HCN hyperpolarization-activated cation channels inhibit EPSPs by interactions with M-type K + channels

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1 HCN hyperpolriztion-tivted tion hnnels inhiit EPSPs y intertions with M-type K + hnnels Meen S George 1, L F Aott 1,2 & Steven A Siegelum 1,3,4 29 Nture Ameri, In. All rights reserved. The proessing of synpti potentils y neuronl dendrites depends on oth their pssive le properties nd tive voltge-gted hnnels, whih n generte omplex effets s result of their nonliner properties. We hrterized the tions of HCN (hyperpolriztion-tivted yli nuleotide-gted tion) hnnels on dendriti proessing of suthreshold exittory postsynpti potentils (EPSPs) in mouse CA1 hippompl neurons. The HCN hnnels generted n exittory inwrd urrent (I h ) tht exerted diret depolrizing effet on the pek voltge of wek EPSPs, ut produed prdoxil hyperpolrizing effet on the pek voltge of stronger, ut still suthreshold, EPSPs. Using omined modeling nd experimentl pproh, we found tht the inhiitory tion of I h ws used y its intertion with the delyed-retifier M-type K 1 urrent. In this mnner, I h n enhne spike firing in response to n EPSP when spike threshold is low nd n inhiit firing when spike threshold is high. Neurons tively proess nd integrte synpti potentils through the tions of wide rry of voltge-gted ion hnnels tht re often differentilly expressed throughout neuron s dendriti tree 1. In some instnes, the effets of voltge-gted hnnels on dendriti proessing re reltively strightforwrd nd well understood. For exmple, dendriti voltge-gted sodium nd lium hnnels n mplify synpti potentils 2 through the genertion of lol or propgted dendriti tion potentils 3,4. In ontrst, dendriti voltge-gted or lium-tivted K + hnnels n redue EPSP mplitude nd dmpen dendriti exitility 5 7. In other ses, however, nonliner intertions etween dendriti voltge-gted hnnels give rise to omplex effets tht re less esily understood. Here, we foused on the prdoxil effets of the HCN hnnels on the proessing of EPSPs in the pil dendrites of CA1 pyrmidl neurons, in whih these hnnels re expressed in grdient of inresing density with inresing distne from the som Unlike most voltge-gted hnnels, HCN hnnels tivte with hyperpolriztion nd detivte with depolriztion. Their mixed permeility to K + nd N + ions results in reversl potentil (E h ) of pproximtely 3 mv, using these hnnels to generte n exittory inwrd urrent (I h ) t suthreshold potentils. These iophysil properties underlie the role of I h s pemker urrent in rdi myoytes nd thlmoortil rely neurons, in whih tivtion of I h following tion potentil repolriztion genertes depolrizing urrent tht drives spontneous, rhythmi firing 12,13. In neurons tht re not spontneously tive, I h ontriutes 5 1-mV depolrizing influene on the resting memrne potentil (RMP) nd inreses the resting memrne ondutne (tht is, it lowers the input resistne), therey regulting the sptil nd temporl integrtion of synpti inputs 1, Despite the ft tht I h provides depolrizing urrent t suthreshold potentils, results from severl studies hve indited tht it hs prdoxil inhiitory effet on the ility of n EPSP to trigger n tion potentil. Thus, enhnement of I h y the ntionvulsnt lmotrigine 17, pplition of dopmine 18 or indution of long-term potentition 19,2 dereses exitility nd spike firing. Conversely, downregultion of I h vi geneti deletion of HCN1 (ref. 21), phrmologil lokde using esium 15,22 or the orgni ntgonist ZD7288 (refs. 15,16), or following indution of long-term depression 23 or seizures 24 inreses EPSP mplitude, temporl summtion nd spike firing. The