Early depolarizing GABA controls critical-period plasticity in the rat visual cortex

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1 Erly depolrizing GABA ontrols ritil-period plstiity in the rt visul ortex Gbriele Deidd,5, Mnuel Allegr,,5, Chir Cerri, Shovn Nskr, Guillume Bony, Giuli Zunino 4, Yuri Bozzi,4, Mtteo Cleo,6 & Lur Cnedd,6 npg 5 Nture Ameri, In. All rights reserved. Hyperpolrizing nd inhibitory GABA regultes ritil periods for plstiity in sensory orties. Here we exmine the role of erly, depolrizing GABA in the ontrol of plstiity mehnisms. We report tht brief interferene with depolrizing GABA during erly development prolonged ritil-period plstiity in visul ortil iruits without ffeting the overll development of the visul system. The effets on plstiity were ompnied by dmpened inhibitory neurotrnsmission, downregultion of brinderived neurotrophi ftor (BDNF) expression nd redued density of extrellulr mtrix perineuronl nets. Erly interferene with depolrizing GABA deresed perintl BDNF signling, nd phrmologil inrese of BDNF signling during GABA interferene resued the effets on plstiity nd its regultors lter in life. We onlude tht depolrizing GABA exerts long-lsting, seletive modultion of plstiity of ortil iruits by strong rosstlk with BDNF. Sensory systems re immture t birth nd undergo experiene-dependent mturtion during development. In prtiulr, during ritil period (CP) of postntl development, neuronl iruits re plsti nd re esily shped by sensory experiene to reh the mture struturl nd funtionl fetures of the dult,. In the visul system, experiene strongly regultes the mturtion of bsi properties of the visul ortex (for exmple, visul uity), s well s both the opening nd losure of the CP,4. Hyperpolrizing nd inhibitory GABA signling through Cl -permeble ionotropi GABA A reeptors is ritil for experienedependent development nd plstiity of the visul ortex,5 7. Nevertheless, GABA exerts depolrizing nd minly exittory tion during erly development (until the first or seond postntl week in rodents) 8,9. Indeed, immture neurons umulte Cl intrellulrly through the N + -K + -Cl (NKCC) o-trnsporter, resulting in net depolrizing efflux of Cl ions fter GABA A reeptor opening. Conversely, in dolesent nd dult nimls, it is mostly the o-trnsporter K + -Cl (KCC) tht is expressed, lowering the intrellulr Cl onentrtion, [Cl] i, nd onverting GABA A ergi trnsmission from depolrizing nd minly exittory to hyperpolrizing nd inhibitory. Although hyperpolrizing nd inhibitory GABAergi trnsmission is well-known regultor of visul system development nd plstiity, nd depolrizing GABAergi signling is fundmentl for erly physiologil mturtion of vrious brin res, nothing is known bout the role of depolrizing GABA in the regultion of ortil plstiity. Here we found tht brief interferene with depolrizing GABA during erly postntl development prolongs the CP of plstiity in the visul ortex without ffeting the overll development of the visul system. This effet is ompnied by hnges in the fundmentl regultors of plstiity nd is resued by inresing BDNF signling during depolrizing GABA interferene. RESULTS Erly depolrizing GABA ontrols CP plstiity To hmper depolrizing GABA A signling, we treted rt pups with the NKCC inhibitor bumetnide (intrperitonel (i.p.) dministrtion,. mg/kg body weight twie dy beuse of its phrmokinetis ) from postntl dy (P) to P8 (Fig. ), when NKCC is the min K + -Cl o-trnsporter in the erebrl ortex (Supplementry Fig. ) 8. Indeed, bumetnide rosses the brin-blood brrier nd effetively shifts the reversl potentil of GABA (E GABA ), onverting GABA A signling to hyperpolrizing in young rts,,. To ssess whether perintl interferene with depolrizing GABA ffets the time ourse of the CP for visul ortex plstiity, we used lssil prdigm of experiene-dependent plstiity (i.e., dys of monoulr deprivtion (MD)) in bumetnide- nd vehile-treted rts (Fig. ). When performed during the CP (but not in dults), brief MD results in mrked shift of the oulr dominne (OD) of ortil neurons in fvor of the nondeprived eye 4. To define the time ourse of the sensitive period for plstiity, we performed MD experiments t different ges spnning the norml opening nd losing of the CP 5. To quntify the OD shift, we performed in vivo eletrophysiologil reordings nd mesured the ontrlterl-to-ipsilterl (C:I) visulevoked potentil (VEP) rtio (i.e., the rtio of VEP mplitudes reorded by stimulting the ontrlterl or ipsilterl eye) in bumetnide- nd vehile-treted nimls either with MD or left undeprived (nonmd). Bseline binoulrity ws omprble between nonmd bumetnidend vehile-treted nimls t ll tested ges (C:I VEP rtio of.5 for both groups; Fig. h), inditing no speifi effet of erly bumetnide tretment on the generl development of binoulrity. The MD experiments indited tht lthough the onset of CP plstiity ws unffeted by bumetnide tretment, its losure ws signifintly Neurosiene nd Brin Tehnologies Deprtment, Istituto Itlino di Tenologi, Genov, Itly. Suol Normle Superiore, Pis, Itly. CNR Neurosiene Institute, Pis, Itly. 4 Lbortory of Moleulr Neuropthology, Centre for Integrtive Biology (CIBIO), University of Trento, Trento, Itly. 5 These uthors ontributed eqully to this work. 6 These uthors jointly direted this work. Correspondene should be ddressed to L.C. (lur.nedd@iit.it). Reeived September; epted November; published online 8 Deember 4; doi:.8/nn.89 nture NEUROSCIENCE VOLUME 8 NUMBER JANUARY 5 87

