Tonic excitation or inhibition is set by GABA A conductance in hippocampal interneurons

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1 Reeive Apr Aepte 8 Jun Pulishe Jul DOI:.8/nomms77 Toni exittion or inhiition is set y GABA A onutne in hippompl interneurons Inseon Song, Leoni Svthenko & Alexey Semynov Inhiition is physiologil proess tht ereses the proility of neuron generting n tion potentil. The two min mehnisms tht hve een propose for inhiition re hyperpolriztion n shunting. Shunting results from inrese memrne onutne, n it reues the neuron-firing proility. Here we show tht mient GABA, the min inhiitory neurotrnsmitter in the rin, n exite ult hippompl interneurons. In these ells, the GABA A urrent reversl potentil is epolrizing, mking seline toni GABA A onutne exittory. Inresing the toni onutne enhnes shunting-meite inhiition, whih eventully overpowers the exittion. Suh iphsi hnge in interneuron firing les to orresponing hnges in the GABA A -meite synpti signlling. The esrie phenomenon suggests tht the exittory or inhiitory tions of the urrent re set not only y the reversl potentil, ut lso y the onutne. RIKEN Brin Siene Institute (BSI), Wko, Sitm -98, Jpn. Deprtment of Clinil n Experimentl Epilepsy, UCL Institute of Neurology, Lonon WCN BG, UK. Corresponene n requests for mterils shoul e resse to A.S. (emil: semynov@rin.riken.jp). nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

2 nture ommunitions DOI:.8/nomms77 GABA is the min inhiitory neurotrnsmitter in the ult rin. Synptilly relese GABA tivtes postsynpti GABA A reeptors, whih trigger fst inhiitory postsynpti urrents (IPSCs). Amient GABA proues persistent tivtion of extrsynpti GABA A reeptors, whih re responsile for the toni urrent. The toni urrent is meite y heterogeneous n plsti GABA A reeptors, n is expresse in ell-type speifi mnner in vrious rin strutures, suh s the hippompus 6, thlmus 7, neoortex 8 n stritum 9. Cell type-speifi expression of the toni urrent inites the importne of this phenomenon for neuronl network opertion, espeilly when the extrellulr GABA onentrtion hnges. Inee, hnges in mient GABA levels re reporte in oth physiologil (for exmple, uring explortory ehviour) n pthologil (for exmple, epilepsy, pin) onitions. The effet of GABA on ell exitility is lrgely etermine y the reversl potentil for Cl, whih is the mjor ion responsile for the GABA A urrent. In ult neurons, the intrellulr Cl onentrtion is kept low y evelopmentlly regulte otrnsporters, n sets reversl potentil for GABA A urrents (E GABA ) lose to the resting memrne potentil (RMP). The RMP n E GABA vry mong ell types n even ell omprtments,6 ; therefore, the effet of GABA n e either hyperpolrizing or epolrizing. Wheres hyperpolriztion is the primry synpti tion of GABA on ortil prinipl neurons 7, epolrizing effets of GABA re oserve in interneurons in numerous iruits, inluing the hippompus 8, ereellum, n solterl mygl ; s well s in stritl neurons 9, n peripherl nervous system neurons. Beuse this epolriztion is set y smll riving fore for Cl, the preominnt effet of GABA A tivtion is thought to e shunting inhiition. Shunting inhiition is onutne-epenent phenomenon n my not provie strong inhiitory effets on the ell t low GABA A onutnes. In ontrst, even smll epolriztion of the neuronl memrne n trigger tivtion of some voltge epenent hnnels. In turn, tivtion of these hnnels n potentilly ring the RMP to the tion potentil threshol. This rises the possiility tht low epolrizing GABA A onutne n e exittory wheres high onutne is inhiitory though shunting 6. Previous reports suggeste tht voltge-epenent osilltions of the memrne potentil in hippompl interneurons oul reh the tion potentil threshol n trigger ell firing 7 9. Inee, hippompl CA strtum (str.) ritum interneurons oul fire spontneous tion potentils even when exittory neurotrnsmission ws loke. Here we foun tht the firing frequeny of these ells is iiretionlly regulte y mient GABA onentrtions. Low GABA onentrtions inrese oth memrne potentil flututions n ell firing; high onentrtions erese these two prmeters elow the ontrol vlues. Results Biphsi effet of toni onutne on interneuron firing. We investigte the effet of toni GABA A onutne on CA str. ritum interneurons in mouse hippompl slies n loke ionotropi glutmte n GABA B reeptors. Consistent with previous reports, grmiiin-perforte pth reorings showe tht these ells hve epolrizing E GABA (RMP, 7. ±.7 mv n = ; E GABA, 6. ±.7, n =, P =. pire t-test; Fig.,) 8. As expete, exogenous GABA proue onentrtion-epenent inrese in the toni GABA A onutne ( g GABA,. ±.8 ns,.7 ±.9 ns, n. ±. ns for,, n µm GABA, respetively, n = 8; Fig.,),6. In ell-tthe reorings, the interneurons oul fire spontneous tion potentils espite the loke of ionotropi glutmte reeptors (Supplementry Fig. S). In ontrst to the ommonly hel notion tht GABA meites inhiitory toni onutne,,, however, µm GABA inrese e g Norm. firing frequeny I toni (pa)... 6 µm GABA +PTX. na. s s RMP V m (mv) 8 6 GABA ms µm pa µm Threshol g GABA (ns) Potentil (mv) g GABA (ns) f Firing freq. (Hz) µm RMP (mv) RMP 7 Bseline E GABA GABA µm Figure The effet of mient GABA on interneuron memrne properties n firing. () Top, Grmiiin-perforte pth reorings t voltge steps from 9 to mv in µm GABA n, therefter µm pirotoxin ( + PTX) ws e. Toni GABA A urrents t ifferent voltges were lulte y sutrting the + PTX -urrents from orresponing µm GABA -urrents. Bottom: Toni urrent (I toni ) versus memrne potentil (V m ). Points were fitte with seon-orer polynomil funtion to etermine E GABA. Arrow mrks RMP. () Summry t of RMP n E GABA showing t from iniviul ells (n =, empty irles) n men vlues (lk irles). () Five verge urrent tres inue y mv voltge step in ontrol (trl) n in µm GABA, emonstrting hnge in memrne onutne. () Summry plot of the GABA-inue hnge in memrne onutne ( g GABA ) (n = 7 for µm GABA, n = for µm GABA, n = for µm GABA). (e) Cell-tthe reorings from interneurons in ontrol n in n µm GABA. (f) Summry plot of the hnge in firing frequeny inue y GABA. (g) Summry plot of normlize firing frequeny versus toni GABA A onutne (g GABA ) with seon orer polynomil fit (she line). Dotte lines inite the threshol onutne. (h) Effet of µm GABA on RMP in iniviul ells (n =, empty irles) n men vlues (lk irles). Error rs, s.e.m., P <., P <., P >., pire t-test. h nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

