Laminar sources of synaptic input to cortical inhibitory interneurons and pyramidal neurons

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1 rtiles Lminr soures of synpti input to ortil inhiitory interneurons nd pyrmidl neurons J. L. Dntzker nd E. M. Cllwy Systems Neuroiology Lortories, The Slk Institute for Biologil Studies, N. Torrey Pines Rd., L Joll, Cliforni, 9237, USA, nd Deprtment of Biology, University of Cliforni Sn Diego, L Joll, Cliforni, USA Correspondene should e ddressed to J.L.D. (dntzker@slk.edu) The funtionl role of n individul neuron within ortil iruit is lrgely determined y tht neuron s synpti input. We exmined the lminr soures of lol input to sutypes of ortil neurons in lyer of rt visul ortex using lser snning photostimultion. We identified three distint lminr ptterns of exittory input tht orrespond to physiologil nd morphologil sutypes of neurons. Fst-spiking inhiitory sket ells nd exittory pyrmidl neurons reeived strong exittory input from middle ortil lyers. In ontrst, dpting inhiitory interneurons reeived their strongest exittory input either from deep lyers or lterlly from within lyer. Thus, differentil lminr soures of exittory inputs ontriute to the funtionl diversity of ortil inhiitory interneurons. The ererl ortex ontins mny types of neurons elieved to serve unique funtionl roles in informtion oding. Notly, ortil inhiitory interneurons hve diverse morphologil, moleulr nd physiologil properties 7. In ddition to these intrinsi hrteristis, eh ortil neuron s funtion is shped y its speifi exittory nd inhiitory synpti inputs 8,9. Thus, orrelting neuron s soures of input with other fetures unique to eh ell type is ruil step towrd understnding its role in ortil iruit. Here we sk whether inhiitory interneurons nd exittory neurons in ortil lyer of rt visul ortex reeive exittory synpti input from different ortil soures. Cortil iruits re est understood t the levels of lminr nd modulr orgniztion of exittory onnetions,. For exmple, xons of ortil exittory neurons onnet within 3 µm olumn to prtiulr ortil lyers. However, eh of these lminr omprtments ontins mny ell types. Do exittory onnetions trget speifi ell types within ortil lyer? Previous eletron mirosopy studies exmined this question in lyer of t visul ortex y orrelting synpti outon morphologies on individul neurons to lol nd thlmi fferent soures 2,3. These studies found no differene in the proportion of exittory inputs reeived y inhiitory nd exittory neurons in the sme ortil lyer. In ontrst, other evidene suggests tht inhiitory interneurons prtiipte in ell-type-speifi onnetions. For exmple, suortil rin regions tht innervte ortex trget speifi interneuron types 7. Additionlly, inhiitory interneurons form seletive onnetions to other types of interneurons or to prtiulr postsynpti elements on exittory neurons To ddress whether exittory ortil onnetions distinguish etween neuron sutypes within their trget lyers, we exmined the soures of exittory ortil input tht onverge on individul inhiitory interneurons nd their neighoring exittory pyrmidl neurons in lyer. We mpped synpti input using whole-ell reordings omined with fol relese of ged glutmte (lser snning photostimultion) 2,25, physiologil method tht llows fine-resolution stimultion of neurons. We quntified nd ompred exittory synpti input from ortil lyers 2 mong neuron types. For suset of neurons, we lso relted ptterns of exittory input to inhiitory synpti input. This nlysis reveled three distint lminr ptterns of exittory input tht orrelted with prtiulr ell types. In ontrst, lminr ptterns of inhiitory input were indistinguishle mong ell types. RESULTS We reorded synpti urrents in individul neurons trgeted with infrred differentil interferene ontrst mirosopy (IR-DIC) in oronl rin slies ( µm thik) from rts ged postntl dys P2 to P3, ner the plteu of ortil mturtion 2. For eh postsynpti neuron, we photostimulted 2 to over 5 presynpti sites ross ortil lyers 2 y folly unging glutmte. We onstruted mps of lotions providing exittory synpti input to individul pyrmidl nd inhiitory interneurons nd ompred input strength ross ortil lyers sed on the mplitude nd numer of evoked postsynpti urrents (PSCs). For suset of these neurons, we lso mpped inhiitory synpti input. Sptil resolution of photostimultion Neurons within the ortex re highly interonneted; dendriti nd xonl proesses often spn ross severl ortil lyers. Thus, mpping funtionl soures of synpti inputs with lyer-speifi resolution requires method tht sptilly onfines tion potentil (AP) genertion to few presynpti neurons, ut rrely stimultes pssing xonl nd dendriti proesses from distnt neurons. Here we show tht photostimultion genertes APs in sptilly lolized popultion of neurons y unging glutmte with quik flsh of foused ultrviolet light. Neurons reorded in whole-ell, urrent-lmp mode were photostimulted diretly nture neurosiene volume 3 no 7 july 2 7