2 b MD in vivo or vehile injetions P P P8 Depolrizing GABA Eye opening Biohemistry Behvior Eletrophysiology Biohemistry Antomy Eletrophysiology Biohemistry Antomy Eletrophysiology P P7 P 6 P45 P75 Degree of plstiity Postntl dy C:I rtio P7 d C:I rtio P e C:I rtio P6 f C:I rtio npg 5 Nture Ameri, In. All rights reserved. NonMD MD NonMD MD NonMD MD NonMD MD Figure Erly GABAergi interferene prolongs ritil-period plstiity in the visul ortex in vivo. () Shemti rtoon of the experimentl protool. (b) Averge level of plstiity (quntified s desribed in the Online Methods) ± s.e.m. derived from experiments performed t different ges (shown in h). The level of plstiity ws higher in bumetnide-treted nimls thn in vehile-treted ontrols t (P =.9 (), post two-wy nlysis of vrine (ANOVA) Holm-Sidk test) but ws similr between the two groups t the other time points (P =.998 (P7), P =.96 (P), P =.999 (P6), P =.988 (P45), P =.999 (P75)). ( h) C:I VEP rtios mesured t the ges indited in vehile- nd delyed (Fig. b). Indeed, OD plstiity rose similrly from P7 to P nd ws mximl t P6 in both bumetnide- nd vehile-treted rts (Fig. e). Conversely, t, onsistent nd signifint OD plstiity ould be indued in bumetnide-treted nimls only (Fig. b,f). Notbly, the shift in eye preferene of bumetnide-treted nimls fter MD t involved depression of responses driven by the deprived eye, n effet tht is typil of juvenile-like plstiity 6 (Supplementry Fig. ). The CP ws over in both groups by P45, s plstiity ws not signifint t either P45 or P75 (Fig. b,g,h). To strengthen the in vivo dt, we ssessed the level of in vitro plstiity in the visul system of nimls previously treted with bumetnide or vehile. In prtiulr, we performed field-potentil reordings in ute visul ortil slies to ssess long-term potentition (LTP) of lyer II/III synpti responses by thet-burst stimultion () of the white mtter (WM-LTP). This is lssil in vitro model of visul ortil plstiity tht is known to undergo n ge-dependent developmentl deline tht orreltes losely with the developmentl deline in suseptibility to MD in vivo,,5. Consistent with our MD experiments, we found tht erly GABAergi interferene prolonged the CP for WM-LTP indution (Fig. ) (this effet is exemplified in Fig. b g, whih shows the verge time ourse of field potentil responses before nd fter t different ges). Wheres both bumetnide- nd vehile-treted nimls opened the CP fter P7 nd showed mximl plstiity t P P6 (Fig. d), only bumetnidetreted nimls showed signifint WM-LTP t (Fig.,e). g C:I rtio NonMD MD NonMD MD P45 NonMD MD NonMD MD h C:I rtio NonMD MD NonMD MD Conversely, neither experimentl group showed signifint plstiity t older ges (P45 nd P75; Fig.,f,g). Altogether, these results indite tht interfering with depolrizing GABA during erly development signifintly extends the sensitive period for tivity-dependent rerrngements of ortil iruits lter in life. Erly bumetnide tretment does not ffet overll ntomy To exlude the possibility tht the effet on plstiity ws due to defets in the ntomil development of the visul system,7, we investigted the segregtion of eye-speifi inputs in the dorsl lterl geniulte nuleus (dlgn) nd the migrtion nd morphologil mturtion in the visul ortex of nimls perintlly treted with bumetnide or vehile, s these proesses our erly in rt perintl life 7,8. To visulize retinl fferents from both eyes, we performed intrvitrel injetions of holer toxin bet (CTB) frgment onjugtes of different olors nd nlyzed oronl dlgn setions t (Fig. ). We found ler segregtion of fibers from the two eyes in both bumetnide- nd vehile-treted rts (Fig. b), inditing norml development of retinogeniulte projetions. To investigte the struturl development of the visul ortex, we performed in utero eletroportion of progenitors of exittory neurons ommitted to lyer II/III with plsmid enoding for enhned GFP (egfp) t embryoni dy (E) 7.5 (Fig. ). This llowed for visuliztion of neuronl migrtion nd morphologil mturtion of the neuronl progeny of trnsfeted ells by egfp fluoresene imging. P75 NonMD MD NonMD MD bumetnide-treted nimls in either nonmd or MD onditions. At ll ges, bseline binoulrity ws superimposble between bumetnide- nd vehiletreted rts (post two-wy ANOVA Tukey test; P =. (), P =.97 (d), P =.974 (e), P =. (f), P =.87 (g), P =.758 (h)). Response to d of MD ws lso similr throughout development, with the exeption of. At this ge, bumetnide- but not vehile-treted nimls showed n OD shift in fvor of the ipsilterl (open eye) s shown by lower C:I VEP rtios (post two-wy ANOVA Tukey test, P <.). The histogrms depit the verge vlues ± s.e.m., nd irles represent dt from single nimls. P = VOLUME 8 NUMBER JANUARY 5 nture NEUROSCIENCE

3 b P7 P d P6 WM-LTP in vitro Postntl dy e 6 8 (6,) (6,) 4 f 6 8 (6,4) (,4) P45 4 g 6 8 (8,4) (6,) P75 4 npg 5 Nture Ameri, In. All rights reserved. Figure Erly GABAergi interferene prolongs ritilperiod plstiity in the visul ortex in vitro. () Averge level of plstiity (lulted s the pek mplitude response normlized to the verged bseline) ± s.e.m. of the mplitude of lyer II/III field synpti potentils fter of the WM from experiments performed t different ges (shown in b g). We found tht ll egfp-positive neurons rehed lyer II/III in both bumetnide- nd vehile-treted nimls t (Fig. d). Moreover, morphologil reonstrution of egfp-positive neurons did not revel ny signifint differene in morphologil mturtion between the two groups (totl brnh number, P =.5; totl brnh length, P =.646; Fig. d nd Supplementry Fig. ). Next, to ssess the development of GABAergi interneurons, we exmined their lminr distribution by immunostining for the GABA biosyntheti enzyme GAD67 in ortil setions from bumetnide- nd vehile-treted rts t. We found no signifint differenes in the distribution of interneurons between the two groups (Fig. e,f). Erly bumetnide tretment does not ffet visul funtion Next we investigted whether bumetnide tretment ould ffet the overll development of the visul system t the funtionl level. In generl, bumetnide-treted nimls developed normlly, s their gin of body weight nd timing of eye opening were omprble to those of ontrols (Fig. 4 nd Supplementry Fig. 4). Moreover, bumetnidend vehile-injeted nimls performed eqully in lerning visul disrimintion tsk (Prusky visul wter box; Fig. 4b). Using the sme test, we mesured visul uity longitudinlly strting from P4 nd found no defiits in its mturtion in bumetnidetreted nimls (Fig. 4). We