Selective reconfiguration of layer 4 visual cortical circuitry by visual deprivation

Transcription

1 Seletive reonfigurtion of lyer 4 visul ortil iruitry y visul deprivtion Arinn Mffei, Sh B Nelson & Gin G Turrigino Visul deprivtion during developmentl sensitive period mrkedly lters visul ortil response properties, ut the hnges in intrortil iruitry tht underlie these effets re poorly understood. Here we use slie preprtion of rt primry visul ortex to show tht 2 d of prior visul deprivtion erly in life inreses the exitility of lyer 4 iruitry. Slie reordings showed tht spontneous tivity of lyer 4 str pyrmidl neurons inresed 25-fold fter 2 d of visul deprivtion etween postntl dys (P) 5 nd P7. This effet ws medited y inresed net exittory nd deresed net inhiitory synpti drive. Pired reordings showed tht exittory onnetions etween str pyrmidl neurons douled in mplitude, wheres inhiitory onnetions deresed or inresed depending on the interneuron lss. These effets reversed when vision ws restored. This dynmi djustment of the exittion-inhiition lne my llow the networks within lyer 4 to mintin stle levels of tivity in the fe of vrile sensory input. Visul deprivtion hs een powerful tool for investigting the ntomil nd physiologil orreltes of experiene-dependent plstiity 3. Visul deprivtion during developmentl sensitive period indues profound hnges in visul response properties, inluding redued ortil responsiveness to visul stimultion,4 6, redued visul uity 7 nd impired orienttion tuning of ortil neurons 5,8. Mny of these physiologil hnges n e indued quite rpidly 9, nd n e reversed if deprivtion is lifted within the sensitive period,2. Despite the long history of these mnipultions, the detiled hnges tht loss of vision produes in intrortil synpti strengths, s well s the plstiity mehnisms tht underlie these hnges, re not fully understood. Here we exmine how synpti onnetivity in rodent lyer 4 is ltered y rief periods of monoulr lid suture. Lyer 4 is the min input lyer to visul ortex, nd exittory str pyrmidl neurons in lyer 4 reeive diret exittory thlmi drive, s well s reurrent exittory onnetions from other str pyrmidl neurons 3,4. This reurrent exittory iruitry is kept in hek y oth feed-forwrd nd feedk inhiition, whih re medited y distint lsses of inhiitory GABAergi interneuron 5,6. Erly in life, 2 d of monoulr deprivtion inreses the quntl mplitude of exittory synpses on str pyrmidl neurons in lyer 4 (ref. 7). In ddition, ortil inhiition my e redued y prolonged visul deprivtion 8,9, lthough how rpidly this ours nd how these hnges re expressed t the level of individul inhiitory onnetions remin unknown. Here we exmine the possiility tht visul deprivtion rewires intrortil lyer 4 iruitry through highly seletive hnges in the strengths of exittory nd inhiitory synpses. To omplish this, one eye of rt ws sutured shut etween P4 nd P7, nd ute slies of primry visul ortex were ut from the deprived nd ontrol hemispheres. We then rried out detiled nlysis of onnetivity in monoulr lyer 4 using qudruple whole-ell reordings. Beuse most of the rodent visul ortex is driven exlusively y the ontrlterl eye 2,2, monoulr deprivtion will lower visul drive to one hemisphere of the monoulr portion of visul ortex, while leving the other hemisphere unffeted 22,23. We found tht visul deprivtion drmtilly inresed the spontneous firing of str pyrmidl neurons in lyer 4, through net inrese in exittory nd net derese in inhiitory synpti drive. This ltertion in the lne etween exittion nd inhiition ws hieved through douling in the strength of exittory onnetions etween str pyrmidl neurons nd derese in the strength of feedk, ut not feed-forwrd, inhiition. This inrese in spontneous lyer 4 tivity ws ompletely reversed y reopening the eye for 2 d efore slie preprtion. These