inhiitory effets of I h, y whih we men the inhiition tht is seen when I h is enhned, hve generlly een ttriuted to its ility to inrese the resting memrne ondutne. This so-lled shunting effet on the exittory postsynpti urrent dereses the mplitude of n EPSP 1,22, with EPSP mplitude (DV EPSP ) eing defined s the differene etween the pek voltge of n EPSP (V pek ) nd the resting potentil. However, the effet of n EPSP depends not on its mplitude, ut on the voltge rehed t its pek, whih determines whether n EPSP is suprthreshold 25.Notly,I h exerts two opposing influenes on V pek : its shunting effet dereses EPSP pek voltge nd its diret depolrizing effet inreses V pek (Fig. 1). We used simple omputtionl model to show tht, in the sene of other voltge-gted ondutnes, I h should lwys e exittory for EPSPs tht re negtive to the I h reversl potentil; tht is, the depolrizing tion of I h on V pek is lwys greter thn its shunting effet. This implies tht ny inhiitory effet of I h on V pek must e used y its intertions with other voltge-gted ondutnes. One suh intertion results in n inhiitory effet of I h on the durtion of C 2+ tion potentils in the distl dendrites of CA1 1 Deprtments of Neurosiene, 2 Physiology nd Cellulr Biophysis nd 3 Phrmology, 4 Howrd Hughes Medil Institute, Columi University, New York, New York, USA. Correspondene should e ddressed to S.A.S. (ss8@olumi.edu). Reeived 28 Otoer 28; epted 4 Mrh 29; pulished online 12 April 29; doi:1.138/nn.237 NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 5 [ MAY

2 29 Nture Ameri, In. All rights reserved V EPSP V EPSP RMP V min V ss V pek V pek + I h I h Time (s) 1.2 pyrmidl neurons 26. In this instne, the depolrizing effet of I h on the resting memrne potentil inreses the resting intivtion of N- nd T-type voltge-gted C 2+ hnnels, thus inhiiting the C 2+ spikes. In priniple, this effet of I h on resting potentil nd resting intivtion ould lso explin how I h suppresses the firing of N + tion potentils. However, it remins unler whether I h n exert n inhiitory effet on V pek for suthreshold EPSPs. We found tht I h, through intertions with voltge-gted K + hnnels, n indeed produe n inhiitory effet on the pek voltge of suthreshold EPSPs. Notly, the influene of I h on V pek depended on synpti strength. Thus, lthough I h shifted V pek to more positive potentils for wek EPSPs, I h inhiited V pek for stronger, ut still suthreshold, EPSPs. In other words, the effets of I h on V pek rossed over from depolrizing to hyperpolrizing s funtion of inresing synpti strength, with the rossover potentil ourring elow oth the reversl potentil for I h nd the tion potentil threshold. This indited tht the net effet of I h is essentilly inhiitory, s it mde it more diffiult for n EPSP to reh threshold. Both our omputtionl nd experimentl results indite tht the inhiitory effet of I h is used y its ility to depolrize the resting memrne, whih enhned the resting tivtion of the delyed-retifier M-type K + hnnels. Beuse the M-hnnels re under neuromodultory ontrol 27, the influene of I h on dendriti integrtion my swith from inhiitory to exittory depending on the stte of M-hnnel regultion. Suh modultion my hve importnt implitions for regultion of long-term synpti plstiity tht ontriutes to lerning nd memory 21,28 nd for the tretment of epilepti disorders in whih oth I h nd M-hnnels my e involved RESULTS Dul influene of I h on EPSPs in CA1 pyrmidl neurons We first exmined the influene of I h on neuronl tivity in mouse hippompl CA1 pyrmidl neurons y rrying out whole-ell urrent-lmp reordings of oth resting memrne properties nd somti EPSPs tht were evoked y stimultion of the Shffer ollterl pthwy (Fig. 1). In response to hyperpolrizing urrent steps injeted in the CA1 neuron som, the memrne voltge showed depolrizing sg tht is hrteristi of I h tivtion (Fig. 1). We then pplied fol synpti stimultion of inresing strength to eliit EPSPs of inresing mplitude to determine the reltionship etween V pek nd stimulus strength (Fig. 1d). In d Stimulting eletrode Exittory Shffer ollterl inputs Reording eletrode Figure 1 Experimentl proedure nd the effets of phrmologil lokde of I h.