Receptive field structure varies with layer in the primary visual cortex

Transcription

1 2 Nture Pulishing Group Reeptive field struture vries with lyer in the primry visul ortex Luis M Mrtinez, Qingo Wng 2, R Cly Reid 3, Cinthi Pilli 2, José-Mñuel Alonso 4, Friedrih T Sommer & Judith A Hirsh 2 Here we sk whether visul response pttern vries with position in the ortil miroiruit y ompring the struture of reeptive fields reorded from the different lyers of the t s primry visul ortex. We used whole-ell reording in vivo to show the sptil distriution of visully evoked exittory nd inhiitory inputs nd to stin individul neurons. We quntified the distriution of On nd Off responses nd the presene of sptilly opponent exittion nd inhiition within the reeptive field. The thlmoreipient lyers (4 nd upper ) were dominted y simple ells, s defined y two riteri: they hd seprted On nd Off suregions, nd they hd push-pull responses (in given suregion, stimuli of the opposite ontrst evoked responses of the opposite sign). Other types of response profile orrelted with lminr lotion s well. Thus, onnetions unique to eh visul ortil lyer re likely to serve distint funtions. How does onnetivity in strite ortex orrelte with reeptive field struture nd, ultimtely, with neurl seletivity for elements of the visul sene? Antomil studies show tht eh of the six ortil lyers hs unique pttern of inputs nd outputs 4. Thus, it is possile to investigte the funtion of speifi omponents of the ortil miroiruit y ompring neurl response ptterns t different lminr positions 2. We took this pproh to sk whether there re response properties exlusive to the first stge of ortil integrtion, where new response properties suh s orienttion sensitivity emerge 2. Erly studies suggested tht orienttion seletivity depends on the struture of the simple reeptive field, n rrngement of elongted On nd Off suregions with n ntgonisti effet on one nother 2,2 23. This ide me from oservtions of responses evoked y stimuli pled t different positions in visul spe. For instne, right ontour ligned lengthwise with n On suregion produed strong exittion tht diminished when the stimulus ws rotted towrds the orthogonl ngle or ws moved sidewys to over lrger portions of n djent Off suregion 2. The geometry of the simple ell s response ws thought to result from n orderly pttern of onvergene from On nd Off thlmi rely ells 2,23 2. Lter studies suggested tht the two min physiologil types of ell in the visul ortex, simple nd omplex, were generted t ll levels of ortil proessing nd represented two ends of ontinuous spetrum (M.S. Jo et l., So. Neurosi. Astr. 9.3, 23). An rgument mde to dvne this view is tht vlues for some prmeters used to distinguish simple from omplex ells re distriuted unimodlly rther thn imodlly 29,32. Yet, if the distriution of vlues for given set of prmeters is unimodl, ut ll ses tht fll to one side of utoff re restrited to prtiulr lyer, it ould nonetheless e possile to orrelte type of visul response with lotion in the ortil iruit. Thus, to mesure quntittively the reeptive fields of neurons t estlished lminr positions, we omined intrellulr stining, whole-ell reording nd sptil mpping protool. Over time, we were le to otin informtion out ntomilly identified ells in eh ortil lyer. We used two min mesures to desrie reeptive field struture. First, we used n overlp index to ssess the sptil segregtion of On nd Off suregions 33. Seond, we used push-pull index to determine the presene nd reltive weight of ntgonisti responses to stimuli of the opposite ontrst within individul sufields 2 23, Our finding is tht ells with simple reeptive fields, s judged y sores for oth indies, re found exlusively in thlmoreipient zones, where they re the mjority. Complex ells re found throughout the ortil depth, though their response hrteristis hnge with lminr lotion. All told, we show tht the simple reeptive field is unique feture of regions tht reeive thlmi input. More generlly, our results support the view tht eh stge of the ortil miroiruit is designed to nlyze different spets of the visul stimulus. RESULTS To explore how ortil reeptive fields vry with position in the ortil miroiruit, we mpped the sptil distriution of exittion nd inhiition in the reeptive fields of neurons t identified ntomil sites. We lso studied thlmi rely ells, whih supply visul ortex. Our smple, 88 ells in 8 dult ts, inluded neurons in the thlmus (n = 2), lyer 4 nd its orders (n = 34), lyers (n = 2), lyer (n = ) nd lyer (n = ). Deprtment of Mediine, Cmpus de Oz, Universidd A Coruñ,, Spin. 2 Deprtment of Biologil Sienes, University of Southern Cliforni, 34 Wtt Wy, Los Angeles, Cliforni , USA. 3 Deprtment of Neuroiology, Hrvrd Medil Shool, 22 Longwood Ave., Boston, Msshusetts 2, USA. 4 Deprtment of Biologil Sienes, Stte University of New York College of Optometry, 33 West 42nd St., New York, New York 3, USA. Redwood Neurosiene Institute, El Cmino Rel, Menlo Prk, Cliforni 942, USA. Correspondene should e ddressed to J.A.H. (jhirsh@us.edu). Pulished online 3 Ferury 2; doi:.38/nn VOLUME 8 NUMBER 3 MARCH 2 NATURE NEUROSCIENCE

