Inhibitory control of neostriatal projection neurons by GABAergic interneurons

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1 rticles Inhiitory control of neostritl projection neurons y GABAergic interneurons Tior Koós nd Jmes M. Tepper Center for Moleculr nd Behviorl Neuroscience nd Progrm in Cellulr nd Moleculr Biodynmics, Rutgers, The Stte University of New Jersey, 197 University Ave, Newrk, New Jersey 07102, USA Correspondence should e ddressed to J.M.T. (tepper@xon.rutgers.edu) The sl gngli re highly interconnected network of nuclei essentil for the modultion nd execution of voluntry ehvior. The neostritum is the principl input nd one of the principl controllers of the output of the sl gngli. Neostritl projection neurons seem to e dynmiclly nd powerfully controlled y GABAergic inputs, ut the source(s) nd physiologicl properties of these inputs remin uncler. Here we use pired whole-cell recordings to show tht this inhiition derives from smll popultions of GABAergic interneurons tht re themselves interconnected through functionl electrotonic synpses. Inhiitory synptic potentils generted from single interneurons re sufficiently powerful to dely or entirely lock the genertion of ction potentils in lrge numer of projection neurons simultneously. The neostritum is criticlly involved in the control nd execution of voluntry ehvior. Mlfunctions of this system underlie severl neurologicl nd psychitric disorders, including Prkinson s disese nd Huntington s disese. Understnding informtion processing in the neostritum requires elucidtion of the processes tht control the sptiotemporl pttern of ctivity of its GABAergic spiny projection neurons, which in rodents mke up 90 95% of the neuronl popultion 1,2. Neostritl spiny projection neurons nd the diverse clsses of interneurons re interconnected in highly orgnized synptic microcircuitry nd communicte using numerous neurotrnsmitters nd neuromodultors 1,2. Despite the importnce of this circuitry, the electrophysiologicl opertion of the neostritum hs so fr een understood mostly in terms of the interction etween memrne currents of individul spiny projection neurons nd their excittory corticostritl input 2 5. In contrst, very little is known out the nture nd function of intercellulr interctions nd in prticulr of fst synptic signling mong stritl neurons. GABAergic fferents re mjor inputs to spiny projection neurons 6 9 nd primry determinnts of their ctivity, s locl phrmcologicl lockde of GABA A receptors in vivo increses the firing rte of these cells y more thn 300% (ref. 10). Furthermore, these inhiitory inputs re ehviorlly significnt ecuse lockde of GABAergic trnsmission in the neostritum results in significnt ctivtion nd/or disruption of motor ehvior 11,12. Therefore, GABAergic control of spiny projection neurons my e one of the most powerful determinnts of the output of the neostritum, ut its functioning is poorly understood. This is primrily ecuse the identity nd physiologicl properties of the functionl fferent sources of GABAergic inputs to spiny projection neurons re not known. Thus we cnnot determine the temporl nd sptil vriility of this input nd its dynmic or ehviorl contingencies. Lrgely on the sis of ntomicl dt 13, GABAergic inhiition of spiny projection neurons trditionlly hs een ttriuted to lterl inhiition mong these cells vi their locl xon collterls. Tests of this hypothesis hve filed to demonstrte ny evidence for lterl inhiition (C.J. Wilson, H. Kit & Y. Kwguchi, Soc. Neurosci. Astr. 15, 360.1, 1989), nd recent dul recording study 14 hs provided compelling evidence tht this form of interction is wek or sent in the neostritum. These findings led to the lterntive hypothesis tht certin GABAergic spiny interneurons, which mke up only 3 5% of the neurons in the rodent neostritum, provide the ulk of the inhiitory control of spiny projection cells 14,15 (C.J. Wilson, H. Kit & Y. Kwguchi, Soc. Neurosci. Astr. 15, 360.1, 1989). We exmined the synptic responses of spiny projection neurons to spiking in GABAergic interneurons to test their possile contriution to the strong inhiitory control of the output of the stritum oserved in vivo. To this end, we dpted recently developed techniques of visulized whole-cell