() Digrm illustrting the opposing effets of I h on suthreshold EPSPs, involving positive shift of the RMP nd derese in the EPSP mplitude (DV EPSP ). Red indites the presene of I h nd lue indites its sene. DV EPSP ¼ V pek RMP. () Shemti of experimentl setup. Whole-ell urrent-lmp reordings were otined from CA1 pyrmidl neurons (reording pth eletrode). An extrellulr stimulting pth eletrode ws pled B15 mm wy from the som in strtum rditum under visul guidne. () Somti voltge response to hyperpolrizing urrent step in the sene (ontrol) nd in the presene of ZD7288 (1 mm). Note the RMP hyperpolriztion fter pplition of ZD7288. Under ontrol onditions, the memrne voltge rehed n initil minimum vlue (V min ) nd then showed depolrizing sg to stedy-stte vlue (V ss ) s result of tivtion of I h. The sg ws eliminted y ZD7288. (d) Smple EPSPs evoked in response to rnge of stimulus strengths under ontrol onditions nd fter lok of I h using ZD7288. ll of these experiments, inhiitory synpti trnsmission ws loked using GABA A nd GABA B reeptor ntgonists. Next, we pplied the orgni ntgonist ZD7288 to lok I h nd repeted the mesurements desried ove of resting memrne properties, voltge sg nd the EPSP input-output urve. Reltively low onentrtions of ZD7288 (1 mm) nd short exposure times (1 15 min) were used to minimize nonspeifi effets of this drug on synpti trnsmission 33. These onditions were suffiient to eliminte the voltge sg in response to hyperpolrizing urrents, inditing n effetive lok of I h (Fig. 1). The verge sg rtio deresed from 1.2 ± 1.% under ontrol onditions to 3.1 ±.5% in the presene of ZD7288 (n ¼ 7, P o.1, pired t test). Applition of ZD7288 lso shifted the RMP y B5 mv to more negtive voltges (RMP under ontrol onditions equled 68.9 ± 1.5 mv, RMP in ZD7288 equled 74. ± 1.4 mv, n ¼ 7, P o.1, pired t test) nd inresed the input resistne y more thn twofold (ontrol, ± 8.2 MO; ZD7288, ± 25.1 MO; n ¼ 7, P o.1, pired t test), onsistent with previous findings 19,23,24,26,34. A omprison of EPSP input-output urves in the presene nd sene of ZD7288 showed tht the effets of I h on pek EPSP voltge depended on EPSP size (Fig. 2). For smll EPSPs, the presene of I h inresed V pek, shifting it to more positive potentils, s expeted for n inwrd, exittory urrent (3-mA stimulus; Fig. 2). However, s the stimulus strength ws inresed to evoke lrger EPSPs, V pek in the sene of I h pprohed its vlue in the presene of I h (45-mA stimulus; Fig. 2). Eventully, with even stronger stimuli, rossover ourred, where the presene of I h deresed the pek EPSP voltge to vlues tht were more negtive thn those rehed in the sene of I h (6-mAstimulus;Fig. 2). This depolrizing/hyperpolrizing rossover effet ws lerly seen when V pek ws plotted s funtion of stimulus strength in the presene nd sene of I h (Fig. 2). To our surprise, the rossover ourred for suthreshold EPSPs whose pek voltges were well elow the I h reversl potentil of 3 mv, tht is, t voltges t whih I h provided n inwrd, depolrizing urrent. We oserved suthreshold hyperpolrizing effets of I h on V pek in six out of the