ccepted 14 Jnury 15; published online 9 Februry 15; doi:.38/nn.3946 nture NEUROSCENCE dvnce online publiction

2 RESULTS Mpping the ntomicl connectivity of developing corticl Re + neurogliform interneurons Mture neurogliform interneurons locted in the superficil lyers of the somtosensory cortex receive innervtion from locl pyrmidl cells nd GABAergic interneurons 12. However, how this pttern of innervtion develops is unknown. To determine the development pttern of fferent connectivity to these interneurons, we dpted the monosynptic rbies trcing technique,13,14 such tht we could trget individul Re + interneurons. We electroported t embryonic dy (E) 15.5 construct in which the expression of histone2b.egfp (hgfp) fusion protein reporter, TA receptor nd B19G glycoprotein ws directed to interneurons through the use of n Dlx5/6 enhncer element 9 (Dlx5/6-hGFP-TA-B19G). Monosynptic trcing of the fferents to this popultion ws chieved through stereotctic injection of recombinnt rbies virus (SAD G_mCherry(EnvA)) into the somtosensory brrel field 1 re (SSBF1) t postntl dy (P) 3, followed by nlysis t P8 P (Fig. 1 nd Supplementry Fig. 2). The focl rbies injection together with the sprse electroportion of ventrlly generted interneurons further diluted by their dispersion during tngentil migrtion to the cortex 15 llowed us to trget one strter cell in ech experimentl brin (Fig. 1 d nd Supplementry Fig. 2). Thus, this technique llowed us to mp the fferent connectivity to individul Re + interneurons during erly postntl periods. The identity of strter cells nd presynptic trgets ws confirmed through immunohistochemistry of excittory cell nd interneuron mrkers, combined with nlysis of their morphologicl fetures (Supplementry Fig. 2; see lso Online Methods). We found tht ech Re + interneuron received inputs from 196 ± 48 (s.e.m.) presynptic neurons locted in corticl nd subcorticl structures. Surprisingly, considerble proportion of these inputs originted in the ventroposteromedil (PM) nd posteromedil (POm) nuclei of the thlmus (PM nd POm: 56 ± 6%). n ddition, Re + interneurons received locl fferents from pyrmidl nd subplte cells (14 ± 2%), s well s from other interneurons (30 ± 8%) (Fig. 1c). Notbly, unlike in other interneuron popultions we hve investigted, such s P- nd SST-expressing interneurons (Supplementry Fig. 3 nd dt not shown), we did not observe pprecible inputs onto the Re + interneurons from scending neuromodultory fibers such s those from the dorsl rphe nd the nucleus bslis (lthough, on the bsis of our previous findings 16, we infer such innervtion must ultimtely be estblished). As our previous experimentl findings hd suggested prominent role for glutmtergic ctivity in driving the integrtion of Re + interneurons into corticl circuits 9, we focused our studies on the contributions of thlmic nd intrcorticl excittory fferents. Figure 1 Determining the fferent inputs onto developing Re + interneurons in lyers /. () Schemtic representtion of the experimentl strtegy. (b) Presynptic prtners to single strter Re + interneuron in the somtosensory (SSBF1) cortex nd thlmus (see lso Supplementry Fig. 2). (c) Quntifiction of the identity of presynptic inputs. Men vlues (±s.e.m.) were obtined from 5 mice. (d) The pttern of presynptic connectivity reveled by rbies trcing to single Re + interneuron in wild-type P8 mouse brin. (e,f) Recording of AMPAR-medited monosynptic responses from Re + interneurons fter in vitro light stimultion in Emx1 Cre ; Ros26 LSL-ChR2-EYFP (Emx1-ChR2) (e) nd Glut2 Cre ;Ros26 LSL-ChR2-EYFP (Glut2-ChR2) (f) mice. Thl, thlmic neurons; Ecx, corticl excittory neurons; cx, corticl interneurons. Scle brs in b, 50 µm (SSBF1) nd 500 µm (thlmus); in e nd f, 500 µm. Re + interneurons receive functionl connectivity from somtosensory thlmic nuclei To confirm tht the ntomicl pttern of connectivity reveled by the retrogrde monosynptic trcing corresponds to functionl connectivity, we crried out chnnelrhodopsin-2 (ChR2)-ssisted optogenetic mpping. To direct the expression of ChR2 to corticl pyrmidl cells, we used Emx1 Cre or Bhlhb5 Cre (Bhlhe22 Cre ) mouse driver lines in combintion with loxp.stop.loxp (LSL) ChR2 (Ros26 LSL.ChR2-EYFP ) line. To chieve expression of ChR2 in thlmic fferents, we used vesiculr glutmte trnsporter (Glut2, lso known s Slc176) Cre driver mouse line in combintion with ChR2 reporter: Glut2 Cre ;Ros26 LSL.ChR2.EYFP or Olig3 Cre ;Ros26 LSL.ChR2.EYFP (Fig. 1e,f nd Supplementry Fig. 4). Re + interneurons were trgeted for whole-cell recordings either by using the sme electroportion pproch we used for monosynptic rbies or by recording unlbeled Dlx5/6-hGFP-TA-B19G b mcherry DAP d A e Electroportion E15.5 P S1 Light SSBF1 Rec SAD G-mCherry(EnvA) Subplte POm Emx1-ChR2 Stereotctic injection P3 40 pa ms PM Cortex f Light S1 Rec c % of totl trced cells PM/POm Cortex Hippocmpus Strter Monosynptic trcing P8 P Thl Ecx cx Strter cell (Re + ) Projection neuron nterneuron Glut2-ChR2 40 pa ms dvnce online publiction nture NEUROSCENCE

3 Figure 2 Perturbtion of sensory inputs during the first postntl week disrupts the xo-dendritic development of Re + interneurons. () Protocol for sensory deprivtion. (b) Norml lyering but diffuse brrel boundries re observed in the somtosensory cortex of sensory-deprived nd Olig3 Cre/+ ; Ros26 LSL. mice. : wild-type, non-sensory-deprived Ros26 LSL. mice. (c) Severe morphologicl defects re present in Re + interneurons fter whisker plucking nd in Olig3 Cre/+ ;Ros26 LSL. () mice. (d) Representtive exmples of neurolucid reconstructions of P + interneurons in control nd sensory-deprived mice. (e g) Quntifiction of neurite defects. Men vlues (±s.e.m.) were obtined from reconstructed Re + interneurons ech in Dlx5/6-eGFP electroported control (, n = 7 interneurons), sensory-deprived (, n = 5 interneurons; versus : xonl length, P = 0.004; xonl nodes, P = 0.011; dendritic length, P = ) nd mice (n = 6 interneurons; versus : xonl length, P = 0.003; xonl nodes, P = 0.005; dendritic length, P = 0.004). (h j) Quntifiction of length nd complexity of dendritic tree nd xonl rbors of P + interneurons (men vlues ±s.e.m.; n = 12 interneurons; n = 4 interneurons; xonl length, P = 0.335; xonl nodes, P = 0.273; dendritic length, P = 0.685). *P < 0.05; P < 0.01; *P < 0.001, P < Unpired t-test. Scle brs for b d, 50 µm. Axons re shown in red, dendrites in blue. lyer cells. n ccordnce with the ntomicl trcing, we recorded light-evoked monosynptic excittory responses from these neurons in the presence of tetrodotoxin (TTX) to block polysynptic events nd 4-minopyridine (4AP) to enhnce presynptic vesiculr relese (Fig. 1e,f nd Supplementry Fig. 5). n seprte set of experiments, we further vlidted these results by recording NMDAR-dependent monosynptic responses in the presence of AMPA nd kinte receptor ntgonists (Supplementry Fig. 5b). n these experiments the observed EPSC ltencies were round ms, which mtched the ltencies of the AMPAR-medited responses in the bsence of TTX nd 4AP (Supplementry Fig. 5,c,d). These vlues re consistent with wht hs been reported in previous studies using very similr pproches Tken together, the monosynptic rbies trcing nd optogenetic mpping reveled tht, during the first postntl week, developing Re + interneurons positioned in the superficil lyers of the cortex receive functionl glutmtergic innervtion not only from the expected locl network, but lso from somtosensory thlmic nuclei. Attenution of thlmic ctivity leds to bnorml differentition of Re + but not P + subtypes Becuse thlmic fferents provide excittory drive to Re + interneurons during the first postntl week (see lso Supplementry Fig. 6), we ssessed their contribution to the morphologicl development of these neurons. To mnipulte sensory ctivity relyed through the thlmus, we used sensory-deprivtion protocol. n mice where the Re + popultion hd previously been lbeled using in utero electroportion with Dlx5/6-eGFP plsmid, we crried out chronic bilterl whisker plucking during the first postntl week beginning t birth (Fig. 2). Thlmic terminls were visulized through their expression of Glut2 nd, in control conditions, were found to typiclly form c Reconstruction e n utero electroportion 7,000 6,000 5,000 E15.5 f Number of xonl nodes Whisker plucking P0 P1 P2 P3 P4 P5 P6 P7 P8 Reelin ived * g 1, 1, Anlysis Olig3 Cre/+ ; Ros26 LSL. d b Glut2 Ctip2 1, brrels in lyer (Fig. 2b). n contrst, these terminls exhibited n bnormlly diffuse termintion pttern fter sensory deprivtion (Fig. 2b). While this mnipultion did not perturb corticl lyering or interneuron migrtion (Fig. 2b nd Supplementry Fig. 7,b), nlysis of the morphologicl mturtion of Re + interneurons reveled severely truncted xonl rbors nd dendritic trees (Fig. 2c,e g nd Supplementry Fig. 7c). This effect ws specific to this subtype, s P + interneurons, which we hve previously shown to be unffected by other ctivity mnipultions 9, developed proper xo-dendritic rbors in this setting (Fig. 2d,h j nd Supplementry Fig. 7d). To further chrcterize the role of thlmic ctivity, we geneticlly blocked thlmic trnsmission nd ssessed the effect of this mnipultion on interneuron mturtion. As the Glut2 Cre driver lso trgets some corticl neurons (Supplementry Fig. 4g), we crossed Olig3 Cre with Ros26 LSL. mice 21, which restricted the expression of the tetnus toxin light chin () to thlmic nuclei. The use of the Ros26 LSL. mouse line s n effective method for blockde of synptic relese hs been previously documented 22. Consistent with the expression of the toxin, we found mrked reduction in the levels of vesicle-ssocited protein 2 (AMP2), synptic protein cleved by nd required for synptic vesiculr relese (Supplementry Fig. 8). This genetic blockde of thlmic ctivity led to more robust disorgniztion of defined brrels in lyer s compred to sensory deprivtion (Fig. 2b). Furthermore, similrly to those in the whisker-plucked nimls, Re + interneurons showed morphologicl impirment fter blockde of thlmic glutmte relese (Fig. 2c,e g). These results suggest tht thlmic ctivity is fundmentl to the xo-dendritic development of Re + interneurons nd re consistent with our previous findings indicting tht, in contrst to P + subtypes, Re + interneurons re ffected by ltertions in ctivity 9. P ived Olig3 Cre/+ ; Ros26 LSL. ived h i j Number of xonl nodes , 1, nture NEUROSCENCE dvnce online publiction

4 Figure 3 Severe ttenution of intrcorticl glutmte relese does not interfere with Re + interneuron morphologicl development. () Schemtic representtion of the experimentl strtegy. (b) Whole-mount epifluorescence imge showing corticl expression of.egfp surrounding the injection site in n Emx1 Cre/+ mouse. (c) Coronl section t P9 t the level of the injection site in SSBF1 shows tht egfp expression is restricted to corticl neurons. (d) Electroported interneurons (mcherry + ) re surrounded by pyrmidl cells expressing.egfp (green). (e) Reduced levels of AMP2 protein in the ipsilterl, but not in the contrlterl, SSBF1 or the ipsilterl PM t P8 fter AA-flex-.eGFP injection t E15.0. (f h) Morphologicl nlysis of Re + interneurons fter injection of AA-flex-.eGFP in the bsence (, n = 9 interneurons) nd presence (, n = interneurons) of the Emx1 Cre llele. Since comprison of Re + interneuron morphology fter E15.5 nd P0 injections did not show significnt difference (P > 0.05), we pooled these groups for the nlysis in g (xonl length: P = 0.254; xonl nodes: P = 0.36) nd h (dendritic length: P = 0.37; dendritic nodes: P = 0.548). (i) A mrked reduction of AMP2 levels is observed in SSBF1 but not the stritum in Emx1- mice. (j) ntrcorticlly evoked EPSCs recorded t 70 m in lyer interneurons in mouse (top) nd in the Emx1- mouse (bottom). The red trce shows smll response tht ws obtined upon moving the stimulting electrode from lyer to lyer in the ltter mice. (k) Averge EPSC mplitude t 70 m in lyer interneurons fter stimulting lyer in control nd Emx1- mice ( = 14 cells; Emx1- n = 4 cells, P = , Mnn-Whitney test). (l) Representtive reconstructions of biocytin filled Re + interneurons in lyer. (m) Quntifiction of length of dendritic rbors nd xonl trees ( n = 14 interneurons; Emx1- n = 8 interneurons; xonl length, P = 0.725; dendritic length: P = 0.24). (n) Reduction of corticl thickness in Bhlhp5 Cre/+ ;Munc18 fl/fl compred to Munc18 fl/fl Dlx5/6-mCherry Electroportion E15.5 d g j Tbr1 DAP.eGFP mcherry 3,500 2,500 1, n Munc18 fl/fl / AA-flex-.eGFP e.egfp AMP2 3.2 ma/60 µs in lyer 60 pa ms Emx1-3.2 ma/60 µs in lyer 3.2 ma/60 µs in lyer Bhlhb5 Cre/+ ; Munc18 fl/fl njection E15.5, P0 psilterl SBF1 Number of xonl nodes k EPSC mplitude (pa) control mice t P9. (o) Representtive reconstructions of biocytin filled Re + interneurons in lyer. (p) Quntifiction of length of dendritic rbors nd xonl trees. (men vlues ±s.e.m.; n = interneurons; Bhlhp5 Cre/+ ;Munc18 fl/fl, n = 4 interneurons; xonl length, P = 0.486; dendritic length, P = 0.194). *P < Unpired t-test. Scle brs in b nd c, µm; in d, f, i, n, l nd o, 50 µm; in e, 0 µm. / o Emx1- Emx1 Cre/+ Cortex Hippocmpus Anlysis P8 P Contrlterl