I h -mediated depolarization enhances the temporal precision of neuronal integration

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1 Reeived 5 Apr 2 Aepted 9 Jn 2 Pulished 5 Fe 2DOI:.38/nomms22 I h -medited depolriztion enhnes the temporl preision of neuronl integrtion Ivn Pvlov, Annlis Simemi,, Leonid Svthenko, Dimitri M. Kullmnn & Mtthew C. Wlker Feed-forwrd inhiition medited y ionotropi GABA A reeptors ontriutes to the temporl preision of neuronl signl integrtion. These reeptors exert their inhiitory effet y shunting exittory urrents nd y hyperpolrizing neurons. The reltive roles of these mehnisms in neuronl omputtions re, however, inompletely understood. In this study, we show tht y depolrizing the resting memrne potentil reltive to the reversl potentil for GABA A reeptors, the hyperpolriztion-tivted mixed tion urrent (I h ) mintins voltge grdient for fst synpti inhiition in hippompl pyrmidl ells. Phrmologil or geneti ltion of I h rodens the depolrizing phse of fferent synpti wveforms y hyperpolrizing the resting memrne potentil. This inreses the integrtion time window for tion potentil genertion. These results indite tht the hyperpolrizing omponent of GABA A reeptormedited inhiition hs n importnt role in mintining the temporl fidelity of oinidene detetion nd suggest previously unreognized mehnism y whih I h modultes informtion proessing in the hippompus. Deprtment of Clinil nd Experimentl Epilepsy, UCL Institute of Neurology, London WCN 3GB, UK. Present ddress: Synpti Physiology Setion, Ntionl Institute of Neurologil Disorders nd Stroke, Ntionl Institutes of Helth, Bethesd, Mrylnd , USA. Correspondene nd requests for mterils should e ddressed to M.C.W. (emil: mwlker@ion.ul..uk ). NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved.

2 M iroeletrode nd eletroenephlogrphy studies show tht groups of neurons n fire synhronously with milliseond preision. To mintin temporl fidelity of informtion enoding, neurons t predominntly s oinidene detetors rther thn neuronl integrtors 2. The preision of oinidene detetion in pyrmidl ells depends ritilly on feed-forwrd inhiition 3,4. Suh inhiition, medited y GABA A reeptors, ts oth y shunting exittory urrents (shunting inhiition) nd y hyperpolrizing neurons (voltge inhiition) 5. Shunting inhiition redues the mplitude nd durtion of exittory postsynpti potentils (EPSPs) y inresing the memrne ondutne. The hyperpolrizing tion of inhiitory postsynpti potentils (IPSPs), on the other hnd, offsets the depolriztion medited y EPSPs nd is long lsting, resulting in iphsi EPSP IPSP sequene in mny neurons 5. It hs een suggested tht the temporl preision of neuronl integrtion lso depends on I h, ut this effet hs een ttriuted to HCN-medited shunting of exittory inputs 6. Ih is mixed tioni urrent with reversl potentil of ~ 3 mv present in neurons throughout the rin (for reviews see refs 7, 8). As I h is present t resting memrne potentil, it depolrizes neurons 9. I h hs mrked effet on dendriti proessing y diretly shunting exittory inputs 2,3 nd through intertions with other memrne ondutnes 4,5. Consequently, loking I h filittes the temporl summtion of EPSPs nd tion potentil firing during repetitive stimultion 2,3,6. As Ih n lso ffet IPSP kinetis 7,8, we sked wht effet I h lokde hs on oinidene detetion, when inhiition is left intt. Here, we report tht loking I h results in signifint rodening of the window for neuronl integrtion. This, however, is not due to the influene of I h on PSP kinetis, ut rther is seondry to the hyperpolrizing effet of I h lokde. Indeed, I h is required to mintin the hyperpolrizing tion of synptilly relesed GABA, nd so, loking I h rodens the exittory phse of the EPSP IPSP sequene evoked y fferent stimultion. These results show n essentil role for I h in determining the reltive vlues of the resting memrne potentil ( V R ) nd the reversl potentil for GABA A reeptor-medited urrents ( E GABA(A) ), nd lso show tht hyperpolrizing inhiition is neessry for temporlly preise neuronl integrtion of synpti inputs. Results Bloking I h inreses the integrtion time window. We ss ess e d oinidene detetion y reording from CA pyrmidl ells Figure Blok of I h widens the oinidene-detetion time window. ( ) Two Shffer ollterl pthwys ( nd ) were stimulted on either side of the reorded neuron ~ 3 μ m wy from the som to evoke EPSP IPSP sequenes. ( ) Both pthwys were then tivted t different interstimulus intervls. Tres show smple responses. Stimultion intensities were djusted so tht the proility of evoking tion potentil ws 5 % when the two stimuli were delivered simultneously. ( ) Summry grph of proility of evoking n tion potentil ginst the intervl etween stimultions ( n = 9). ( d ) Smple experiment demonstrting hnge in the oinidene-detetion time window following I h lokde with ZD Top: Smple tres reorded in response to stimultion of two pthwys in ontrol nd fter pplition of ZD-7288; middle: rster plots of spike genertion; ottom: frequeny histogrms showing spike proility t different intervls etween stimuli. ( e ) Summry grph of reltive proility of firing t different interstimulus intervls in ontrol onditions (open olumns) nd in the presene of ZD-7288 (shded olumns; n = 6 ; P =. for differene). ( f ) The proility of evoking n tion potentil y simultneous tivtion of oth pthwys under seline onditions nd fter the redjustment of stimulus intensity following I h lok (open irles: individul reordings; losed irles: verged dt; n = 6 ). Error rs represent s.e.m. NATURE COMMUNICATIONS DOI:.38/nomms22 using grmiidin perforted-pth in urrent-lmp mode 9. We stimulted two seprte popultions of Shffer ollterls ( Fig. ) representing wek nd strong synpti inputs (see Methods). The stimulus intensities were djusted so tht simultneous tivtion of the two pthwys resulted pproximtely in 5 % hne of the neuron spiking. We then mesured the spike proility while systemtilly vrying the interstimulus intervl. As previously reported, the spike proility deresed s the intervl inresed ( Fig., ). We used μ M ZD-7288 to lok I h. Agin onsistent with previous studies 9,, this resulted in hyperpolriztion, n inrese in input resistne nd omplete dispperne of the hrteristi depolrizing sg of the memrne potentil following hyperpolrizing step urrent injetion ( Supplementry Fig. S ). We then redjusted the stimultion intensities to mth the spiking proility for simultneous stimultion oserved under ontrol onditions. Bloking I h resulted in signifint rodening of the 2 ms 9 ms 6 ms 3 ms ms +3 ms +6 ms +9 ms +2 ms d e Normlized spike proility Cyle Normlized spike proility mv 2 ms 2 mv 2 ms ±3 ±6 ±9 ±2 2 ms Normlized spike proility f ZD Mximum spike proility ZD NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI:.38/nomms22 ARTICLE time window for integrtion of the two input stimuli ( Fig. d f ; n = 6; repeted mesures nlysis of vrine (ANOVA): F (,5) = 5.5, P =.). We further onfirmed this effet using ell-tthed reordings t ner physiologil temperture nd with the sme stimultion prdigm ( Supplementry Fig. S2 ; n = 4; repeted mesures ANOVA: F(,3) = 37.3, P =.9 for the effet of ZD-7288). V R (mv) ZD-7288 ZD PTX Bloking I h prolongs exittion through hyperpolriztion. Input summtion in the ove experiments depends on the time ourse of the EPSP IPSP sequene 4,9,2. The long memrne time onstnt of hippompl prinipl ells permits EPSP summtion over lrge time window, ut disynpti feed-forwrd inhiition limits the temporl summtion of the exittory inputs y urtiling the EPSPs 4,9. I h ould influene the EPSP IPSP sequene profile y ltering memrne ondutne 2,22, interneuron reruitment 23 nd / or V R 9. To ddress the reltive roles of these effets of I h, we evoked n EPSP IPSP sequene in CA pyrmidl ells y stimulting Shffer ollterls, nd then loked I h with nd without orreting the memrne voltge ( Fig. 2 ). Bloking I h with ZD-7288 ompletely olished the hyperpolrizing omponent of the EPSP IPSP sequene nd resulted in onsiderle rodening of the hlf-width of the depolrizing phse of the response to 25 ± 27 % of ontrol ( n = 4; P =.3; Fig. 2, ). We first exmined whether the effet of I h lok on the EPSP IPSP sequene ould e entirely ounted for y hyperpolriztion of V R. Following the pplition of ZD-7288, V R ws returned to the seline level y injeting onstnt urrent into the reorded neuron nd the EPSP IPSP sequene ws gin reorded ( Fig. 2 ). Almost full reovery of the hyperpolrizing phse of the response ws oserved (8 ± 8 % of the seline mplitude efore ZD-7288 pplition; n = 4 ; P =.). In these experiments, there ws only reltively smll inrese in pek mplitude (36 ± % ; P =.4) nd hlf-width (39 ± 9 % ; P =.2) of the depolriztion, onsistent with some diret effet of I h on the EPSPs; these effets were signifintly smller thn those oserved with the ddition of ZD-7288 without urrent injetion (tht is, without orreting the neuronl hyperpolriztion; P =.2 nd P =.4 for mplitude nd hlf-width of depolrizing phse of the EPSP IPSP sequene, respetively ( Fig. 2 )). The hnges in the EPSP mplitude nd hlf-width following ZD-7288 pplition nd diret urrent injetion ( Fig. 2 ) were similr to those oserved when testing the effet of ZD-7288 on EPSPs when GABA A reeptors were lso loked with μm pirotoxin ( Fig. 2, V R ws fixed t the ontrol level with diret urrent injetions). These results rgue ginst signifint ontriution of non-speifi effets of ZD-7288 on presynpti funtion 24. In ddition, euse of the limited spe lmp in our experimentl onditions (see Methods), reovery of the hyperpolrizing omponent of the postsynpti response y urrent injetion indites tht the mjority of the inhiitory synpses reruited y Shffer ollterl stimultion impinge either lose to the som or on proximl dendrites of pyrmidl ells. This is in line with previous findings tht feed-forwrd inhiition in hippompl CA pyrmidl neurons underlying temporlly preise synpti integrtion is predominntly perisomti 4. We further tested the effet of I h lok on GABA A