then mesured bsi physiologil prmeters in the primry visul ortex using VEP reordings t (i.e., when these prmeters reh dult levels in nive nimls) 9. We found tht sptil resolution (visul uity), ontrst threshold nd VEP mplitude nd lteny in bumetnide-treted rts were within the norml rnge nd did not differ from those of ontrols (Fig. 4d g). Thus, briefly interfering with depolrizing GABA during erly development prolongs the CP for OD plstiity nd WM-LTP into 6 8 (,8) (4,6) (6,4) (6,4) 4 dulthood without ffeting the bsi ntomil nd physiologil development of the visul system. effets re not due to regultion of osmolrity To ensure tht the effets of bumetnide tretment on visul ortil plstiity were due speifilly to regultion of NKCC rther thn to side effets of phrmologil tretment, we treted rt pups perintlly (P P8) with nother NKCC inhibitor (furosemide; i.p., mg/kg body weight, twie dy) nd ssessed the level of plstiity t. In ft, furosemide bloks both NKCC nd KCC; nevertheless, beuse of low KCC expression t the ge of tretment (Supplementry Fig. ), furosemide tretment mimis the effet of the NKCC-speifi bloker bumetnide on hloride regultion under our experimentl onditions. Aordingly, we found tht furosemidetreted nimls displyed higher levels of plstiity thn vehiletreted ontrols t, s they responded to brief MD with robust OD shift nd displyed signifint WM-LTP in slies (Fig. 5,b). These plsti hnges were extly omprble to those observed in bumetnide-injeted rts (Fig. 5,b), inditing the involvement of erly hloride homeostsis in regulting plstiity lter in life. Nevertheless, diret regultion of NKCC lso results in neuronl shrinkge, s bumetnide nd furosemide re diuretis pproved by the US Food nd Drug Administrtion tht regulte blood osmolrity (Fig. 5). Thus, we verified tht the effets of bumetnide nd furosemide on ortil plstiity re not simply due to n ltertion in the volume of the extrellulr spe. To this im, we treted rt pups perintlly (P P8) with the osmoti diureti mnnitol (i.p., 7.5 g/kg body weight),, whih regultes the volume of the extrellulr spe without ffeting NKCC tivity,. Mnnitol-treted nimls displyed the sme levels of plstiity s vehile-treted 6 8 (4,) (9,) The level of plstiity ws higher in bumetnide-treted nimls thn in vehile-treted ontrol nimls t (post two-wy ANOVA on rnks Holm-Sidk test, P <.) but ws similr between the two groups t the other time points (P =.969 (P7), P =.87 (P), P =.74 (P6), P =.86 (P45), P =.599 (P75)). (b g) Averge time ourse of the inrese in mplitude of field potentils in vehile- nd bumetnide-treted nimls t the ges indited. Insets show n verge of ten tres reorded from slie of ontrol vehile- or bumetnide-treted niml before (ontinuous lines) nd min fter (dshed lines). Stimulus rtifts hve been deleted from the tres for lrity. Sle brs,. mv (vertil); ms (horizontl). Numbers in prentheses re the number of slies proessed, number of nimls. Error brs represent the s.e.m. 4 nture NEUROSCIENCE VOLUME 8 NUMBER JANUARY 5 89

4 or vehile injetions Introulr injetions of nterogrde trers Tripolr in utero eletroportion (egfp) or vehile injetions Migrtion nd morphology ssessment b P P P8 Depolrizing GABA d E7.5 P P P8 Depolrizing GABA Inset Inset npg 5 Nture Ameri, In. All rights reserved. Totl dlgn re (µm ) 5 75 () (5) Contr re (%) 5 () (5) ontrols, both in vivo nd in vitro (Fig. 5,b), inditing tht bumetnide nd furosemide regulte ortil plstiity through modultion of Cl homeostsis but not ell volume. Vritions in CP regultors ompny bumetnide s effets Next we investigted the possible mehnisms responsible for the higher levels of plstiity t in bumetnide-treted nimls. First, s inhibitory GABAergi trnsmission is mjor regultor of losure of the CP 5,7, we used whole-ell pth-lmp reordings to exmine bsl GABAergi trnsmission in visul ortil slies from nimls tht hd been treted perintlly with bumetnide or vehile. Although the bsi membrne properties (membrne resistne, membrne pitne nd membrne resting potentil) of lyer II/III visul ortil neurons were omprble in the two experimentl groups (Supplementry Fig. 5 ), the frequeny of miniture inhibitory postsynpti urrents (mipscs) ws drstilly redued in the bumetnide-treted nimls (bumetnide,.6 ±.5 (men ± s.e.m.) Hz; vehile,.6 ±.6 Hz; Fig. 6). Conversely, there ws no differene in the mplitude (Fig. 6b) or kinetis (τ nd rise time; Supplementry Fig. 5d,e) of these events. Notbly, we found no signifint effet of perintl bumetnide tretment on the frequeny Ipsi re (%) () (5) Figure Erly GABAergi interferene does not lter the overll struturl development of the visul system. () Shemti rtoon of the experiment for lbeling retinogeniulte projetions. (b) Top, representtive oronl setions of the left nd right sides of the dlgn from vehile- nd bumetnide-treted nimls fter intrvitrel injetions of green nd red CTB frgment onjugtes t. Sle br, µm. Bottom, quntifition of the totl dlgn re, the re oupied by ontrlterl or ipsilterl eye inputs Overlp (%) 4 () (5) I II/III IV V VI WM I II/III e Inset Inset GAD67 I II/III IV V VI f Cortil lyers II/III IV V/VI IV V VI WM 6 GAD67-positive ells/mm (6) () nd the overlp of retinl projetions from the two eyes, showing no signifint differenes between the two groups (Student s t test, P =.8, P =.7, P =.66, P =.666 from left to right). The histogrms depit the verge vlues ± s.e.m. Numbers in prentheses re the numbers of nimls proessed. () Shemti rtoon of the experiment in d. (d) Representtive imges (n = vehile-treted nimls; n = 4 bumetnide-treted nimls) of egfp fluoresene in oronl setions of visul orties t fter in utero trnsfetion (t E7.5) with pcag-ires-egfp of nimls treted perintlly with vehile or bumetnide. Sle br, µm. The insets show higher-mgnifition imges of the fields outlined with boxes in the imges to the left. Sle br, µm (insets). The top dshed line indites the surfe of the slie, nd the bottom solid line outlines the WM with the typil rrow shpe in orrespondene