SBF1 * h 1, 1, 1, 1, l Munc18 fl/fl Bhlhb5 Cre/+ ;Munc18 fl/fl b psilterl PM Number of dendritic nodes Ros26 LSL. Emx1 Cre/+ ;Ros26 LSL. p.egfp f Reconstruction 3,500 2,500 1, cp9 AA-flex-.eGFP m i AMP2 DAP 3,500 2,500 1, Emx1- Munc18 fl/fl Bhlhb5; Munc18 fl/fl.egfp Emx1 Cre/+ + P0 AAflex-.eGFP Ros26 LSL. Emx1 Cre/+ ;Ros26 LSL. SSBF1 SSBF1 Stritum / 1, 1, 1, 1, 1, 1, 1, 1, / Emx1- Munc18 fl/fl Bhlhb5; Munc18 fl/fl Severe ttenution of corticl glutmtergic ctivity does not perturb Re + interneuron differentition Since the rbies trcing experiments indicted tht Re + interneurons lso receive locl corticl inputs, we sought to ssess the impct of ltering intrcorticl ctivity on interneuron development. We begn by electroporting Emx1 Cre mice with Dlx5/6-mCherry plsmid t E15.5 to lbel Re + interneurons. Subsequently, we injected n denossocited virus (AA) contining Cre-dependent genetic switch (FLEx switch) nd.egfp 23 in the somtosensory nd other corticl res t P1 nd nlyzed their brins t P8 P (Fig. 3). This experimentl strtegy hs been successfully used to block synptic output from hippocmpl interneurons 23 nd corticl neurons 22. We found tht the.egfp fusion protein ws robustly expressed in pyrmidl cells t P8 s reflected by the high levels of egfp expression (Fig. 3b d). n n effort to mximize the level of virl expression, we lso crried out the virl injections t the time of the electroportion t E15.5. We indeed found tht these erly injections cused mrked decrese in the AMP2 protein levels (Fig. 3e). We then reconstructed the morphology of lbeled Re + interneurons surrounded by pyrmidl cells expressing (Fig. 3f h) in both sets of experiments nd found no significnt chnges in the length or complexity of xonl rbors nd dendritic trees (Fig. 3g,h). Although we observed mrked decrese in AMP2 levels upon injection with the AA-flex-.eGFP virus (Fig. 3e), we could not formlly exclude the possibility tht residul corticl glutmtergic signling to Re + interneurons persisted. To chieve more complete blockde of intrcorticl glutmte relese, we crossed Emx1 Cre to Ros26 LSL. mice. Although the lethlity of this cross is high, dvnce online publiction nture NEUROSCENCE

5 b c egfp Glut2 Cre/+ ;RCE fl/+ PM Experimentl design AA1.Ef1.dflox. hchr2.mcherry. WPRE.hGH Hippocmpus P2 Glut2 Cre/+ SSBF1 Glut2 Cre/+, AA1.Ef1. DO.hChR2.mCherry we obtined single survivor Emx1 Cre ;Ros26 LSL. (herefter, Emx1-) mouse (out of 15 crosses). t ppered helthy nd ws undistinguishble from its littermtes. Consistent with the corticl expression of tetnus toxin in this mouse, we observed reduction P mcherry DAP SSBF1 d Light NMDA Emx1-ChR2 NMDA + ifenprodil +40 m AMPA 70 m 40 pa ms Figure 4 Enrichment of NR2B-contining NMDARs ctivted by thlmic fferents onto Re + interneurons. () egfp expression in neuronl cell bodies in the PM in Glut2 Cre/+ ;RCE fl/+ (where RCE is n R26R CAG-boosted EGFP) mice. (b) Experimentl design for virl injections. (c) A representtive section of thlmic injection showing mcherry expression in xons reching SSBF1 in Glut2 Cre/+ -injected mice. (d) AMPAR- nd NMDAR-medited monosynptic responses recorded from Re + interneurons fter light stimultion of Emx1 Cre+ ;Ros26 LSL.ChR2-EYFP or Bhlhb5 Cre/+ ;Ros26 LSL.ChR2.EYFP (n = 11 interneurons), Glut2 Cre/+ ;Ros26 LSL.ChR2.EYFP (n = 12 interneurons) or Glut2 Cre/+ thlmic injection (n = 9 interneurons) slices. Superimposed exmple trces of ifenprodil (3 µm) blockde of NMDAR medited currents (red). (e) NMDAR/AMPAR chrge rtio (±s.e.m.) (Emx1-ChR2 versus Glut2-ChR2, P = ; Emx1-ChR2 versus Glut2 Cre/+ thlmic injection, P = , Mnn-Whitney test). Emx1-ChR2 Bhlhb5-ChR2 indictes tht these dt sets were pooled (see Online Methods). (f) The percentge reduction of NMDAR-dependent current mplitude fter ifenprodil ppliction (Emx1-ChR2 versus Glut2-ChR2, P = ; Emx1-ChR2 versus Glut2 Cre/+ thlmic injection, P = 0.01, Mnn-Whitney). P < Scle br in, 0 µm; in c, 500 µm. Error brs represent s.e.m. Electroportion Dlx5/6 Pmin Cre loxp + NR1/ 2B Anlysis E15.5 P8 P Cre Dlx5/6 Pmin Cre Cre loxp No NMDA or NR2B receptor egfp egfp egfp Light NMDA e NMDA/AMPA chrge rtio Glut2-ChR2 NMDA + ifenprodil +40 m AMPA in AMP2 levels in the cortex but not in ventrl structures (Fig. 3i). To confirm the bsence of corticlly evoked synptic responses, we performed in vitro electrophysiology nd t the sme time filled lyer Re + interneurons with biocytin to revel their morphology. P = 0.05 Emx1-ChR2 Bhlhb5-ChR2 Glut2- ChR2 70 m Glut2 Cre thlmic injection 40 pa ms pa b +40 m ms c d Dlx5/6 Dlx5/6 GABA A R blocked NR2B fl/fl GABA A R + AMPAR blocked NMDAR current (pa) m * e 1, Reelin f g P h 1, 1, 1, 1, * * f Light NMDA % reduction by ifenprodil Glut2 Cre thlmic injection NMDA NMDA + ifenprodil +40 m AMPA 4,500 1,500 P = 0.01 Emx1-ChR2 Bhlhb5-ChR2 70 m 40 pa ms Glut2- ChR2 Glut2 Cre thlmic injection 3,500 2, Reelin P = 0.01 NR2B fl/fl NR2B fl/fl P Reelin P Figure 5 NR2B-contining NMDARs re required for proper Re + but not P + interneuron development. () A schemtic representtion of the genetic strtegy for bltion of NMDARs nd NR2B-contining NMDARs in Re + interneurons. (b) The bsence of NMDAR-medited currents in cells nd the presence of robust AMPAR-dependent currents (n = 7 control () interneurons ( ) nd n = 3 interneurons; Mnn-Whitney test, P = ). GABA A receptors were blocked with SR (c e) Anlysis of neuronl morphology in control (n = 25) interneurons ( or NR2B fl/fl ) nd in (n = ) nd NR2B fl/fl (n = 13) Re + interneurons (men vlues ± s.e.m.; xonl length,, unpired t-test; P = 0.0; NR2B fl/fl, P = 0.003; dendritic length,, P = 0.0; NR2B fl/fl, P = 0.016). (f h) The sme nlysis performed in control (n = 9) nd (n = 6) P + interneurons (xonl length, NR2B fl/fl, P = 0.656; dendritic length:, P = 0.678). *P < 0.05; P < Scle brs, 50 µm. Axons re shown in red, dendrites in blue. nture NEUROSCENCE dvnce online publiction