3 nture ommunitions DOI:.8/nomms77 ARTICLE interneuron firing y.68 ±.8 Hz (n = 7, P =.8 pire t-test). Cell firing i not signifintly hnge with µm GABA (inrese y.6 ±.6 Hz, n =, P =. pire t-test), n signifintly erese with µm GABA y.9 ±. Hz (n =, P =. pire t-test; Fig. e,f). These finings suggest iphsi effet of toni GABA A onutne on ell exitility. Therefore, we estlishe the inhiitory threshol onutne t whih GABA swithes its tion from exittory to inhiitory n the pek exittion onutne t whih GABA proues mximl exittory effet. We plotte the ell firing rte versus the orresponing toni GABA A onutne (g GABA = seline toni onutne + g GABA ). The inhiitory threshol onutne (.7 ns) n the pek exittion onutne (.6 ns) were otine from seonorer polynomil fit (Fig. g). These two prmeters hrterize the ell response to the hnge in mient GABA onentrtion n re potentilly importnt for omputtion within iniviul interneurons n lol networks. Aoring to the ove efinition of the pek exittion onutne n inrese in the toni GABA A onutne further from this point shoul erese the ell firing rte n vie vers. Inee, low ose of the GABA A ntgonist pirotoxin ( µm) reverse the inhiitory effet of µm GABA y prtil reution of the toni GABA A onutne, presumly setting it loser to the pek exittion (Supplementry Fig. S). It shoul e note tht pirotoxin t this onentrtion hs only minor effet on IPSCs 6. This fining is onsistent with our previous report emonstrting n inrese in hippompl interneuron firing in guine pigs y prtilly reuing the toni onutne 6. Bseline toni GABA A onutne in guine pig interneurons is lmost twie tht in mie (guine pigs,. ±. ns, n = 7 versus mie,.6 ±.6 ns, n = 7) 6 n oul e ove the exittion pek onutne in these nimls, even if still elow the inhiitory threshol onutne (exittory GABA tion). The intivtion of N + hnnels is n lterntive explntion for the iphsi effet of toni GABA A epolriztion. This explntion for the inhiitory effet of epolrizing GABA ws previously suggeste for neurons in the peripherl nervous system. In ft, GABA oul not provie strong epolriztion to hippompl interneurons, even t high onutnes (E GABA ~ mv ove RMP). µm GABA proue only. ±. mv (n =, P =. pire t-test) of epolriztion in perforte-pth experiments (Fig. h). In wholeell urrent lmp reorings, we teste the effet of epolrizing lok on the interneuron firing y injeting ifferent mounts of onstnt urrent. At first, ell firing inrese with the epolriztion, ut fter the epolriztion rehe 8.7 ±. mv (~6 mv ove RMP, n = ), the firing rte egn to erese, presumly ue to intivtion of N + hnnels. This fining inites tht the smll mount of epolriztion proue y mient GABA is unlikely to proue signifint epolrizing lok in hippompl interneurons. We nnot ompletely rule out this possiility, however, euse the epolrizing effet of GABA n e lrger in the xon initil segment, s it hs een emonstrte in priniple ells in the ontext of xo-xoni synpti onnetions 6,. The smll epolriztion proue y toni GABA A onutnes rises the question tht whether it is suffiient to reh the tion potentil threshol. Consistent with previous reports, the tion potentil threshol (the vlue of memrne potentil when the first erivtive of potentil ws mv/ms) estimte in whole-ell urrent lmp reorings ws.9 ±. mv (n = 8) 7. Inee, the smll epolriztion proue y toni GABA A onutne nnot reh this vlue. The tion potentil mesurements, however, re ommonly otine in the ell som lthough the tion potentil threshol n e lower in the xon trigger zone. Moreover, neuron firing n e triggere not y onstnt epolriztion, ut through the enhnement of voltge-epenent memrne potentil flututions hrteristi of interneurons 9,. Inee, GABA proue iphsi effet on the stnr evition (s..) of the RMP reore in perforte-pth experiments: µm GABA inrese s.. y ± 8% (n =, P =. pire t-test), µm proue n insignifint erese of ± % (n = 7, P =.9, pire t-test), n µm erese the s.. y ± 9% (n = 6, P =. pire t-test). The oserve erese in the RMP flututions proue y µm GABA ws onsistent with the previously reporte oservtions in ell tthe reorings. Thus, our results point tht low toni GABA A onutne epolrizes the ell n enhnes voltge-epenent flututions of the RMP, whih n trigger tion potentils. When the onutne inreses, however, it shunts the flututions n prevents ell firing. If this is the se, suh mehnism shoul work not only in hippompl interneurons, ut lso in ny neuron with epolrizing GABA. We resse this issue y eveloping simple omputer simultion on ylinril moel ell lking ny interneuron-speifi fetures. The exitility of this ell ws se on the Hogkin Huxley moel with the kinetis of N + n K + hnnels ssuming stohsti opening n losing (Methos). The ell lso ontine stohsti A-type K + hnnels. Uner seline onitions, the ell reeive no synpti input n generte no tion potentils, ut i show smll flututions in the RMP (Fig. ). A toni GABA A onutne (g GABA =. ms m - ) with epolrizing E GABA (. mv ove RMP) ws