with the primry visul ortex. (e) Representtive imges (n = 6 vehile-treted nimls; n = bumetnide-treted nimls) quired from oronl setions of the visul ortex from vehile- nd bumetnide-treted nimls t lbeled for GAD67. Sle br, µm. The insets show higher-mgnifition imges of the fields outlined with boxes in the imges on the left. Arrows point to exmples of GAD67-positive ells. Sle br, 5 µm (insets). (f) Quntifition of the density of GAD67-positive ells ross ortil lyers, showing no signifint differenes (two-wy ANOVA on rnks, P =.45). The histogrm depits the verge ± s.e.m. The numbers in prentheses re the numbers of nimls proessed. of miniture exittory AMPA postsynpti urrents t (bumetnide,.4 ±.7 Hz, n = ells; vehile,.79 ±.4 Hz, n = 9 ells; Student s t test, P =.5). Thus we investigted whether we ould resue the persistent OD plstiity of bumetnide-treted nimls t by enhning GABA A ergi trnsmission with i.p. injetion of dizepm,4 during the period of MD in group of rts treted perintlly with bumetnide (Supplementry Fig. 6). We found tht the OD shift ws signifintly (P =.5) mitigted by dizepm infusion (Supplementry Fig. 6b), inditing tht redued inhibitory tone ounts in prt for the effet of bumetnide on plstiity t nd tht other regultors of plstiity my lso hve role. Thus, s BDNF ontrols the losure of the CP in the visul system by influening inhibitory iruits 5,4, we investigted its expression in bumetnide- nd vehile-treted nimls t. Using western blot experiments, we found signifintly lower levels of BDNF in the visul orties of bumetnide-treted nimls (Fig. 6). Components of the extrellulr mtrix, suh s hondroitin sulfte proteoglyns (CSPGs), lso limit experiene-dependent plstiity by ondensing mostly round prvlbumin (PV)-positive interneurons in the form of perineuronl nets (PNNs) 4,9,5. Therefore, we investigted whether erly bumetnide tretment ffets PNN development 9 VOLUME 8 NUMBER JANUARY 5 nture neuroscience

5 npg 5 Nture Ameri, In. All rights reserved. Pups with open eyes (%) 5 (4) () Postntl dy b Corret hoies (%) P9 P4 (7) () Dys of trining round PV-positive ells by performing immunostining using Wisteri floribund letin (WFA; to lbel CSPG glyosminoglyn hins) nd n ntibody to PV t (Fig. 6d). Visul ortil slies derived from bumetnide-treted nimls showed lower PNN density ompred to slies from ontrols (Fig. 6d,e). Notbly, there ws no signifint differene in the bsolute number of PV-positive ells (Fig. 6e) or in the density of PV-positive boutons onto pyrmidl neurons between bumetnide- nd vehile-treted rts (Supplementry Fig. 7). Nevertheless, double lbeling for WFA nd PV onfirmed tht lower proportion of PV-positive interneurons were surrounded by PNNs in bumetnide-treted nimls (Fig. 6d,e). Altogether, these findings indite tht the inresed plstiity in bumetnide-treted nimls t is ompnied by modultion of well-known plstiity brkes, suh s GABAergi inhibitory neurotrnsmission, BDNF nd PNNs 4. If relese of the plstiity brkes were responsible for the higher degree of plstiity in bumetnide-treted nimls t, then one.9 (4) (4) Postntl dy Figure 4 Erly GABAergi interferene does not lter the overll funtionl development of the visul ortex. () Averge perentge (± s.e.m.) of vehile- nd bumetnide-treted pups tht opened their eyes t the ges indited. The time of eye opening ws similr for three independent litters (χ test, P =.99). The numbers in prenthesis re the numbers of nimls nlyzed. (b) Performne in lerning the Prusky visul wter disrimintion tsk (P9 P4) expressed s the perentge of orret hoies ± s.e.m. The lerning urves reveled equivlent visul performnes for bumetnide- nd vehile-treted nimls (two-wy repeted mesures ANOVA, P =.968). () Development of behviorl visul uity ssessed with the Prusky visul wter disrimintion tsk. Two-wy repeted mesures ANOVA demonstrted similr ge-dependent mturtion of visul uity in bumetnide- nd Visul uity (/deg). f VEP mplitude (µv) d Visul uity (/deg) 7 5. (5).6 (7) (7) Bin Contr would expet no differene in the levels of these sme plstiity brkes t ges when the potentil for plstiity is omprble between bumetnide- nd vehile-treted nimls. Therefore, we ssessed mipscs nd BDNF levels t P6 (the pek of plstiity for both bumetnide- nd vehile-treted nimls) nd mipscs, PNNs nd BDNF levels t P75 (when the CP is over in both groups). Reordings t P6 nd P75 indeed showed no differenes in either the frequeny or mplitude of mipscs between bumetnide- nd vehile-treted nimls (Supplementry Figs. 8,b nd 9,b). The frequeny of miniture exittory AMPA postsynpti urrents ws lso superimposble between the groups (P6, bumetnide,. ±.6 Hz, n = 6 ells; vehile,.6 ±. Hz, n = 9 ells; Student s t test, P =.67; P75, bumetnide,.87 ±. Hz, n = 7 ells; vehile,.5 ±.9 Hz, n = 9 ells; Student s t test, P =.644). In keeping with the dt on mipscs, we found omprble levels of BDNF t P6 nd P75 in bumetnide- nd vehile-treted nimls (Supplementry Figs. 8 nd 9). We ssessed the density of PNNs t P75 nd gin (8) Ipsi e Contrst threshold (%) g VEP lteny (ms) 7. (5).5 5 (7) 75 (7) (7) vehile-treted rts (P =.5). The grph depits the verge ± s.e.m. The numbers in prentheses re the numbers of nimls tested. /deg, yles per degree. (d g) In vivo eletrophysiologil reordings in the primry visul ortex showing tht bumetnide tretment did not ffet the development of bsl visul properties suh s visul uity (d), ontrst threshold (e), VEP mplitude of binoulr (bin), ontrlterl (ontr) nd ipsilterl (ipsi) responses (f) nd VEP lteny of binoulr responses (g) (Student s t test; P =.466 (d); P =.56 (e); P =.85, P =.545, P =.757 (f, left to right); P =.99 (g)). The histogrms depit the verge ± s.e.m. The numbers in prentheses indite the numbers of nimls reorded. Figure 5 The effet of erly bumetnide tretment on visul ortil plstiity is due to regultion of tion-cl o-trnsporters rther thn the regultion of osmolrity. () Averge ± s.e.m. of the level of OD plstiity in vehile-, bumetnide-, furosemide- nd