reeptor-medited trnsmission, s I h hs een reported to e present in some interneurons 23,25,26. We stimulted Shffer ollterls nd reorded in whole-ell voltge-lmp onfigurtion with the memrne potentil lmped to mv (the reversl potentil for glutmtergi urrents) to isolte inhiitory postsynpti urrent (IPSCs). Consistent with the results otined in urrent lmp, ZD-7288 only redued the IPSC mplitude y 7.5 ± 3 % (n = 3). The effet of I h on the EPSP IPSP sequene n therefore e lrgely ttriuted to the I h -dependent depolriztion of V R nd to the loss of the hyperpolrizing tion of the IPSPs. Does shunting inhiition lso modify the EPSP IPSP sequene? To test this, we pplied the GABA A reeptor ntgonist V m (mv) ZD-7288 ZD-7288+DC 2 ms % Chnge ZD-7288+DC 25 ms pirotoxin with I h loked. This resulted in further prolongtion of the EPSPs, inditing n dditionl role of shunting inhiition in regulting the shpe of the EPSP IPSP sequene ( Fig. 2 ). One predition from these results is tht geneti ltion of I h should similrly hyperpolrize neurons, hnge the driving fore for hloride nd prolong the depolrizing phse of the EPSP IPSP sequene. We exmined HCN knokout mie nd ompred them with wild-type littermte ontrols. Consistent with previous studies 27,28, HCN knokouts lked the I h -medited memrne potentil sg following hyperpolrizing step urrent injetion ( Fig. 3 ). The resting memrne potentil of pyrmidl ells ws more hyperpolrized in the knokout mie ( 72.6 ± 2.) ompred with 3 2 Depolriztion mplitude % Chnge Amplitude Depolriztion hlf-width 4 ms Hlf-width Figure 2 Ih lok olishes the hyperpolrizing phse of the EPSP IPSP sequene. ( ) EPSP IPSP sequenes were evoked in CA pyrmidl ells y Shffer ollterl stimultion s in Figure. Conseutive tres from representtive experiment showing shift in V R following ZD pplition, ssoited olition of the GABA A reeptor-medited hyperpolrizing omponent of the EPSP IPSP sequene (GABA B reeptors re loked with 5 μ M CGP52432) nd the overll ontriution of inhiition reveled y susequent pplition of pirotoxin (PTX). ( ) Summry of ZD-7288 effet on EPSP IPSP hrteristis with (diret urrent (DC)) nd without onstnt urrent injetion to ompenste for ZD-7288-indued shift in V R (n = 4 ells; shded olumns: ZD-7288, open olumns: ZD DC). Overlpped verged tres re shown on the left (prepulse seline is normlized). ( ) Effet of pplition of ZD-7288 on the mplitude nd durtion of phrmologilly isolted EPSPs, while mintining V R with urrent injetion ( n = 7; reordings were mde in the presene of 5 μ M CGP52432 nd μ M pirotoxin); this effet is similr to the effet of ZD-7288 on the EPSP IPSP sequene + DC ( ). Br hrts represent perentge hnge of ontrol vlues; error rs represent s.e.m.; P <.5, P <., P <.. NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved. 3

4 V m (mv) WT KO d WT KO e V R (mv) WT 5 ms E GABAA (mv) KO wild-type littermte ontrol nimls ( 6.9 ±.6 mv; P =.37; Fig. 3 ); however, E GABA(A) ws similr in oth genotypes ( Fig. 3,d ). As predited, the hyperpolrizing phse of the EPSP IPSP sequene ws either sent or redued in the knokout nimls, nd the width of the EPSPs ws rodened to 93.4 ± 8.4 % of the wildtype vlue ( P =.9; Fig. 3e ). I h mintins V R more depolrized thn E GABA(A). The results thus fr indite tht I h -dependent neuronl depolriztion is neessry to mintin hyperpolrizing effet of GABA A reeptor urrents. We diretly tested this hypothesis using grmiidin perfortedpth urrent-lmp reordings, whih minimize perturtion of the internl Cl onentrtion. E GABA(A) ws determined from the reversl potentil of evoked IPSPs, nd ws uniformly negtive to V R (men E GABA(A) V R : 5.2 ±. mv; n = 2; P =.3; Fig. 4 ). In ll ells, pplition of ZD-7288 resulted in signifint negtive shift in V R ( 8.6 ±.9 mv; P = ; n = 2; Fig. 4, ). In ontrst, inhiition of I h led to only smll depolrizing shift in E GABA(A) (2. ±.8 mv; P =.3; Fig. 4d ). The net effet of loking I h ws to mke V R more negtive thn E GABA(A) in out of 2 ells (men E GABA(A) V R : 5.5 ±.3 mv; n = 2; P =.3; Fig. 4e ). This demonstrtes mjor role of I h in mintining hyperpolrizing driving fore for fst GABAergi trnsmission. Correting V R fter I h lok restores oinidene detetion. Although the rodening of input integrtion is onsistent with the hypothesis tht GABA A reeptor-medited hyperpolriztion is required to mintin the nrrow time window for oinidene detetion, n lterntive potentil explntion is tht I h hs profound effet upon dendriti exitility nd temporl summtion of exittory inputs 2,3. To distinguish etween these hypotheses, we repeted the oinidene-detetion experiments, ut depolrized the neuron following ddition of ZD-7288 to return the resting IPSP mplitude (mv) V m (mv) WT KO 2 V R KO V R WT 8 WT mv 6 t V R +DC t V R 4 KO 4 ms +DC Figure 3 HCN deletion results in the loss of the hyperpolrizing effets of GABA A reeptor-medited inhiition. ( ) Exmple tres showing voltge responses to urrent steps injetions in wild-type (WT) mie nd HCN knokout (KO) mie. ( ) An exmple