seven ells tht we exmined. The one exeption ourred in ell whose resting potentil in the presene of I h ws unusully positive ;( 64 mv) nd spikes were thus evoked with smll urrent stimuli (35 ma). In some ells, rossover ourred well elow the tion potentil threshold, providing ler evidene tht I h n exert n unmiguously inhiitory influene on suthreshold EPSP pek voltge (Fig. 2). In other ells, the rossover from depolrizing to hyperpolrizing effets ourred ner threshold (Fig. 2). In suh ells, V pek in the presene of ZD7288 pprohed or overlpped with V pek in the sene of drug up to potentils very ner spike threshold. A slight inrese in stimulus intensity ould then evoke spikes in 578 VOLUME 12 [ NUMBER 5 [ MAY 29 NATURE NEUROSCIENCE

3 29 Nture Ameri, In. All rights reserved. 4 3-µA stimulus µA stimulus µA stimulus Stimulus strength (µa) Stimulus strength (µa) the presene ut not sene, of ZD7288. Suh results re onsistent with previous findings tht I h hs prdoxil effet of inhiiting spiking 17 19,23. I h is purely exittory when it is the sole tive ondutne How n we explin the inhiitory effet of I h to redue the pek EPSP voltge t potentils tht re negtive to the I h reversl potentil? As disussed ove, lthough the depolrizing urrent rried y I h should mke V pek more positive, the shunting effet of the I h ondutne is expeted to derese V pek. To determine whether these two opposing effets of I h ould yield net inhiitory influene on V pek,wefirst exmined single-omprtment omputtionl model ontining only pssive lek ondutne, physiologilly relisti model of I h 1,17 nd liner exittory synpti ondutne modeled s n lph funtion 35 (Fig. 3). We determined the V pek tht ws ttined for different strengths of synpti input when either the mximl ondutne of I h or the voltge t whih I h ws hlf-mximlly tivted (V 1/2 ) ws vried ross rnge of physiologilly nd experimentlly relevnt vlues 1,17. When we inresed I h y inresing its mximl ondutne, the RMP ws shifted to more positive vlues, s expeted. Similrly, depolrizing shifts of V 1/2, whih lso inresed resting I h, lso depolrized the memrne (Fig. 3). Enhning I h y either method diminished the EPSP mplitude (DV EPSP ) for ll of the synpti Figure 3 Computtionl model predits exittory role for I h in the sene of other voltge-gted hnnels. () Digrm of the single-omprtment model ontining I h (HCN hnnels), liner lek ondutne nd synpti ondutne (lph funtion). () Effet of I h on RMP. Inresing mximl I h ondutne (ḡ h ) or depolrizing shifts of the I h V 1/2 produed positive shifts in RMP. () Effets of I h on EPSP mplitude, defined s the differene etween V pek nd RMP. Inresing ḡ h or depolrizing shifts of V 1/2 used redution in EPSP mplitude. Synpti strength ws set to produe n B8-mV EPSP in the sene of I h.(d) Smple EPSPs from our model for three synpti input strengths (ḡ syn in ms) in the sene of I h (lk tres) nd in the presene of I h for two levels of ḡ h (in units of S m 2 ). (e) The effet of I h on the reltionship etween V pek nd synpti strength (V 1/2 ¼ 9 mv in d nd e). Inresing ḡ h to the indited vlues hd depolrizing effet on V pek for ll memrne potentils elow E h ( 3 mv). The y interept shows the effet of I h on RMP. 9 8 Figure 2 Dul effets of I h on pek voltge of suthreshold EPSPs. () Representtive EPSP responses for three stimulus strengths reorded t the som under ontrol onditions (red) nd in the presene of ZD7288 (1 mm, lue). Top, I h hd depolrizing effet on V pek for smll EPSP tht ws eliited y wek stimulus. Middle, for n intermedite strength stimulus, V pek ws similr in the presene or sene of I h. Bottom, with stronger stimulus, I h hd n inhiitory effet nd its lokde thus led to more depolrized V pek.