mnnitol-treted rts t. Higher levels of OD plstiity were found in bumetnide- nd furosemide-treted nimls (post ANOVA Holm-Sidk test: vehile ompred to bumetnide, P <.; vehile ompred to furosemide, P =.4) but not in mnnitol-injeted rts (vehile ompred to mnnitol, P =.958; bumetnide ompred to mnnitol, P =.9; furosemide ompred to mnnitol, P =.8). The numbers in Degree of plstiity (,) (8,) In vivo (,4) Furosemide prentheses re the numbers of nimls reorded (nonmd nd MD). (b) Averge ± s.e.m. nd single-slie vlues (irles) of the level of LTP min fter in slies from nimls treted s in. Plstiity ws greter in bumetnide- nd furosemide-treted nimls (post ANOVA Dunn s test: vehile ompred to bumetnide, P <.; vehile ompred to furosemide, P =.) thn in vehile- or mnnitol-injeted rts (vehile ompred to mnnitol, P =.784; bumetnide ompred to mnnitol, P =.4; furosemide ompred to mnnitol, P =.9). () Averge serum osmolrity ± s.e.m. mesured min fter ute injetion of bumetnide t P8. Osmolrity ws inresed in bumetnide-treted nimls in omprison to vehile ontrols (Student s t test, P =.4). The histogrm depits the verge ± s.e.m., nd irles represent dt from single nimls. P <.5, P <., P.. (4,4) Mnnitol b 8 8 In vitro Furosemide Mnnitol Serum osmolrity (%) 5 5 P8 nture NEUROSCIENCE VOLUME 8 NUMBER JANUARY 5 9

6 npg 5 Nture Ameri, In. All rights reserved. Figure 6 Erly GABAergi interferene dereses the ortil inhibitory tone, redues BDNF expression nd impirs the mturtion of PNNs t. () Top, mipscs in nimls treted perintlly with vehile or bumetnide. Bottom, quntifition of mipsc frequeny (Student s t test, P =.45). The histogrm represents the verge ± s.e.m., nd irles indite dt from single ells. Sle brs, pa (vertil); s (horizontl). (b) Top, verge tres of 5 mipsc events from vehile- nd bumetnide-treted nimls. Bottom, quntifition of mipsc mplitudes (Student s t test, P =.46). The histogrm depits the verge ± s.e.m., nd irles indite dt from single ells. Sle brs, pa (vertil); ms (horizontl). () Top, ropped imges of immunoblotting for BDNF on protein extrts from visul orties of vehile (Veh)- or bumetnide (Bum)-treted nimls. α-tubulin ws used s the internl stndrd. Full-length blots re presented in Supplementry Figure. Bottom, verge ± s.e.m. nd dt from single nimls (irles) (Student s t test, P =.9). (d) Coronl setions of the visul ortex from vehile- nd bumetnide-treted nimls double lbeled for PNNs (WFA, red) nd PV (green). Arrows indite PV-positive ells not surrounded by PNNs. Sle br, µm. (e) Quntifition of the density of PNN-positive ells (left; Mnn-Whitney rnk sum test, P =.9) nd PV-positive ells (middle; Student s t test, P =.5) nd the perentge of PV nd PNN double-lbeled ells (right; Mnn-Whitney rnk sum test, P <.). The dt in the left nd right plots re summrized by box hrt, nd the histogrm in the middle represents the verge ± s.e.m. The horizontl lines in the box denote the 5th, 5th nd 75th perentile vlues. The error brs denote the 5th nd 95th perentile vlues. The squre symbol in the box denotes the men of the olumn of dt. The numbers in the prentheses in the left nd right box hrts re the numbers of nimls proessed. found no signifint hnges in the bumetnide-treted nimls (P =.766; Supplementry Fig. 9d). Erly DHF o-tretment resues bumetnide s effets t Wht re the possible mehnisms tht link the effet of erly bumetnide tretment with the prolongtion of CP plstiity? Depolrizing GABA nd BDNF re involved in positive-feedbk reiprol regultion during erly development 6,7. In prtiulr, tivtion of GABA A reeptors triggers ugmented relese of BDNF nd enhned phosphoryltion of its reeptor TrkB (ptrkb), whih in turn redues the internliztion of GABA A reeptors from the plsm membrne 6. Therefore, we hypothesized tht perintl bumetnide tretment my disrupt the positive loop desribed bove, thus reduing BDNF signling in response to tivtion of the GABA A reeptor. To test this hypothesis, we olleted visul ortex smples from pups treted with bumetnide or vehile perintlly (P P8) nd systemilly hllenged t P8 by ute injetion of GABA A reeptor gonist (midzolm; i.p., 5 mg/kg body weight; Fig. 7). Using quntittive RT-PCR, we found signifint upregultion of Bdnf mrna expression in vehile- but not bumetnide-treted nimls injeted with midzolm (Fig. 7b). Next we ssessed TrkB tivtion by western blot nlysis with n ntibody to ptrkb (Y86) in bumetnide- nd vehile-treted pups fter ute midzolm injetion. In keeping with the RT-PCR dt, we found tht GABA A reeptor tivtion enhned TrkB phosphoryltion in vehile- but not bumetnide-treted pups (Fig. 7). Together these results suggest tht erly interferene with depolrizing GABA by bumetnide tretment impirs both the synthesis nd relese of BDNF. To investigte whether the perintl derese in BDNF signling medited the effets of erly bumetnide tretment on visul ortil plstiity lter in life, we performed resue experiment by inresing BDNF signling onomitnt to bumetnide tretment. In prtiulr, we injeted bumetnide together with the BDNF mimeti drug 7,8-dihydroxyflvone (DHF; i.p., 5 mg/kg body weight 8 from P to P8; Frequeny of mipscs (Hz) d WFA PV Merge e WFA-positive ells/mm 4 5 (6) (5) b Amplitude of mipscs (pa) 6 8 PV-positive ells/mm 5 75 Veh Bum Fig. 7d), whih indues TrkB phosphoryltion in the ortex in vivo 8. Infusion of DHF nd bumetnide together during erly development normlized the level of plstiity t both in vivo (Fig. 7e,f) nd in vitro (Fig. 7g,h). Notbly, DHF tretment per se did not interfere with plstiity, s WM-LTP indution t P6 in rts treted perintlly with DHF or vehile showed omprble levels of plstiity (Supplementry Fig. ). Notbly, tretment of DHF together with bumetnide lso resued inhibitory GABAergi trnsmission (Fig. 8), BDNF expression (Fig. 8b) nd PNNs (Fig. 8). Thus, disruption of positive feedbk regultion between depolrizing GABA nd BDNF during erly development ounts for the effets of erly bumetnide tretment on the plstiity of the visul system