2 rtiles Fig.. Sptil resolution of lser-snning photostimultion. During photostimultion, most APs our from diret stimultion ner neuron s som, not from stimultion of distl dendrites or synpti tivtion vi distnt neurons. () Cytohrome-oxidsestined oronl setion ontining ioytin-leled lyer pyrmidl neuron reorded in urrent lmp nd stimulted t the ortil lotions shown y the superimposed grid of irles. Colored irles indite the numer of APs reorded t the som within 5 ms fter photostimultion t eh site (olor r sle elow). Most APs (ottom tres) ourred within 75 µm rdius ner the neuron s som nd were mintined mostly within lyer. However, this neuron fired single AP when stimulted t one lotion on its pil dendriti tuft in lyer. This ws typil for ll neurons tht hd pil tufts in lyer. Thus, lyer stimultion sites were not inluded in the nlysis of input mps. The onset of the UV flsh is indited y n rrow (durtion, ms). () The men numer of APs generted per stimultion site (± s.e.m.) with vertil distne from som, inluding sites where no APs were generted, demonstrtes lyer speifiity of photostimultion (n = 2 neurons from lyers 2 ). () Diret photostimultion urrents were distinguished from synpti urrents during voltge-lmp experiments sed on lk of lteny etween onset of glutmte unging nd inwrd urrent. This exmple demonstrtes single-tril voltge-lmp reording in whih diret urrent () is followed y six synpti urrents. Photostimultion-evoked synpti urrents were loked y infusing 2 µm TTX into the slie, reveling solely the diret urrent. nd up to severl hundred mirons wy from their somt (Fig. ). Photostimultion eliited multiple APs from eh neuron (men,.9 ±.7 APs per stimultion site). Neurons from ll ortil lyers (n = 2) fired 8% of their APs when stimulted t lotions within µm of their somt (Fig. ). On verge, ll APs fired were within rdius of 5 ± 23 µm from the som (men ± s.d). One exeption ws tht lyer 2 5 neurons with pil dendriti tufts in lyer lso fired tion potentils when their tufts were stimulted in lyer (Fig. ). Therefore, lyer ws not inluded in this study euse the lminr origin of the tivted neuron ould not e determined during experimentl trils. These sptil resolution experiments indite tht, with the exeption of lyer, the lotions of presynpti neurons tht re photostimulted to fire APs n e determined with n effetive resolution of 5 75 µm. Neurons only fire APs when diretly stimulted, ruling out the possiility of polysynpti APs from photostimulted neurons in other distnt ortil lyers. For exmple, lthough lyer provides strong onnetions to lyer 5 neurons 27, photostimultion in lyer did not result in AP genertion in lyer 5 neurons. Finlly, these experiments indite tht presynpti neurons fire multiple APs when photostimulted, inresing the proility of deteting wek input to postsynpti ell 28. Exittory input mps Exittory input mps, otined y mesuring the mplitude nd numer of inwrd synpti urrents from postsynpti neurons voltge-lmped t 5 mv, show severl ptterns of lminr input to distint types of lyer neurons. Within 5 µm of somt nd proximl dendrites, diret urrents due to glutmte unging on the reorded neuron exeeded pa nd deyed over ms so tht the entire window ws omitted from nlysis (Fig. ). Therefore, our mesurements of lyer onnetions re ised towrd input t lest 5 µm wy from the reorded neuron. We egin y summrizing exittory input ptterns, with representtive exmple of eh neuron type (Fig. 2). We then desrie the morphologil nd physiologil hrteristis of ells nd wm 2µm 235 mv 5 ms # APs present the synpti input nlyses etween ell groups. For lyer pyrmidl neurons (n = 23), the lrgest mplitude nd numer of exittory postsynpti urrents (EPSCs) were reorded fter stimulting in lyer (Fig. 2). Exittory input to pyrmidl neurons ws similr for ll neurons exmined, wheres exittory input to interneurons (n = 37) fell into t lest three different lminr ptterns. Fst-spiking inhiitory interneurons reeived strong exittory input in pttern similr to pyrmidl neuron inputs (Fig. 2). Adpting interneurons (known s omodting or