then introue. This proue iphsi hnge in the ell firing frequeny tht ws epenent on the g GABA mplitue, similr to the effet oserve in hippompl interneurons (Fig.,). The ensity of N + hnnels ws then reue from µm (in initil moel) to µm to prevent the moel ell from spiking, whih llowe us to oserve the effet of g GABA on the RMP flututions (Fig. ). The RMP s.. initilly inrese with ell epolriztion, n then erese regrless of the memrne potentil (Fig. ). Interestingly, the iphsi hnges in the ell firing frequeny n in the RMP flututions were not ue to the intivtion of N + hnnels. The epolrizing urrent injetions i not proue ny iphsi effet on the ove prmeters t the sme RMP rnge tht ws proue y g GABA (Fig. e h). This moel emonstrtes tht the esrie phenomenon oes not require ny speifi ell ntomy, suellulr grients of E GABA, or intivtion of N + hnnels; n n e hieve with limite numer of voltge-epenent onutnes. Synpti GABA A urrents re inhiitory. Our finings suggest tht wheres low toni GABA A onutne n e exittory, synpti signlling meite y high GABA A onutne shoul provie shunting inhiition in hippompl interneurons. To test this, we proue stimultions with n extrellulr eletroe ple proximlly to the reore ell in the str. ritum. The reorings were performe in the presene of ionotropi glutmte n GABA B reeptor ntgonists. First, in whole-ell moe, we reore the evoke GABA A -meite postsynpti urrent (PSC) inue in CA interneurons y -µa, -µs stimulus (Supplementry Fig. S). We then estimte the postsynpti onutne (PSG) require to generte suh PSC oring using the following formul: PSG = PSC/(E GABA V hol ), where V hol is the holing potentil ( 7 mv). The PSG pek onutne.6 ±.8 ns (n = 7, Fig. ) ws signifintly lrger thn the onutne require to reh the inhiitory threshol onutne from the seline toni GABA A onutne (. ns, lulte s the ifferene etween the inhiitory threshol onutne n seline toni GABA A onutne, Fig. g). Moreover, the synpti onutne lulte from PSCs, reore in the som, ws proly unerestimte euse some synpti urrents originte from the istl enrites. Seon, we reore CA interneuron firing in the ell-tthe moe n proue extrellulr stimultion s esrie ove. The time etween the stimulus n preeing spike (t ps ) ws ssume to e nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

4 nture ommunitions DOI:.8/nomms77 N v hnnels ( µm ).... g GABA (ms m ) mv ms N v hnnels ( µm ) ms g GABA (ms m )... mv RMP (mv) Firing frequeny (Hz) g GABA (ms m ) g GABA (ms m ) e SD (mv ) g GABA I inj (mv) (mv) mv ms g g (mv) GABA (mv) I inj mv ms f Firing frequeny (Hz) h SD (mv ) RMP (mv) 6 6 RMP (mv) Figure The effet of epolrizing toni GABA A onutne on moel neuron. () Smple tres of the ell firing inue y g GABA. () Summry plot of the ell firing frequeny versus g GABA (n = ) () Smple tres of the effet of g GABA on RMP flututions set y stohsti hnnel openings. The N + hnnel ensity ws reue y hlf ompre with pnel to voi ell firing. () Summry plot of RMP (lk squres, n = ) n RMP stnr evition (s.., white squres, n = ) versus g GABA. (e) Representtive tres of memrne potentil t ifferent RMPs proue y g GABA (left pnel) n y I inj (right pnel). (f) Summry t of the hnge in the moel ell firing t ifferent RMPs set y g GABA (empty irles, n = ) n y I inj (lk squres, n = ). (g) Representtive tres of memrne potentil t ifferent RMPs proue y g GABA (left pnel) n y I inj (right pnel). g N ws reue y hlf to prevent the ell from firing. (h) Men stnr evition of RMP (s..) t ifferent RMPs set y g GABA (empty irles, n = ) n y I inj (lk squres, n = ). Error rs, s.e.m. rnom; the time etween the stimulus n the sueeing spike (t ss ) ws influene y the PSG. The men t ps n t ss were ompre for eh ell (Fig. ). Consistent with the inhiitory synpti effet, the t ss ws lmost twie s lrge s the t ps (t ss /t ps,.96 ±., n = 6, P =. pire t-test). The ifferene etween t ps n t ss ws ompletely loke y µm pirotoxin (t ss t ps, 6 ±.7 ms in ontrol n 7.8 ± ms in pirotoxin, n =, P =. pire t-test; Fig. ). These t support the notion tht GABA A -meite PSGs in interneurons re inhiitory (IPSGs). Therefore, we use the term spontneous inhiitory PSCs (sipscs) in the following setions for onsisteny. An lterntive explntion for the inhiitory effet of synpti GABA is tht the site of tion is ifferent from tht of toni onutne. In seprte set of experiments, we teste the effet of GABA puff ( µm ms). Similr to synpti stimultion, the GABA puff inrese the t ss y ftor of two (t ss /t ps,.99 ±., n =, P =. pire t-test; Fig. ). Beuse puff pplition proues lol trnsient inrese in GABA, whih tivtes oth synpti n extrsynpti GABA A reeptors, this fining suggests tht the onutne mgnitue is more importnt thn the site of tion. Biphsi hnges in GABAergi synpti signlling. Toni GABA A onutne is ommonly referre s toni inhiition,, n therefore its funtion in neuronl networks is thought to e uniiretionl. Here we emonstrte tht toni GABA A onutne hs iphsi effet on the hippompl interneuron firing rte. Interneurons re iverse group of ells n eh lss of interneurons hs ifferent funtion in the lol network. The mount of toni GABA A onutne lso vries mong interneuron types 8. Thus, our smple of interneurons oes not hrterize the toni GABA A urrent in ll lsses of interneurons n nnot e use to preit the effet of mient GABA on the CA network. Therefore, we reore the sipsc frequeny iretly in CA pyrmil neurons, whih re the finl trgets of the CA interneurons n generte the mjor hippompl output. Consistent with the iphsi hnge in interneuron firing, µm GABA inrese sipsc frequeny in the pyrmil ells y. ±. Hz (n =, P =. pire t-test), µm GABA i not proue signifint hnge (inrese y. ±. Hz; n = 9, P =. pire