lter in life. DISCUSSION Depolrizing GABA lso ontrols CP plstiity Hyperpolrizing nd inhibitory GABA hs mjor role in regulting CP plstiity in the visul ortex 5 7,4. In prtiulr, opening of the CP requires rehing threshold level in the development of GABAergi inhibition 6, wheres omplete development of GABAergi inhibition leds to the losure of the time window of mximl plstiity 5,7. Unlike in dults, GABA depolrizes neuronl ells during erly development, representing the min exittory drive for immture ortil networks 8. Consequently, depolrizing GABA hs been implited in series of developmentl proesses, inluding neuronl migrtion nd morphologil differentition, s well s synpse mturtion in the ortex 8,9. To our knowledge, we report for the first time key role for erly depolrizing GABA in the long-lsting ontrol of the plstiity of neuronl iruits. To interfere with erly GABA tion, we used systemi dministrtion of the NKCC inhibitor bumetnide, with timing nd doses tht re effetive in bolishing GABA-indued depolriztion. We evluted the plstiity of ortil iruits using two independent mesures, BDNF expression (%) KD PV-WFA ololiztion (%) 7 (6) BDNF α-tubulin (5) 9 VOLUME 8 NUMBER JANUARY 5 nture neuroscience

7 npg 5 Nture Ameri, In. All rights reserved. d P P Midzolm injetion or vehile injetions P Bdnf mrna expression P8 Bum, or vehile injetions P P8 Depolrizing GABA TrkB reeptor tivtion b.4 Bdnf/Gpdh mrna expression. Eletrophysiology Bsl Figure 7 DHF tretment during erly GABAergi interferene resues the bumetnide-indued effet on plstiity. () Crtoon of the experimentl protool. (b) Bdnf mrna expression in P8 vehile- nd bumetnide-treted pups in bsl onditions or fter indution by i.p. injetion of the GABA A reeptor gonist midzolm (Mid). Gpdh ws used s the internl stndrd. Two-wy ANOVA showed tht bumetnide tretment bloked (post Tukey test, P =.4) the Midzolm-indued inrese (post Tukey test, P =.) of Bdnf mrna expression. The histogrm depits the verge ± s.e.m., nd irles indite single-niml dt (nlyzed in triplite). () Rtio of ptrkb to TrkB protein expression in P8 vehile- nd bumetnide-treted pups in bsl onditions or fter indution by midzolm. Two-wy ANOVA showed tht bumetnide tretment bloked (post Tukey test, P =.9) the midzolm-indued inrese (post Tukey test, P =.6) of ptrkb/trkb protein expression. The histogrm depits the verge ± s.e.m., nd irles indite Mid Bsl Mid e g Filed potentil (% of bseline) C:I rtio ptrkb/trkb expression (%) 5 5 NonMD MD NonMD MD NonMD MD Bsl In vivo In vitro 4 f Degree of plstiity h 6 8 Mid Bsl Mid (8,) (4,6) single-niml dt. (d) Crtoon of the experimentl protool. Bum, bumetnide. (e) C:I VEP rtios in nimls treted perintlly with vehile, bumetnide or bumetnide + DHF in either nonmd or MD onditions. Two-wy ANOVA reveled tht plstiity in nimls treted with bumetnide + DHF ws negligible (P =.55). P <.. The histogrm depits the verge ± s.e.m., nd irles indite dt from single nimls. (f) Averge degree of OD plstiity ± s.e.m. for the dt in e (Student s t test, P =.). The dshed ornge line represents the verge level of plstiity in vehile-treted rts. The numbers in prentheses re the numbers of nimls reorded (nonmd, MD). (g) Averge time ourse of WM-LTP in nimls treted with vehile, bumetnide or bumetnide + DHF. Error brs represent the s.e.m. The insets show the verge of ten tres reorded from nimls treted with vehile, bumetnide or bumetnide + DHF before (ontinuous line) nd min fter (dshed line). Sle brs,. mv (vertil); ms (horizontl). (h) Dt from the single slies (irles) reorded in g nd the verge ± s.e.m. of the plstiity level (normlized to ontrols, dshed ornge line; Student s t test, P =.5). i.e., suseptibility to brief MD in vivo nd indution of LTP in brin slies in vitro. Both pprohes lerly indited protrted time window for plstiity in nimls treted perintlly with bumetnide. Tretment with nother NKCC inhibitor, furosemide, yielded similr effet on CP plstiity t. Nevertheless, s hnges in the volume of the extrellulr fluid n potently ffet brin exitbility, we tested whether the effets of NKCC inhibition were seondry to ltertions of ell volume desribed fter diureti tretment rther thn regultion of Cl homeostsis. We found tht erly tretment with the osmoti diureti mnnitol whih does not regulte NKCC tivity filed to replite the effet on plstiity. Altogether, our experimentl evidene dds to the vst literture on the role of GABA in the ontrol of visul ortil plstiity, inditing tht not only hyperpolrizing but lso depolrizing GABA hs n importnt role. Erly depolrizing GABA ontrols plstiity speifilly The deline of suseptibility to MD nd WM-LTP indution temporlly orreltes with the development of visul funtions, ssessed in terms of the mturtion of visul uity,5. In number of studies, mnipultions ffeting the time ourse of the CP lso ffeted the overll development of the visul system. For exmple, rering nimls in drkness onstrins the visul system in n immture stte with deresed visul uity, whih is oupled to prolongtion of the window of suseptibility to MD nd WM-LTP 5,9. Conversely, mnipultions leding to preoious losure of the CP for plstiity, suh s environmentl enrihment, dministrtion of IGF (ref. ) or BDNF overexpression 5,9, lso use elerted ortil development (i.e., erly mturtion of visul uity). Thus, the physiologil development nd plstiity of the visul ortex hve been lssilly onsidered s two strongly intermingled proesses, with mturtion of visul funtion being ineludibly linked to wning of the potentil for experiene-dependent modifitions. Our results indite tht t lest under ertin onditions, the two proesses my be regulted independently, s hs been seen for other individul response properties in developing ortil networks,. Indeed, we found tht erly GABA interferene prolonged the time nture NEUROSCIENCE VOLUME 8 NUMBER JANUARY 5 9