plot showing tht, unlike WT mie, HCN KO mie lked hyperpolrizing GABA A reeptor-medited driving fore. Dt points were fitted with seond-order polynomil funtion. ( ) Comprison of V R in HCN KO mie nd WT mie (WT: n = 5; KO: n = 8). (d ) Summry plot of E GABA(A) in oth genotypes (WT: n = 5; KO: n = 4). (e ) Smple tres showing tht the KO mie displyed no hyperpolrizing phse t V R, wheres WT nimls hd iphsi EPSP IPSP sequene. The hyperpolrizing IPSPs eme pprent in the KO mie when ells were depolrized y DC injetion. Conversely, the IPSP ws less evident when pyrmidl ells from WT mie were hyperpolrized. Error rs represent s.e.m.; P <.. % V R hnge V R (mv) e NATURE COMMUNICATIONS DOI:.38/nomms E GABA A (mv) V m (mv) ms 2 Time (min) ZD-7288 V R mv ZD-7288 ( μm) memrne potentil to the seline level. Using this protool, we oserved no hnge in the time window for integrtion ( Fig. 5 ; n = 5; repeted mesures ANOVA: F (,4) =.374, P =.57). This implies tht it is primrily the depolrizing effet of I h nd loss of the hyperpolrizing effet of GABA A reeptor urrents tht mintins the nrrow oinidene detetion for input integrtion. A further 5 5 IPSP mplitude (mv) V R (mv) d E GABA A (mv) E GABAA > V R 5 3 E GABAA < V R ZD-7288 Figure 4 Bloking I h hnges the V R E GABA(A) reltionship in CA pyrmidl ells. ( ) In ontrol onditions, ll ells hd negtive E GABA(A) ompred with V R (n = 2). Smple IPSPs t different memrne potentils from typil experiment re shown in the inset. Dt points were fitted with seond-order polynomil funtion. ( ) Time ourse of the hnges in V R following the pplition of ZD-7288 ( n = 2). ( ) Effet of ZD-7288 on V R (filled irles re men vlues, open irles re dt from individul experiments; n = 2). (d ) Effet of I h lok on E GABA(A) (filled irles re men vlues, open irles re dt from individul experiments; n = 2). ( e ) Summry grph showing the effet of ZD-7288 on E GABA(A) nd V R. E GABA(A) eme more depolrized thn V R in out of 2 ells (open irles: ontrol, filled irles: in ZD-7288). Error rs represent s.e.m.; P <.5, P <.. 4 NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI:.38/nomms22 ARTICLE Cyle Normlized spike proility Normlized spike proility ms ±3 ±6 ±9 predition from this is tht simple hyperpolriztion with I h intt would widen the oinidene-detetion time window, whih is indeed the se ( n = 5, repeted mesures ANOVA: F(,4) = 8.2, P =.46; Supplementry Fig. S3 ). Hyperpolrizing inhiition underlies temporl preision. A possile onfounder in the ove experiments is tht in order to mintin 5 % spiking proility, we hd to djust the stimultion intensity to ompenste for the memrne hyperpolriztion, potentilly ffeting interneuron reruitment. This would tend to inrese the interneuron reruitment nd therefore would e expeted to nrrow the integrtion time window (in ontrst to the rodening tht we oserved). Nevertheless, to ontrol for this nd to ddress the question of whether loss of the hyperpolriztion effet of GABA A ±2 Mximum spike proility ZD DC ms ZD+DC Figure 5 Repolriztion restores temporl preision when I h is loked. ( ) Spike proility t different intervls etween stimuli in ontrol onditions nd in the presene of ZD-7288 when neurons were repolrized to their initil V R y DC injetion, therefore restoring the iphsi shpe of the EPSP IPSP sequene. Top: smple tres; middle: rster plots of spikes initited y stimultion of the two pthwys t different dely intervls; ottom: spike proility distriution grphs. ( ) Averged dt from five experiments (open olumns: ontrol, shded olumns: ZD DC; P =.57). ( ) Proility of tion potentil genertion for synhronous stimultion of oth pthwys efore nd fter pplition of ZD-7288 ( n = 5). Error rs represent s.e.m. reeptor urrents is suffiient to explin our results, we onstruted simple integrte-nd-fire model of neuron tht reeives two inputs. Eh input onsisted of n exittory followed y n inhiitory ondutne, nd the prmeters were djusted to simulte the kinetis of the experimentlly otined EPSP IPSP sequene wveform (see Methods; Fig. 6 ). In greement with the experimentl findings, systemti vrition of the dely etween the two inputs reveled nrrow integrtion time window for spike genertion ( Fig. 6 ). Hyperpolriztion of V R rodens the integrtion time window. R emov ing I h from the modelled ell, hyperpolrized the memrne from 7 to 8 mv nd led to n inrese in the width of the integrtion time window ( Fig. 6 ; the re under the spike proility urve ws inresed y 26.3 ± 2.3 % ; P <.). This effet ws roust nd reltively insensitive to vrying EPSP nd IPSP kineti prmeters, dely times etween exittory nd inhiitory synpse tivtion, s well s synpse lotions on the dendrites ( Supplementry Fig. S4 ). We lso systemtilly hnged the mximl proility of tion potentil genertion to test whether this ould ffet our experimentl results. The effet of memrne potentil hyperpolriztion ws onstnt ross wide rnge of tested proility vlues ( Supplementry Fig. S5 ). To determine whether the effet on the integrtion window in the