t-test), n µm GABA reue the frequeny y. ±. Hz (n = 6, P =.6 pire t-test; Fig.,). In this set of experiments, we use reltively high onentrtions of exogenous GABA (up to µm). Although it is unlikely tht ll the GABA rehe the neurons eep in the tissue euse of its effiient uptke, it is still possile tht some of the synpti GABA A reeptors were reruite or esensitise, n the tivtion of presynpti GABA A reeptors ffete the proility of GABA relese. To rule out these possiilities, we reore tion-potentil inepenent miniture IPSCs (mipscs) uner ontrol onitions in the presene of µm tetrootoxin (N + hnnel loker) n then e, or µm GABA (Supplementry Fig. S). Neither the mplitue nor frequeny of the mipscs hnge signifintly, suggesting the lk of synpti GABA A reeptor tivtion/esensitistion n the lk of n effet on GABA relese proility, respetively. Beuse of effiient GABA uptke, the toni GABA A urrent is either sent in pyrmil ells or signifintly smller thn tht in interneurons,6. Amient GABA, however, n inrese toni GABA A urrent in these ells. Inee,, n µm GABA proue ose-epenent inrese in the toni GABA A urrent (Fig. ). This inrese ws two orers of mgnitue lrger thn the hnge in the time-verge urrent meite y sipscs (I spont ), whih ws lulte s men hrge trnsfer of sipscs multiplie y their frequeny 6 (Fig. ). Thus, the overll effet of th-pplie GABA on the exitility of CA pyrmil neurons ws ominte y toni inhiition (Disussion). We next investigte the onitions uner whih mient GABA is erese. Reue toni onutne orreltes with low firing rte. We use mouse lking one of the two mjor GABA synthesizing enzymes, 6 kd glutmi i eroxylse (GAD6 / ). nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

5 nture ommunitions DOI:.8/nomms77 PSG (ns) t ss t ps (ms) 6 ns PTX. s PTX t (ms) This mouse evelops n mient GABA efiit in the rin fter the first month of life 6,7. Consistent with the reue extrellulr GABA, the toni GABA A urrent ensity ws two times smller in CA str. ritum interneurons of GAD6 / mie thn in wiltype () mie interneurons (GAD6 /,. ±. pa m, n = 7 versus,. ±.6 pa m, n = ; P =.7; Fig.,). Surprisingly, we etete no ifferene in the mgnitue of the toni urrents etween the n GAD6 / CA pyrmil neurons (Fig.,). It is possile tht uner seline onitions, mient GABA oes not reh the reeptors on these ells 6 n the smll toni urrent is meite y gonist-inepenent spontneous openings of GABA A reeptors 8,9. GABA ( µm) inue similr rise in the holing urrent in GAD6 / n interneurons, initing tht the smller toni urrent in knokout nimls ws not ue to hnge in their sensitivity to mient GABA (Fig. 6,). Consistent with our previous report 6, nm zolpiem, enzoizepine site gonist, enhne the toni GABA A urrent n inrese the sipsc ey time in interneurons, suggesting tht these urrents were meite y γ-suunit-ontining GABA A reeptors (Fig. 6 f). Zolpiem h similr effet in GAD6 / interneurons, pointing to n unhnge phrmologil profile of GABA A reeptors in these nimls. Also onsistent with previous report, the mplitue, kinetis n frequeny of mipscs ws not signifintly ifferent in GAD6 / interneurons ompre with interneurons (Supplementry Fig. S), suggesting tht synpti signlling ws intt uner resting onitions. This fining oes not exlue, however, t (ms). s t ps t ps.s Stim t ss GABA puff Figure Evoke GABA A PSG n mm GABA puff re inhiitory. () Top: smple tre of lulte GABA A meite PSG. Bottom, Summry plot of verge pek PSG in ontrol (trl) n in µm pirotoxin (PTX) (n = 7). () Top: Five superimpose ell-tthe reorings with synpti stimultion (rrow). Bottom: Plot of verge preeing spike (t ps ) n sueeing spike (t ss ) intervls in 6 iniviul ells. () Differene etween t ss n t ps in ontrol (trl) n in µm pirotoxin (PTX) (n = ). () Top: superimpose ell-tthe reorings with µm GABA puff. Bottom: plot of verge preeing spike (t ps ) n sueeing spike (t ss ) intervls in four iniviul ells. Error rs, s.e.m., P <., pire t-test. t ss Norm.sIPSC freq. I toni ensity ( pa m ) min ARTICLE the possiility tht synpti GABA A signlling iffers etween the two genotypes uring sustine synpti tivtion inue y prolonge eletril stimultion, s previously reporte. Beuse we i not eliver prolonge stimultion to the slies, this phenomenon is unlikely to ontriute to the ifferene in the toni urrents. In ontrst to GABA A reeptor knokout nimls, we foun no hnges in the non-gaba-meite holing urrent in GAD6 / interneurons, whih suggests lk of ompenstory hnges in the resting memrne onutnes (Fig. 7). The RMP ( 7.6 ±. mv, n = ) n E GABA ( 9. ±.8 mv, n =, P =.9 ompre with RMP, pire t-test) in GAD6 / interneurons were not signifintly ifferent from the RMP n E GABA in ells. Consistent with the exittory effet of seline toni GABA A onutne, the firing frequeny of GAD6 / interneurons (. ±. Hz, n = 7) ws signifintly lower thn tht of ells (. ±.7 Hz, n = 7; P <. ompre with ) (Fig. 7,). When GABA A reeptors were loke, the exitility of the GAD6 / interneurons ws not ifferent from the ells, ruling out the possiility tht ompenstory hnges were responsile for the reue firing rte of these ells (Fig. 7). If reue firing rte of GAD6 / interneurons results from the erese toni GABA A onutne, exogenous GABA shoul resue it. Inee, th pplition of µm GABA inrese the firing rte of GAD6 / interneurons y.8 ±. Hz (n = 6, P =. pire t-test). In ontrst to interneurons, µm GABA further inrese the firing rte of GAD6 / interneurons y. ±. Hz (n = 7, P =. pire t-test; Fig. 7e,f). This effet ws ue to lower seline toni onutne in GAD6 / (. ±. ns, n = 9) thn tht in (.6 ±.6 ns, n = 7, P =. for ifferene with GAD6 / ) interneurons. Both n µm GABA resulte in n inrese in toni onutne ( g GABA,.8 ±.7 ns, n = 9 n.7 ±. ns, n = 9, respetively) within the rnge of exittory GABA tion (Fig. g). This fining further supports the notion tht the erese firing rtes of GAD6 / interneurons re ue to erese toni GABA A exittion. I spont ensity sipsc freq. (Hz) ( pa m ).... Figure Effet of mient GABA on sipsc n toni urrent in CA pyrmil neurons. () Normlize sipsc frequeny in CA pyrmil neuron in ontrol (trl) n fter pplition of, n µm GABA. () Summry plot of the hnge in sipsc frequeny inue y GABA (n = for µm GABA, n = 9 for µm GABA, n = 6 for µm GABA). () Summry plot of the hnge in toni GABA A urrent ( I toni ) ensity reore in, n µm GABA (n = ). () Summry plot of the hnge in time-verge phsi urrent ( I spont ) ensity reore in, n µm GABA (n = ). Error rs, s.e.m., P <., P >., pire t-test. nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