2 ARTICLES 2 Nture Pulishing Group Figure Reeptive fields with push-pull rrngement of synpti inputs. ( d) Reeptive fields of thlmoortil neuron in the lterl geniulte nuleus (), two spiny ells (,d) nd smooth ell (), ll in lyer 4. The reeptive fields re shown s rrys of tre pirs in whih eh position in the stimulus grid is represented y verges of the orresponding responses to drk (lk tres) nd right (gry tres) squres. The oundries of On (red) nd Off (lue) suregions re pproximted y dshed irles or ovls. In ll pnels, stimuli of the reverse ontrst evoked responses of the opposite sign (push-pull) in eh suregion. The smll vertil dshes indite the onset of the stimulus, whih ws flshed for 3 or 47 ms; stimulus size ws.8 or.7 nd grid sping ws.8 (tht is, eh squre in the rry represents.8 of visul ngle). d mv 2 ms Synpti strutures of reeptive fields An overview of the reeptive fields we reorded is provided y Figures nd 2. Eh figure is orgnized ording to sttion in the miroiruit, from the thlmus, to lyer 4, to the superfiil lyers (lyer ) to the deep lyers (lyers nd ). The stimulus ws sprse noise (individully flshed right nd drk squres). The reeptive fields re shown s grids in whih eh oordinte is represented y pir of tres tht show the verged response to right nd drk stimuli. The dshed lue nd red ontours outline the generl shpe of the Off nd On suregions, respetively. The reeptive field of n Off-enter thlmi rely ell is shown in Figure. Within eh suregion, enter nd surround, stimuli of the reverse ontrst evoked responses of the opposite sign: push-pull pttern2 23,3 37. Drk squres t the enter oordintes evoked n initil depolriztion followed y hyperpolriztion tht orresponded to withdrwl of the stimulus. Bright squres flshed t the sme positions evoked the opposite response: hyperpolriztion sueeded y depolriztion. The responses from the surround, though wek (smll spots re suoptiml stimuli for the surround), showed push-pull pttern s well. Figure 2 Reeptive fields with push-push or push-null onfigurtions. ( ) Reeptive fields of spiny stellte ell () nd smooth ell () in lyer 4, pyrmidl ell in lyers () nd pyrmidl ell in lower lyer (d); onventions s for Figure. Exittion to right nd drk stimuli ws sptilly overlpping (push-push) in the reeptive fields from lyer 4 (,). Outside lyer 4, ells rrely responded to oth polrities of the stimulus, so reeptive fields often hd just one suregion (push-null) (,d) or ould not e mpped with the sprse noise (not shown). Stimulus size ws.8 or.7 nd grid sping ws.8. The mjority of reeptive fields (2 of 38) in thlmoreipient zones, lyer 4 nd its orders, nd upper lyer, were uilt of djent On nd Off suregions; eh subright region hd push-pull pttern, s in the Drk thlmus. In ortex, however, the suregions ly side y side. This qulittive rrngement resemles simple reeptive fields s first desried2 (Figs. d). A reeptive field of ell in lyer 4 (Fig. ) hd strong Off suregion flnked y smller On suregion. Throughout the Off suregion, drk squres evoked strong initil depolriztion wheres right squres flshed in the sme positions produed hyperpolriztion. A omplementry pttern ws seen in the On suregion. Push-pull ws present for ells with different numers of suregions or ntomil profiles. For exmple, pushpull ws seen in ll three suregions of the reeptive field of spiny stellte ell (Fig. d) nd throughout the reeptive field of sket ell (Fig. ; see refs. 3,3). Most remining ells (n = 37) lked djoining On nd Off suregions, sptil profile often termed omplex9,2,34. Suh ells responded in one of three min wys to the sprse-noise stimulus (Fig. 2). One pttern, typil of thlmoreipient zones, is shown for spiny stellte neuron mv Bright 2 ms Drk NATURE NEUROSCIENCE VOLUME 8 NUMBER 3 MARCH 2 d 373