simultions ws due to the ondutne or voltge effet of I h removl, we repolrized the neuronl som k to 7 mv y introduing depolrizing urrent. In greement with experimentl oservtions, this restored the width of the time window for integrtion, inditing ruil role for the depolrizing tion of I h (Fig. 6d ). We next sked whether the effet tht we oserved ws due to the voltge hnge itself rther thn the hnge in the polrity of GABA A reeptor-medited trnsmission. We therefore ssessed the effets of memrne potentil on the integrtion time window in the sene of GABAergi trnsmission. As expeted 4, removing feed-forwrd inhiition rodened the integrtion time window y 6.8 ± 6.7 %. However, the width of the window ws minimlly ffeted y hnges in V R in the rnge oserved in our experiments nd simultions ( Fig. 7 ; n inrese of 3. ±.4 % with mv hyperpolriztion). In the ove simultions, the mgnitude of the inhiitory synpti ondutne ws fixed to mth the simulted EPSP IPSP wveform with tht oserved experimentlly. We therefore tested the effet of different inhiitory synpti ondutnes. In ll instnes, hyperpolrizing V R, so tht the IPSPs eme depolrizing, widened the integrtion time window. This effet initilly inresed with inreses in the IPSP mplitude ( Fig. 7 ) up to mximum nd then deresed proly euse of the inresing shunting versus voltge effet of inhiitory synpses. Finlly, we onsidered the possile onfounding effet of vriility in the size of the inhiitory ondutne. At high stimultion intensity, the inhiitory ondutne is reltively invrint 29. However, t lower stimultion intensities, inhiitory ondutnes vry with stimultion intensity (nd mgnitude of exittory ondutne). We therefore ovried the ondutnes of exittory nd inhiitory synpses (see Methods) to model this sitution. Under these onditions, the window of integrtion ws still inresed following hyperpolriztion of the neuron y 38. ±.4 % ompred with 2.2 ± 2.6 %, with onstnt inhiitory synpti ondutnes ( Fig. 7 ; P <.5). Disussion We hve shown tht I h is neessry to mintin hyperpolrizing driving fore for GABA A reeptor-medited trnsmission y depolrizing the resting memrne potentil of pyrmidl ells. Hyperpolrizing the resting memrne potentil through phrmologil inhiition of I h or deletion of HCN results in depolrizing GABA A reeptor-medited potentils nd rodening of the exittory phse NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved. 5

6 NATURE COMMUNICATIONS DOI:.38/nomms22 Normlized spike proility d Normlized spike proility Normlized spike proility Experimentl EPSP EPSP IPSP sequene 2 3. ±.5 ±.. ±.5 Simulted 2 ±. ±.5 2 ms 3 ±.5 ±2. ±2.5 ±3. ±2. ±2.5 ±3. Figure 6 I h -medited hyperpolriztion rodens the integrtion time window in n integrte-nd-fire neuronl model. ( ) Suessive tivtion of exittory nd inhiitory ondutnes in simple integrtend-fire neuron ws used to produe n EPSP IPSP sequene similr to tht oserved experimentlly. ( ) Simultion of two pthwys tivted with different delys (see Methods) resulted in nrrow integrtion time window. Synpti strength ws djusted so tht the pek proility of tion potentil genertion remined t 5 %. ( ) Removl of I h from the dendrites hyperpolrized the modelled neuron y mv. The grph shows spike proility t different intervls etween stimuli with (open rs) nd without I h (red rs) in the modelled neuron. ( d ) The effet of I h removl ws olished when the neuronl som ws repolrized y DC injetion (open rs: ontrol with I h, lue rs: no I h repolrized). Dt re presented s mens of 3 rounds of simultions, error rs represent s.e.m. Normlized spike proility AUC (% hnge) AUC (% of ontrol) ± ±2 of the EPSP IPSP sequene, leding to prolonged time window for synpti integrtion. These results lso demonstrte n importnt nd unexpeted role of voltge inhiition. I h hnnels re densely expressed in the dendriti rour of CA pyrmidl ells 2. A signifint proportion of these hnnels is tive ± IPSP mgnitude (g i ) Constnt G IPSP Adjusted G IPSP Figure 7 The effets of hnging the mgnitude of inhiitory ondutnes on the width of the integrtion time window. ( ) In the sene of feed-forwrd inhiition (no IPSPs), the integrtion time window eme roder. Under suh onditions, memrne hyperpolriztion did not hnge the width of oinidene detetion, inditing tht hnges in the polrizing effet of GABA A reeptor-medited urrents, rther thn the V R vlue itself, determine neuronl integrtion (open rs: V m = 7 mv no IPSP; hthed rs: V m = 8 mv no IPSP; red rs: V m = 7 mv with IPSP). ( ) The effet of vrying the strength of inhiitory onnetions on the width of the integrtion time window ssessed s the hnge in the re under the spike proility urve (AUC). ( ) The effet of hyperpolriztion on the oinidene-detetion time window when inhiitory synpti ondutnes were sled with exittory ondutnes, mimiking the sitution when inhiitory inputs onto neuron re not sturted. Dt re presented s mens of 3 rounds of simultions, error rs represent s.e.m.; P <.5, P <., P <.. ± NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved.