6 pa PTX s Biuulline pa s We then ompre the seline frequeny of spontneous tion potentil-epenent IPSCs (IPSCs = sipscs mipscs) in n GAD6 / neurons to estimte the effet of n mient GABA efiit on synpti GABA A signlling riven y interneuron firing. Consistent with interneuron firing rtes, the frequeny of IPSCs in pyrmil ells (. ±. Hz, n = ) ws signifintly higher thn the frequeny of the prtilly sent IPSCs in GAD6 / pyrmil ells (. ±. Hz, n = 6, P =. ompre to ; Fig. 8). A similr ifferene in the frequeny of the IPSCs ws oserve in the interneurons (,. ±. Hz, n = ; GAD6 /,. ±. Hz, n = 9, P =.; Fig. 8). Exogenous GABA inrese the frequeny of sipscs in GAD6 / pyrmil ells ( µm, y. ±. Hz, n = 8, P <. pire t-test, n µm, y. ±. Hz, n = 6, P <. pire t-test, Fig. 8,). Interestingly, this inrese in the frequeny of sipscs ws not proportionl to the inrese in GAD6 / interneuron firing, wheres µm GABA inrese firing frequeny severl times more thn µm (Fig. 7e,f). There re t lest two explntions for suh isrepny. First, using somti reorings, we were unle to etet sipscs from istl enrites, n thus the frequeny hnge might e unerestimte. Seon, interneurons trget oth pyrmil ells n other interneurons. The output of interonnete interneurons to pyrmil ells might not extly follow the verge firing rte of iniviul interneurons reore in the str. ritum. These onsiertions point to the omplexity of the interneuron network n the nee for future stuies to isset the effet of toni exittion on ifferent lsses of interneurons. The present t, however, re suffiient to rw the generl onlusion tht resting levels of mient GABA re set to mintin the tone of synpti GABA-meite signlling in the hippompl CA region. I toni ensity ( pa m ) Figure Toni GABA A urrent in GAD6 / interneurons n pyrmil ells. () Chnge in I hol in n GAD6 / interneurons proue y µm pirotoxin (PTX). Right pnels: Current histogrms with Gussin fits of reore urrent. Dshe lines inite the I hol otine from the peks of the fits. () Summry plot of toni GABA A urrent (I toni ) ensity in interneurons (n = 7 for GAD6 /, n = for ). () Chnge in I hol in n GAD6 / pyrmil ells proue y µm iuulline. Right pnels: sme s in. () Summry plot of toni GABA A urrent (I toni ) ensity in (n = 6) n GAD6 / (n = ) pyrmil ells. Error rs, s.e.m., P >., P <., unpire t-test. I toni ensity ( pa m ) e pa s pa nture ommunitions DOI:.8/nomms77 s ms GABA Zolpiem I hol ensity ( pa m ) I hol ensity ( pa m ) f τ Dey (%) Disussion Chnges in extrellulr GABA etermine the mgnitue of toni GABA A onutne, whih hs iphsi effet on the exitility of hippompl interneurons. A low-toni GABA A onutne epolrizes the ell n results in the onset of ell firing. This epolriztion lso enhnes voltge-epenent RMP flututions, whih re riven y stohsti N + hnnel opening 9. It is likely tht these flututions re involve in the inrese in the ell firing rte, euse they get lrger following GABA A -meite epolriztion. When the toni GABA A onutne inreses, the exittory effet of epolriztion is overpowere y inhiition. Both ell firing frequeny n RMP flututions erese. This inhiition n e expline y the shunting effet, whih inreses with the toni GABA A onutne Figure 6 n GAD6 / interneurons eqully respon to mm GABA n nm zolpiem. () Chnge in I hol in n GAD6 / interneurons proue y µm GABA. Right pnels: urrent histogrms with Gussin fits of reore urrent. Dshe lines inite the I hol otine from the peks of the fits. () Summry plot of the hnge in I hol ensity inue y µm GABA in (n = ) n GAD6 / (n = ) interneurons. () Chnge in I hol in n GAD6 / interneurons proue y nm zolpiem. Right pnels: sme s in. () Summry plot of the hnge in I hol ensity inue y zolpiem in (n = 7) n GAD6 / (n = 7) interneurons. (e) Men pek sle sipscs reore from n GAD6 / interneurons without (thin tre) or with nm zolpiem (thik tre). (f) Summry plot of the hnge in the sipsc ey time onstnt (τ ey ) inue y zolpiem in (n = 7) n GAD6 / (n = 7) interneurons. Error rs, s.e.m., P >., unpire t-test. nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