3 2 Nture Pulishing Group Figure 3 The sptil rrngement of On nd Off suregions in ortil reeptive fields. (,) Contour plots of the reeptive fields of five simple () nd four omplex ells () ompre mps onstruted from synpti potentils to those mde from spikes. For simple ells, On (red) nd Off (lue) responses re shown in the sme plot, nd for omplex ells, mps of On nd Off responses re shown in seprte pnels. Eh ontour ws smoothed nd represents % derement reltive to the pek (rightest) vlue; the mps were thresholded y %. The responses onstruted from spikes were normlized seprtely from those mde from synpti potentils. Stimulus size ws.8 or.7 nd grid sping ws.8 (tht is, the spe etween eh line on the overly is.8 ). Overlp index vlues for simple ells from left to right were (synpti potentils nd spikes, respetively).2,.34;.7,.8;.,.29;.22,.82;.8,.4. For omplex ells, they were.8,.3;.79,.7;.82,.; polrity, polrity. () Histogrm showing the distriution of vlues of overlp index (in size =.) for the entire popultion with grphil explntion of the index elow. Only ells tht responded to oth polrities of the stimulus were inluded. The distriution of vlues ws not unimodl (proility of rejetion.99, Hrtign s dip test). (d) The histogrms t top nd right show index vlues for synpti exittion (s in ) nd spikes (s in ), respetively. The entrl stter plot ompres the two sets of vlues for eh ell; the red line indites unit slope. (Fig. 2) nd for smooth ell (Fig. 2). For oth neurons, right nd drk squres produed exittory responses throughout the field: push-push rther thn push-pull profile. As for ll ells t the first stge of ortil proessing, the time ourse of the response followed the temporl envelope of thlmi tivity. A seond group of ells, in lyers, or, responded only to one polrity of the stimulus: push-null profile (Fig. 2,d). The responses of these ells re rief nd irregulr, s is typil of ells tht do not reeive ontt from the thlmus. Lst, mny ells filed to respond to the sprse noise, though they responded vigorously to moving stimuli. Suh ells oupied lter ortil stges: the upper tier of lyers, lyer or the ottom hlf of lyer. Thus, the response profile of omplex ells t the thlmoortil level distinguishes them from omplex ells in regions tht do not reeive diret fferent input. The synpti struture of the reeptive field predited the suprthreshold pttern of response. For simple ells (Fig. 3), ontour plots of the reeptive field re shown in single plot. For omplex ells (Fig. 3), mps of right nd drk responses re shown seprtely, in djent plots, so tht On nd Off responses n e ompred. The reeptive fields mde with spikes were often smller thn those mde with synpti potentils, ut the overll shpe ws similr. In rre instnes, however, wek suregions remined suthreshold. Sptil distriution of On nd Off responses We used n overlp index 33 to mesure the sptil segregtion of suregions within the reeptive field (Figs. 3,d). Vlues indite seprted suregions nd those denote symmetrilly overlpped suregions (Fig. 3, legend). The index did not resolve potentil overlp etween the outermost regions of eh sufield (see Methods), nd only ells tht responded to drk nd right spots ould e inluded. The Synpti potentils Spikes Synpti potentils Spikes 7. Overlp index Overlp index (grphil explntion) On suregion Off suregion On-off field d Overlp index (spikes) Overlp index (PSPs) ells qulittively desried s simple ells hd vlues etween.4 nd.9 (.9 ±.2, men ± s.d.; n = 2) nd ells with sores from.32 to.84 (.7 ±.; n = ) orresponded to suset of those desried qulittively s omplex. We next ompred the overlp indies from synpti responses to those mesured from spikes (Fig. 3d). (We were le to use only suset of the popultion; for some ells, tion potentils were loked with QX-34, nd in rre ses, suregions remined elow spike threshold.) The distriutions of the vlues for suthreshold (Fig. 3d, top) nd suprthreshold (Fig. 3d, right) responses were similr (orreltion oeffiient r =.9; P <.). Still, most points in the stter plot (Fig. 3d, enter) tht ompres the two vlues for eh ell fell elow the line of unit slope; it is likely tht the redued width of the spike sufields emphsized even smll disprities etween the peks of lrgely osptil On nd Off sufields nd widened the distne etween segregted On nd Off suregions. We further nlyzed reeptive field struture to inlude inhiition, using push-pull index (mesurements were restrited to the enter of eh sufield; see Methods). If stimuli of the opposite ontrst evoked omprle mounts of push nd pull, the index vlue ws ; vlue indited push-null (numers re solute vlues) nd sore 2 denoted push-push (Fig. 4, ottom). Cells with seprted On nd Off suregions (Fig. 3) hd index vlues << (histogrm, Fig. 4; rnge,..7; men,.22 ±.7; n = 2). Conversely, lmost ll ells tht lked segregted On nd Off suregions hd vlues (rnge,.9.; men,.97 ±.7; n = 8) or vlues pprohing 2 (rnge.27.93; men,.2 ±.2; n = ); the two outliers hd strong push-pull ut only single prominent response re. We then ompred the pttern of push nd pull for simple ells to tht for thlmi rely ells (Fig. 4,), whih re widely held to hve 374 VOLUME 8 NUMBER 3 MARCH 2 NATURE NEUROSCIENCE