dt indite tht lyer 4 exitility my e dynmilly djusted to ompenste for ltertions in sensory drive through highly seletive hnges in intrortil synpti strength. This homeostti regultion of the exittion-inhiition lne is likely to e importnt for mintining stle ortil tivity during norml development nd my lso ontriute to the pthologil hnges in visul response properties tht re indued y sensory deprivtion. RESULTS Monoulr deprivtion inreses spontneous tivity To ssess the effets of monoulr deprivtion on lol iruit tivity, we ompred spontneous firing rtes in the monoulr region of visul ortil slies from the deprived nd nondeprived hemispheres. We initilly trgeted str pyrmidl neurons, whih re the mjor lss of exittory neuron in lyer 4 of rt primry visul or- Deprtment of Biology nd Volen Ntionl Center for Complex Systems, Brndeis University, Wlthm, Msshusetts 2454, USA. Correspondene should e ddressed to G.G.T. (turrigino@rndeis.edu). Pulished online 4 Novemer 24; doi:.38/nn35 NATURE NEUROSCIENCE VOLUME 7 NUMBER 2 DECEMBER

2 6 mv 6 mv 6 mv 5 s 5 s WC tex 3. In stndrd rtifiil ererl spinl fluid (ACSF), there ws little or no spontneous tivity. Chnging the solution to modified ACSF, whih is loser to the omposition of rt CSF in situ 24,25, initited low levels of spontneous firing of str pyrmidl neurons (Fig. ), similr to wht hs een reported in ferret prefrontl ortil slies 26. Whole-ell reordings showed tht str pyrmidl neurons in slies from the deprived hemisphere hd firing rtes tht were 25-fold higher thn those from the ontrol hemisphere (Fig.,; ontrol:.5 ±.3 Hz, n = ; deprived:.28 ±.24 Hz, n = ; P <.6). A similr inrese ws oserved using loose-pth reordings to mesure str pyrmidl neuron firing rtes extrellulrly, to void modifying firing properties through whole-ell dilysis (Fig. ; ontrol:. ±.6 Hz, n = 8; deprived: 2.9 ±.8 Hz, n = 8; P <.3). Firing tivity in modified ACSF ws ompletely olished y loking synpti trnsmission with APV, DNQX nd iuulline (Fig. ), inditing tht spontneous firing ws driven y synpti tivity. This inresed tivity in the deprived hemisphere ws highly seletive for str pyrmidl neurons. Monoulr deprivtion elevted spiking in lyer 4 regulr-spiking nonpyrmidl (RSNP) neurons y only.6-fold (Fig. ; ontrol:.57 ±.9 Hz, n = 8; deprived: 2.47 ±.38 Hz, n = 7; P <.), muh smller inrese in firing thn tht produed in str pyrmidl neurons. In ddition, the effets of monoulr deprivtion on pyrmidl neuron tivity were seletive for lyer 4: the firing rtes of lyer 2/3 pyrmidl neurons were unffeted y monoulr deprivtion t this ge (ontrol:.85 ±.45 Hz, n = ; deprived:.77 ±.25 Hz, n = 8; P =.88). When 2 d of monoulr deprivtion ws followed y removl of the suture for 2 d, str pyrmidl neuron tivity in lyer 4 ws restored to ontrol vlues (Fig. ; ontrol:.8 ±.3 Hz, n = 8; previously deprived:. ±.2 Hz, n = 8; P =.55). This reversiility suggests tht these hnges in exitility represent dynmi, ompenstory response to lowered sensory drive nd is onsistent with ehviorl dt showing tht the effets of visul deprivtion re reversile if deprivtion is lifted erly in life,2. Norm. frequeny 4 2 LP Modified ACSF Modified ACSF Modified ACSF APV, DNQX, i. RSNP REV Figure Spontneous tivity of lyer 4 str pyrmidl neurons ws inresed fter 2 d of visul deprivtion. () Representtive wholeell reordings from str pyrmidl neurons in slies from ontrol nd deprived hemispheres, illustrting the elevtion in tivity in the deprived hemisphere. () Averge spontneous firing rtes from str pyrmidl neurons () in whole-ell (WC) nd loose-pth (LP) onfigurtions, RSNP ells in the whole-ell onfigurtion nd from str pyrmidl neurons 48 h fter reopening the eye (REV). Firing rtes in the deprived hemisphere re represented reltive to the ontrol hemisphere to show the fold hnge. Asterisk indites signifint