8 npg 5 Nture Ameri, In. All rights reserved. Figure 8 DHF tretment during erly GABAergi interferene resues the bumetnide-indued effet on regultors of plstiity. () Top, representtive mipscs of neurons from nimls treted with vehile, bumetnide or bumetnide + DHF. Bottom, quntifition of mipsc frequeny (normlized to vehiletreted ontrol verge; Student s t test, P =.). The histogrm depits the men ± s.e.m., nd the irles indite dt from single ells. Sle brs, pa (vertil); 5 s (horizontl). The ornge dshed line indites the verge mipsc frequeny in vehile-treted rts. (b) Top, ropped imges of representtive immunoblotting for BDNF on protein extrts from visul orties of nimls treted with vehile, bumetnide or bumetnide + DHF (drk red). α-tubulin ws used s the internl stndrd. Full-length blots re presented in Supplementry Figure b. Bottom, quntifition of BDNF levels in nimls treted with bumetnide- or bumetnide + DHF normlized to vehile-treted ontrol verge (Mnn-Whitney rnk-sum test, P =.4). The histogrm represents the verge ± s.e.m., nd irles indite dt from single nimls. The ornge dshed line indites the BDNF level in vehile-treted rts. () Top, representtive imges of PNNs from oronl setions of the visul ortex from nimls treted with vehile, bumetnide or bumetnide + DHF. Sle br, µm. Bottom, quntifition of the density of PNN-positive ells (normlized to the medin of vehile-treted ontrols; Student s t test, P =.9). Dt re summrized by box hrt. The horizontl lines in the box denote the 5th, 5th nd 75th perentile vlues. The error brs denote the 5th nd 95th perentile vlues. The squre symbol in the box denotes the men of the olumn of dt. The ornge dshed line represents the medin density of WFA-positive ells in vehile-treted rts. The numbers in the prentheses re the numbers of nimls proessed. window for plstiity while leving the overll ntomil nd physiologil mturtion of the visul system unffeted. We doumented ler segregtion of retinogeniulte fibers in the dlgn, no ltertion in migrtion or morphologil mturtion of lyer II/III pyrmidl neurons nd norml lminr distribution of interneurons in the visul ortex. Visul uity, binoulrity, ontrst threshold, VEP mplitude nd lteny were within the norml rnge t, the sme ge t whih onsistent nd signifint plstiity ould be indued in bumetnide-treted nimls only. The ombined regultion of CP plstiity nd overll visul system development found in previous studies (i.e., drk rering from birth 5, BDNF overexpression in trnsgeni mie 5,9, environmentl enrihment from birth or IGF- dministrtion fter wening ) my depend on the ft tht these long-lsting experimentl protools set in motion number of different pthwys ting in prllel to regulte physiologil mturtion nd simultneously delimit experiene-dependent plstiity. Conversely, we found tht erly nd seletive NKCC inhibition left speifi mrk on developing ortil iruits, ffeting only the durtion of the plstiity window. At the funtionl level, the dissoition between CP plstiity nd overll visul system development my be onvenient, s ortil networks my remin ustomizble by experiene even t the ompletion of the period of physiologil mturtion. This ide is onsistent with the robust plstiity desribed in fully mture mie fter prolonged MD 4. The bsene of gross neurontomil defets in bumetnidetreted nimls is seemingly in ontrst with the reported ltertion in ortil neuron morphology fter tretment with bumetnide strting from the embryoni stge to the first postntl week. This disrepny my be due to longer time period of the previous tretment (E5 P7) in omprison to our mnipultion (P P8). Moreover, tretment from E5 to P7 inludes the time of delivery. As delivery is ssoited with mrked shift of GABA tion from depolrizing to hyperpolrizing,4, prt of the disrepny with our results ould depend on the ft tht mnipultion from E5 to P7 inludes time period in whih GABA is not depolrizing. Frequeny of mipscs (% of vehile) 7 5 b BDNF expression (% of vehile) KD Veh Bum BDNF α-tub Erly depolrizing GABA nd BDNF exert long-term ontrol of CP regultors Critil-period plstiity in the visul ortex is ontrolled by fundmentl regultors suh s inhibitory GABAergi trnsmission, BDNF nd the extrellulr mtrix 4. In prtiulr, GABAergi inhibition nd PNNs re onsidered to be lssil plstiity brkes 4 beuse their downregultion in dulthood effetively restores the sensitivity of ortil networks to MD nd WM-LTP 7,5,5. In keeping with this ide, the high plstiity of bumetnide-treted rts t ws ompnied by derese in inhibitory neurotrnsmission, the number of PNNs nd BDNF levels. Notbly, we found tht the GABA inhibitory tone ws involved in the higher OD plstiity of bumetnide-treted rts t, s enhning GABAergi trnsmission with dizepm signifintly dmpened the response to MD. We lso found no differene in the expression of these brkes t ges when plstiity ws the sme between bumetnide- nd vehile-treted nimls (P6, pek of the CP; nd P75, dulthood). The mplitude nd frequeny of GABAergi events tht we reorded t ll ges were lower thn those reported in other studies using ess resistnes lower thn ours 6 9, lthough the frequeny tht we report here is in greement with reent study using n ess resistne similr to ours. Besides their oordinted effet on visul ortex plstiity, GABAergi trnsmission, BDNF nd the extrellulr mtrix lso intert with one nother in the ontrol of visul ortex development 5,5,4. For exmple, tivtion of GABA A reeptors by depolrizing GABA during erly development triggers the synthesis nd relese of BDNF 6,7. The onsequent tivtion of TrkB, in turn, promotes ell-surfe expression of GABA A reeptors, leding to positive feedbk loop 6. We found tht disruption of this positive loop ws key event in the plstiizing effets of bumetnide tretment. Indeed, BDNF signling downstrem of GABA A reeptor tivtion ws signifintly impired in bumetnide-treted pups, nd the long-term onsequenes of NKCC inhibition (i.e., inresed plstiity t ) were ounterted by TrkB tivtion through DHF tretment. Notbly, restortion of norml losure of the CP by erly tretment with DHF nd bumetnide together ws lso ompnied by reovery of the norml levels of GABAergi inhibition, PNNs nd BDNF, WFA-positive ells (% of vehile) Veh Bum 8 (5) (5) 94 VOLUME 8 NUMBER JANUARY 5 nture neuroscience