4 2 Nture Pulishing Group 2. Push-pull index Push-pull index (grphil explntion) 2 2 (NR) Pull-pull Null-pull Push-pull Push-null Push-push Null-null segregted On nd Off suregions (Fig. ). The vlues for the push-pull index, mesured from the enter suregion of the thlmi field, were..9 (. ±.4; n = 2; Fig. 4). To estimte the reltive sptil distriution of push nd pull for simple nd rely ells, we expnded the use of the overlp index (Fig. 4). For eh suregion, we ompred the re of push response (exittion evoked y stimuli of the preferred ontrst, fitted with n elliptil Gussin) with the orresponding pull response (inhiition evoked y stimuli of the opposite ontrst, fitted with n elliptil Gussin). The results show tht the push nd pull lrgely overlp; vlues rnged from.3.9 (.9 ±.7; n = 2) for simple ells nd.7.9 (.72 ±.9; n = 2) for rely ells. Note tht these vlues my underestimte the tul overlp euse wek inhiition ws sometimes diffiult to visulize. Overll, the sores for simple nd rely ells were similr ut not identil; the slight disprity might Lminr lotion Lminr lotion Lminr lotion 4 (+ orders) 4 (+ orders) 4 (+ orders) Simpleness Simpleness Complexness 3 2 NR Sufield segregtion NR 7 Thlmi reeptive fields Simple reeptive fields Push-pull index..p NR Overlp index Simpleness 7. Overlp index (push-pull) Complexness Complexness NR Push-pull index NR Figure 4 Exittion nd inhiition within single suregions of the reeptive field. () Histogrm of solute vlues of the push-pull index (in size =.) with grphil explntion of the index elow. Filled rs, ells with segregted On nd Off suregions (overlp index.9); open rs, ells with overlpping On nd Off sufields (overlp index >.3) or with just one suregion; NR indites tht there ws no response to the flsh stimulus. The sterisk mrks pyrmid in lyer whose dendrites extended into lyer 4 nd whose reeptive field hd push-pull in only one of two suregions. The distriution of vlues ws not unimodl, proility of rejetion.99 (Hrtign's dip test). () Comprison of vlues of push-pull index for thlmi reeptive fields (gry) nd simple ortil reeptive fields with segregted On nd Off suregions (lk); in size =.. () Overlp index vlues of exittory nd inhiitory responses to stimuli of the opposite ontrst in thlmi reeptive field enters (gry) nd in the individul sufields of ortil ells with seprted suregions (lk); in size =.. reflet mild symmetries in the rrngement of exittion nd inhiition in the two types of reeptive fields. Lminr distriution of reeptive fields How do these different reeptive field profiles orrelte with position in the ortil miroiruit? We plotted the distriution of the overlp index (Fig. ), numer of segregted On nd Off suregions (Fig. ) nd push-pull index (Fig. ) ording to lminr lotion. The profiles show tht vlues for eh prmeter vry with depth in the ortil olumn. Cells whose reeptive fields hd sores tht indited simpleness (smll vlues of overlp nd push-pull indies nd multiple suregions) were loted only in lyer 4 nd the upper hlf of lyer, where the thlmi fferents terminte (see Fig. 7). By ontrst, neurons whose sores indited omplexness were found in ll lyers, though response pttern vried with lminr lotion. For exmple, omplex ells in thlmoreipient zones lwys responded to oth right nd drk sprse-noise stimuli, so the push-pull nd overlp indies were ner the mximum vlues. Conversely, ells in positions frther removed from the thlmus (lyers, lyer nd lower lyer ) seldom responded to the sprse noise; when they did respond, the push-pull index vlues were euse responses were limited to stimuli of one polrity: either right or drk. The reltionship etween sores for the overlp nd push-pull indies for ells in the different ortil lyers is depited in stter plot (Fig. ; olor-oded for lminr lotion). The resulting distriution forms two louds tht represent sttistilly signifint groups. If simple reeptive fields re defined s hving seprted On nd Off suregions with Figure Correltion etween reeptive field struture nd ortil lyer. ( ) Histogrms show the distriution of vlues for the overlp index (), numer of suregions () nd push-pull index () in the different ortil lyers. P indites ells tht responded to only one polrity of the sprse noise, nd NR denotes ells tht did not respond to the stti stimulus t ll; in size ws. in nd. In eh histogrm, for eh lyer, the in with the gretest numer of ells is shded lk, nd the gry level in the remining ins is normlized to tht mximum. For, the ins re leled y the numer (2 or 3) of seprted On nd Off suregions; inludes ells with overlpped On nd Off suregions nd those tht responded to only one stimulus polrity; sterisk sme s for Figure 4. NATURE NEUROSCIENCE VOLUME 8 NUMBER 3 MARCH 2 37