recording to simultneously record pirs of identified interneurons nd projection cells in mture slice preprtion. RESULTS Chrcteristics of interneurons nd projection cells Recorded neurons were identified using primrily physiologicl nd, in some cses, morphologicl nd/or immunocytochemicl criteri 17, Spiny projection neurons were redily recognized y their typicl memrne properties (Fig. 1 nd ). Spiny projection cells (recorded from nimls dys old) were mture s indicted y their memrne properties, including memrne potentil ( 94.1 ± 1.6 mv, n = 7), input resistnce (67.4 ± 10.2 MW, n = 8), inwrd rectifiction, ction potentil threshold (47.3 ± 1.3 mv ove rest) nd ction potentil mplitude (80 ± 2.7 mv, n = 8), ll of which were similr to those oserved in slices tken from dults 17, In ddition, ll intrcellulrly stined nd recovered spiny projection neurons hd nture neuroscience volume 2 no 5 my

2 rticles c d Spiny projection * Fig. 1. Chrcteriztion of the memrne properties of recorded neurons. () Spiny projection neurons. Note the strong inwrd rectifiction nd the depolrizing rmp. () Differentil current voltge (I V) reltionships of the recorded neuron types. Note the strong inwrd rectifiction of spiny projection neurons nd the reltively liner I V curves of the interneurons. (c) FS interneurons were chrcterized y very high mximl firing frequency (upper pnel) nd episodic urst firing with suthreshold memrne potentil oscilltions (green trce) in response to depolrizing current injection. (d) LTS interneurons exhiited regenertive depolrizing hump resemling n LTS (sterisks) tht lwys elicited fst spikes. LTS cells hd iphsic fterhyperpolriztions. Note the sence of sustined depolrizing potentil (clirtion rs pply to, c nd d). DV m (mv) Vm [mv] 60 Spiny FS LTS I (na) 0.6 I [na] FS LTS densely spiny dendrites (Fig. 2), which is sensitive indictor of their morphologicl nd electrophysiologicl mturity 23,24. Two physiologiclly distinct types of interneurons were nlyzed. Of the eight interneurons recorded in intercting pirs ( totl of eleven pirs), six hd the electrophysiologicl properties of the prvlumin-contining (PV + ), fst-spiking (FS) interneurons descried previously 17, including nrrow ction potentils (< 0.5 ms), ruptly peking, lrge-mplitude spike fterhyperpolriztions, reltively liner current voltge reltionship (Fig. 1) nd very high mximl sustined firing frequencies with little or no dpttion (200 ± 23.4 Hz; n = 5; Fig. 1c). FS interneurons lso showed episodic urst firing nd suthreshold memrne potentil oscilltions (Fig. 1c). FS interneurons were morphologiclly heterogeneous. Four FS interneurons hd morphologicl chrcteristics previously reported for PV + FS neurons 17 (Fig. 2). These neurons were chrcterized y som of 80 ms medium to lrge dimeter (16.2 ± 1.6 mm; rnge mm; n = 5), frequently rnching, non-vricose or only slightly vricose spiny dendrites nd very dense locl xon collterl system. The xon collterl ror overlpped with the dendritic field ut tended to extend frther from the som nd covered 20 mv ovl re with mximl extent of 388 ± 32 mm (n 1.6 na = 3) nd miniml dimeter of 294 ± 39 mm (n = 3). The xon collterl system ws more dense in the inner two thirds of the extent of the roriztion. Two other FS neurons hd prominent dendritic vricosities nd significntly less dense xonl ror with lrger outons (Fig. 2). Two FS interneurons were immunopositive for prvlumin. These cells elonged to the more common morphologicl clss (dt not shown). Their synptic ction could not e tested ecuse of the limit on recording time imposed y the rpid loss of cytoplsmic ntigens during whole-cell recording. Two other interneurons monosynpticlly connected to spiny projection cells hd electrophysiologicl properties not previously descried in the neostritum 17,25. Their defining feture ws the presence of pronounced regenertive depolrizing potentil resemling low-threshold spike (LTS cells; Fig. 1d). Like FS interneurons, LTS neurons hd nrrow ction potentils nd could fire t frequencies up to 300 Hz. However, they hd higher input resistnce thn FS interneurons (179 ± 114 MW, n = 2, versus 93.2 ± 26.3 MW, n = 4; Fig. 1) nd iphsic spike fterhyper- Fig. 2. Cmer lucid reconstruction of spiny projection nd FS neurons. () The more common