differene from ontrol. Here nd for ll susequent figures, error rs represent the s.e.m. () Exmple reording from str pyrmidl neuron from the deprived hemisphere during wsh-in (middle pnel) nd wshout (right pnel) of synpti lokers (APV, DNQX nd iuulline). Inresed firing is due to ltered synpti drive At lest three mehnisms ould ontriute to the inresed spontneous tivity of str pyrmidl neurons: inresed intrinsi exitility 27, inresed exittory synpti drive 7,28 nd deresed inhiitory synpti drive 29. To test hnges in intrinsi properties, we generted firing rte versus urrent (F I) urves in the presene of the synpti reeptor lokers APV (5 µm), DNQX (2 µm) nd iuulline (2 µm; Fig. 2,). No signifint differenes were oserved in the slopes of the liner portion of these urves (ontrol,.53 ±.7; deprived,.49 ±.4; P =.49) or in the spike threshold (Fig. 2). Monoulr deprivtion lso did not signifintly ffet resting input resistnes (ontrol: 38 ± 4 MΩ, n = ; deprived: 36 ± 56 MΩ, n = ; P =.56) nd resting memrne potentils (ontrol: 66.4 ±.6 mv, n = ; deprived: 67.3 ±.7 mv, n = ; P =.63) mesured in the presene of synpti lokers. To test whether hnges in exittory nd inhiitory synpti drive ould explin the inresed firing rtes, we reorded spontneous exittory nd inhiitory urrents onto str pyrmidl neurons y leving ongoing iruit tivity intt nd voltge lmping individul postsynpti neurons to different potentils. Exittory nd inhiitory urrents were seprted y holding the postsynpti neuron t the mesured reversl potentil (E rev ) for inhiitory postsynpti urrents (IPSCs; 4 mv; Fig. 3) or t the mesured E rev for exittory postsynpti urrents (EPSCs; + mv; Fig. 3). Integrting spontneous exittory nd inhiitory urrents over 5-min period showed tht monoulr deprivtion produed 75% inrese in exittory synpti hrge (Fig. 3,; ontrol, n = 9; deprived, n = 9; P <.) nd 46% derese in inhiitory synpti hrge (Fig. 3,; ontrol, n = 9; deprived, ms Spike rte (Hz) Figure 2 Visul deprivtion did not ffet the intrinsi exitility of str pyrmidl neurons. () Representtive reordings of lyer 4 str pyrmidl neurons in slies from ontrol nd deprived hemispheres in response to DC depolrizing urrent injetion (5 ms, 8 pa). Reordings were otined in the presene of synpti lokers. () Top pnel: verge urrent versus firing rte from str pyrmidl neurons in ontrol nd deprived hemispheres. Bottom pnel: verge spike threshold (the interpolted memrne potentil t whih dv/dt equled 2 V/s) for the ontrol nd deprived onditions. 8 pa 8 pa Spike threshold (mv) I (pa) 354 VOLUME 7 NUMBER 2 DECEMBER 24 NATURE NEUROSCIENCE

3 4 mv 4 mv 2 pa s + mv + mv pa s Figure 3 The lne etween exittory nd inhiitory synpti drive ws ltered y visul deprivtion. () Representtive reordings of spontneous EPSCs (reorded t the Cl reversl potentil) from str pyrmidl neurons in slies from ontrol nd deprived hemispheres in modified ACSF. () Representtive reordings of spontneous IPSCs (reorded t the reversl potentil for spontneous EPSCs); exmples re from the sme ontrol nd deprived neurons s in. () Averge exittory nd inhiitory hrge (otined y integrting spontneous EPSC or IPSC urrent over 5 min for eh neuron) for str pyrmidl neurons from ontrol or deprived hemispheres. dt re expressed reltive to ontrol to show the fold hnge. Asterisk indites signifint differene from ontrol. n = 9; P <.). These results show tht the lne etween exittion nd inhiition in the reurrent iruitry of lyer 4 hs shifted to fvor exittion. Monoulr deprivtion strengthens exittory onnetions Lyer 4 str pyrmidl neurons in the visul ortex reeive diret thlmi input nd reurrent exittory onnetions from other str pyrmidl neurons within lyer 4. The verge quntl mplitude of exittory synpses on str pyrmidl neurons is inresed fter 2 d of monoulr deprivtion 7. To investigte in detil how monoulr