6 Figure 6 Morphologicl development proceeds normlly in NR2A / Re + interneurons. (,b) Neurolucid reconstructions of Re + interneurons in control () nd in NR2A / (b) mice. (c) Quntifiction of length nd complexity of dendritic rbors nd xonl trees (control (NR2A +/+ nd NR2A +/ ), n = 7 nd NR2A /, n = 6 interneurons; men vlues ± s.e.m.; unpired t-test; xonl length: P = 0.345; xonl nodes: P = 0.243; dendritic length: P = 0.439; dendritic nodes: P = 0.272). P > 0.05, ns. Scle brs, 50 µm. Axons re shown in red, dendrites in blue. Reelin b NR2A / n the sme session, we recorded from littermte Ros26 LSL. (Cre ) niml. We subsequently recorded from dditionl Ros26 LSL. (Cre ) nimls to increse c 7,000 the number of dt points for both the electrophysiologicl nd morphologicl nlyses. 6,000 To test for the blockde of intrcorticl inputs 5,000 onto Re + interneurons, we plced bipolr stimulting electrode under the recorded cell in lyer nd stimulted t incresing intensities ( ma) with 60-µs-long squre pulse (Fig. 3j). All control cells displyed evoked EPSCs t 70 m, but only one of the cells recorded in the Emx1- niml showed ny response, nd it ws very smll, consistent with the reduced levels of AMP2 protein (Ros26 LSL. : ± pa, n = 14 cells; Emx1-: 3.60 ± 3.60 pa, n = 4 cells; P = , Mnn-Whitney test) (Fig. 3j,k). nterestingly, though, we were ble to evoke EPSCs fter plcing the stimulting electrode in lyer, presumbly by stimulting thlmic fferents, consistent with the specific expression of in corticl but not thlmic terminls (Fig. 3j,k). Despite the blockde of intrcorticl excittion, we found tht Re + interneurons developed norml morphologies in the Emx1- mouse (Fig. 3l,m). n n effort to increse the smple size for this nlysis, we generted mice contining Bhlhb5 Cre llele nd loxp-flnked (fl) Munc18 (Stxbp1) lleles 24. Bhlhb5 Cre ;Munc18 fl/fl mice were born t Mendelin frequencies nd survived until P15. Despite mrked reduction in corticl thickness (Fig. 3n), Re + interneurons in lyer exhibit norml morphology in Bhlhb5 Cre ;Munc18 fl/fl mice compred to Munc18 fl/fl controls (Fig. 3o,p). These results show tht we cn substntilly reduce the fferent intrcorticl drive to Re + interneurons without noticebly impiring their development. Tken together, our results suggest tht erly thlmic ctivity is uniquely criticl for the proper morphologicl development of Re + interneurons. Hence, while it ppers likely tht, under physiologicl circumstnces, secondry ctivtion of excittory corticl neurons ugments the developmentl excittory drive tht neurogliform cells receive from the thlmus, the direct thlmic excittory drive to neurogliform cells lone is sufficient to llow them to develop normlly. Thlmo-corticl inputs preferentilly ctivte NR2B-contining NMDARs Wht, then, re the moleculr nd functionl distinctions tht result in the differentil requirement of thlmic versus intrcorticl ctivity for Re + neurogliform interneuron development? n the hippocmpus, inputs from the perfornt nd Schffer collterl pthwys onto CA1 pyrmidl cells cn be distinguished by the selective enrichment of Axonl nodes , 1, NR2A / NR2A / NR2A / NR2A / NR2B-contining NMDARs 25. However, whether similr mechnism could operte in Re + neurogliform interneurons, which possess smller dendritic rbors, remined unknown. NMDAR-medited currents re prominent in severl neuronl popultions, including mturing corticl pyrmidl cells 26, olfctory grnule cells 27 nd hippocmpl interneurons 28. To dissect the receptor composition of thlmic nd intrcorticl synpses, we used Ros26 LSL.ChR.EYFP line in combintion with either Emx1 Cre nd Bhlhb5 Cre driver lines, which exclusively trget excittory corticl neurons, or Glut2 Cre driver line, which preferentilly (but not exclusively) induces recombintion in thlmic neurons (herefter Emx1- ChR2 nd Glut2-ChR2, respectively) (Fig. 4 nd Supplementry Fig. 4). We then performed optogenetic stimultion of the different fferents nd recorded NMDAR-dependent responses from Re + interneurons (Fig. 4d f). Most corticl NMDARs re heterotetrmers composed of two obligtory NR1 nd two NR2 (NR2A or NR2B) subunits, with hippocmpl interneurons expressing both 2A nd 2B 28,29. Notbly, depending on the NR2 subunit present, the receptors my disply differentil downstrem signling pthwys 26. We therefore ssessed the NMDAR composition in Re + interneurons ctivted in Glut2-ChR2 nd in Emx1-ChR2 mice, by nlyzing their degree of blockde by the NR2B-specific ntgonist ifenprodil. We found tht light-evoked NMDAR-medited synptic responses showed lrger NR2B-component in Glut2-ChR2 thn in Emx1-ChR2 mice (Fig. 4d f). Bsed on the observed ptterns of ChR2 expression in these two compound genotypes, we infer tht the thlmocorticl projection preferentilly ctivtes NR2B-contining receptors on the Re + interneuron popultion. n n effort to ssess the robustness of the results obtined by our experimentl design, we performed two more sets of experiments. First, to ctivte the intrcorticl inputs onto Re + interneurons, we used bipolr stimulting electrode plced close to the recorded cell (Supplementry Fig. 9). Second, in n effort to overcome the potentilly Dendritic nodes dvnce online publiction nture NEUROSCENCE

7 Figure 7 NMDAR bltion reconfigures fferent connectivity onto Re + interneurons. (,b) The distribution of fferent inputs onto control (; Cre, ) nd (b; Cre +, ) interneurons s reveled by monosynptic rbies trcing (see Fig. 1). (c) Representtive imges of corticl presynptic neurons to control nd interneurons. (d) Quntifiction of the totl number of presynptic neurons per strter cell in control (; n = 6) nd (n = 3) mice (men vlues ± s.e.m.; unpired t-test; P = 0.646). (e) Representtive imges of thlmic presynptic neurons to control nd interneurons. (f) The proportion of thlmic (Thl), corticl excittory (Ecx) nd corticl interneuron (cx) inputs to control (n = 6 mice) nd interneurons (n = 3 mice) (Thl, P = 0.002; Ecx, P = 3.8E-06; cx, P = 0.3). P < 0.01; P < Scle brs in c, 50 µm; in e, 500 µm. b A P Subplte Cortex Cortex Strter cell (Re + ) Projection neuron nterneuron confounding effect of the corticl expression of Glut2, we performed thlmic injections with n AA1.Ef1.dflox.hChR2.mCherry.WPRE. hgh (where Ef1 represents humn elongtion fctor-1α (EEF1A), hchr2 represents n H134R vrint nd hgh represents the humn growth hormone polydenyltion sequence) virus in Glut2 Cre mice t P2 nd performed recordings t P9 P12 (Fig. 4b). This strtegy llowed robust virl expression in the thlmus without impinging on the cortex (Fig. 4c). Although there ws more vribility in the AMPA-medited responses mong the thlmiclly injected mice (possibly due to vribility in the levels of ChR2 expression nd/or number of thlmic neurons infected with the virus), there ws no sttisticl difference between them nd the responses from Glut2-ChR2 mice (P = 0.54 for chrge, Mnn-Whitney), nd the results we obtined using the two pproches were comprble (Fig. 4 nd Supplementry Fig. 9). Abrogtion of NMDAR ctivity leds to berrnt Re + interneuron differentition To ssess the contribution of NMDARs versus AMPARs to the development of Re + interneurons, we crried out genetic nd phrmcologicl blockde of the different types of