FS interneuron morphology in our smple (dendrites in red, xons in lck). These FS interneurons from 21-dy-old niml hd spiny dendrites with little or no vricosities nd dense xonl roriztion overlpping with nd extending eyond the dendritic field. () A synpticlly connected FS interneuron (red nd lck) nd spiny projection neuron (green) from 25-dy-old niml. The interneuron is morphologiclly different from the one shown in (), exhiiting pronounced dendritic vricosities (rrows), lrger xonl outons nd n only modertely dense xonl ror. The light microscopiclly identified puttive contct sites re perisomtic (circles). The dendrites of the spiny projection cell re densely covered with spines, indicting tht the neuron is mture 23,24. Axons of the spiny projection neuron re not shown. 468 nture neuroscience volume 2 no 5 my 1999

3 rticles polriztion. LTS neurons were considered to e different from the persistent depolriztion, low-threshold spike (PLTS) interneurons descried previously 17 ecuse of the sence of depolrizing plteu potentil (Fig. 1d). Neither of the LTS neurons ws recovered, nd their morphologicl nd neurochemicl identity is unknown. We note however, tht the physiologicl properties of LTS neurons were not very different from PLTS cells, nd therefore they my e vrints of the sme neurochemicl cell type. However, neurons showing the distinctive properties of PLTS cells were encountered in our preprtion t ll ges, which rgues ginst the possiility tht LTS neurons of this study differ from previously descried PLTS cells simply ecuse of developmentl or other differences of the preprtions used. The presence of gp junctions etween FS interneurons 26 indictes unique mode of opertion for this cell type if these junctions provide significnt electrotonic coupling. We tested for significnt electrotonic coupling etween pirs of FS interneurons. Two of the six pirs of FS interneurons recorded showed evidence of electrotonic coupling (Fig. 3). The coupling rtios were 3% nd 20%. This direct demonstrtion of functionl electrotonic coupling mong FS interneurons suggests tht they my operte in syncitil fshion. Biophysicl properties of interneuronl IPSPs FS nd LTS neurons provided powerful inhiition to spiny projection neurons within the territory of their xon ror (n = 11 pirs). Approximtely 25% of spiny projection neurons recorded within 250 mm from n interneuron showed synptic response to ctivtion of the interneuron. The postsynptic responses elicited from FS nd LTS neurons did not differ noticely with respect to ny of the exmined properties, nd therefore dt from the two popultions were pooled. Action potentils evoked in the interneurons elicited n inhiitory postsynptic potentil (IPSP; Fig. 4 nd ), which ws medited y GABA A receptors, s it ws reversily locked y the selective ntgonist icuculline, (20 mm; n = 3; Fig. 4c) nd hd reversl potentil close to the theoreticl equilirium potentil for chloride ions ( 74 ± 7.2 mv; n = 4; dt not shown). Single presynptic ction potentils evoked vrile mplitude IPSPs in the postsynptic spiny projection neurons. The coefficient of vrition of the IPSP mplitude rnged from 0.31 to 1.0. The trnsmission ws very relile with n verge rte of filures of only 0.11 ± 0.06 (n = 4). At the resting memrne potentil, (pproximtely 94 mv), the IPSP hd n verge mplitude of 0.42 ± 0.13 mv, (n = 5; rnge, mv). In contrst, the IPSP mplitude ws more thn twice the resting vlue ( 1.06 ± 0.22 mv; rnge, 0.33 to 2.13 mv; n = 7) when mesured t the most depolrized stle memrne potentil (7.6 ± 1.8 mv elow spike threshold; Fig. 4). The IPSP lso hd longer rise time nd slower decy t this memrne c c 20 ms Cont. Control Bicuclline Bic. Wsh 50 ms Fig. 3. Electricl coupling etween two FS interneurons. Depolrizing or hyperpolrizing current injection into FS 1 (I 1 ) induces memrne potentil deflections in oth cells (V m1, V m2 ). The response is much smller in FS 2, pproximtely 3% of tht in FS 1. The dshed line in the upper pnel (V m2 ) is the response of FS 1 normlized to the mplitude of the response in FS 1. Note the sigmoid shpe of the response of FS 2 due to electrotonic filtering long the dendrites etween the two recording sites. (Trces re the verge of single sweeps). potentil (Fig. 4). The decy phse of the IPSPs ws well fitted with single exponentils in ll ut one cse. The verge decy time constnt ws 15.0 ± 2.3 ms t rest nd 36.5 ± 3.5 ms (n = 3) t threshold. The lrger mplitude (nd slower time course) of the IPSP t this memrne potentil is due to the higher input resistnce (nd concomitntly longer time constnt) of the spiny projection neurons (Fig. 1 nd ), s the driving force of the IPSP is even lrger t rest thn ner threshold. 