deprivtion ffets the properties of unitry exittory synpti onnetions within lyer 4, we rried out qudruple reordings from str pyrmidl neurons to find monosynptilly onneted pirs (Fig. 4). All pired reordings here nd elow were done in stndrd ACSF so there ws miniml kground synpti tivity. We found 25 onneted pirs out of 234 tested (.6%) from ontrol hemispheres nd 45 onneted pirs out of 238 tested (8.9%) from deprived hemispheres (Fig. 4; P <.25, χ 2 ). In ddition to this inrese in onnetion proility, the verge mplitude of monosynpti pyrmidl-to-pyrmidl onnetions pproximtely douled in the deprived hemisphere (Fig. 4,d; Reltive hrge _ 4 mv + mv P <.), with no signifint hnges in EPSC lteny (ontrol,.2 ±.2 ms; deprived,. ±.2 ms; P =.3) or kinetis (2 8% rise times: ontrol,.69 ±.4 ms; deprived,.64 ±.9 ms; P =.63 nd dey time onstnts: ontrol, 7.2 ±.8 ms; deprived, 8.9 ±.6 ms; P =.37). A smll ut signifint hnge ws oserved in the short-term plstiity of pyrmidl-topyrmidl onnetions in response to trins of five preisely timed presynpti spikes t 2 Hz. Connetions from the deprived hemisphere showed slightly more depression (Fig. 4,e; n = ontrol nd 7 deprived pirs; P <., one-wy ANOVA). Filure rtes nd oeffiients of vrition of EPSC mplitude were not, however, signifintly different (Fig. 4d; P =.56), suggesting tht proility of relese ws not muh ltered. Similrly, only modest hnges in short-term plstiity hve een oserved in rodent somtosensory ortex fter whisker deprivtion,3, nd tivity deprivtion in ortil nd spinl ultures inreses exittory synpti strength primrily through postsynpti hnges in reeptor umultion 28,32. A higher onnetion proility etween str pyrmidl neurons, ompnied y douling of EPSC mplitude etween onneted pirs, should ooperte to inrese the gin of exittory feedk within lyer 4. Interneurons respond differently to monoulr deprivtion Different ortil interneurons re funtionlly distint within the ortil iruit 33. A numer of studies hve suggested tht tivity deprivtion in ulture 29,34 or visul deprivtion in speies rnging from rodents to primtes 8,35 n redue ortil inhiition. Whether this redution differentilly ffets inhiition tht rises from different lsses of interneuron hs not een ssessed. Here we exmined hnges in unitry onnetions from two lsses of interneuron, fst-spiking nd RSNP ells, onto str pyrmidl neurons. Fst-spiking neurons re nondpting, high frequeny firing GABAergi inhiitory interneurons (Fig. 5), whih generte feedk inhiition onto str pyrmidl neurons 36. There were 6 onneted pa µm II/III IV 2 ms Conn. proility (%) d 2 2 I (pa) 5.5 Fil. (%) CV e Chnge (%) EPSC numer pa 5 ms Figure 4 Visul deprivtion inresed the mplitude of monosynpti onnetions etween str pyrmidl neurons. () Top: mer luid reonstrution of representtive monosynptilly onneted pir of str pyrmidl neurons in lyer 4, showing lminr position. Bottom: exmple of the typil firing pttern of str pyrmidl neurons in response to 5-ms depolrizing urrent step of pa. () Connetion proility in ontrol nd deprived hemispheres. () Representtive reording of monosynpti EPSCs (reorded t 8 mv) in response to preisely timed presynpti spikes t 2 Hz. Eh tre is the verge of repetitions. (d) EPSC mplitude (urrent; I), filure rte (Fil.) nd oeffiient of vrition (CV), verged ross onnetions from ontrol nd deprived hemispheres. (e) Averge EPSC mplitude for eh response in the 2-Hz trin (plotted s perentge of the first EPSC mplitude) for ontrol nd deprived pirs. Asterisk indites signifint differene from ontrol. NATURE NEUROSCIENCE VOLUME 7 NUMBER 2 DECEMBER