5 2 Nture Pulishing Group Figure Comprison of suregion overlp nd push-pull. A stter plot of suregion overlp versus push-pull; the results re olor oded for lyer. The points ove the plot show push-pull index vlues for ells tht responded to only one polrity of the stimulus (P); the lel NR indites ells (upper right) tht did not respond to the sprse noise. The intersetion of the rosses in eh luster of points orresponds to the men, nd the length of eh line to the 9% onfidene intervls (lulted with ootstrp method). Conentri symols re used when multiple ells shred the sme oordinte; sterisk s for Figure 4. push-pull, the plot shows tht simple ells re onfined to thlmoreipient zones. The remining heterogeneous group of ells, whih we ll omplex, is distriuted through the ortil depth. Notly, lyer 4 nd ordering regions ontin the ells with the gretest degree of seprtion etween On nd Off suregions ( of 9 smooth ells nd 7 of 2 spiny neurons, mostly spiny stellte ells from lyer 4) s well s those with the highest degree of overlp 7, (4 of 9 smooth ells nd of 2 spiny neurons, 3 pyrmids t the orders of lyer 4 nd 2 spiny stellte ells). The remining neurons hd reeptive fields omposed of single region, 2 (in lyer 4) with push-pull profile nd (t the 4 order) with push-null profile. Lst, we used Person s orreltion oeffiient to ompre the rw responses to right nd drk stimuli point y point, s others 29,32 hve done to mesure the segregtion of On nd Off suregions. The resulting distriution (not shown) ws similr to tht for the overlp index (r =.94783, P <.) s well s to tht for the push-pull index (r =.9322, P <.). A plot of the lminr position of ells with simple reeptive fields (Fig. 7) gives the strong impression tht simple ells in lower lyer 4 hve more ompt suregions thn those in the middle or upper prts of the lyer, lthough our smple ws not lrge enough to llow us to estlish definitive sulminr ptterns. Also, multiple suregions were more ommon in the middle to upper hlf of lyer 4. Lst, reeptive fields uilt of very long suregions were found in lyers 4 nd, s indited in n erlier physiologil study 39. Morphology nd reeptive field struture Finlly, we found no systemti ssoitions etween reeptive field struture nd generl ntomil lss, exept for stereotyped lminr vritions in morphology,3,4, (see summry of reonstrutions, Fig. 8). We found simple (Fig. 8) nd omplex (Fig. 8) spiny stellte ells nd smooth ells in lyer 4, simple pyrmids t the orders of lyer 4 or the upper hlf of lyer, nd omplex pyrmids nd smooth ells throughout the ortil depth. On more sutle level, our pst work 4 hs shown tht simple pyrmidl ells in lyer hve different dendriti rnhing ptterns nd xonl termintion zones from omplex 4 pyrmidl ells in lyer. Perhps future studies will show similr trends for neurons in other lyers. Overlp index NR P. 2 Push-pull index extrellulr reordings. First, it ws possile to lel the ells from whih we reorded to sertin their lminr lotion nd, hene, their position in the miroiruit. Seond, the method reveled the synpti struture of responses y showing suthreshold exittion s well s inhiition. Two min indies, n overlp index 33 nd push-pull index, llowed us to quntify the sptil reltionship etween On nd Off responses nd the presene of exittion (push) nd/or inhiition (pull) within eh reeptive field. Cells with simple reeptive fields (djent On nd Off suregions with push-pull) were restrited to the first stge of ortil proessing. The reeptive fields of the remining neurons, nmely the omplex ells, were heterogeneous, lthough stereotyped ptterns orrelted with seprte positions in the miroiruit. Thus, different neurl iruits ply distint roles in ortil proessing. Reeptive field struture t the first ortil stge By omining morphologil identifition with quntittive mpping of the reeptive field, our experiments show tht simple ells re onfined to regions tht reeive diret thlmi input: lyer 4, its orders nd upper lyer. Simple reeptive fields hve sores for the overlp index (<<.) inditing segregted On nd Off suregions. Additionlly, the sores for the push-pull index (<<) show tht stimuli of the reverse ontrst evoke responses of the opposite NR Lyers Lyer 4 (+ orders) Lyer Lyer DISCUSSION Eh lyer of ortex is hrterized y unique profile of onnetions,3,4,,4. The gol of our study ws to understnd the struture of the reeptive fields tht these different iruits uild. The pproh we used, wholeell reording with dye-filled eletrodes, provided two key dvntges over trditionl Figure 7 Lminr distriution of reeptive fields with push-pull. Reeptive fields with push-pull orgniztion were found in lyer 4, its orders or in upper lyer, with one exeption: pyrmid in lyer with dendrites extending into lyer 4. The reeptive fields re ordered from left to right ording to depth of the som. All ut three of the reeptive fields with push-pull hd 2 suregions; On nd Off suregions re red nd lue, respetively, nd sterisks indite ells with only one ovious suregion. The sle r ( ) indites the size of the reeptive fields. o 37 VOLUME 8 NUMBER 3 MARCH 2 NATURE NEUROSCIENCE