0.5 mv 1 mv -94 mv Fig. 4. Properties of IPSPs elicited in spiny projection cells y FS nd LTS interneurons. () Four superimposed sweeps show vrile mplitude IPSPs evoked in spiny projection neuron t rest y n FS interneuron. Presynptic spikes re indicted y the rrow (stimulted) nd rrowheds (spontneous). () The mplitude nd time course of the IPSP is strongly influenced y the postsynptic memrne potentil. IPSPs evoked y n FS cell in spiny projection neuron t rest (lower green trce; 94 mv) nd ner threshold (upper green trce; 47 mv). The IPSP is depolrizing t rest, nd it is reversed in sign elow threshold. The IPSP mplitude is smller t rest. (c) The IPSP cn e reversily locked with 20 mm icuculline pplied in the perfusion medium, indicting tht it is medited y GABA A receptors. V m2 FS 2 (recorded) V m1 I 1 FS 1 (stimulted) τ = 39.2 ms τf = 7.6 ms τs = 35.2 ms 20 ms -47 mv 0.3 mv -94 mv nture neuroscience volume 2 no 5 my

4 rticles 20 mv 40 ms 10 mv 1, 2 Projection neuron FS neuron 2 mv 20 ms Temporl summtion of IPSPs in response to ursts of ction potentils in the interneuron (2 5 ction potentils occurring within ms) resulted in lrge-mplitude compound responses in spiny projection cells (Fig. 5 nd ). At the most depolrized stle potentil, the verge mplitude of the compound IPSP ws 2.7 ± 0.78 mv (rnge, 0.8 to 4.6 mv; n = 5). Compound IPSPs with significntly lrger mplitudes (up to 6 7 mv) could e elicited during spike fterhyperpolriztions of spiny projection neurons mde to fire y intrcellulr depolrizing current injection (Fig. 5). We tested the cell pirs for the presence of synptic responses evoked in interneurons y spiny projection neurons under numer of conditions. Single spikes or ursts were elicited in spiny projection neurons while the spike-triggered verge of the postsynptic (interneuronl) memrne potentil ws recorded t different levels of depolriztion. The possiility of oth slow ( ms) nd fst responses ws investigted, ut none ws detected, suggesting the sence of feedck from spiny projection neurons to FS or LTS cells. This oservtion ws supplemented with recordings from eleven dditionl pirs of synpticlly connected FS nd spiny projection neurons in slices from younger (16 23 dy old) nimls. The upper nd lower limits of the numer of spiny projection cells innervted y single FS interneurons (divergence) nd the numer of FS cells synpsing on individul spiny projection neurons (convergence) were estimted sed on clcultions of the volume of xon rors of FS interneurons nd known stritl cell densities (see Methods). This nlysis yielded divergence vlues of spiny projection cells per FS neuron nd convergence of 4 27 FS neurons per spiny projection cell. 1, 2 10 mv -50 mv 2,4 1, 3 2, 4 40 mv 40 ms 1,3 Projection neuron LTS neuron Fig. 5. Interneuronl modultion of ction potentil genertion in spiny projection neurons. () A single ction potentil elicited in spiny projection neuron y current injection (upper lck trce) is delyed y IPSPs evoked y single spikes (lower green trce) or spike doulet (lower red trce) of FS interneuron. The dely is vrile (compre green spikes 1, 2), nd the spike doulet (red trces) is more effective thn single spikes. The inset shows the IPSPs t higher gin. () The sme experiment s in () conducted in pir of n LTS interneurons (lower trce, sme cell s in Fig. 1d) nd spiny projection cell (upper trces). The LTS of the interneuron elicits three fst spikes (lower trce) evoking compound IPSPs (upper green trces 1, 3) which prevent the firing of the spiny projection cell (lck trces 2, 4) for pproximtely 20 ms. The momentry firing rte is decresed y 35%. The trils were performed in the order of numering, indicting the stility of the postsynptic cell nd the reliility of the inhiition. Interneuronl inhiition of ction potentil genertion The functionl