regulr-spiking non-pyrmidl 29 ) reeived either dominnt input from lyer 5 (Fig. 2) or dominnt lterl input from lyer (Fig. 2d, see lso Fig. ). Although these input mps re representtive of evoked EPSCs, they lso inlude spontneous PSCs (see elow). Interneuron sutypes Interneurons tht reeived the three different ptterns of synpti input displyed rnge of morphologies nd orresponding firing properties (Fig. 3 nd ), ompred with n exmple pyrmidl neuron tht displyed the typil regulr firing pttern 3 (Fig. 3). Lyer interneurons trgeted using IR-DIC mirosopy hd round or ovoid somt without n ovious dendrite extending towrd the pil surfe. Morphology ws verified y reonstruting ioytin-leled neurons, nd firing properties were exmined during steps of intrsomti urrent injetion. Differenes in spiking ptterns etween interneurons divided them into two distint groups. Fst-spiking (FS) inhiitory interneurons (n = 7) were distinguished from other types of interneurons y lk of dpttion in spike rte during intrsomti urrent injetion (Fig. 3, lte-phse dpttion lulted s the rtio of the interspike intervl of the penultimte pir of spikes nd finl pir of spikes ner threshold urrent levels,.3 ±.; rnge,.97 to.) nd liner inrese in spike rte with inresing urrent,3. All fst-spiking interneurons tht we reonstruted (/7) were sket ells 9,32 with most of their xons spreding horizontlly within lyer (Fig. 3). Numer of APs Distne from som (µm) efore TTX fter TTX pa 5 ms 72 nture neurosiene volume 3 no 7 july 2

3 rtiles Fig. 2. Lminr exittory input to lyer ortil neurons flls into three ptterns. Pseudo-olored input mps demonstrte representtive ptterns of exittory input to four individul neurons. These input mps re liner interpoltions of the sum of EPSC mplitude vlues mesured following photostimultion t sites sped 5 µm prt. Colored vertil sle rs demonstrte the orresponding sum of EPSC mplitude vlues for input mps to the left nd right. () Pyrmidl neuron tht reeives its strongest exittory input from lyer nd wekest input from lyers (totl numer of stimultion trils, t = 5). () Fst-spiking inhiitory interneuron tht reeives its strongest input from lyers nd nd modertely strong input from lyer (t = 35). () Adpting interneuron tht reeives its strongest input from lyer nd moderte input from lyers 2 (t = 37). (d) Adpting interneuron tht reeives strong input solely from lyer (t = 8). Below eh input mp for ll pnels re exmple voltge-lmp reordings ( 5 mv holding potentil) mde while stimulting presynpti regions tht resulted in low ( ) nd high (+) EPSC sum vlues. Short dshes ove eh tre show the onset of glutmte tivtion. The urrent mrked with is diret urrent from glutmte unging nd ws omitted. Horizontl lines rossing the input mps represent the ntomil lminr orders. The djent tles give the men EPSC mplitude sum (± s.e.m.) for eh lyer nd the men spontneous EPSC sum vlues mesured during no-stimultion ontrols. Antomil reonstrutions of dendriti nd xonl rors (only dendrites in ) re shown in white. Sle rs pply to ll pnels. Pyrmidl Neuron Lyer sepscs Adpting Interneuron Lyer EPSC mplitude 35±3. 8±. 32±5. 5±2. ±.9 ±.2 EPSC mplitude 29±3. 7±5. 9±7. 59±5.9 23±2. sepscs ±.5 pa µm 2 pa 5 ms pa µm 2 pa 5 ms d Fst-spiking Interneuron Lyer EPSC mplitude sepscs 3±.2 Adpting Interneuron 2±3 2±2 2±23 78±9.2 8±3.2 Lyer EPSC mplitude 5±5. ±.5 3±2. ±.2 7±.7 sepscs 9±.3 Adpting interneuron properties nd input The remining interneurons were dpting, identified y their deresing spike rte during urrent injetion (n = 2; Fig. 3, ltephse dpttion,.9 ±., rnge,.79.97). Antomilly reonstruted dpting interneurons were most similr to smll or nest sket ells nd Mrtinotti ells 7,29,33 ; however, vrition in xonl nd dendriti morphologies etween neurons ws too high for definitive ssignment of dpting interneurons into distint ntomil lsses. Nevertheless, two distint groups of dpting interneurons ould e lerly differentited sed on the perentges of exittory synpti input they reeived from lyer (Fig., see Methods for lultion of perent input) either they reeived less thn 37% (for exmple, Fig. 3, first 2 neurons) or over 53% (Fig. 3, lst 2 neurons) of their input from lyer. Those tht reeived little lyer input reeived etween 28% nd 7% of their input from lyer 5 (Fig. ), wheres those tht reeived strong lyer input reeived less thn 2% of their input from lyer 5. Adpting interneurons tht reeived stronger input from deeper lyers will e referred to s dpting type (AD, n = 9). Those tht reeived most of