receptors. To blte ll NMDARmedited responses, we co-electroported Dlx5/6-eGFP nd Dlx5/6- Cre plsmids into (Grin1 fl/fl ) mice (Fig. 5). By removing the NR1 subunit, we efficiently blted NMDAR-dependent currents in Re + interneurons while mintining AMPARdependent currents (Fig. 5b). To selectively blte NR2B-contining receptors, we electroported NR2B fl/fl mice (Fig. 5). As judged by comprison of their pssive electrophysiologicl properties nd bility to dischrge ction potentils to control cells, Re + interneurons with NMDAR deletion ppered helthy (Supplementry Fig. ). Re + interneurons migrted to their norml position in the somtosensory cortex (dt not shown). n both nd NR2B fl/fl mice, however, we found tht, s with mnipultions tht ttenute thlmocorticl output, Re + (but not P + ) interneurons filed to mture properly nd displyed both truncted xonl rbors nd dendritic trees (Fig. 5c h nd Supplementry Fig. ). n prllel experiment, we observed similr morphologicl defects fter subdurl dministrtion of ifenprodil (0.5 nm) t P3 (Supplementry Fig. 11). These defects were specific to the loss of NR2B-contining receptors, s Re + interneurons developed normlly in NR2A / (Grin2 / ) mice (Fig. 6), s well s fter blockde of AMPA receptors in vivo (Supplementry Fig. 12). Thus, the ctivtion of NR2B-contining NMDARs is selectively required for the proper mturtion of Re + interneurons llocted to the superficil lyers of the cortex. A criticl period for the requirement of NMDARs in morphologicl development Our previous observtions indicte tht ctivity is required for morphologicl development of Re + interneurons fter P3 (ref. 9). c mcherry DAP e mcherry DAP A P POm PM / Subplte POm PM / Strter cell (Re + ) Projection neuron nterneuron To ssess whether the requirement for NMDAR ctivtion follows similr time course, we selectively removed the receptor t different developmentl time points. After electroporting Dlx5/6-Cre ER (estrogen receptor) nd CAG-STOP-GFP plsmids in mice t E15.5, we subsequently dministered tmoxifen t P3 or P6 nd nlyzed Re + interneuron morphology t P8. Tmoxifen dministrtion t P3 but not t P6 phenocopied the morphologicl defects observed in the embryonic Dlx5/6-Cre bltion experiments (Supplementry Fig. 13). These results indicte tht NR2B-contining NMDARs driven by the thlmic inputs re required in Re + interneurons between P3 nd P6 for their proper morphologicl development. Severe reduction of thlmic inputs fter brogtion of NMDAR function Does the morphologicl impirment observed in NMDAR-blted Re + interneurons preclude their proper integrtion into nscent corticl circuits? To determine whether the fferent connectivity onto NMDAR-blted interneurons ws ffected, we performed monosynptic rbies trcing fter cell-utonomous removl of NMDARs. We first electroported Dlx5/6-hGFP-TA-B19G nd Dlx5/6-Cre plsmids into mice nd subsequently injected the SAD G_ mcherry(enva) virus t P3 (Fig. 7). At P8, we observed single strter cell (Re + ) per brin in the control (Cre, ; n = 6 mice) nd the NMDAR-blted (Cre +, ; n = 3 mice) groups, d Totl trced cells f % of totl trced cells Thl Ecx cx nture NEUROSCENCE dvnce online publiction

8 corroborting previous results tht bltion of these receptors does not ffect Re + cell survivl. Furthermore, the totl number of presynptic trced cells ws not significntly different between control (190 ± 55) nd (150 ± 47; P = 0.646) interneurons (Fig. 7d). However, the distribution of presynptic prtners ws significntly ltered by NMDAR bltion. interneurons showed severe reduction in thlmic fferent connectivity (: 60% ± 6 versus : 16% ± 2, P < 0.01) nd concomitnt increse in intrcorticl pyrmidl cell innervtion (: 14% ± 1 versus : 42% ± 2, P < ) (Fig. 7c,e,f). n contrst, interneuron fferent connectivity ws not significntly chnged in interneurons (: 27% ± 6 versus : 42% ± 4, P = 0.3) (Fig. 7f). t is interesting tht, despite developing rudimentry dendritic tree, interneurons received exubernt yet berrnt intrcorticl innervtion. Remrkbly, even in the presence of substntil glutmtergic signling through AMPARs, interneurons filed to develop proper xonl processes nd to integrte properly into nscent corticl circuits. Together these dt suggest tht ctivtion of NMDARs medites the proper integrtion of mturing interneurons in the brin. DSCUSSON Our results indicte tht specific types of erly ctivity ply n ctive nd crucil role in directing the formtion of selective connectivity ptterns. We found tht Re + neurogliform interneurons respond differentilly to distinct glutmtergic inputs, which regulte their selective synptic connectivity. Our observtions complement recent findings indicting tht glutmtergic trnsmission from retinl gnglion cell (RGC) xons from the ipsilterl eye is fundmentl in preventing berrnt contrlterl RGC xon innervtion in the dorsl lterl geniculte nucleus (dlgn) 30. n ddition, mnipultion of spontneous cholinergic retinl ctivity, while spring overll ctivity levels, prevents the eye-specific segregtion nd refinement of RGC projections in the dlgn 31. Similrly, our ctivity mnipultions ffecting NMDAR-medited signling, even under conditions tht preserve both intrinsic electrophysiologicl properties nd AMPAR-medited responses, resulted in profound neuronl mturtion defects. Re + interneurons in the somtosensory cortex receive bundnt locl innervtion from pyrmidl cells, s reveled by glutmte uncging in dult corticl slices 12. n ddition to this pttern of locl connectivity, recent experimentl evidence hs reveled tht Re + interneurons of lyer in the prefrontl cortex receive monosynptic connections from the thlmic ventromedil nucleus nd in turn provide feedforwrd inhibition to lyer pyrmidl cells 17. Together these findings suggest tht the pproprite connectivity of Re + interneurons (nd perhps other subtypes) my depend on criticl specific interctions occurring when interneurons cese migrting nd ttin their finl position. Our in vivo nlysis provides one exmple in which Re + interneurons llocted to lyers through receive strong thlmic drive during development, which is essentil in determining their mture connectivity. n ddition to providing specific insights regrding the development of connectivity onto Re + interneurons, our findings suggest tht monosynptic trcing using the rbies virus provides n effective method for determining the fferent connectivity of corticl neurons t different developmentl time points. Furthermore, to rule out bis of the rbies trcing to GABAergic nd glutmtergic synpses, we crried out the sme experiment in lrger