() Reltionship etween V pek nd stimulus urrent strength in the presene (ontrol, red) or sene (, lue) of I h.all EPSPs were suthreshold. The ma points (dshes) denote the resting potentil. Note tht I h hd n inhiitory effet on V pek over lrge rnge of suthreshold stimulus strengths. () V pek versus stimulus strength plot for ell in whih I h hd depolrizing effet on V pek for lrge rnge of wek stimuli. An inhiitory effet of I h ws only oserved for strong stimulus tht ws just suthreshold. strengths tht we exmined (Fig. 3). These results onfirmed previously reported findings for I 1,17,21 h. Despite the effet of I h to derese DV EPSP, the inrese in I h lwys hd depolrizing effet on the pek EPSP voltge s long s the memrne potentil ws negtive to the I h reversl potentil of 3 mv (Fig. 3d,e). As expeted, I h did shift V pek to more negtive potentils for very lrge synpti ondutnes tht drove V pek positive to E h, where I h provided n outwrd urrent tht hyperpolrized the memrne. Beuse the threshold for firing spike is negtive to 3 mv (typilly round mv), our results indite tht I h,inthesene of other voltge-gted ondutnes, should lwys e exittory for suthreshold EPSPs. This exittory effet of I h persisted even when its mximl ondutne ws vried 1-fold (Fig. 3e), mimiking the rnge of I h ondutnes tht hve een reported long the somtodendriti grdient in CA1 nd lyer V neoortil pyrmidl neurons 8 11,36. We lso oserved n exittory effet on V pek when the RMP (mv) V EPSP (mv) 4 V 1/2 mv mv 9 mv.5.1 (S m 2 ) Exittory synpti input Lek HCN.5 (S m 2 ) V 1/2 9 mv mv mv.1 e d E h g syn =.1 g syn =.25 g syn =.1 =.1 =.1 = NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 5 [ MAY

4 29 Nture Ameri, In. All rights reserved. d g syn =.1 54 g syn =.3 35 g syn = = =.1 = τ K = E h τ K = e mgnitude of I h ws inresed y shift in its voltge-dependent tivtion to more positive potentils (Supplementry Fig. 1 online). One well-hrterized effet of I h is to redue temporl summtion during urst of EPSPs, n effet tht hs een ttriuted to the tion of the I h ondutne to derese the memrne time onstnt nd to the hyperpolriztion used y the detivtion of I h during the urst of EPSPs 14,15. Our simultions onfirmed tht I h did derese the extent of temporl summtion during 1-Hz urst of five EPSPs. Even during the urst, however, the net effet of I h on memrne voltge ws still exittory, with the pek EPSP voltge during the urst rehing more positive potentils in the presene of I h thn in its sene (Supplementry Figs. 2 nd 3 online). Previous studies reported prtiulrly strong inhiitory effet of I h on EPSP pek voltge when its tion when its ility to depolrize the resting memrne is ompensted y injetion of hyperpolrizing urrent or redution in externl K + onentrtion 17 19,23,34. Suh results re not surprising, euse when hnges in resting potentil re prevented, the inhiitory effet of I h to enhne the memrne ondutne should predominte. Indeed, when we simulted this protool y djusting the lek ondutne reversl potentil to keep the resting potentil onstnt, n inrese in I h hd mrked inhiitory effet on V pek (Supplementry Fig. 1). The more importnt question, however, is how does I h exert its inhiitory effet on V pek when the resting potentil is free to dopt its intrinsi vlue, s ours under physiologil onditions. I h n inhiit EPSPs y intertions with K + urrent Our omputtionl results indite tht I h lone hs purely exittory effet on suthreshold EPSPs nd thus suggest tht the inhiitory effet of I h on the lrge EPSPs tht ws oserved in our experiments must involve n