their input from lyer will e referred to s dpting type 2 interneurons (AD2, n = ). Mesured differenes in dpting interneurons intrinsi physiologil hrteristis did not orrelte with the two types of input ptterns. For exmple, mny prmeters used to distinguish the spiking ptterns of neurons in other studies 3,3 were lso tested in our popultion. Mesures of input resistne, spike mplitude nd width, interspike intervl nd fterhyperpolriztion mplitude nd re were not signifintly different etween the two types of dpting interneurons (dt not shown). Quntittive omprisons of exittory lminr input Lminr synpti input ws quntified y omining two prmeters, the numer nd the mplitude of EPSCs ourring within 5 ms following photostimultion t eh stimultion site (Fig., Tle ). This ws done to llow reltively equl weighting of frequent ut smll-mplitude EPSCs versus infrequent, lrge-mplitude EPSCs. For eh neuron, the produt of the numer nd men mplitude of EPSCs (EPSC sum) within lyer were ollpsed into one men vlue for the lyer (for exmple, Fig. 2) nd normlized to spontneously ourring PSCs (sepscs) during no-stimultion ontrols for eh neuron, resulting in normlized evoked input (NEI, Tle, see Methods). nture neurosiene volume 3 no 7 july 2 73

4 rtiles Fig. 3. Types of lyer interneurons nd pyrmidl neurons for whih lminr synpti input ws mpped in rt visul ortex. Antomil reonstrutions nd spiking ptterns to low ( to 2 pa, top tres) nd higher (25 to 5 pa, ottom tres) intrsomti urrent injetion demonstrte the rnge of postsynpti neuron types reorded in P2 3 rts. () Pyrmidl neuron demonstrtes typil regulr dpting firing pttern. (, ) Interneurons with smll, round somt trgeted with DIC mirosopy were either sket ells with spike rtes tht did not derese during urrent injetion, similr to lssi fst-spiking interneuron (), or sutypes of sket ells with spike rtes tht dpted during urrent injetion (). Among these dpting interneurons, morphology nd spiking hrteristis were highly vrile, so tht further sulsses ould not e distinguished. Those shown here resemle smll nd nest sket ells. Dendrites for ll neurons re shown s lk proesses nd xons s gry. (Axons re omitted for pyrmidl neuron.) Sle rs pply to ll pnels. Beuse neurons usully fire smll numer of APs within long time window ( 5 ms) following photostimultion (for exmple, Fig. ), individul EPSCs likely represent unitry responses from single presynpti neuron. Thus, lrge-mplitude responses re suggestive of onnetions tht re either more relile nd/or omposed of multiple relese sites. Likewise, euse stimultion prmeters were held onstnt in our experiments, when ompring etween neuron types, differenes in the numer of reorded EPSCs suggest differenes in the numer of neurons onneted to neuron sutypes (lthough it ould lso suggest inresed relese proility). Sttistil omprisons of lminr inputs using NEI nd perentge input reveled signifint differenes mong pyrmidl neurons, FS interneurons nd dpting interneurons. Pyrmidl neurons (n = 23) nd FS interneurons (n = ) reeived the highest frtion of their input from lyer (Fig. ; one FS interneuron omitted euse of insuffiient stimultion sites in lyer ). This input ws signifintly greter to pyrmidl neurons (7.5 ± 2.7%) thn the proportion of lyer input to either AD Perent lyer 5 NEI Perent lyer Exittory Input FS AD AD2 Pyrmid FS AD AD2 Lyers Perent exittory input 8 2 Pyrmid FS AD AD2 Pyrmid µm 2 mv 5 ms Adpting smll nd nest sket ells l. l. l.5 l. Fst spiking sket ells µm 2 mv 5 ms (3. ± 3.7%) or AD2 (9.5 ± 3.3%) interneurons (p.5, onewy ANOVA) nd greter to FS interneurons (3.7 ± 3.8%) thn to AD2 interneurons (9.5 ± 3.3%, p.). AD interneurons (n = 9) reeived out threefold more of their input from lyer 5 (.8 ±.7) thn ny other group (pyrmidl ells, 5.2 ±.7%; FS, 5.2 ±.8%; AD2, 5.3 ±.9%, p <.; Fig. ). This greter proportion of lyer 5 input ws not simply due to less input from other lyers; the solute mgnitude of lyer 5 input (NEI) reeived y AD interneurons ws greter thn tht reeived y either pyrmidl neurons or AD2 interneurons (ut not FS interneurons, p.5; Tle ). AD2 interneurons (n = ) reeived 72.8 ±.% of their input from lyer (Fig. ). This ws signifintly higher frtion thn reeived y ny other group (pyrmidl neurons, 27.3 ± 3.3%; FS,. ± 3.