6 ARTICLES Simple ells n 4 2t 2 2 n Nture Pulishing Group Figure 8 Morphology nd reeptive field struture. (,) The figure shows smple of our threedimensionl reonstrutions tken from the simple ell () nd omplex ell () popultions. The figure shows oronl views (from left to right, top) of pyrmid in upper lyer, pyrmid t the 4 order, spiny stellte ell in lyer 4, smooth ell in lyer 4 nd pyrmid t the 3 4 order; nd (from left to right, ottom) of pyrmid in mid lyer, pyrmid in lyer, two pyrmids in the superfiil lyers; sket ell in lyer 4 nd spiny stellte ell in the sme lyer. Cell odies nd dendriti rors re gry, nd xons re lk. Complex ells 4 sign. Thus, ler view of the simple reeptive field emerges when the sptil distriution of exittion nd inhiition re tken into ount. Furthermore, we hve found tht the vlues of the push-pull index mesured from simple ells re similr to those mesured from the enters of thlmi rely ells, inditing tht this rrngement is rried forth from geniulte to ortex. Our urrent study, whih ples simple ells in thlmoreipient zones, is onsistent with the ide tht simple reeptive fields re uilt y the onvergene of thlmi inputs2,23 2,4. Thus, our results n e understood in the ontext of feed-forwrd models of orienttion seletivity2,37,42. Lyer 4 nd its orders lso ontin seond, smller popultion of ells (24% of the spiny neurons nd 44% of the smooth ells) whose reeptive fields hve lrgely superimposed On nd Off sufields (overlp index >.3; push-pull index >.34). Indeed, these ells hve sores for the overlp nd push-pull indies t the upper ounds of oth distriutions. Suh first-order omplex ells my well orrespond to types of nonsimple ells (omplex nd/or non-oriented onentri ells) tht reeive synpti ontts from the lterl geniulte nuleus7,43. Although the lyout of the reeptive fields of these first-order omplex ells is different from tht of neighoring simple ells, oth groups shre ommon synpti physiology; s well the responses of ll ells in lyer 4 follow the time-ourse of thlmi drive. A nturl question is whether the first-order omplex ells re orienttion seletive. Reent studies hve shown tht inhiitory omplex ells in lyer 4 were not tuned for stimulus orienttion nd hve suggested tht they might serve vrious roles in the glol regultion of exitility3,37,43. As yet, it is unler whether spiny omplex ells in lyer 4 re orienttion seletive nd wht their funtionl role might e. Finlly, we wonder whether the sulminr differenes in reeptive field struture tht we hve oserved relte to the ntomil orgniztion of the primte s visul ortex. Speifilly, we hve found tht simple ells with ompt sufields re more ommon in the deeper spet of lyer 4, wheres those with nrrower suregions re more frequent in the upper hlf of the lyer. In the monkey, reeptive fields in lower lyer 4 re rounder thn in the higher tiers, differene NATURE NEUROSCIENCE VOLUME 8 NUMBER 3 MARCH 2 tht ovries with the distriution of prvoellulr versus mgnoellulr inputs3,44. Reeptive field struture t lter ortil stges The roust On nd Off responses tht sprsenoise stimuli routinely evoke in lyer 4 re rre in lyers, nd lower. At these lter stges of ortil proessing, most ells respond primrily to flshed stimuli of only one polrity (pushnull) or do not respond to the stti stimulus t ll. Evoked responses re riefer nd less relile thn in lyer 4. Although ells in ll lyers respond vigorously to moving rs, these stimuli never evoked push-pull pttern of response in lyers, or lower (ref. ). In generl, responses t lter stges of proessing seem heterogeneous, unlike the sitution in lyer 4, where reeptive fields divide into one of two lusters (Fig. ). Similr differenes in omplex ell profile hve een reported erlier, notly etween the lsses C2 nd C (ref. 22). Here we extend this oservtion y demonstrting orreltion etween response type nd ortil lotion. Lminr distriution of simple nd omplex ells Our results support some erlier studies in the t tht hve pled simple ells t the first stge of ortil proessing nd hve found roder distriution of vrious lsses of omplex ells7,9,2. Other studies hve reported simple nd omplex ells throughout the ortil depth,32 (M.S. Jo et l., So. Neurosi. Astr. 9.3, 23). We elieve this disrepny reflets vritions in the nomenlture or riteri used for lssifition. First, there re different definitions of simple reeptive fields, some of whih inlude ells tht re exited y only one polrity of the stimulus (S). Thus, ell lssified s S y some investigtors ould e lssified s omplex y others, inluding ourselves. Indeed there re mny terms for ells tht respond to only one stimulus polrity, inluding omplex9,2, A4, C or S (refs. 7,,22,4), disrete omplex38, Eon or Eoff 4 nd monoontrst3. It seems likely, therefore, tht the push-null responses tht we hve reorded from lyers, or might hve een lled simple y others. A seond method of distinguishing simple from omplex ells is sed on response linerity rther thn sptil struture of the reeptive field. Erlier results hve suggested tht drifting sinusoidl grtings evoke liner responses from ells with seprte On nd Off suregions ut nonliner responses from ells with osptil On nd Off sufields38,47. Reent work in primtes, however, hs hllenged the ssumption tht the sptil struture of the reeptive field nd linerity of response neessrily orrelte3. Also, we think tht the ells sensitive to only one stimulus ontrst (suh s push-null) would proly respond to grtings in liner fshion. Hene, the oservtion tht ells with liner responses re found throughout the ortil depth is not in pprent onflit with 377