4 8 pa FS µm II/III IV 2 ms Conn. proility (%) d pirs of fst-spiking nd pyrmidl neurons out of 25 tested in ontrol hemispheres (64.%) nd of 6 in deprived hemispheres (68.8%; P =.9, χ 2 test; Fig. 5). Monoulr deprivtion indued 5% derese in the mplitude of monosynpti onnetions etween fst-spiking nd str pyrmidl neurons (Fig. 5,d; ontrol, n = 6; deprived, n = ; P <.3). No signifint hnges were oserved in synpti delys (ontrol,.8 ±.2 ms; deprived, 2. ±.3 ms; P =.43), rise times (ontrol,.4 ±.2 ms; deprived,.4 ±. ms; P =.85) nd dey time onstnts (ontrol, 2.4 ±.4 ms; deprived,.7 ±.9 ms; P =.69). This derese in mplitude ws ompnied y drmti hnge in short-term plstiity. Although ontrol onnetions showed short-term depression, onnetions from the deprived hemisphere showed pronouned short-term filittion (Fig. 5,e; n = 6 ontrol nd deprived pirs; P <., ANOVA), nd the oeffiient of vrition ws signifintly inresed in the deprived hemisphere (Fig. 5e; P <.), onsistent with redution in relese proility ontriuting to the redution in IPSC mplitude. Beuse ontrol onnetions depressed while deprived onnetions filitted, the solute mplitude of the lst IPSC in 2-Hz trin ws omprle etween onditions (ontrol, 3.4 ±.3 pa; deprived, 3.2 ±.5 pa)..5 Chnge (%) I (pa) CV IPSC numer e 4 5 pa 5 ms Figure 5 Visul deprivtion deresed the mplitude of monosynpti onnetions etween fst-spiking nd str pyrmidl neurons. () Top: mer luid reonstrution of representtive monosynptilly onneted fst-spiking nd str pyrmidl neuron pir. Bottom: typil firing pttern of fst-spiking interneuron in response to 5-ms depolrizing urrent step of 8 pa. () Connetion proility in ontrol nd deprived hemispheres. () Representtive reording of monosynpti IPSCs (reorded t 8 mv, so they pper s inwrd urrents) in response to preisely timed presynpti spikes t 2 Hz. (d) IPSC mplitude (urrent; I) nd oeffiient of vrition (CV), verged ross onnetions from ontrol nd deprived hemispheres. (e) Averge IPSC mplitude for eh IPSC in the 2-Hz trin (plotted s perentge of the first IPSC mplitude) for ontrol nd deprived pirs. Asterisk indites signifint differene from ontrol. RSNP neurons in rt visul ortil lyer 4 re regulr-spiking, modertely dpting GABAergi interneurons with ipolr morphology (Fig. 6). They reeive diret thlmi drive nd generte feed-forwrd inhiition onto str pyrmidl neurons 37. The proility of finding onneted pirs in the deprived hemisphere ws less thn hlf tht of the ontrol (Fig. 6; ontrol, 6 of 5 or 32.%; deprived, 8 of 58 or 3.8%; P <.5, χ 2 test). In ontrst to the effets of monoulr deprivtion on fst-spiking-to-pyrmidl onnetions, the mplitude of RSNP-to-pyrmidl onnetions more thn douled fter monoulr deprivtion (Fig. 6,d; P <.). Like pyrmidl-to-pyrmidl onnetions, deprivtion produed modest inrese in short-term depression with no signifint hnge in filure rte or oeffiient of vrition (Fig. 6,e; P =.62). No hnges were oserved in synpti delys (ontrol,.8 ±.3 ms; deprived,.7 ±.2; P =.9), rise times (ontrol,.2 ±.2 ms; deprived,.3 ±.2 ms; P =.73) nd dey time onstnts (ontrol,.4 ±.7; deprived,.9 ±.5 ms; P =.3). These dt indite tht str pyrmidl neurons reeive inhiition from smller frtion of RSNP neurons, ut tht eh unitry onnetion is more potent. Figure 6 Visul deprivtion inresed the mplitude of monosynpti onnetions etween RSNP nd str pyrmidl neurons ut deresed the onnetion proility. () Top, mer luid reonstrution of representtive monosynptilly onneted RSNP nd str pyrmidl neuron pir. Bottom, typil firing pttern of RSNP interneuron in response to 5-ms depolrizing urrent injetion. () Connetion proility for ontrol nd deprived hemispheres. () Representtive reording of monosynpti IPSCs (reorded t 8 mv, so they pper s inwrd urrents) in response to preisely timed presynpti spikes t 2 Hz. (d) IPSC mplitude (urrent; I), filure rte (Fil.) nd oeffiient of vrition (CV), verged ross onnetions from ontrol nd deprived hemispheres. (e) Averge IPSC mplitude for eh IPSC in the 2-Hz trin (plotted s perentge of the first EPSC mplitude) for ontrol nd deprived pirs. Asterisk indites signifint differene from ontrol. pa RSNP µm II/III IV 2 ms Conn. proility (%) d I (pa) Fil. (%) CV.5 e Chnge (%) IPSC numer 5 pa 5 ms 356 VOLUME 7 NUMBER 2 DECEMBER 24 NATURE NEUROSCIENCE