7 nture ommunitions DOI:.8/nomms77 ARTICLE I hol (pa) 8 6 Firing freq. (Hz) CsCl KCl s mv ms Firing freq. (Hz) 6 I inj (pa) e s f Firing freq. (Hz) Figure 7 Anlysis of GAD6 / interneurons. () Summry of I hol reore in (she olumns) n GAD6 / (open olumns) interneurons voltge-lmpe t 6 mv loe with CsCl (GAD6 / n = 8; n = ) n with KCl (GAD6 / n = ; n = 7) in the presene of µm pirotoxin. () Cell-tthe reorings from n GAD6 / interneurons (without pirotoxin). () Summry plot of firing frequeny in n GAD6 / interneurons (n = 7) otine uner the sme onitions s for. () Left pnel: Ation potentils reore in response to epolrizing urrent steps (9 pa; ms) in whole-ell urrent-lmp moe from n GAD6 / interneurons in the presene of µm pirotoxin. Right pnel: Summry plot of tion potentil frequeny reore uring epolrizing steps vrying from to 8 pa (input output hrteristis) in GAD6 / (n = ) n (n = ) interneurons. (e) Cell-tthe reorings from GAD6 / interneuron in ontrol (trl) n in n µm GABA (without pirotoxin). (f) Summry plot of the hnge in firing frequeny inue y GABA in GAD6 / interneurons otine uner the sme onitions s for e. Error rs, s.e.m., P <., P <., P >., unpire ( n ) n pire (f) t-test. IPSCs freq. (Hz) Norm.sIPSCs freq min Consistent with the effet of shunting, evoke synpti GABA A signlling ws inhiitory in interneurons, euse of the lrge pek onutne. This fining, however, oes not rule out the possile sipsc freq. (Hz).... Figure 8 GABA A meite synpti signlling in GAD6 / CA neurons. (, ) Summry plots of IPSCs frequeny in n GAD6 / pyrmil ells (n = for, n = 6 for GAD6 / ) n interneurons (n = for, n = 9 for GAD6 / ), respetively. () Normlize sipsc frequeny in GAD6 / pyrmil neuron in ontrol (trl) n fter n µm GABA pplition. () Summry plot of the hnge in sipsc frequeny y GABA in GAD6 / pyrmil ells (n = 8 for µm GABA, n = 6 for µm GABA). Error rs, s.e.m., P <., unpire (, ) n pire () t-test. IPSCs freq. (Hz) exittory effet of reltively smll spontneous GABA A -meite postsynpti urrents. Nevertheless, the present fining s one more istintion to the funtionl role of toni n phsi GABAmeite signlling in the rin. A iphsi effet of toni GABA A onutne on neuronl firing hs importnt implitions for omputtion within neuronl network. The lssi view hols tht neurons generte vrile output in response to the integrtion of synpti inputs. Here we show tht hippompl interneurons n generte vrile output in response to hnges in extrsynpti GABA, regrless of exittory synpti rive. Beuse these neurons re prt of the lol iruit, extrsynpti input n e onsiere s one of the inputs into the lol neuronl network tht etermines the network opertion n output. An inrese in mient GABA hs een reporte for prtiulr types of ehviour (for exmple, explortion ) or in pthologil onitions,. We use th pplition of GABA to mimi suh n inrese. Exogenous GABA, however, lso inue ose-epenent toni urrent with mgnitue of two orers lrger thn the time-verge urrent proue y the hnge in sipscs in CA pyrmil neurons. Beuse these ells re reporte to hve hyperpolrizing E GABA (ref. 7), the toni GABA A urrent my e their min soure of inhiition. This rises the question of whether iphsi hnge in sipsc frequeny is of ny importne for the exitility of pyrmil neurons. In ft, th pplition of GABA my not fithfully reproue the hnges in mient GABA onentrtions uner physiologil onitions. A reent report using n innovtive GABA imging tehnique emonstrte lyer-speifi hnges in mient GABA resulting from inrese neuronl tivity. Moreover, the funtionl roles of toni n phsi GABA A signlling re lrgely ifferent,. For exmple, toni GABA A onutne etermines the memrne time onstnt n hrteristi length onstnt, n moultes neuronl offset in CA pyrmil neurons. GABA A -meite IPSCs intert in time-epenent mnner with glutmtergi EPSCs, whih hs speifi impt on neuronl nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

8 omputtion (for exmple, integrtion time winow of neuronl inputs ), n re involve in network synhroniztion n osilltory ehviour 6. Thus, the tul effet of physiologil hnges in mient GABA onentrtion on the hippompl network funtion requires further investigtion. In summry, we suggest tht the esrie phenomenon hs role in ifferent types of exitle ells. The mount of onutne ts to swith etween exittion n inhiition when ertin onitions re met: the urrent hs low epolrizing reversl potentil n the proue epolriztion is mplifie y other voltgeepenent onutnes. Here we emonstrte these onitions in hippompl interneurons, ut they n e potentilly oserve in the other neurons with epolrizing E GABA (refs 9, ). It is lso likely tht E GABA etermines the exittion pek onutne n the inhiitory threshol onutne. Thus, the iphsi tion of toni GABA A onutne shoul e onsiere in the roer ontext of the eterminnts of E GABA. First, the expression of the min Cl trnsporters (NKCC n KCC) is evelopmentlly regulte, whih iffers in vrious ell types. For exmple, in hippompl pyrmil neurons, the rtio of KCC/NKCC inreses uring evelopment, mking GABA hyperpolrizing. In ontrst, this rtio is fixe in hippompl interneurons n GABA ontinues to hve epolrizing effets throughout evelopment 9. Seon, tivity-epenent regultion of Cl trnsporters ontriutes to E GABA in ifferent ells. A reent report emonstrte tht NMDA reeptor tivity ownregultes KCC, resulting in epolrizing GABA A - meite urrent in rt issoite neurons 7. Aitionlly, the esrie phenomenon n hve role t suellulr level. We i not systemtilly investigte the effet of low GABA onentrtions t ifferent sites within interneurons. A previous report, however, emonstrte tht epolrizing GABA n proue inhiition in the som n exittion in enrites of ortil pyrmil ells 8. This n e expline tht shunting inhiition of the xon initil segment is lrger for som thn for more remote enriti sites. Tht is, the inhiitory threshol onutne inreses with istne from the xon initil segment. This oservtion prompts further investigtion of toni GABA A onutnes t the suellulr level. Methos Eletrophysiology in hippompl slies. All experimentl proeures were performe in orne with the guielines of the Animl Experiment Committee of the RIKEN Brin Siene Institute. Hippompl slies ( 8 µm) were otine from - to 6-week-ol mle or GAD6 / littermtes 9. Heterozygous knokout mie with C7BL/6J geneti kgroun were interre to proue n GAD6 / mie in speifi pthogen-free fility. Mie were house t C with /-h light/rk yle n liitum ess to foo n wter. Genomi PCR ws use to ifferentite the