7 NATURE COMMUNICATIONS DOI:.38/nomms22 ARTICLE t rest, reduing the memrne resistne nd neuronl time onstnt. These effets of I h hve een shown to determine the time ourse of EPSPs onto CA pyrmidl ells 2. The previous studies hve onentrted on the impt of I h on the integrtion properties of exittory inputs, with GABA A reeptor-medited inhiition loked. However, GABAergi inhiition hs profound effet on the width of the integrtion time window of exittory inputs 4. The reltive roles of shunting nd hyperpolrizing inhiition in mintining preise oinidene detetion hve not een ddressed. Although one study hs onsidered the effet of I h on the integrtion time window without GABA A reeptor lokde 6, tht work ws performed in whole-ell mode, with the memrne potentil of the neurons fixed nd the reversl potentil of GABA A responses set to e depolrizing. In hippompl neurons, E GABA(A) is negtive to V R y the end of the seond postntl week 3, so tht GABA A reeptor-medited potentils predominntly hyperpolrize the postsynpti memrne in dult tissue, for exmple, see ref. ( 3 ). In dult neurons, E GABA(A) is minly determined y the Cl otrnsporter KCC2, whih uses the trnsmemrne K + grdient to mintin low intrellulr Cl onentrtion, therey lmping the reversl potentil for Cl (E Cl ) loser to tht of K + (E K ) 3. E GABA(A) is, however, more depolrized thn E Cl due to the permeility of the GABA A reeptors to HCO 3 ions 32. Therefore, to mintin hyperpolrizing IPSPs, V R lso hs to e depolrized reltive to E GABA(A). A numer of ondutnes my ontriute to this depolriztion (suh s persistent sodium urrents nd lek ondutnes); in CA pyrmidl ells, I h provides signifint depolrizing drive t resting memrne potentil. In our study, this effet of I h ws more evident with phrmologil lok thn with geneti ltion, perhps euse of ompenstory hnges in other ondutnes in the HCN knokout mie. We hve shown tht I h sets the polrity of GABAergi events y mking V R more depolrized thn E GABA(A), nd most of the effets of I h on the EPSP IPSP sequene n e resued y repolrizing V R to its seline vlue, rguing for mjor role of GABAergi voltge inhiition in regulting the integrtion properties of synpti inputs of CA pyrmidl ells. As oserved in our study nd in other studies, for exmple, see ref. ( 33 ), GABA A reeptor tivtion n still shunt EPSPs even when E GABA(A) is more depolrized thn V R. However, the reltive importne of shunting nd hyperpolrizing effets of inhiition for neuronl omputtions nd preise timing of tion potentil genertion hs reeived muh less ttention. Some experimentl nd theoretil studies hve suggested tht the hyperpolrizing effets of GABAergi neurotrnsmission re ritil for network synhroniztion, for exmple, see ref. ( 34 ), wheres others emphsize the role of shunting inhiition in osilltory ehviour of interonneted neurons 35. The ility of I h to swith the inhiitory mode of GABAergi signls from hyperpolrizing to shunting or depolrizing dds further omplexity in defining the omputtionl properties of hippompl neurons 36. Further, it might e expeted tht the lrge, erly shunting effet of GABAergi inhiition hs the predominnt prt in urtiling the EPSPs. Our results, however, undersore the ontriution of GABA A reeptor-medited hyperpolriztion in determining the temporl preision of oinidene detetion y hippompl pyrmidl neurons. How does hnging the GABA A reeptor-medited response from hyperpolrizing trnslte to n inresed integrtion time? Although shunting is mintined regrdless of the polriztion of GABA A reeptor-medited responses (see Fig. 2 ) ttenuting the EPSPs generted in response to the seond stimultion, this ttenution would e muh stronger when the IPSP produes hyperpolriztion. More importntly, euse the voltge effet of n IPSP outlsts its shunting effet 33, it would hve n impt on the exittory potentils over n extended period. In the sitution when the hyperpolrizing phse of the GABAergi response is olished (nd even more so if the polrity of the GABA A response is hnged), this would filitte EPSP summtion. Therefore, the hyperpolriztion tht results from inhiition of I h results in onsiderle inrese in the hlf-width of the time window for oinidene detetion. Inhiition of other memrne ondutnes through hyperpolriztion nnot ount for this, s voltge hnges of similr mgnitudes, when I h is loked, result in miniml hnge in the memrne resistne or time onstnt. The ritil role of hyperpolriztion for oinidene detetion hs extensive implitions, s nything tht lters the polrity of GABA A reeptor-medited trnsmission will ffet the time window for input integrtion. This would inlude either shift in E GABA(A) (for exmple, due to loss or inhiition of KCC2) or hnges in resting memrne potentil medited through, for exmple, hnges in memrne potssium ondutnes. We hve therefore shown the ritil role of GABA A reeptor-medited hyperpolriztion in informtion proessing nd n unexpeted mehnism y whih I h n modulte oinidene detetion. As I h is regulted y developmentl 37, physiologil 38 nd pthologil proesses 6,39 42, suh proesses my onsequently ffet the properties of GABA A reeptor-medited signlling nd the temporl fidelity of oinidene detetion. This my ontriute to ognitive nd psyhitri onsequenes of epilepsy y inresing the proility of errnt ssoitions. Moreover, the mgnitude of somti I h is dependent upon exittory synpti tivity, nd this hs een proposed to e homeostti mehnism regulting neuronl exitility 38. We further hypothesize tht suh mehnism my hve n dditionl homeostti role y nrrowing the time window for oinidene