5 DISCUSSION Here we show tht pronouned effet of erly visul deprivtion in lyer 4 is highly seletive djustment of intrortil synpti strengths tht results in inresed lyer 4 exitility. Two dys of lid suture just t eye opening douled the mplitude of lol exittory onnetions nd douled the proility of finding onneted exittory pirs. In ontrst, inhiitory synpti drive ws redued through seletive hnges in inhiitory iruitry, nd these two effets ooperted to drmtilly inrese spontneous tivity within lyer 4. This indites tht reduing sensory drive erly in life inreses the exitility of lol lyer 4 iruitry. Theoretil work hs suggested tht ompenstory, or homeostti, plstiity mehnisms tht stilize neuronl or network tivity re essentil for the proess of tivity-dependent refinement 38,39, nd severl forms of homeostti plstiity hve een identified experimentlly 4. The dt presented here indite tht, in highly reurrent lyer 4 networks, this stiliztion my e hieved through oordinted set of hnges in exittory nd inhiitory iruitry. When sensory drive is redued, reurrent exittory onnetions re inresed in strength, wheres feedk inhiition is redued in strength (Supplementry Fig. online). These synpti hnges re likely to t synergistilly to produe the drmti inrese (25-fold) in spontneous str pyrmidl firing rtes tht ws oserved, lthough we nnot rule out tht other synpti hnges lso ontriute to this inresed exitility. This ompenstory inrese in lyer 4 exitility in response to lowered sensory drive my enhne the ility of lyer 4 to mplify the sensory signls it does reeive. Prolonged periods of visul deprivtion (using lid suture, drk rering or tetrodotoxin injetions) hve long een known to redue inhiition in primry visul ortex of rodents nd primtes 8,9,35,4, ut it ws not known whether different lsses of interneuron re differentilly ffeted y sensory deprivtion. Here we show tht rief visul deprivtion erly in life hs fundmentlly different effets on different lsses of inhiitory synpse. Fst-spiking interneurons generte lssi feedk inhiition on str pyrmidl neurons 36,42, nd this lss of onnetion ws redued in the deprived hemisphere. Interestingly, the short-term plstiity of this synpse underwent drmti hnge from depressing to filitting. This suggests tht fst-spiking onnetions re preferentilly redued in mplitude t low firing rtes, ut tht t high firing rtes this synpse will filitte nd more inhiition will e reruited. This my help to set n upper limit on spontneous tivity. In ontrst to fst-spiking neurons, RSNP neurons reeive diret thlmi input nd generte feed-forwrd inhiition onto str pyrmidl neurons 37. The verge mplitude of RSNP-to-pyrmidl onnetions more thn douled in the deprived hemisphere, wheres the onnetion proility ws redued to less thn hlf of ontrol vlues. This indites tht totl inhiition from RSNP neurons remins roughly onstnt ut tht str pyrmidl neurons reeive more potent inhiition from fewer RSNP neurons. These results show tht funtionlly distint lsses of inhiitory onnetion re djusted in highly seletive mnner y ltered sensory drive, nd tht the effets of erly sensory deprivtion in lyer 4 depend ritilly on the identity of the synpse nd on its role in ortil funtion. The effets of sensory deprivtion re strongly developmentlly regulted, nd different ortil lyers n hve different sensitive periods during development. For exmple, in rodents the sensitive periods for oulr dominne plstiity in inoulr visul ortex nd for whisker deprivtion plstiity in somtosensory ortex lose erly in lyer 4 (fter the first few weeks of life) ut persist signifintly lter in lyer 2/3. The effets of visul