mie. Hippompl slies were mintine t room temperture in sumerge hmer with solution ontining (in mm): 9 NCl,. KCl,. MgSO, CCl, 6. NHCO, NH PO, n gluose, n sturte with 9% CO /% O. After h of inution, slies were trnsferre to the reoring hmer n superfuse t C with the solution esrie ove, ut ontining. mm CCl. Exept for the experiment shown in Supplementry Figure S, AMPA/kinte, NMDA, n GABA B reeptors were loke with µm NBQX, µm APV, n µm CGP (Toris Cookson), respetively. CA pyrmil ells n str. ritum interneurons were visully ientifie with n infrre ifferentil interferene ontrst mirosope (Olympus BXWI). The t were quire with Multilmp7B (Axon Instruments), filtere t khz, n igitize t khz using NI PCI-6 t quisition r (Ntionl Instruments). The t were nlyse without further re-smpling. Smple tres were lso tken t the sme rte, exept for the long tres ( > s), whih were re-smple t Hz. Whole-ell, grmiiin-perforte pth, n ell-tthe reorings were performe in seprte ells. Whole-ell experiments. Whole-ell pipettes use in voltge-lmp reorings ontine (in mm): CsCl, 8 NCl, Cs-HEPES, EGTA,. MgCl, MgATP,. N GTP, n QXBr (ph 7., osmolrity 9 mosm, liqui juntion potentil. mv). In the experiments for Figure 7, KCl ws use inste of CsCl (liqui juntion potentil. mv). Whole-ell urrent-lmp reorings were performe with pipettes ontining (in mm):. K-gluonte, 7.7 KCl, NCl,. CCl, HEPES, EGTA, MgATP, n. N GTP. nture ommunitions DOI:.8/nomms77 Series resistne (R s ), input resistne (R i ), n memrne pitne ( m ) were monitore throughout the reorings. These prmeters were otine from the urrent in response to hyperpolrizing voltge steps (V step = mv). Rs = Vstep / Ipek Ri = Vstep / Iss Rs m = t( / Rs + / Ri ) where, I pek is the pek mplitue of the urrent trnsient immeitely fter the step is pplie, τ is the ey time onstnt of the urrent, n I ss is the stey-stte urrent. The R s ws not ompenste n t were isre if R s hnge y more thn % uring the reoring. There ws no signifint ifferene in m etween GAD6 / (7.9 ± 7.9 pf, n = 9) n (69. ± 6.9 pf, n = ) interneurons (P =.). Toni GABA A urrent (I toni ) ws lulte s the ifferene etween the seline I hol n the I hol in the presene of GABA A reeptor ntgonist. The hnge in the toni GABA A urrent ( I toni ) ws lulte s the ifferene etween the seline I hol n the I hol in the presene of exogenous GABA. I toni ensity ws otine y iviing I toni y the surfe re of the neuron. The surfe re ws otine y iviing m y the memrne pitne onstnt (.9 µf m - ). Toni GABA A onutne ws lulte s the ifferene etween the seline onutne (/R i ) n the onutne in the presene of GABA A reeptor ntgonist (g GABA ) or exogenous GABA ( g GABA ). Synpti GABA A onutne in response to eletri stimultion (PSG, Fig. ) ws lulte oring to PSG = PSC/( EGABA Vhol ) where PSC postsynpti urrent in the presene of ionotropi glutmte n GABA B reeptor ntgonists; V hol = 7 mv holing urrent; E GABA =.7 mv ws lulte y RT PCl [ Cl ] o + PHCO [ HCO ] o EGABA = ln F PCl [ Cl ] i + PHCO [ HCO ] i where P Cl = n P HCO =. reltive permeilities for Cl n HCO, respetively. mispcs were reore in the presene of µm tetrootoxin. sipscs n mipscs were ompletely loke y µm pirotoxin. Frequeny, men mplitue, n ey time onstnt of sipscs n mipscs were nlyse off-line with the MiniAnlysis (Synptosoft) n Clmpfit (MDS Anlytil Tehnologies). IPSC frequeny ws lulte s the ifferene etween the sipsc n mipsc frequenies. Time-verge urrent meite y sipscs (I spont ) ws lulte s the men hrge trnsfer of sipscs multiplie y their frequeny 6. Grmiiin-perforte pth experiments. The pipette tips were fille with the solution ontining (in mm) KCl, HEPES, ATP-Mg,. GTP-N, QX, n EGTA to prevent lekge of the ntiioti while pprohing the ell. The pipettes were k-fille with the sme solution tht lso ontine µg ml grmiiin-d (Sigm). Fresh grmiiin solution ws me every h. After the pipette tip ontte the ell memrne, the R s ws monitore. Reorings were strte when the R s stilize t MΩ (usully within min). All mesurements were orrete for liqui juntion potentil n voltge rop ross the R s (ref. ). RMP ws mesure in urrent lmp moe (I hol ws pa). Men RMP n its stnr evition were otine uner ontrol onitions n in the presene of GABA. The epolrizing effet of GABA ws lulte s ifferene etween men RMPs. In some experiments, the ells were voltge-lmpe t 6 mv. Memrne I V hrteristis were then otine with,-ms voltge steps elivere from 9 to 7 mv with mv intervls, n from to mv with -mv intervls. I V hrteristis for GABA A toni urrent were otine s the ifferene etween memrne I V hrteristis in µm GABA n in µm pirotoxin, e sequentilly. Cell-tthe experiments. Pipettes were fille with superfusion solution n loosely tthe to the ell memrne to mesure interneuron firing without perturing intrellulr ioni onentrtions. Reorings were performe in the voltge-lmp moe from the interneurons in whih tion urrents were etetle (pproximtely % of ells),7. The ommn potentil ws set to the potentil t whih the holing urrent ws pa to voi iret ell stimultion y the eletroe. The evoke GABA A -meite PSGs were triggere y -µs urrent pulses t µa elivere y tungsten monopolr eletroe ple in the str. ritum. GABA A tivtion ws lso proue with -µm GABA puff ( ms, psi). Preeing (t ps ) n sueeing (t ss ) spike intervls were lulte from the eginning of the synpti stimultion or GABA puff. Computtionl moel. The ylinril neuron (length µm; imeter µm; totl surfe 68 µm ) ws rete with the Neuron 7 simultor. The ell exitility ws esrie oring to Hogkin Huxley s moel with orresponing N + (N v ) n K + (K v ) hnnels (single-hnnel onutne: γ N = ps n () () () () () nture ommunitions :76 DOI:.8/nomms77 Mmilln Pulishers Limite. All rights reserve.