%; AD, 23. ± 2.7%; p.). Additionlly, oth AD2 interneurons nd FS interneurons reeived more solute input (NEI) from lyer thn pyrmidl neurons (p <.5; Tle ). AD2 interneurons reeived, t Fig.. Lminr exittory input is homogeneous to fst-spiking interneurons, ut dpting interneurons fll into two groups. () Perentge of exittory input from lyers versus lyer 5 ( nd omined) plotted for individul fst-spiking (FS) nd dpting interneurons. Input vlues re expressed s perentge of totl input reeived from ll lyers. Exittory input to dpting interneurons fell into two non-overlpping lsses. Adpting interneurons reeived either < 37% (AD type, AD) or > 53% (AD type 2, AD2) exittory input from lyer. FS interneurons were intermedite etween the two types of dpting interneurons. () Perentge of input from ll lyers to the four ell types. Pyrmidl neurons nd FS interneurons reeived similr proportion of input from lyer. () Normlized evoked input (NEI; Tle ) from superfiil lyer 5 (top 5 µm, lyer ) versus deeper lyer 5 (). All neuron types exept AD2 interneurons reeived signifint lyer input ove kground spontneous tivity (p <., repeted-mesures ANOVA). Only AD interneurons reeived signifint lyer input. 7 nture neurosiene volume 3 no 7 july 2

5 rtiles Tle. Normlized exittory input (NEI) lulted from numer nd mplitude of EPSCs reorded following photostimultion in eh ortil lyer for pyrmidl neurons nd inhiitory interneurons. Pyrmidl neurons Fst-spiking interneurons Adpting type interneurons Adpting type 2 interneurons Stimulus Numer Amplitude NEI Numer Amplitude NEI Numer Amplitude NEI Numer Amplitude NEI lotion Lyer. ±.2 2. ±. 2. ±.3. ± ±. 5.9 ±..9 ±.3.8 ±..9 ±. 2.8 ±.3 2. ± 3.5. ±.5 Lyer 2. ± ±.9. ±. 5.2 ± ±.7.5 ±.2 2. ± ±. 2.9 ±.8. ± ± 2.. ±.2 Lyer 5.2 ±. 7. ±.8.3 ±.2 3. ±. 8. ±.9 2. ± ±.5 9. ± ±.7.2 ± ±.8.5 ±.2 Lyer.9 ±.. ±.5. ± ± ±.2.5 ±.3.2 ±.2 3. ±..3 ±.2.9 ±. 5. ±.8. ±. sepscs. ±.. ±.8 2. ±.3.8 ±.7. ±.2. ±.8.9 ±..7 ±. NEI lulted s the men produt of the numer nd mplitude of EPSCs mesured t ll sites within lyer minus the sme vlue mesured for spontneous EPSCs (sepscs) for eh ell (Methods). Amplitude vlues used to lulte the NEI were divided y the verge mplitude of sepscs to ompenste for vritions in series resistne etween reordings. Signifint ove kground spontneous vlues (p <.5); tested using repeted-mesures ANOVA nd pirwise omprisons to spontneous ontrol vlues for eh neuron. est, wek input from lyers other thn lyer, s only lyer provided signifint input ove spontneous synpti tivity sed on mplitude nd numer of EPSCs (Tle ). Deep-lyer input (lyers 5 nd ) ws reltively wek to ll neuron types exept AD interneurons, lthough pyrmidl neurons, AD nd FS interneurons reeived signifint input from lyer 5 (Tle ). Lyer 5 n e divided into sulyers, nd. Lyer omprises the top 5 75 µm of lyer 5 nd stins lightly for ytohrome oxidse (for exmple, Fig. ). All neuron types exept AD2 interneurons reeived signifint input from lyer. In ontrst, only AD interneurons reeived signifint input from lyer (Fig. ). Beuse the effetive resolution of our stimultion ws 5 µm, the strong lyer input my e due to the stimultion of sl dendrites from lyer neurons extending into lyer 5. Although some individul neurons reeived signifint lyer input (dt not shown), on verge lyer input ws wek nd not signifintly different etween groups. Wek input from lyer is supported ntomilly, s most of our neurons re in the top hlf of lyer, nd lyer neurons in rt ortex tend to projet xons only s high s deeper lyer (refs. 35, 3). Inhiitory lminr input Cortil inhiition n shpe neuronl tivity in lyer-speifi fshion 37,38. In our experiments, we did not lok inhiition, so it is theoretilly possile tht insted of differentil exittory input, neurons reeived differentil lminr inhiitory input tht shunted exittory input from ertin lyers. For exmple, AD interneurons my hve reeived less inhiition from lyer 5 thn other neuron types, resulting in lrger pprent exittory input from lyer 5. Likewise, AD2 interneurons my hve reeived signifint inhiition from lyers 5 tht shunted exittory input from those lyers. To test these possiilities, we mesured inhiitory input in suset of the neurons (n = 27, Fig. ) y hnging the holding potentil to etween nd 35 mv, inresing the driving fore for inhiitory urrents. Inhiitory urrents were distinguished from exittory urrents y their outwrd diretion (Fig. ). In ontrst to exittory input, inhiitory input did not fll into distint lminr ptterns mong neuron sutypes. Most lyer neurons, regrdless of type, reeived most of their inhiitory input from neighoring interneurons in lyer (Fig. ; pyrmidl neurons, 78. ± 5.5%, n = ; FS, 7.8 ± 9.9%, n = 5; AD, 2.8 ± 3.