subset of superficil interneurons, including P- nd clretinin-expressing subtypes. We found tht, in contrst to Re + interneurons, these popultions of superficil interneurons received innervtion from the nucleus bslis t P6, indicting tht the rbies trcing strtegy does not preclude per se the trcing of these synpses (Supplementry Fig. 3). These results re in greement with recently published dt on rbies trcing of mture P + interneurons 32. P + interneurons present in the visul cortex receive substntil innervtion from intrcorticl popultions nd the nucleus bslis, with minor contribution from the dlgn, indicting tht the rbies virus does not preferentilly lbel thlmic synpses over corticl or cholinergic ones. Our findings extend previous work demonstrting tht sensory experience shpes the topogrphy of the sensory cortices directly nd not through second order corticl neurons. To exclude generlized effects of ctivity on corticl cytorchitecture, we performed more restricted mnipultion of thlmic ctivity, whisker-plucking, which does not cuse lyering defects, nd confined our nlysis to the first postntl week. ndeed, the totl number nd the distribution of pyrmidl cells in superficil nd deep lyers were similr in control nd sensory-deprived brins (Fig. 2b nd Supplementry Fig. 7,b). Similr results were obtined fter genetic blockde of thlmic trnsmission (Fig. 2b g nd Supplementry Fig. 8). n spite of the norml lyering of the cortex t P8, these mnipultions induced profound defects in the morphologicl development of Re + interneurons. How do different fferent sources of excittion differentilly impct development? Re + interneurons distinguished between seemingly similr inputs on the bsis of the postsynptic receptors tht the fferents impinge on. The prominent requirement for NMDARs during CNS mturtion is well estblished 26. n corticl excittory neurons, the NR2B subunit is required for proper dendritic ptterning 38 nd is lso differentilly used by hippocmpl interneuron subtypes shring the sme embryonic origin s the Re + interneurons studied here 28. Our results extend these observtions nd suggest tht there is preferentil lloction of NR2B-contining NMDARs to the postsynptic sites in the dendrites of Re + interneurons tht receive thlmic input. The present results, combined with findings from both our lbortory 9 nd others 39, suggest tht the roles of ctivity in interneuron development re both specific to subtype nd multifceted, nd ffect both migrtion nd differentition. Re + but not P + interneurons filed to migrte to pproprite lmine nd displyed impired morphologicl mturtion fter n overll dmpening of neuronl excitbility. Similrly, Re + but not P + interneurons filed to develop norml xonl rbors nd dendritic trees fter sensory deprivtion, genetic blockde of thlmic glutmte relese or blockde of NMDAR-dependent currents. These results indicte tht Re + neurogliform interneurons re criticlly relint on electricl ctivity to mture nd integrte into corticl circuits, wheres P + interneurons my use either intrinsic genetic progrms or s-yet-unidentified sources of ctivity. Thus, n intriguing combintion of intrinsic nd extrinsic mechnisms is used for the integrtion of distinct interneurons into corticl circuits. n summry, our results indicte tht emergent electricl ctivity is crucil in shping the ssembly of select corticl neuronl circuits. These observtions emphsize tht understnding how brin wiring is chieved will require cse-by-cse exmintion of how different cell types nd the corticl microcircuits they contribute to re ssembled. This in turn suggests tht developmentl insults re likely to differentilly ffect the formtion of specific circuit configurtions. As such, understnding the rules governing the integrtion of different interneurons subtypes into corticl circuits my provide insights into the pthogenesis of neuropsychitric disese. dvnce online publiction nture NEUROSCENCE

9 Methods Methods nd ny ssocited references re vilble in the online version of the pper. Note: Any Supplementry nformtion nd Source Dt files re vilble in the online version of the pper. Acknowledgments We re grteful to S. Arber, B. Benedetti, J. Burrone, X. Jglin, J. Kltschmidt, S. Lee, D. Pispi nd S. Shi for comments on the mnuscript. We thnk E. Cllwy (Slk nstitute for Biologicl Sciences) for providing the recombinnt rbies virus; M. Tripodi, A. Ponti nd S. Arber for guidnce with the rbies method nlysis; nd L. Yin, J. Deng nd J. Di for technicl ssistnce. N..D.M.G. is recipient of NARSAD Young nvestigtor Awrd nd is lso supported by grnts from the US Ntionl nstitutes of Helth (5 K99 MH ; 3 K99 MH S1). T.K. hs been supported by the Ptterson Trust postdoctorl fellowship in brin circuitry nd Roche postdoctorl fellowship. Reserch in the Fishell lbortory is supported by the US Ntionl nstitutes of Helth, Ntionl nstitute of Mentl Helth, Ntionl nstitute of Neurologicl Disorders nd Stroke, New York Stte Stem Cell Science nd the Simons Foundtion. AUTHOR CONTRBUTONS N..D.M.G., T.K. nd G.F. conceived the project. N..D.M.G., T.K. nd R.P. performed the experiments. S.N.T. prepred the rbies virus. N..D.M.G. wrote the mnuscript with the help of ll uthors. COMPETNG FNANCAL NTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is vilble online t reprints/index.html. 1. Lien, A.D. & Scnzini, M. Tuned thlmic excittion is mplified by visul corticl circuits. Nt. Neurosci. 16, (13). 2. M, T. et l. Subcorticl origins of humn nd monkey neocorticl interneurons. Nt. Neurosci. 16, (13). 3. Hnsen, D.. et l. Non-epithelil stem cells nd corticl interneuron production in the humn gnglionic eminences. Nt. Neurosci. 16, (13). 4. Pfeffer, C.K., Xue, M., He, M., Hung, Z.J. & Scnzini, M. nhibition of inhibition in visul cortex: the logic of connections between moleculrly distinct interneurons. Nt. Neurosci. 16, (13). 5. Pi, H.J. et l. Corticl interneurons tht specilize in disinhibitory control. Nture 503, (13). 6. Lee, S., Kruglikov,., Hung, Z.J., Fishell, G. & Rudy, B. A disinhibitory circuit medites motor integrtion in the somtosensory cortex. Nt. Neurosci. 16, (13). 7. Plmer, L.M. et l. The cellulr bsis of GABA B -medited interhemispheric inhibition. Science 335, (12). 8. Letzkus, J.J. et l. A disinhibitory microcircuit for ssocitive fer lerning in the uditory cortex. Nture 480, (11). 9. De Mrco Grcí, N.., Krynnis, T. & Fishell, G. Neuronl ctivity is required for the development of specific corticl interneuron subtypes. Nture 472, (11).. Wickershm,.R., Finke, S., Conzelmnn, K.K. & Cllwy, E.M. Retrogrde neuronl trcing with deletion-mutnt rbies virus. Nt. Methods 4, (7). 