intertion with other voltge-gted ondutnes. One possiility tht we onsidered is tht the resting memrne M urrent Figure 4 Computtionl model inluding Hodgkin-Huxley voltge-gted K + ondutne predits suthreshold inhiitory effets of I h on pek voltge of strong EPSPs. () Model EPSPs for three synpti input strengths (ḡ syn )in the sene or presene of I h for two levels of ḡ h s indited (I h : V 1/2 ¼ 9 mv; I K : ḡ K ¼.36 S m 2, V 1/2 ¼ 52 mv, where I K is the delyedretifier K + urrent nd ḡ K is its mximl ondutne). () EffetofI h on the reltionship etween V pek nd synpti strength. Inreses in ḡ h hd depolrizing effet on V pek for wek synpti inputs nd n inhiitory effet for strong inputs. Notly, the inhiitory regime ourred negtive to the I h reversl potentil E h ( 3 mv). The rrow indites the rossover voltge t whih I h hnged from depolrizing to hyperpolrizing influene. () EffetsofI h on V pek when the kinetis of delyed-retifier K + ondutne tivtion were mde infinitely slow. (d) Effets of I h on V pek when the kinetis of delyed-retifier K + ondutne tivtion were mde infinitely fst. (e) Effets of I h on V pek in model ontining the mmmlin M-type K + urrent (V 1/2 ¼ 35 mv). Inresing ḡ h in this model lso produed mixed depolrizing nd hyperpolrizing effets on V pek with rossover voltge (rrow). depolriztion used y the presene of I h my inrese the tivtion of low-threshold voltge-gted K + ondutne nd tht this intertion my hve net inhiitory effet on the pek voltge of the EPSP. To test this, we extended our omputtionl model y dding Hodgkin-Huxley delyed-retifier K + ondutne 35,37 nd repeted our simultions. In the presene of the delyed-retifier K + ondutne, n inrese in I h lwys depolrized the resting memrne nd diminished EPSP mplitude (DV EPSP ), s seen in our single-ondutne model (Supplementry Fig. 4 online). For smll EPSPs, I h still exerted net depolrizing influene on V pek, s oserved in the model with I h lone. However, inlusion of the voltge-gted K + ondutne reveled ler inhiitory effet of I h on the pek voltge of lrger, ut still suthreshold, EPSPs (Fig. 4). This inhiitory effet ws oserved even when the pek EPSP voltge ws negtive to oth the reversl potentil of I h ( 3 mv) nd typil vlues for tion potentil threshold ( mv) (Fig. 4). Thus, these omputtionl results reprodued the mjor findings of our synpti stimultion experiments, inluding the presene of suthreshold rossover voltge t whih I h shifted from hving depolrizing effet on V pek to hving hyperpolrizing influene (Fig. 4). As oserved in the model with I h lone, when the resting potentil ws held fixed (y ltering the lek ondutne reversl potentil), I h hd purely inhiitory effet, reduing V pek t ll synpti strengths (Supplementry Fig. 4). Whih iophysil properties of the voltge-gted K + ondutne re required to enle the suthreshold inhiitory effets of I h?wefirst exmined the importne of the K + ondutne kinetis y mking the rte of tivtion infinitely slow, suh tht the K + ondutne remined t its initil equilirium vlue set y the resting potentil during the entire time ourse of the EPSP. Under these onditions, I h still exerted dul depolrizing/hyperpolrizing influene (Fig. 4), inresing V pek for smll EPSPS, ut reduing V pek for lrger suthreshold EPSPs. In ontrst, when we mde the tivtion rte infinitely fst, so tht the K + ondutne ttined its stedy-stte level of tivtion instntneously throughout the EPSP, I h now exerted purely exittory effet (Fig. 4d), shifting V pek to more depolrized vlues for ll suthreshold EPSP sizes. Thus, the ility of I h to inhiit V pek requires tht the I h -dependent enhnement in stedy-stte resting K + ondutne persists throughout the EPSP. Next, we exmined how shifting the stedy-stte voltge dependene of K + urrent tivtion ffets the ility of I h to influene the EPSP. We found tht the rossover voltge t whih I h hnges from hving depolrizing influene on V pek to hving hyperpolrizing influene eme more negtive s the voltge dependene of K + ondutne 58 VOLUME 12 [ NUMBER 5 [ MAY 29 NATURE NEUROSCIENCE