significnce of inhiition rising from single interneurons ws investigted y exmining their influence on the firing of postsynptic spiny projection neurons. Single or compound IPSPs evoked y FS or LTS interneurons hd profound effect on the genertion of ction potentils in spiny projection cells. The effect of interneuronl inhiition ws tested on spiny projection cells mde to fire etween 30 nd 50 Hz y depolrizing current injection. IPSPs evoked y single presynptic ction potentils in either FS or LTS cells were sufficient to dely the elicited spikes of spiny projection neurons y 5.7 ± 1.4 ms (n = 4; rnge, ms; Fig. 5). IPSP doulets were more effective thn single spikes (Fig. 5). The effect of compound IPSPs elicited y ursts of ction potentils in presynptic interneurons ws studied y evoking n LTS-driven urst in LTS neurons or depolriztion-induced urst of Hz consisting of 2 5 spikes in FS interneurons. Burst firing in the interneurons ws le to cuse very long postsynptic spike delys (24.6 ± 3.8 ms; n = 6; rnge, ms; Fig. 5). Furthermore, the genertion of postsynptic spikes could e locked relily y ctivtion of single interneurons if the IPSPs rrived shortly efore the expected onset of the firing of the spiny projection cell (Fig. 5). The durtion of the dely in the postsynptic spike correlted with the interspike intervl. The dely ws shorter t higher postsynptic firing frequencies nd ws pproximtely constnt frction of the interspike intervl. Therefore the inhiitory effect of single interneurons could e expressed s frctionl decrese of momentry firing rte, which ws pproximtely 10% for single nd 40% for compound IPSPs. DISCUSSION The min finding of the present study is tht GABAergic interneurons cn exert powerful control on the ctivity of projection neurons in the neostritum. The dt demonstrte tht even single ction potentils of one interneuron cn significntly decrese the momentry firing rte of its postsynptic trgets. Single FS interneurons re likely to innervte over one hundred spiny projection neurons; therefore the firing of n FS cell will simultneously lock or dely the firing of ction potentils in lrge popultion of spiny projection neurons. Furthermore, lthough our estimtes of the convergence nd divergence etween FS nd spiny projection neurons re lile to error, it is likely tht ech spiny projection neuron is innervted y t lest four FS cells. Consequently, inhiition y FS interneurons lone cn ccount for mjor frction of the GABAergic control of spiny projection cells oserved in vivo. FS s well s other GABAergic interneurons re known to form feedforwrd link etween the corticostritl projection nd spiny projection cells 17,25,27,28. Tken together with the evidence for the sence or wekness of collterl inhiition 14, the strength of the synptic effect of interneurons indictes tht stritl inhiition is primrily or exclusively feedforwrd. A similr functionl orgniztion hs een previously inferred from recordings in orgnotypic cocultures of stritum, cortex nd sustnti nigr 48. This conclusion is t vrince with mny forml 470 nture neuroscience volume 2 no 5 my 1999

5 rticles models of neostritl informtion processing 29,30. The present dt suggest numer of dditionl fetures of the microcircuitry of neostritl neurons. First, the lck of inhiition of interneurons y spiny projection cells in our smple of 11 mture nd 11 young synpticlly connected cell pirs suggest tht there is no significnt reciprocl feedck from projection neurons to interneurons. Second, there seems to e no inhiition mong (FS) interneurons, lthough our smple of pirs of FS cells tested ws very smll. Finlly, FS interneurons re likely to communicte through electrotonic coupling. The iophysicl properties of these vrious (synptic or electrotonic) interctions of interneurons my e criticl determinnts of their popultion ctivity, possily resulting in complex ctivity ptterns 31,32. The present dt demonstrte tht spiny projection neurons receive strong inhiitory input from t lest two physiologiclly distinct types of interneurons. Becuse of their different intrinsic memrne properties, FS nd LTS neurons re likely to hve dissimilr response to synptic inputs. In prticulr, the higher input resistnce of LTS neurons nd their expression of regenertive