11. Petersen, C.C. The functionl orgniztion of the brrel cortex. Neuron 56, (7). 12. Xu, X. & Cllwy, E.M. Lminr specificity of functionl input to distinct types of inhibitory corticl neurons. J. Neurosci. 29, (9). 13. Tripodi, M., Stepien, A.E. & Arber, S. Motor ntgonism exposed by sptil segregtion nd timing of neurogenesis. Nture 479, (11). 14. Miymichi, K. et l. Corticl representtions of olfctory input by trns-synptic trcing. Nture 472, (11). 15. Fishell, G. & Rudy, B. Mechnisms of inhibition within the telencephlon: where the wild things re. Annu. Rev. Neurosci. 34, (11). 16. Lee, S., Hjerling-Leffler, J., Zgh, E., Fishell, G. & Rudy, B. The lrgest group of superficil neocorticl GABAergic interneurons expresses ionotropic serotonin receptors. J. Neurosci. 30, (). 17. Cruikshnk, S.J. et l. Thlmic control of lyer 1 circuits in prefrontl cortex. J. Neurosci. 32, (12). 18. Petrenu, L., Mo, T., Sternson, S.M. & Svobod, K. The subcellulr orgniztion of neocorticl excittory connections. Nture 457, (9). 19. Mo, T. et l. Long-rnge neuronl circuits underlying the interction between sensory nd motor cortex. Neuron 72, (11).. Tod, T. et l. Birth regultes the initition of sensory mp formtion through serotonin signling. Dev. Cell 27, (13). 21. Zhng, Y. et l. 3 spinl neurons estblish robust nd blnced locomotor rhythm during wlking. Neuron 60, (8). 22. Xu, W. & Sudhof, T.C. A neurl circuit for memory specificity nd generliztion. Science 339, (13). 23. Murry, A.J. et l. Prvlbumin-positive CA1 interneurons re required for sptil working but not for reference memory. Nt. Neurosci. 14, (11). 24. Dudok, J.J., Groffen, A.J., Toonen, R.F. & erhge, M. Deletion of Munc18 1 in 5-HT neurons results in rpid degenertion of the 5-HT system nd erly postntl lethlity. PLoS ONE 6, e28137 (11). 25. Arrigoni, E. & Greene, R.W. Schffer collterl nd perfornt pth inputs ctivte different subtypes of NMDA receptors on the sme CA1 pyrmidl cell. Br. J. Phrmcol. 142, (4). 26. Wng, C.C. et l. A criticl role for GluN2B-contining NMDA receptors in corticl development nd function. Neuron 72, (11). 27. Kelsch, W., Li, Z., Eliv, M., Goengrich, C. & Monyer, H. GluN2B-contining NMDA receptors promote wiring of dult-born neurons into olfctory bulb circuits. J. Neurosci. 32, (12). 28. Mtt, J.A. et l. Developmentl origin dicttes interneuron AMPA nd NMDA receptor subunit composition nd plsticity. Nt. Neurosci. 16, (13). 29. Snz-Clemente, A., Nicoll, R.A. & Roche, K.W. Diversity in NMDA receptor composition: mny regultors, mny consequences. Neuroscientist 19, (13). 30. Koch, S.M. et l. Pthwy-specific genetic ttenution of glutmte relese lters select fetures of competition-bsed visul circuit refinement. Neuron 71, (11). 31. Xu, H.P. et l. An instructive role for ptterned spontneous retinl ctivity in mouse visul mp development. Neuron 70, (11). 32. Fu, Y. et l. A corticl circuit for gin control by behviorl stte. Cell 156, (14). 33. Morishit, H. & Hensch, T.K. Criticl period revisited: impct on vision. Curr. Opin. Neurobiol. 18, 1 7 (8). 34. Li, H. et l. Lminr nd columnr development of brrel cortex relies on thlmocorticl neurotrnsmission. Neuron 79, (13). 35. Ktz, L.C. & Shtz, C.J. Synptic ctivity nd the construction of corticl circuits. Science 274, (1996). 36. Li, H. & Crir, M.C. How do brrels form in somtosensory cortex? Ann. NY Acd. Sci. 1225, (11). 37. Dunn, F.A., Dell Sntin, L., Prker, E.D. & Wong, R.O. Sensory experience shpes the development of the visul system s first synpse. Neuron 80, (13). 38. Espinos, J.S., Wheeler, D.G., Tsien, R.W. & Luo, L. Uncoupling dendrite growth nd ptterning: single-cell knockout nlysis of NMDA receptor 2B. Neuron 62, (9). 39. Bortone, D. & Polleux, F. KCC2 expression promotes the termintion of corticl interneuron migrtion in voltge-sensitive clcium-dependent mnner. Neuron 62, (9). nture NEUROSCENCE dvnce online publiction

10 ONLNE METHODS Mouse strins. Pregnnt mice were electroported t 15 d of gesttion (E15.5). Strins from Jckson lbortories used in this study include Swiss Webster, NR1 fl, Emx1 Cre, Glut2 Cre, RCE fl nd Ros26 LSL.ChR2.EYFP (Ai32). n ddition, we used Olig3 Cre ( gift from Y. Nkgw, University of Minnesot), Bhlhb5 Cre ( gift from L. Gn, University of Rochester Medicl Center), R26 floxstop-tent (which we clled Ros26 LSL. for consistency with the virl tools; gift from M. Goulding, Slk nstitute for Biologicl Studies), NR2A / ( gift from S. Nknishi, Kyoto University), NR2B fl ( gift from H. Monyer, University of Heidelberg), Munc18 fl/fl ( gift from M. erhge, U University Amsterdm) nd 5HT3A Cre ( gift from N. Henitz, Rockefeller University) mouse lines. Tmoxifen ( mg/ml in corn oil; 50 0 µl per pup) ws dministered by orl gvge t selected time points (P3, P6). nformtion bout the mouse strins including genotyping protocols cn be found t nd elsewhere 21,24,27,40,41. n utero electroportion. The protocol for mouse in utero electroportion hs been described elsewhere 9,42,43. The plsmids used in the electroportion experiments were generted using stndrd cloning techniques. To generte the Dlx5/6-hGFP-TA-B19G plsmid, the H2BGFP-F2A-TA-T2A-B19 frgment ( gift from M. Goulding, Slk nstitute for Biologicl Studies) ws cloned into Dlx5/6-Pmin-polyA plsmid. Mouse colony mintennce nd hndling ws performed in complince with the protocols pproved by the nstitutionl Animl Cre nd Use Committee of the New York University School of Medicine. irl injections. Adeno-ssocited virus contining the egfp-tgged tetnus toxin light chin () reding frme inverted in Flip-excision (flex) cssette 23 (AA1/2.flex..eGFP) ( gift from P. Wulff, University of Aberdeen) ws injected unilterlly into the cortex of electroported (Dlx5/6.mCherry) wildtype mice t E15.5 or P0. For the E15.5 virl injections, we first performed the electroportion nd promptly injected the AA virus in the developing cortex of Emx1 Cre embryos. For ll postntl injections, nimls were nesthetized by inducing hypothermi on ice nd kept on either ice wrpped in cloth or n ice-cold cly mold. For rbies trcing experiments, recombinnt mcherryexpressing, ASL-A envelope glycoprotein (EnvA)-pseudotyped, glycoproteindeleted rbies virus SADG-mCherry(EnvA) (gift from E. Cllwy, Slk nstitute for Biologicl Studies) 44,45 ws injected unilterlly into the cortex of electroported wild-type, or 5HT3A Cre mice between postntl dys 0 nd 3. To chieve ChR2 expression in thlmic terminls, we unilterlly nd stereotcticlly injected AA1.Ef1.dflox.hChR2.mCherry.WPRE.hGH (University