5 29 Nture Ameri, In. All rights reserved. 2 I M V 1/ g M tivtion ws shifted to more hyperpolrized potentils. Conversely, depolrizing shifts in K + urrent tivtion properties moved the rossover voltge to more positive potentils (dt not shown). Notly, suthreshold rossover voltge, nd hene n inhiitory effet of I h, ws oserved over wide voltge rnge of K + urrent tivtion prmeters, inditing roust effet. I h interts with the M-type K + urrent to inhiit EPSPs Beuse our omputtionl results relied on the squid xon K + ondutne, it ws importnt to explore whether model inorporting mmmlin voltge-gted K + ondutne ould lso intert ppropritely with I h to yield suthreshold inhiitory effets. We resoned tht the K V 7 M-type K + urrent ws good ndidte for mediting the inhiitory effets of I h, s the M urrent is present in CA1 pyrmidl neurons, tivtes t suthreshold voltges, hs slow time ourse of tivtion nd shows non-intivting gting properties We therefore exmined model ontining I h, pssive lek ondutne, n exittory synpti input nd model of the M urrent sed on previous studies in mmmlin pyrmidl neurons 4,42,43. We found tht the M urrent lso enled I h to exert dul depolrizing/hyperpolrizing effet on the pek voltge of suthreshold EPSPs. As oserved with the Hodgkin-Huxley K + ondutne model, I h interted with M urrent to depolrize the pek voltge of wek EPSPs nd to hyperpolrize V pek for stronger, ut still suthreshold, EPSPs (Fig. 4e). Also similr to the Hodgkin-Huxley K + ondutne, shifts in the V 1/2 of M-urrent tivtion (Fig. 5,) orhngesin M-urrent mximl ondutne (Fig. 5,d) ltered the rossover voltge nd synpti strength t whih I h egn to exert n inhiitory influene. In the presene of M urrent, I h ws lso le to exert net inhiitory effet on pek voltge during urst of strong EPSPs, s oserved previously 14,15, lthough the influene of I h y the finl EPSP ws smll s result of its detivtion during the urst (Supplementry Figs. 2 nd 3). In ontrst with the rossover in the EPSP input-output urves in response to hnges in I h, omprison of EPSP input-output urves with or without M urrent reveled tht this K + urrent exerted purely inhiitory effet, shifting pek EPSP voltge to more negtive potentils t ll synpti strengths. This inhiitory tion of M urrent ws seen in either the sene or presene of fixed level of I h (dt not shown). Suh n effet is in ord with previous results showing tht the M urrent onsistently inhiits neurl tivity 4,41. V pek t rossover (mv) d V pek t rossover (mv) Exittory I M V 1/2 (mv) Inhiitory Exittory g M (S m 2 ) Inhiitory Figure 5 Computtionl results demonstrting tht hnges in M-urrent properties lter the rossover voltge t whih I h first exerts n inhiitory influene on EPSP V pek.() The effet of I h on the reltionship etween V pek nd synpti strength depends on M-urrent V 1/2 vlues (the mximl ondutne for I M equls.35 S m 2 throughout). The rossover voltge in response to n inrese in ḡ h ws shifted to more negtive vlues s M-urrent V 1/2 ws mde more negtive. () The effets of M-urrent V 1/2 vlues on synpti ondutne mgnitude (ornge line, right xis) or V pek (lue line, left xis) t whih rossover ourred (from dt in ). Regions ove the lue or ornge lines orrespond to V pek or synpti strength vlues t whih I h ws inhiitory; regions elow the line orrespond to V pek or synpti strengths for whih I h depolrized V pek.