depolrizing potentil suggest tht these neurons my require less strong excittory input for the firing of ction potentils thn do FS neurons nd tht their response my e short stereotypicl urst. Without ntomicl nd neurochemicl identifiction, the fferents of LTS neurons nd their plce in the microcircuitry of the neostritum remin unknown. Severl ntomicl fetures of FS interneurons re inconsistent with role of providing strictly sptilly loclized inhiitory input to spiny projection cells. FS interneurons mke up only smll frction (3 5%) of the rodent neostritl neuronl popultion 17,25,26 nd hve widely divergent output 17,25, ech innervting over hundred spiny projection neurons. Furthermore, we found evidence of electricl coupling in one third of the tested pirs of FS neurons (Fig. 3) which is consistent with previous ultrstructurl dt. This locl syncitil orgniztion suggests more generl regultory function for neostritl inhiition. Studies using focl corticl stimultion nd immunocytochemicl detection of immedite-erly gene expression s mesure of stritl ctivity reveled tht FS neurons re indeed ctivted in wider neostritl territories, from more extended corticl res nd t lower threshold thn spiny projection cells 33. By integrting the level of ctivity from wider corticl re, interneuronl inhiition my suppress the firing of suoptimlly excited spiny projection neurons nd selectively enle the ctivtion of spiny projection neurons tht receive ove verge corticl excittory input. In ddition, the electriclly coupled network of interneurons my synchronously inhiit projection neurons, influencing the temporl reltionship of their spike trins 32,34,35. The strong postsynptic effect nd syncitil orgniztion of FS interneurons mke them well suited for distriuting nd mplifying the effects of vrious stritl fferents, including neuromodultory inputs. Interneuronl medition of neuromodultory control of the functioning of locl neuronl circuits hs een descried in other rin structures such s the cererl cortex 36 or the hippocmpus 34. It is of prticulr interest tht the dopminergic nd cholinergic control of the firing of spiny projection neurons my in prt e medited y interneuronl inhiition, ecuse PV + interneurons express dopmine receptors 37, IPSPs in spiny projection cells re presynpticlly inhiited y dopmine 38,39, nd PV + interneurons receive cholinergic synptic input from locl interneurons 40. In ddition, PV + nd NOS + neostritl GABAergic interneurons my lso medite the effect of other stritl fferents such s the pllidostritl input 41. In conclusion, the present dt demonstrte tht, despite their smll numer, neostritl GABAergic interneurons re powerful determinnts of the ctivity of projection neurons in the neostritum. This findings potentilly open new directions to understnding vrious norml nd pthologicl processes involving the sl gngli. METHODS Visulized whole-cell recordings were otined in slices from young dult (24 32 dy) nd in some cses juvenile (17 21 dy) Sprgue-Dwley rts. Surgicl nd other procedures were done with the pprovl of the Rutgers University Institutionl Reserch Bord nd in ccordnce with the NIH Guide to the Cre nd Use of Lortory Animls. Eleven synpticlly connected pirs of interneurons nd spiny projection neurons were recorded in oth the older nd the younger group ( totl of 22 pirs). Older nimls were used to ensure the physiologicl nd morphologicl mturity of recorded neurons nd their connections, nd ll reported physiologicl dt were otined from the older group. Six of the interneurons from the older group nd four from the younger were nlyzed morphologiclly. Phrmcologicl lockde of the IPSP ws demonstrted in slices from the younger group. Slice preprtion. Rts were deeply nesthetized with 80 mg per kg ketmine nd 15 mg per kg xylzine i.p., trnscrdilly perfused with 4 5 ml ice-cold modified Ringer s solution (see elow) nd µm olique horizontl or coronl sections were cut in the sme medium on Viroslice (Cmpden Instruments). The norml Ringer s solution contined 125 mm NCl, 2.5 mm KCl, 2.5 mm CCl 2, 1.5 mm MgCl 2, 26 mm NHCO 3, 1.25 mm NH 2 P0 4 nd 9 mm glucose (ph ). In the modified solution, NCl ws sustituted for choline chloride, nd the solution contined 0.3 mm CCl 2 nd 3.7 mm MgCl 2. Slices were