7 2 Nture Pulishing Group our results. Rther, it seems likely tht there my e multiple wys to generte liner responses in the different ortil lyers. Are simple ells distint popultion? There is urrent dete out whether simple nd omplex ells divide into two generl lsses or represent two ends of ontinuous distriution 29,32,48. Although the prmeters we hve mesured re not unimodlly distriuted, we understnd the potentil for nonliner output effets to hnge the shpe of the distriutions nd urrent limittions of reording nd sptil mpping tehniques (see Methods). In ny se, the strength of our result is tht ells with low sores for oth the overlp nd the push-pull indies re found only in lyer 4. Hd we otined unimodl distriutions of oth indies ut found tht ll ells tht fell elow ertin utoff were in lyer 4, wheres ll others were distriuted in other ortil lyers, our onlusions would e the sme: tht simple reeptive fields represent n exlusive feture of the first stges of ortil proessing. Thus, model iruits for simple ells in the t should e onstrined to omponents of the lyers where thlmi fferents terminte. Comprle studies of diverse speies nd sensory systems Antomil evidene emphsizes the importne of lminr speiliztion: the projetion ptterns of xons nd dendrites grow more sptilly preise with progression from rodent to rnivore to primte 3,. Our results ontriute to the ide tht onnetions in different lyers re speilized for different tsks. Although we do not ntiipte tht reeptive field struture per se will lwys vry with ortil lyer, there is sustntil evidene tht different response properties rise t suessive stges of ortil proessing. The tree shrew s visul ortex provides drmti exmple. There, orienttion tuning (sent in lyer 4) emerges in the superfiil lyers, development thought to rise from speifi ptterns of inter- nd intrlminr onvergene 7,49. Likewise, in the monkey, orienttion tuning nd dynmis hnge s funtion of lminr lotion 2. Furthermore, we hve previously shown tht the reltive orienttion tuning of exittory nd inhiitory inputs vries sustntilly from the superfiil to the deep lyers in the t. Systemti hnges in response properties re oserved in other sensory modlities s well. For exmple, in rodent somtosensory ortex, reeptive field struture, orienttion seletivity nd plstiity hnge from lyer 4 to the superfiil nd deep lyers,,. In the uditory ortex, response properties suh s inhiitory-side nd struture (nlogous to the ntgonisti suregions of the simple reeptive field) nd ndwidth seem to vry with lminr lotion 4. Our hope is tht etter understnding of the struture nd funtion of the visul ortil miroiruit will expose fundmentl priniples of neoortil proessing. METHODS Physiologil preprtion. Anesthetized dult ts (. 3. kg) were prepred s desried erlier 3. All proedures were in ordne with the guidelines of the US Ntionl Institute of Helth nd the Institutionl Animl Cre nd Use Committees of the Rokefeller University nd the University of Southern Cliforni. Reording, dt quisition nd memrne properties. The methods for reordings were identil to those used in erlier studies,,3,3,4. Voltge-urrent reltionships were mesured efore nd fter eh stimulus yle to monitor hnges in ess nd input resistnes, spike threshold nd memrne time onstnt ( 28 ms, smooth ells; 32 ms, spiny ells). Reordings lsted from.3 to h. It ws often imprtil to ssign solute resting potentil, s the rtio of ess to sel resistne led to voltge division in the neurl signl. Reeptive field mpping. Reeptive fields were hnd plotted to position the stimulus monitor nd then mpped quntittively with modified 3 sprse noise 2 ; individul light nd drk squres were flshed riefly (3 47 ms) in pseudorndom order, times eh on squre grid; stimulus size ws.8 or.7, nd ontrst ws or 7%. Plots of reeptive fields were mde in two wys: s ontour plots, where eh onentri line represents % redution in response strength, or s rrys of tre pirs. For ells with sptilly segregted On nd Off suregions, ontour plots were mde y sutrting drk from right responses. For overlpping On nd Off sufields, plots for right nd drk stimuli, entered on the sme sptil oordintes, re shown seprtely. For the rrys of tre pirs, eh position on the stimulus grid is