deprivtion on the quntl mplitude of exittory urrents on pyrmidl neurons lso hve lyer-speifi ritil periods 7. Lowering retinl tivity etween P4 nd P7 sled up exittory quntl urrents in lyer 4 ut not in lyer 2/3, wheres the sme deprivtion rried out etween P2 nd P23 hd no effet in lyer 4 ut insted sled up exittory quntl mplitudes in lyer 2/3 (ref. 7). These dt re onsistent with the findings reported here tht spontneous tivity in lyer 4, ut not in lyer 2/3, is inresed y lid suture etween P4 nd P7. Tken together, these studies indite tht homeostti plstiity egins in lyer 4 ut (like other forms of plstiity) shifts during development to lyer 2/3. The first few weeks of development re likely to represent the period of gretest instility in lyer 4, s thlmoortil nd intrortil synpses re rpidly forming nd there is roust thlmoortil long-term potentition, so homeostti plstiity in lyer 4 my e espeilly importnt during this developmentl window. Whether the synpti mehnisms underlying inresed lyer 4 exitility tht re desried here will generlize to other ortil lyers t lter developmentl times remins to e determined. In ddition to serving homeostti funtion, shifts in the lne etween exittion nd inhiition ould hve lrge impt on tivity-dependent iruit refinement nd funtion. Chnges in this lne lter the ese with whih synpse-speifi forms of ortil plstiity (suh s long-term potentition nd depression) n e eliited 43 nd n modify or prolong the developmentl periods during whih visul response properties re sensitive to experiene 44,45. The initition nd propgtion of tivity in ortil networks is lso influened y the exittion-inhiition lne 46, nd so the seletivity of ortil responses my e strongly ffeted when this lne is ltered. Our dt indite tht homeostti regultion of the exittion-inhiition lne my e importnt in mintining stle ortil tivity during norml development nd my lso ontriute to the pthologil hnges in visul response properties tht re indued y sensory deprivtion. METHODS Monoulr deprivtion. Eyelid suture ws done lte on P4 (just efore eye opening) during rief nesthesi with 7 mg/kg ketmine, 3.5 mg/kg xylzine hydrohloride nd.7 mg/kg epromzine mlete, intrperitonelly. The eyelid ws overed with thin lyer of xyloine gel, nd the lid ws seured with three mttress sutures. Sutures were heked every dy until nimls were killed for reording erly on P7. All methods were pproved y the Brndeis Animl Use Committee nd were rried out in ordne with the Ntionl Institutes of Helth guidelines. Eletrophysiology. Coronl slies ontining primry visul ortex were prepred from the deprived nd ontrol hemispheres, nd visulized pthlmp reordings were otined from lyer 4 of the monoulr region of V, s desried 7. Neurons were trgeted visully using infrred differentil interferene ontrst optis nd were filled with ioytin during the reording session for post ho morphologil reonstrution. Neurons were identified sed on lminr position, morphology, synpti properties nd firing properties. Neurons tht were inluded in the nlyses hd memrne potentil (V m ) elow 6 mv, n input resistne (R in ) ove 2 MΩ nd series resistne elow 5 MΩ; for the inluded neurons, these prmeters did not hnge more thn % during the reording. To mesure spontneous firing rtes, smll mount of DC urrent (generlly depolrizing) ws injeted to djust the interspike potentil to 6 mv. For loose-pth reordings, we used 2 3 MΩ pipettes filled with ACSF. A sel of 6 2 MΩ ws otined y pthing onto visully identified str pyrmidl neurons, nd extrellulr spikes were reorded. Reordings were nlyzed if spikes exeeded 5 mv nd if the sel remined stle for >5 min. For generting F-I urves, series of depolrizing urrent steps ws delivered in the presene of synpti lokers. For mesuring spontneous synpti urrents, the reversl potentil (E rev ) ws determined y voltge lmping the postsynpti neuron to different potentils (in 5 mv inrements) etween 55 nd mv for inhiitory urrents nd etween 5 nd +5 NATURE NEUROSCIENCE VOLUME 7 NUMBER 2 DECEMBER