%, n = 7; one AD2 interneuron omitted euse it lked ny signifint inhiitory input), nd nerly ll the rest of their inhiitory input from lyer (pyrmidl neurons,.2 ±.%; FS, 2.7 ± 9.8%; AD, 3.5 ± 2.2%, no signifint differene etween interneurons nd pyrmidl neurons for lyers or ). Few lyer pyrmidl neurons (/) nd FS interneurons (/5) reeived signifint inhiitory input from lyer 5, suggesting tht exittory input from lyer 5 ws not strongly influened y inhiition from lyer 5 (inhiitory input signifine determined s explined for EPSCs in Tle ). Additionlly, AD interneurons did not reeive more lyer (2/8) or lyer 5 (/8) inhiitory input thn other neuron types. In onlusion, ll ell types reeived most of their inhiition from neighoring inhiitory interneurons within lyer, onsistent with the xonl ntomy of most interneuron types 29. Therefore, exittory input from lyer my e stronger thn mesured here. It is unlikely, however, tht the distint lminr ptterns of exittory input oserved re due to the lminr orgniztion of inhiitory input. DISCUSSION We used physiologil method, fol relese of ged glutmte y lser snning photostimultion, to exmine the soures of lol synpti input to individul inhiitory interneurons nd Perent lyer 8 2 Inhiitory Input Perent lyer Pyrmids FS AD AD pa 5 ms Fig. 5. Lminr ptterns of exittory input re not due to differentil inhiitory input. () Perentge of inhiitory input from lyer versus lyer plotted for individul pyrmidl neurons nd inhiitory interneurons. Unlike exittory input, there ws no signifint systemti vrition in inhiitory input mong the different ell types. Most neurons reeived the mjority of their inhiitory input from lyer nd less from lyer. Neurons tht lie on the midline of the grph reeived no inhiitory input from lyers 5 or. The symol + indites n inhiitory interneuron of unknown type. () Exmple IPSCs mesured in four different ells (V H = to 35 mv) following photostimultion in lyer nd lyer. Inhiitory urrents were distinguished from exittory urrents y their outwrd diretion t ll holding potentils used in this study. Pyrmid Nonpyrmid nture neurosiene volume 3 no 7 july 2 75

6 rtiles pyrmidl neurons in lyer of rt visul ortex rin slies. We found tht pyrmidl nd fst-spiking inhiitory neurons reeived similr lminr distriutions of exittory input nd showed little vrition etween individul neurons. However, ptterns of exittory input to dpting interneurons were unlike input to pyrmidl nd FS interneurons nd formed two seprte lsses. Lminr ptterns of inhiitory input, in ontrst, did not vry signifintly mong neuron sutypes. These findings provide evidene tht distint types of inhiitory interneurons within ortil lyer reeive different ortil informtion, ontriuting to their unique funtionl roles in ortil proessing. All ortil lyers ontin exittory neurons tht send xons to lyer (ref. 3); thus, if ortil exittory onnetivity is predited stritly y xonl density, s suggested previously 39,, distint ell types in lyer should reeive similr distriutions of lminr input. Contrry to this notion, we found tht exittory fferent xons from different lminr soures onnet preferentilly to neuron sutypes within lyer. For exmple, dt presented here nd in other studies 27, indite tht lyer pyrmidl neurons rrely form strong synpses onto pyrmidl neurons in lyer. We showed tht, insted, lyer pyrmidl neurons formed preferentil onnetions onto suset of dpting interneurons (AD) nd less so to FS inhiitory interneurons. Lyer onnetions to lyer lso showed speifiity, s demonstrted y the lk of signifint lyer input to AD2 interneurons. In ontrst to lyers nd 5, lyer seemed to onnet nonspeifilly to ll neuron sutypes exmined. It is importnt to note tht n rtift of utting xons in the sliing proess nnot explin these different input ptterns. If this were the se, then oth FS nd pyrmidl ells would hve shown the sme diversity of input. The funtionl signifine of differentil exittory input to lyer inhiitory interneurons desried here depends, in prt, on the postsynpti trgets of these interneurons. Speifilly, tivtion of inhiitory interneurons ould either derese exittion y diret inhiition of exittory ells or inrese exittion y disinhiition vi other inhiitory interneurons 23. Beuse lyer pyrmidl neurons form the mjor efferent projetions to other ortil res 2, these two senrios would result in very different onsequenes on the flow of informtion etween ortil res. Indeed, interneuron sutype is orrelted with postsynpti trget type. Bsket ells predominntly trget the somt nd proximl dendrites of pyrmidl neurons 9,3, (ut