of Pennsylvni virl vector core) into the thlmus of Glut2 Cre nimls t P2. n these experiments, we did not observe ny ectopic expression of the virus in the neocortex, s judged by immunofluorescence nd the lck of light-induced depolriztion in rndomly picked excittory cells round the injection site. For the rbies experiments, we injected 0 nl of virus; for the thlmic ones, 300 nl. These injections were done over 2-min period using glss micropipette (tip dimeter ~ µm) ttched to Nnolitre 0 pressure injection pprtus (World Precision nstruments). The pipette ws held in plce for 2 min fter ech injection before being completely retrcted from the brin. To llow for dequte virus expression fter the injection, mice were returned to their home cge for 5 9 d before either fixing the brin for cryostt section nlysis or using vibrtome for cute slice preprtion nd electrophysiology. mmunohistochemistry, neuronl morphology nlysis nd subdurl injections. The methodology for these nlyses hs been previously described 9. The identity of presynptic prtners identified by mens of rbies monosynptic trcing ws determined by coexpression of mcherry (SADG-mCherry(EnvA)), excittory cell mrkers (rbbit nti-rorβ, 1:00 ( gift from G. Miyoshi, New York School of Medicine); rbbit nti-ctp2, 1:00, Abcm (b28448); rbbit nti-tbr1, 1:00, Abcm (b31940); rbbit nti-stb2, 1:500, Abcm (3b4735) nd GABAergic interneuron mrkers (guine pig nti-gaba, Abcm (b17413), mouse nti-reelin, 1:500, MBL (D223-3); rbbit nti-dsred, Abcm (b62341)). For ssessing the effectiveness of tetnus toxin expression, we used mouse nti- AMP2 BT ntibody (1:, Synptic Systems 4 211BT). Successful trgeting of the desired Re + nd P + popultions in ll of our experiments ws confirmed through post hoc exmintion of mrker expression (rbbit nti-p 1:00, mmunostr, 77). The morphology of Cre + NR1 fl/+ nd NR2B fl/+ interneurons ws not significntly different thn tht of control Cre or NR2B fl/fl interneurons (Cre- versus Cre-NR2B fl/fl : xonl length, P = 0.30; dendritic length, P = 0.11); therefore, these interneurons were ll grouped in the controls. Similrly, wild-type non-sensory-deprived nd Ros26 LSL. mice were grouped in the control group (xonl length, P = 0.222; xonl nodes, P = 0.793; dendritic length, P = 0.879). Finlly, NR2A +/+ nd NR2A +/ mice were lso grouped in control group (xonl length, P = 0.33; xonl nodes, P = 0.73; dendritic length, P = 0.563; dendritic nodes, P = 1). Anlysis of presynptic inputs. To quntify the number of corticl nd subcorticl inputs, ll cryostt sections from n individul rbies-infected brin were collected for nlysis. Confocl z-stcks were nlyzed with the custom-mde Mtlb plug-in Reference Axes 13. The progrm llowed digitliztion of the imges nd quntifiction of corticl excittory (Tbr1 +, Ctip2 +, RORβ +, Stb2 + ) neurons, thlmic (PM nd POm) nd GABAergic interneurons (GABA + ). Morphologicl criteri were lso used to confirm the identity of the neurons. The identity of presynptic inputs ws determined s the proportion of the totl number of mcherry + neurons (corticl nd thlmic) tht expressed either corticl excittory or GABAergic mrkers or were locted in the PM nd POm nuclei of the thlmus. For identifiction of the strter cells in the rbies trcing experiments, -µm cryostt sections were nlyzed for GFP nd mcherry coexpression under n Olympus upright microscope equipped for multi-fluorescence nlysis before processing the tissue for immunohistochemistry. The sections tht showed cells coexpressing GFP nd mcherry (strter cells) were processed for immunohistochemistry ginst Reelin, GFP nd mcherry. All of the brins used in our experiments exhibited single Re + strter cell (Supplementry Fig. 2). The sections tht did not contin strter cells ( sections per brin) were processed for presynptic prtner nlysis. mmunohistochemistry ws performed on these sections with cocktil of rbbit ntibodies ginst excittory cell mrkers (Stb2, Ctip2, RORβ, Tbr1) nd guine pig nti-gaba ntibody to lbel interneurons. Subsequently, the sections were processed with nti-rbbit Alex 488 nd nti guine pig Cy5 secondries in combintion with mcherry fluorescence to identify mcherry + corticl excittory nd inhibitory neurons. Electrophysiology nd nlysis. Whole-cell ptch-clmp electrophysiologicl recordings were performed on egfp-positive nd egfp-negtive cells of lyers in cute brin slices prepred from P8 P12 nimls. No dt reported hve been previously published with the exception of the control nd Kir2.1-expressing Re + cells used for comprison of the intrinsic electrophysiologicl properties to the NMDA receptor knockout Re + cells 42 (Supplementry Fig. ). Briefly, nimls were decpitted nd the brin ws dissected out nd trnsferred to physiologicl Ringer s solution (ACSF) cooled to 4 C, of the following composition (mm): 125 NCl, 2.5 KCl, 25 NHCO 3, 1.25 NH 2 PO 4, 1 MgCl 2, 2 CCl 2 nd glucose. The brin ws then glued to stge nd slices µm were cut using vibrtome (ibrtome 3000 EP). The slices were llowed to recover in recording ACSF t room temperture for t lest 45 min before recording. Acute slices were then plced in recording chmber mounted on the stge of n upright microscope (Axioscope, Zeiss, Germny) equipped with immersion differentil interference contrst objectives (5, 40 ) coupled to n infrred cmer system (Zeiss), superfused t rte of 1 2 ml/min with oxygented recording ACSF nd mintined t temperture of 31 C. An EGFP filter ws used to visulize the fluorescent interneurons in epifluorescence. Ptch electrodes were mde from borosilicte glss (Hrvrd Apprtus) nd hd resistnce of 4 8 MΩ. For both intrinsic electrophysiologicl properties nd spontneous excittory postsynptic current (sepsc) recordings, the ptch pipettes were filled with solution contining the following (in mm): 128 potssium gluconte, 4 NCl, 0.3 N-GTP, 5 Mg-ATP, CCl 2, HEPES. For cquisition of both AMPA receptor medited nd NMDA receptor medited excittory currents in the sme cell, the pipettes were filled with the following (in mm): 126 cesium methnesulfonte, 4 CsCl, 0.3 N-GTP, 4 Mg-ATP, HEPES, d-trisphosphocretine. n ll cses 5 mg/ml biocytin (Sigm) ws dded in the recording solutions. All experiments tht involved extrcellulr stimultion with n electrode or optogenetic stimultion of chnnel rhodopsin were performed in voltge clmp in the presence of GABA A receptor blockers, either bicuculine ( µm) or SR95531 ( µm), except for slices from the Emx1 Cre ;Ros26 LSL./ niml, where no drugs were dded. AMPAR-medited currents were recorded t 70 m, wheres nture NEUROSCENCE doi:.38/nn.3946