6 mv for exittory urrents. The verge reversl potentils for EPSCs (ontrol, 8.4 ±.5 mv; deprived, 9.2 ±. mv; P =.46) nd IPSCs (ontrol, 4. ±. mv; deprived, 4. ±. mv; P =.32) were not different etween onditions, nd when orreted for juntion potentils, they were within mv of those predited with our internl nd externl solutions. Monosynpti pired reordings were otined in lyer 4 y qudruple whole-ell reordings of nery neurons s desried 47. Firing properties were ssessed in urrent lmp y delivering 5 ms depolrizing urrent steps. Reversl potentils of synpti urrents were determined y stimulting presynpti neurons while voltge lmping the postsynpti neuron to different potentils. To mesure short-term plstiity, trins of 5 presynpti tion potentils were eliited t 2 Hz (every 2 s), nd postsynpti urrents were monitored t 8 mv. Clssifition of neurons. Str pyrmidl neurons were identified s previously desried 7. Bsed on morphology, firing properties nd short-term plstiity of synpti onnetions, we identified two popultions of onnetions etween interneurons nd str pyrmidl neurons. Fst-spiking neurons were multipolr, fst spiking nd nondpting, nd their IPSCs on pyrmidl neurons showed moderte depression 3,5,42,48. RSNP neurons hd ipolr morphology nd were regulr spiking with frequeny dpttion; their IPSCs on pyrmidl neurons showed mrked depression 3,5,42,49. Note tht interneuronl properties in lyer 4 of visul ortex differ in some respets from those reported in lyer 4 of somtosensory ortex 5. Sttistil nlysis. All dt re expressed s the men ± s.e.m. for the numer of neurons (or onneted pirs) speified, nd ll sttistil tests were unpired two-tiled t-tests, exept s noted in text. Solutions. Stndrd ACSF onsists of 26 mm NCl, 3 mm KCl, 2 mm MgSO 4, 2 mm CCl 2, mm NHPO 4, 25 mm NHCO 3 nd 25 mm dextrose. For pired reordings, the Mg 2+ onentrtion ws mm. Modified ACSF (3.5 mm KCl,.5 mm MgCl 2 nd mm CCl 2 ) ws used to reord spontneous firing nd spontneous synpti urrents. The internl solution ws 2 mm KCl, mm potssium gluonte, mm HEPES,.% ioytin, 4 mm mgnesium ATP,.3 mm sodium GTP nd mm sodium phosphoretine. For mesuring spontneous synpti urrents, the internl solution ontined 2 mm KCl, mm esium methylsulfonte, mm HEPES,.% ioytin, 4 mm Mg-ATP,.3 mm N-GTP, mm sodium phosphoretine nd 3 mm QX-34. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk R. Cudmore for help with softwre, J. Brry nd K. Essig for histology nd S. Fusi nd X.-J. Wng for helpful disussions. Supported y the Ntionl Eye Institute (EY4439) nd the Ntionl Institute on Drug Ause (DA6455). COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Reeived Septemer; epted 3 Otoer 24 Pulished online t Huel, D.H. & Wiesel, T.N. 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7 45. Hung, Z.J. et l. BDNF regultes the mturtion of inhiition nd the ritil period of plstiity in mouse visul ortex. Cell 98, (999). 46. Chgn-Amiti, Y. & Connors, B.W. Horizontl spred of synhronized tivity in neoortex nd its ontrol y GABA-medited inhiition. J. Neurophysiol. 6, (989). 47. Sjostrom, P.J., Turrigino, G.G. & Nelson, S.B. Rte, timing, nd oopertivity jointly determine ortil synpti plstiity. Neuron 32, (2). 48. Deuhrs, J. & Thomson, A.M. Single xon fst inhiitory postsynpti potentils eliited y sprsely spiny interneuron in rt neoortex. Neurosiene 65, (995). 49. Peters, A. & Herrimnn, K.M. Enigmti ipolr ell of rt visul ortex. J. Comp. Neurol. 267, (988). 5. Beierlein, M., Gison, J.R. & Connors, B.W. Two dynmilly distint inhiitory networks in lyer 4 of the neoortex. J. Neurophysiol. 9, 2987 (23). NATURE NEUROSCIENCE VOLUME 7 NUMBER 2 DECEMBER