see refs. 7, 23). In ontrst, lyer lretinin-positive inhiitory interneurons predominntly trget other inhiitory interneurons in lyer (refs. 2, 2). It follows tht FS ells in our popultion, ll of whih re sket ells, likely inhiit neighoring lyer pyrmidl neurons when tivted y lyer. The orreltion of input pttern with interneuron sutype is less distint mong our dpting interneurons. However, most AD interneurons resemled smll sket ells, wheres mny AD2 interneurons resemled itufted lretinin-positive ells 2. Thus, lyer neurons my seletively inhiit dendrites in lyer vi AD interneurons. Additionlly, lyer pyrmidl neurons my form positive-feedk iruit y disinhiition through AD2 interneurons. To etter link ortil inputs nd outputs of interneurons, future studies should omine knowledge out lminr inputs with more detiled understnding of the distinguishing fetures of interneuron sutypes 8. In light of mounting evidene showing tremendous vriety of interneuron sutypes sed on differenes in physiologil, moleulr, morphologil nd synpti properties,5,7, it is surprising tht we identified only three lminr ptterns of funtionl input. This my e euse our study is not n exhustive survey of ll possile types of interneurons. (For exmple, FS hndelier ells nd ipolr neurons were not smpled.) It is lso likely tht speifiity of xonl onnetivity demonstrted here is refined y diversity of presynpti neuron types or y timedependent synpti intertions, resulting in differentil tivtion of neuron popultions 7,5,. In onlusion, the experiments presented here demonstrte tht exittory ortil neurons in different lyers onnet preferentilly to distint types of inhiitory interneurons within lyer. Therefore, in ddition to lyer-speifi onnetions, exittory ortil neurons form ell-type-speifi onnetions. These differentil ortil exittory inputs suggest speilized funtionl roles for inhiitory interneuron sutypes. METHODS Slie preprtion. Virtome-ut oronl slies ( µm) were prepred from the primry visul ortex of P2 3 Long-Evns rts. Slies were held sumerged in oxygented rtifiil ererl spinl fluid (CSF, 25 mm NCl, 5 mm KCl, 2.5 mm CCl 2,.3 mm MgCl 2,.25 mm NH 2 PO, 2 mm NHCO 3 nd mm gluose, sturted with 95% O 2 nd 5% CO 2 ) heted to 32 3 C. We used n infrred Olympus DIC mirosope with,.8 NA wter immersion lens to visulize nd trget lyer neurons for whole-ell reordings in living rin slies. Glss miroeletrodes ( 8 MΩ resistne) filled with potssium-gluonte-sed intrellulr solution (7 mm K-gluonte, mm HEPES, 8 mm NCl, mm MgCl 2, mm EGTA,.33 mm CCl 2, 3.5 mm MgATP, mm GTP) ontined.5 % ioytin for ell leling. Photostimultion nd input mps. Brin slies were thed in oxygented CSF ontining 25 µm ged glutmte (γ-(α-roxy-2- nitroenzyl) ester, trifluoroette, L-glutmi id CNB-glutmte ) t room temperture. Ultrviolet light (-ms flsh from n rgon-ion lser) ws foused to smll spot in the plne of the rin slie through mirosope ojetive ove the slie. Whole-ell voltge-lmp reordings ( 5 mv) were mde from postsynpti neuron, nd inwrd EPSCs resulting from photostimultion of presynpti neurons ws mesured. Outwrd IPSCs were mesured y hnging the holding potentils of neurons to etween nd 35 mv. To hold neurons t mv, tion potentils were loked y dding mm QX-3 to the intrellulr solution. To mp the lotions of input to single ell, up to 9 sites were stimulted sequentilly in pseudo-rndom pttern tht overed ll ortil lyers. In ddition, photostimultion trils were interleved with ontrol trils (no stimultion) to otin sepscs. After ompletion of photostimultion nd reordings from ell, the lser ws used to urn lignment sites (< µm) into the slie so tht x y photostimultion oordintes ould e ssigned to their orresponding position in the tissue. Lminr orders were determined using ytohrome oxidse stin. Anlysis of PSCs. We nlyzed EPSCs tht ourred during the first 5 ms following the light flsh. This window ws hosen euse neurons fired most of their APs during this time (Fig. ), inditing tht shorter nlysis windows would exlude lrge perentge of photostimultion-evoked APs. We omitted diret effets of unging glutmte on the reorded ell (see Fig. ). Within 5 µm of the reorded ell, diret urrents often exeeded pa nd deyed over ms, so tht the entire window ws omitted from nlyses. The mplitudes of EPSCs were mesured for every stimultion site nd for the no-stimultion ontrols using pek nlysis softwre from Synptosoft (Leoni, New Jersey) nd other ustom softwre. EPSC mesurements for eh stimultion site were then ssigned to their ortil lyer, nd EPSC mplitudes for ll stimultion sites within lyer were summed together using ustom Mtl (Mthworks) softwre. We ssumed tht sepscs ourred rndomly during the stimultion trils t the sme frequeny s oserved during no-stimultion ontrol trils. We orreted for the influene of sepscs, euse this vried signifintly etween groups (Tle ; p <., ANOVA), y sutrting the EPSC mplitude sum vlues me- 7 nture neurosiene volume 3 no 7 july 2