9 nture ommunitions DOI:.8/nomms77 ARTICLE γ K = ps; hnnel ensity: n.667 µm, giving totl numer of hnnels of N N =, n N K = 9). Aitionlly, A-type K + hnnels (K A ) were introue (single-hnnel onutne γ KA = ps; hnnel ensity: 8 µm giving totl numer of hnnels: N KA =,). The trnsition of the hnnels etween ifferent onformtionl sttes ws esrie s Mrkov proess, wheres the rte onstnts vrie with the voltge,. The memrne lso inlue the liner eterministi toni GABA A onutne (g GABA ), whih ws vrie from to. ms m - with. ms m - steps. On the sis of this set of onutnes, we use the following eqution: V Cm Iinj gn V EN gkv V EK t = ( ) ( ) gka( V EK) ggaba( V EGABA) where C m memrne pitne ( µf m - ), I inj injete urrent ( pa for seline onitions), E N N + equilirium potentil ( mv), n E K K + equilirium potentil ( 77 mv). The reversl potentil for GABA A urrent (E GABA = mv) ws set. mv ove the men RMP without GABA A onutne (6. mv) to mth experimentl t. g N, g K, n g KA N V, K V, n K A onutnes, respetively, were ontrolle y ontinuous vriles tht rnge etween n (m, h, n, k, l). gn = NNg N[ mh ], gk = NKg K[ n ], gk = NKg K[ lk ], where [m h ], [n ], n [l k ] re the proilities of the N V, K V, n K A hnnels to e in the open stte oring to their respetive kineti shemes,. The vriles m, h, n, k, l oeye first-orer ifferentil equtions with voltge epenent rte onstnts (α m, β m, α h, β h, α n, β n, α k, β k, α l ). m = m( m) mm t ( ). V + m = V + exp ( ) exp V 6 m = 8 h = h( h) hh t V h = exp h = + V + exp n = n( n) nn t ( ). + V n = + V exp V n = 6 +. exp 8 k = k( k) kk t k = exp V V + exp( ) ( ) + k = exp V V + exp( ) ( ) + (6) (7) (8) (9) () () () () () () (6) (7) (8) (9) () () l = l( l) t l = exp(. ( V + 6)) The effet of g GABA on flututions of RMP (RMP stnr evition) ws stuie when the ensity of N + hnnels ws reue from to µm to prevent tion potentil genertion. The effet of memrne epolriztion on the ell firing n the RMP flututions ws stuie with the urrent injetions (I inj ) from to na n g GABA = ns. All simultions were run for, ms, n the t were ollete from the intervl etween ms n, ms when the moel rehe qusi-stey stte. All the moel t re mens from inepenent simultions. To voi the orreltion etween t, eh omputtion ws performe with ifferent see otine from the rnom numer genertor. Sttistil nlysis. Dt re presente s mens ± s.e.m. Error rs on the grphs represent s.e.m. Sttistil omprisons were me using unpire Stuent s t-test unless pire t-test is stte. A P-vlue of less thn. ws onsiere sttistilly signifint. () () Referenes. Glykys, J. & Moy, I. Ativtion of GABAA reeptors: views from outsie the synpti left. Neuron 6, (7).. Frrnt, M. & Nusser, Z. 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Aknowlegements We thnk Kunihiko Ot for proviing us GAD6 / mie; Kiyu Zheng for proviing the multimoe luster for omputtions; Dimiti Kullmnn, Mtthew Wlker, Hjime Hirse, Ivn Pvlov, Krri Lms, Thoms MHugh, Kirill Volynski, Stephen F. Heinemnn, Dmitri Ruskov n ll memers of the AS lortory for reing n ommenting on the mnusript. This work ws supporte y RIKEN BSI n The Wellome Trust. Author ontriution A.S. n I.S. esigne the stuy n plnne the experiments; A.S., I.S., n L.S. nlyse the t n prepre the mnusript; I.S. performe ll the experiments; L.S. performe the moelling. Aitionl informtion Supplementry Informtion ompnies this pper t ntureommunitions Competing finnil interests: The uthors elre no ompeting finnil interests. Reprints n permission informtion is ville online t reprintsnpermissions/ How to ite this rtile: Song, I. et l. Toni exittion or inhiition is set y GABA A onutne in hippompl interneurons. Nt. Commun. :76 oi:.8/nomms77 (). 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