incuted in norml Ringer s solution t for 1 2 h immeditely fter sectioning. Visulized whole-cell recording. Recordings were otined with orosilicte pipettes pulled from 1.5 dimeter tuing (W.P.I.) hving 5 8 MW impednces. The intrcellulr solution contined mm K-gluconte, 11.1 mm KCl, 2 mm MgCl 2, 10 mm HEPES, 3 mm N 2 ATP, 0.3 mm GTP nd 0.02 mm EGTA (ph ). E Cl = 58 mv t 35 C. The equilirium potentil for chloride ws set t 58 mv sed on reversl potentil mesurements of GABA A responses in spiny projection cells 23,42. Memrne potentil mesurements were corrected for the liquid junction potentil off line y 8 mv clculted from the generlized Hnderson eqution 43,44. Recordings were mde with Neurodt IR-283 dul chnnel current clmp mplifier. Neurons were visulized under infrred differentil interference contrst (IR-DIC) microscopy using n Olympus BX50 microscope equipped with 40 long-working-distnce wter immersion ojective. Interneurons were trgeted sed on their differentil ppernce under IR-DIC. For neurotrnsmitter/receptor identifiction, 20 mm icuculline methochloride (Sigm) ws pplied in the superfusion medium. Histologicl procedures. Interneurons nd spiny projection neurons were recorded with micropipet filled with the intrcellulr solution contining 0.6% nd 0.15% iocytin, respectively. Visuliztion of iocytin nd douleleling immunocytochemistry were done with modifictions of descried procedures 17,45. Briefly, slices were immersion fixed in 4% prformldehyde nd 0.3% picric cid for 1 4 h t RT nd resectioned t 50 or 60 µm. Sections were then incuted in 1% NBH for 50 minutes nd in mixture of 3% H 2 O 2 nd 10% methnol for 15 minutes. For visuliztion of iocytin lone, sections were incuted in PB contining 0.5% Triton X-100 nd 1:200 ABC regent (Vector) overnight nd rected with 3,-3 -diminoenzidine s chromogen (in most cses using nickel intensifiction). For doule-leling immunocytochemistry, sections were incuted overnight in PB contining 2% ovine serum lumin, 10% norml got serum, 0.5% Triton X-100 nd mouse monoclonl -PV IgG (Sigm) t 1:2000 dilution. Biocytin ws then visulized y incution in 1:300 AMCA conjugted streptvidin (Jkson Lortories) nd prvlumin ws visulized with 1:600 Cy3 conjugted got -mouse IgG (Sigm). The neurons were exmined using n Olympus BX60 epifluorescence microscope with stndrd filter sets. Neurons were reconstructed on Nikon Optiphot microscope under 100 -oil immersion ojective using drwing tue. nture neuroscience volume 2 no 5 my

6 rticles Dt nlysis. Convergence nd divergence vlues were estimted on the sis of the volume of the xon ror of FS cells nd the density of spiny projection neurons. The verge volume of the xon ror of FS neurons ws 6.85 ± 1.19 x 10 3 mm 3 (n = 4) in our smple, ssuming threedimensionlly symmetricl xon ror. The neuron density in the stritum is 84,900 cells per mm 3 (refs. 46, 47), which corresponds to 541 ± 101 spiny projection neurons within the xon ror of n FS cell, the upper limit of divergence. The upper limit of convergence is 27 FS cells per spiny projection neuron if ll spiny projection neurons re innervted within the presynptic xon ror nd if FS cells mke up their estimted mximl popultion of 5% (refs. 25, 26). As pproximtely one qurter of the spiny projection neurons show synptic interction in the vicinity of n FS cell (within 250 mm), miniml divergence of 135 spiny projection neurons per FS cell nd convergence of 4 FS neurons per spiny projection cell cn e clculted for the miniml FS neuron popultion of 3% (refs. 25, 26). ACKNOWLEDGEMENTS We thnk K. Moore nd K. Png for help during the erly phse of this experiments, J.P. Bolm, A. Sík nd L. Záorszky for dvice concerning ntomicl methods, G. Buzsáki, A. Czurkó, A. Göndöcs, C. Pldini nd D. Shohmy for criticlly reviewing the mnuscript nd F. Shh for technicl ssistnce. Supported y MH58885, NS34865 nd Rutgers University Busch-BRSG funds. RECEIVED 19 JANUARY; ACCEPTED 11 MARCH Bolm, J. P. & Bennett, B. D. in Moleculr nd Cellulr Mechnisms of Neostritl Function (eds. Mrjorie, A., Arino, M. A. & Surmeier, D. J.) 1 2 (Springer, Heidelerg, 1993). 2. Gerfen, C. R. & Wilson, C. J. in Hndook of Chemicl Neurontomy Vol. 12 Integrted Systems of the CNS (eds. 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