7 rtiles sured in spontneous ontrol trils from tht mesured in experimentl trils for eh lyer. In ddition, we orreted for differenes in EPSC mplitudes tht ould result from vritions in series resistne tht were not due to differenes in funtionl onnetivity. Therefore, EPSC mplitudes (pa) mesured for eh lyer were divided y the men sepsc mplitude for the sme ell, effetively expressing evoked input s rtio of the sepscs. In summry, normlized evoked input vlue (NEI) ws lulted for eh lyer for eh ell using the following eqution: NEI = f E E fc C C where f E nd fc re the men numer of mesured urrents in the experimentl nd ontrol windows for eh lyer, nd E nd C re the men mesured pek heights in the experimentl nd ontrol windows for eh lyer. Sttistil omprisons of lyer-speifi input etween groups were mde using one-wy ANOVA nd Sheffé s post ho test. Within-group omprisons for individul neurons were mde using repeted-mesures ANOVA. All mens re reported with stndrd errors unless otherwise noted. ACKNOWLEDGEMENTS This work ws supported y n NIH grnt (E.M.C.) nd n NSF grdute reserh fellowship, NIH trining grnts nd the Chpmn Chritle Trust (J.L.D). We thnk A. Swtri for disussions nd tehnil ssistne, A. Burkhlter, M. Dntzker, N. Spitzer, C. Stevens, nd memers of the l for omments on the mnusript, nd E. Hung for providing pek nlysis softwre. RECEIVED 2 APRIL; ACCEPTED 8 MAY 2. Peters, A. & Regidor, J. A ressessment of the forms of nonpyrmidl neurons in re 7 of t visul ortex. J. Comp. Neurol. 23, 85 7 (98). 2. Prr, P., Gulys, A. I. & Miles, R. How mny sutypes of inhiitory ells in the hippompus? Neuron 2, (998). 3. Thomson, A. M. & Deuhrs, J. Synpti intertions in neoortil lol iruits: dul intrellulr reordings in vitro. Cere. Cortex 7, (997).. Kwguhi, Y. Groupings of nonpyrmidl nd pyrmidl ells with speifi physiologil nd morphologil hrteristis in rt frontl ortex. J. Neurophysiol. 9, 3 (993). 5. Culi, B. et l. Moleulr nd physiologil diversity of ortil nonpyrmidl ells. J. 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Development of GABA-ontining neurons in the visul ortex. Prog. Brin Res. 9, (992). 27. Thomson, A. M. & Bnnister, A. P. Postsynpti pyrmidl trget seletion y desending lyer III pyrmidl xons: dul intrellulr reordings nd ioytin filling in slies of rt neoortex. Neurosiene 8, 9 83 (998). 28. Stevens, C. F. & Wng, Y. Filittion nd depression t single entrl synpses. Neuron, (995). 29. Kwguhi, Y. & Kuot, Y. GABAergi ell sutypes nd their synpti onnetions in rt frontl ortex. Cere. Cortex 7, 7 8 (997). 3. MCormik, D. A., Connors, B. W., Lighthll, J. W. & Prine, D. A. Comprtive eletrophysiology of pyrmidl nd sprsely spiny stellte neurons of the neoortex. J. Neurophysiol. 5, (985). 3. Connors, B. W. & Gutnik, M. J. Intrinsi firing ptterns of diverse neoortil neurons. Trends Neurosi. 3, 99 (99). 32. Jones, E. G. & Hendry, S. H. C. in Cererl Cortex (eds. Peters, A. & Jones, E. G.) (Plenum, New York, 98). 33. Kwguhi, Y. & Kuot, Y. 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Britenerg, V. & Shuz, A. Antomy of the Cortex (Springer, Berlin, 99).. Reyes, A. & Skmnn, B. Developmentl swith in the short term modifition of unitry EPSPs evoked in lyer nd lyer 5 pyrmidl neurons of rt neoortex. J. Neurosi. 9, (999). 2. Fellemn, D. J. & Vn Essen, D. C. Distriuted hierrhil proessing in the primte ererl ortex. Cere. Cortex, 7 (99). 3. Kisvrdy, Z. F., Mrtin, K. A., Whitteridge, D. & Somogyi, P. Synpti onnetions of intrellulrly filled luth ells: type of smll sket ell in the visul ortex of the t. J. Comp. Neurol. 2, 37 (985).. Freund, T. F., Mglozky, Z., Soltesz, I. & Somogyi, P. Synpti onnetions, xonl nd dendriti ptterns of neurons immunoretive for holeystokinin in the visul ortex of the t. Neurosiene 9, (98). 5. Reyes, A. et l. Trget-ell-speifi filittion nd depression in neoortil iruits. Nt. Neurosi., (998).. Glrret, M. & Hestrin, S. Frequeny-dependent synpti depression nd the lne of exittion nd inhiition in the neoortex. Nt. Neurosi., (998). nture neurosiene volume 3 no 7 july 2 77