9 npg 5 Nture Ameri, In. All rights reserved. onfirming the tight orreltion between funtionl plstiity nd these plstiity brkes. However, DHF tretment per se did not lter ortil plstiity. Altogether, these findings demonstrte tht the loop between depolrizing GABA nd BDNF during erly development regultes the widely desribed intertion between hyperpolrizing GABA, BDNF nd PNNs, whih re mjor regultors of CP plstiity lter in life. Mehnisms of CP regultion by erly depolrizing GABA Our dt demonstrte tht BDNF signling is ruil meditor of the effets of erly depolrizing GABA on ortil inhibition nd plstiity lter in development. But wht re the mehnisms tht my ount for the long-term, plstiizing effets of trnsient interferene with depolrizing GABA? At lest three different mehnisms (working lone or in ombintion) n be hypothesized. First, depolrizing GABA signling might diretly 4 or indiretly (through BDNF 6,7 ) promote the subsequent mturtion of inhibitory iruitry neessry for the losure of the CP 5,7. Nevertheless, this hypothesis seems unlikely. As GABAergi inhibition is lso neessry for the opening of the CP, if the bove hypothesis were true, one would expet temporl shift of the whole CP rther thn its mere prolongtion, s we found in the urrent study. Seond, spontneous tivity (before the onset of visul experiene) nd visully evoked dishrges represent ontinuum tht ts to determine nd instrut visul iruit development nd plstiity 8,4. By definition, the CP is phse of heightened sensitivity to environmentl stimuli during whih ortil networks re ustomized by visul experiene 4. When network tivity is redued, suh s in drk rering, the visul system rets with n extension of the CP to prolong the effetive time window during whih visul experiene n instrut iruit refinement 5. In this ontext, preoious redution of GABA-medited exittion during erly development my trigger extended plstiity of the visul ortex to ompenste for the erly loss of wiring instrutions oded by the spontneous tivity ptterns. The speifi effet of erly bumetnide tretment on the losure (nd not the opening) of the CP, together with the retention of juvenile-like plstiity t, would fvor this hypothesis. A third possible explntion for the long-term effets of erly GABA interferene resides in epigeneti mehnisms 4,44. Clssi experiments hve shown tht erly environmentl experienes (suh s levels of erly mternl re) influene dult behvior by modifitions in hromtin struture 45. Thus, we speulte tht trnsient interferene with GABA-medited exittion my leve persistent mrks on speifi lsses of ortil neurons, reverberting on ltered funtion nd struture (i.e., redued inhibitory trnsmission nd redued PNN) lter in life. These epigeneti hnges ould disply their effets t speifi developmentl stges (i.e., ) with no pprent impt on the initil, defult developmentl trjetory of plstiity nd its regultors. Therpeuti perspetive is drug pproved by the US Food nd Drug Administrtion tht hs been used extensively in the pst s loop diureti nd is used urrently to tret hert filure nd respirtory disorders in infny. Notbly, beuse of its bility to inhibit the brinspeifi trnsporter NKCC, bumetnide hs been proposed s promising ndidte to tret brin diseses 46 on the bsis of studies in niml models,4,46 nd reent linil trils However, the use of this drug in neontes hs been prtilly questioned,49,5 beuse of the key role of depolrizing GABA during erly development 8. Our dt similrly suggest possible long-term onsequenes of NKCC inhibition during erly development. Our study lso sheds light on the existene of therpeuti windows for phrmologil interventions imed t prolonging CP plstiity without ffeting overll neuronl iruit development 46. Methods Methods nd ny ssoited referenes re vilble in the online version of the pper. Note: Any Supplementry Informtion nd Soure Dt files re vilble in the online version of the pper. Aknowledgments We thnk K. Kil (University of Helsinki) for providing brin tissue from NKCC knokout brins for ontrol experiments. We thnk M. Cristin Cenni (CNR, Pis) for providing the protool for retinogeniulte xon lbeling. We thnk M.V. Cho (New York University Shool of Mediine) for providing nti-ptrkb. We thnk F. Benfenti (Istituto Itlino di Tenologi) for reding the mnusript. The work ws supported by Compgni di Sn Polo (grnt 8.67 to L.C. nd M.C.), Telethon (grnts GGP5 to L.C., GGP6 to M.C. nd GGP87 to L.C.), the Itlin Ministry of Reserh (PRIN - grnt N8PBAA_ to Y.B.) nd the University of Trento (Centre for Integrtive Biology (CIBIO) strt-up grnt to Y.B.). AUTHOR CONTRIBUTIONS G.D. performed in vitro eletrophysiologil reordings, western blot experiments, in utero eletroportion nd immunohistohemistry nd wrote the mnusript. M.A. performed in vivo eletrophysiologil reordings, western blot experiments, immunohistohemistry, behviorl testing nd retinogeniulte xon lbeling nd wrote the mnusript. C.C. ontributed to in vivo eletrophysiologil reordings. G.B. nd S.N. performed pth-lmp experiments. G.Z. nd Y.B. performed RT- PCR nd immunohistohemistry. M.C. nd L.C. designed the experiments nd wrote the mnusript. M.C., Y.B. nd L.C. provided finnil support. All uthors red nd revised the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre ompeting finnil interests: detils re vilble in the online version of the pper. Reprints nd permissions informtion is vilble online t reprints/index.html.. Berrdi, N., Pizzorusso, T. & Mffei, L. Critil periods during sensory development. Curr. Opin. Neurobiol., 8 45 ().. Espinos, J.S. & Stryker, M.P. Development nd plstiity of the primry visul ortex. Neuron 75, 49 ().. Cnedd, L. et l. Aelertion of visul system development by environmentl enrihment. J. Neurosi. 4, (4). 4. 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