detetion with inreses in neuronl tivity nd onversely rodening the time window for integrtion when synpti tivity dereses. The role of I h in restriting temporl input integrtion my lso ontriute to explining the enhned lerning ehviour oserved in HCN knokout mie 27, whih my our t the expense of loss of temporl disrimintion of inputs. Methods Eletrophysiology. We used trnsverse hippompl slies (35 μm) from 3 6- week-old mle Sprgue Dwley rts, or HCN knokout mie nd their wild-type littermte ontrols. The HCN knokout nimls were re-derived from tht desried previously, nd mintined on 29SVEV kground 27,28. Animls were killed ording to shedule of the UK Animls (Sientifi Proedures) At 986. The rins were rpidly removed, disseted nd ut with Lei VTS virtome in ie-old solution ontining (in mm) surose (7), NCl (8), KCl (2.5), MgCl 2 (7), CCl 2 (.5), NHCO 3 (25), NH 2 PO 4 (.25) nd gluose (22), nd were equilirted with 95 % O 2 /5 % CO 2 (ph 7.4). The slies were llowed to reover in n interfe hmer ( > h) t room temperture efore eing trnsferred to the reording hmer. Storge nd perfusion solutions ontined (in mm) NCl (9), KCl (2.5), MgSO 4 (.3), CCl 2 (2.5), NHCO 3 (26.2), NH 2 PO 4 () nd gluose (22), nd were gssed with 95 % O 2 /5 % CO 2. All reordings, exept for those in Figures, 5 nd Supplementry Figure S3, were rried out t 32 C. Current-lmp whole-ell reordings ( Supplementry Fig. S ) were performed using pipettes (3 5 M Ω ) filled with n intrellulr solution ontining (in mm) K-gluonte (45), NCl (8), KOH HEPES (), EGTA (.2), Mg-ATP (2) nd N 3 -GTP (.3); ph 7.2; 29 mosm. Grmiidin (5 μg ml ) ws dded for perforted-pth reordings 43, nd pth pipettes (8 2 MΩ ) were front-filled with grmiidin-free solution; either QX-34 Br (5 mm) ws dded or [Cl ] ws inresed to 26 mm (E Cl = 4 mv) to monitor pth integrity. Series resistne ws monitored throughout. Dt quisition egn when the series resistne ws < 5 MΩ (5 2 min fter otining ell-tthed onfigurtion). To isolte GABA A reeptor-medited IPSPs, NMDA, AMPA nd GABA B reeptors were loked with AP5 (5 μ M), NBQX (2 μ M) nd CGP52432 (5 μ M), respetively. Constnt urrent stimuli were delivered to Shffer ollterls through ipolr stinless steel eletrodes pled in strtum rditum. Monosynpti IPSPs were evoked y positioning the stimulting eletrode lose to the reording site. EPSP IPSP sequenes were evoked y positioning the eletrodes in strtum rditum t lest 3 μ m wy from the reording site; CGP52432 (5 μ M) ws dded to the perfuste. Although pplition of CGP52432 redued the durtion of the hyperpolrizing phse y 6 ± 3 % (Supplementry Fig. S6 ), it did not ffet the depolrizing omponent. Two Shffer ollterl pthwys were stimulted for the oinidene-detetion protool ( Figs nd 5, Supplementry Fig. S3 ). Experiments were performed in the presene of 5 μ M AP5 (to void spike timing-dependent plstiity) nd 5 μm CGP AP5 hd miniml impt on the exittory phse of the EPSP IPSP sequene, deresing the hlf-width y 4 ± 4 % (more thn n order of mgnitude smller thn the impt of hnging the polrity of GABA A responses; Supplementry NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved. 7

8 NATURE COMMUNICATIONS DOI:.38/nomms22 Fig. S6d f ). To ensure reording stility, these experiments were performed t room temperture (23 25 C); this inresed the time ourse of the synpti responses ( Supplementry Fig. S7 ), ut the shpe of the EPSP IPSP wveform ws mintined. Although modelling indited tht this does not qulittively hnge the experimentl findings ( Supplementry Fig. S4 ), we onfirmed this with ell-tthed reordings t 32 C (Supplementry Fig. S2 ). These experiments were performed using pth pipettes (8 2 MΩ ) filled with ACSF in voltge-lmp with the voltge set so tht no urrent ws injeted under seline onditions. As fferent input strength to the hippompus n vry onsiderly, we stimulted wek nd strong input; the mplitude of the response in one pthwy ws djusted to e pproximtely twie thn tht in the other pthwy. In the ell-tthed experiments, the wek pthwy ws stimulted t pproximtely hlf-threshold intensity, wheres the strong pthwy stimultion ws set lose to the firing threshold. Stimultion intensities were djusted to otin ~ 5 % spike proility when the two pthwys were tivted simultneously. The two pthwys were stimulted with n interstimulus dely vrying from 2 to + 2 ms in 3 ms steps (thus the order of inputs ws reversed over the rnge investigted). To void ny onfounding effets of symmetri distriution of spike proilities, the dt re presented s the verged vlues from orresponding points on either side of the mximum. In some experiments, onstnt urrent injetion ws used to repolrize the somti memrne potentil. Under our experimentl onditions, there is spe lmp error in distl dendrites. However, s we oserved experimentlly tht somti urrent injetion restored the hyperpolriztion phse of the EPSP IPSP wveform, we onlude tht feed-forwrd inhiition in our study ws predominntly perisomti ( Supplementry Fig. S8 ). For experiments on the effet of I h lok on the mgnitude of IPSCs, the intrellulr solution ontined (in mm) Cs-methnesulfonte (2), NCl (8), HEPES (), EGTA (.2), MgCl 2 (.2), Mg-ATP (2), N 3 -GTP (.3) nd QX-34 Br (5 mm); 29 mosm. Shffer ollterls were stimulted in the presene of AP5 nd CGP Neurons were voltge-lmped t mv (lose to the reversl potentil for AMPA reeptor-medited response), nd outwrd GABA A reeptormedited urrents were ompred in the sene nd presene of ZD All reordings were otined using MultiClmp 7B mplifier ( Moleulr Devies ), filtered t 2 khz nd