represented y two stked tres showing the (spike-sutrted) verge to ll right nd drk stimuli flshed there. Mesuring synpti exittion nd inhiition. For responses evoked from eh oordinte within the reeptive field, exittion (net depolriztion) ws defined s the re of the synpti response tht ws ove rest (the verged memrne potentil in the prestimulus ondition) within fixed time intervl entered ner the pek response ( 2 8 ms fter stimulus presenttion),3,3. Inhiition (net hyperpolriztion) ws defined s the re etween rest nd more negtive voltges in the sme time window. We sometimes mde reordings t different memrne levels for the sme ell. In those instnes, the visully evoked hyperpolriztion grew smller t more negtive memrne potentils, onsistent with the ide tht it ws produed y inhiitory inputs rther thn withdrwl of exittion,3. Mesuring reeptive field struture. We omined different mesures to nlyze the sptil reltionship etween exittion nd inhiition in the reeptive fields. First, we guged the sptil reltionship of On nd Off exittory responses y mens of n overlp index 33 :.W p +.W n d Overlp index =.W p +.W n + d where W p nd W n re the widths of the On nd Off suregions, respetively, nd d is the distne etween the pek positions of eh suregion (mesured from the synpti response res, s ove). The vlue of the index is for seprted suregions nd pprohes for suregions tht overlp symmetrilly. The prmeters for the overlp index, W p, W n nd d, were determined y seprtely fitting eh On nd Off exittory response region with n elliptil Gussin: A x' f(x,y) = 2 y' 2 exp( ) for whih A determines the mximum mplitude, nd re the hlf xes of the ellipse, nd x nd y re trnsformtions of the stimulus oordintes x nd y, tking into ount the ngle θ nd the offset (x nd y ) of the ellipse. Thus, there were six free prmeters in the fitting proedure: A,,, θ, x nd y. Any mesure of sufield overlp is sujet to nonlinerities tht ould is results towrds greter segregtion (simpleness) or overlp (omplexness). For instne, the method we used is sed on Gussin fits of the suregions; thus, it n overestimte the degree of overlp if On nd Off sufields hve very different mplitudes. Additionlly, it nnot ount for the wek orders of the suregions, whih fll elow the utoff of the fitted Gussin. Nonetheless, we prefer the overlp index to lterntive mesures suh s the orreltion oeffiient 32, whih n exggerte tul overlp euse stimuli tht strddle the orders etween suregions will evoke On nd Off responses simultneously 3. This prolem is exerted y the voltge dependene of the mplitudes of synpti potentils. Still, to relte our mesures to others (e.g., ref. 32), we evluted our results with Person s orreltion oeffiient: Σ n (R oni R on )(R offi R off ) i = r = (3) Σ n (R oni R on ) 2 Σ n (R offi R off ) 2 i = i = () (2) 378 VOLUME 8 NUMBER 3 MARCH 2 NATURE NEUROSCIENCE

8 2 Nture Pulishing Group where R on is the response to ll right squres nd R off the response to ll drk squres tht fell within the reeptive field. Our seond mesure, the push-pull index, guged the presene nd reltive mgnitude of ntgonisti responses to stimuli of the reverse ontrst within eh sufield: Push pull index = P + N (4) where P nd N represent synpti responses to right nd the drk stimuli, respetively. The vlue of the index ws when stimuli of the opposite ontrst evoked exittory nd inhiitory responses of omprle mgnitude (Fig. 4, ottom). Vlues indited tht stimuli of only one ontrst generted signifint responses nd sores 2 indited tht right nd drk stimuli evoked responses of the sme sign nd similr strength. We onfined our mesurement to the enter of eh suregion euse our stimuli were often lrge nd sometimes seemed to overlp suregions 3. We normlized the mesures for push nd pull euse the reltive strength of eh depends strongly on memrne potentil 3. Although we reorded t memrne levels set to show oth exittion nd inhiition, it ws impossile to hieve equivlent reording onditions in every ell. Notly, however, we lulted the index without normlizing nd found little hnge in the shpe of the distriution of vlues (not shown). Lst, we expnded the use of the overlp index to estimte the reltive sptil distriution of push nd pull within individul sufields (tht is, to ompre exittory nd inhiitory responses evoked y right nd drk spots flshed in the sme suregion). If the push nd pull were lrgely osptil, the vlues of the index pprohed. Histology. After histologil proessing 3, leled neurons were drwn using omputerized three-dimensionl reonstrution system (Mirorightfield). ACKNOWLEDGMENTS We thnk T.N. Wiesel for support over the yers nd C.G. Mrshll, K.D. Nik nd J.M. Provost for ssistne with the ntomil reonstrutions. Supported y US Ntionl Institutes of Helth grnt EY993 to J.A.H. 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