digitized t 5 khz. LView ( Ntionl Instruments ) softwre ws used for dt quisition nd off-line nlysis. Chemils were purhsed from Toris Cookson or Sigm-Aldrih. Modelling. All simultions were onduted with NEURON 7. (ref. 44) on multimode luster 45. The Hodgkin Huxley neuron model onsisted of ylindril som (dimeter = length = 2 μ m) nd two identil dendrites (3 μ m dimeter nd 2 μ m long). The xil resistne ( R ) ws 35.4 Ohm m ; memrne pitne ( C m ) ws μf m 2. The somti memrne hd N + nd K + ondutnes with the following pek vlues: g N =.2 S m 2, g K =.36 S m 2 (E K = 77 mv nd E N = + ). The dendriti memrne hd pssive lek urrent ( g ps = ms m 2 nd E ps = 8 mv) nd I h (g Ih = ms m 2, kinetis s in ref. 2). V R ws 7 mv. Removl of I h hyperpolrized the neuron y mv. In suh onditions,.4 na injeted into the som ws neessry to repolrize the memrne potentil to 7 mv. All simultions were performed with either V R = 7 mv (seline onditions) or 8 mv (hyperpolrized onditions). Deterministi tion potentil genertion ws prevented y introduing Poisson-distriuted ondutnes of uniformly distriuted vrile mgnitude (.9. ns; τ rise =. ms; τ dey = ms; reversl potentil = V R to void signifint memrne potentil flututions) into the ell t men frequeny of, Hz. Eh dendrite hd glutmtergi nd GABAergi synpses, loted t 8 nd 4 μ m from the som, respetively. The dul-exponentil formlism in the Exp2Syn funtion of neuron simultor ws used to determine the time ourse of synpti ondutnes: gs( t) = Gm(exp( t/ t) exp( t/ t2)), () where g s (t ) is the synpti ondutne t time t fter tivtion; τ is the rise time onstnt (7 ms for glutmtergi synpses nd 5 ms for GABAergi synpses); τ 2 is the dey time onstnt (35 ms for glutmtergi synpses nd 5 ms for GABAergi synpses) nd G m is the vlue of the mximum synpti ondutne. To simulte the EPSP IPSP sequene, there ws 7 ms dely from EPSPs to IPSPs. The reversl potentils were set t 75 nd mv for GABAergi nd glutmtergi synpses, respetively. Th e left exittory nd inhiitory synpti ondutnes ( G ESL nd G ISL ) were twie the right synpti ondutnes ( G ESR nd G ISR ). The onset time of the left EPSP IPSP sequene ws fixed wheres tht of the right vried from to + ms. To lulte the time distriution of the spike proility, interstimulus intervls were divided into.5 ms ins. The proility of tion potentil genertion ws determined using 3 rounds of ten synpti stimultions with 5 ms intervl etween trils, initited with different seed. The intervl etween stimuli ws suffiient to llow the model neuron to reh initil stedystte onditions. The men numer of tion potentils per in ( M i ) nd s.e. ( ε i ) were lulted. Are under the spike proility urve ws used to quntify hnges in the oinidene-detetion time window. The s.e. of the surfe under the distriution ws defined s: e = N 2 in ei i=, pmx where N is the numer of ins, in is the in size nd p mx is the mximum proility of the distriution. The s.e. of the rtio of surfes S / S2 ws defined s: 2 e e ess = + 2 S S S 2 S. 2 2 The mximl proility of tion potentil genertion (.5) ws set y sling synpti ondutnes with the prmeter Stim: Stim G ESL nd Stim G ESR. The strengths of inhiitory synpses were set to G ISL = 45 ns nd G ISR = 225 ns for left nd right synpse, respetively, for the mjority of simultions. For Figure 7, the strength of inhiitory onnetions vried s indited. For simultions presented in Figure 7 in ddition to exittory ondutnes, we lso sled inhiitory ondutnes: Stim G ISL nd Stim G ISR. Simultions with mximl proilities from.2 to.85 were performed ( Supplementry Fig. S5 ). In these experiments, the tul mximl spike proility ws determined post ho. Sttistis. Two-tiled Student s t -test (pired or independent) nd repeted mesures ANOVA were used for sttistil nlysis. P <.5 for signifint differenes. Dt re presented s men ± s.e.m. Referenes (2) (3). Konig, P., Engel, A. K. & Singer, W. Integrtor or oinidene detetor? The role of the ortil neuron revisited. Trends Neurosi. 9, 3 37 (996 ). 2. Aeles, M. Role of the ortil neuron: integrtor or oinidene detetor? Isrel J. Med. Si. 8, (982 ). 3. Buzski, G. Feed-forwrd inhiition in the hippompl formtion. Prog. Neuroiol. 22, 3 53 (984 ). 4. Pou i l l e, F. & S n zi n i, M. 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Reeptor tions of synptilly relesed glutmte: the role of trnsporters on the sle of nnometers to mirons. Biophys. J. 95, (28 ). Aknowledgments This work ws supported y the Medil Reserh Counil, Epilepsy Reserh UK nd the Europen Reserh Counil. We re grteful to D. Ruskov, R. Surges nd other memers of the lortory for omments nd suggestions. We thnk M. Shh for kindly providing us with the HCN knokout mie nd ritil reding of the mnusript. We thnk K. Zheng for providing the multimode luster for omputtions. Author ontriutions I.P. nd A.S. designed nd performed experiments, nlysed dt nd wrote the pper; L.S. performed modelling; D.M.K. nd M.C.W. designed experiments, direted the projet nd wrote the pper. Additionl informtion Supplementry Informtion ompnies this pper t ntureommunitions Competing finnil interests: The uthors delre no ompeting finnil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to ite this rtile: Pvlov, I. et l. I h -medited depolriztion enhnes the temporl preision of neuronl integrtion. Nt. Commun. 2:99 doi:.38 / nomms22 (2). Liense: This work is liensed under Cretive Commons Attriution-NonCommeril- Shre Alike 3. Unported Liense. To view opy of this liense, visit NATURE COMMUNICATIONS 2:99 DOI:.38/nomms Mmilln Pulishers Limited. All rights reserved. 9