() The effet of I h on the reltionship etween V pek nd synpti strength depends on M-urrent mximl ondutne vlues (in S m 2 ;M-urrentV 1/2 ¼ 35 mv). The rossover voltge in response to n inrese in ḡ h ws shifted to more negtive potentils s I M ws inresed. (d) The effets of vrying the mximl M-ondutne on V pek (lue line) or synpti ondutne (ornge line) t whih rossover ourred. So fr we hve onsidered the intertion of I h nd M urrent in the ontext of single-omprtment model. However, I h is present in grdient of inresing density long the pil dendriti tree of oth CA1 nd lyer 5 pyrmidl neurons, where I h density t the distl tips of the dendrites is up to 5-fold lrger thn tht in the som 8,9,11.In ontrst, the preise suellulr loliztion of the M-type K + hnnels is less ler, with some studies reporting dendriti M urrents 38,41 nd others liming only somti nd/or xonl loliztion 4,44.Toexmine the importne of the suellulr loliztion of these hnnels, we inorported the I h dendriti grdient into multiomprtment model of CA1 neuron in whih exittory inputs were trgeted to the pil dendrites. Notly, n inhiitory effet of I h on the somti pek EPSP voltge ws still oserved, regrdless of whether M urrent ws present in dendrites or restrited to the som (Supplementry Fig. 5 online). When M urrent ws restrited to the som, dendriti I h exerted purely depolrizing effet on the lol dendriti EPSPs reorded t the site of synpti input, 25 mm from the som. However, the dendriti I h still ws le to inhiit the pek somti voltge for lrge, suthreshold EPSPs. Conversely, in multiomprtment models lking n M urrent, I h produed purely depolrizing effet on oth lol dendriti EPSPs nd somti EPSPs (Supplementry Fig. 5). These results indite tht the inhiitory effets of I h on somti EPSPs require the presene of M-type K + hnnels, ut re not very sensitive to the suellulr distriution of either I h or M urrent. Inhiitory effets of I h re prevented y M-urrent lokde We used n experimentl pproh to exmine whether the inhiitory effets of I h in CA1 neurons do indeed rise from its intertion with the M urrent y pplying the speifi M-urrent inhiitor XE991 3,39,4 t onentrtion (1 mm) tht does not lter synpti trnsmission under the onditions of our experiments 44. Applition of XE991 produed vrile depolriztion of the resting memrne tht ws lrge enough to led to spontneous firing in some CA1 neurons, onsistent with the inhiitory role of the M urrent. Suh ells were not studied further euse the spiking interfered with EPSP mesurements. In those ells tht did not fire spontneously, XE991 produed reltively smll 3.4 mv (P ¼.1, n ¼ 7) depolriztion of the resting memrne. We next exmined the effets of I h on somti EPSPs with M urrent loked y mesuring V pek s funtion of stimulus strength, first in the presene of I h nd then following I h lokde with ZD7288. Addition of ZD7288 in the presene of XE991 hyperpolrized the resting memrne y B8 mv (RMP with XE991 lone ¼ 65.5 ± 1.7 mv, RMP with XE991 nd ZD7288 ¼ 73.5 ± 1.8 mv, n ¼ 7, NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 5 [ MAY

6 Vm (mv) ARTICLES 65-µA stimulus XE XE + ZD µA stimulus µA stimulus Figure 6 Phrmologil lokde of M urrent used I h to hve purely exittory influene. () Experimentl EPSPs reorded t the som in response to three stimulus strengths when M urrent ws loked with XE991 (1 mm). EPSPs shown in XE991 (XE, red tres) nd XE991 with 1 mm ZD7288 (XE + ZD, lue tres). The presene of I h hd n exittory effet on V pek for wek (left), intermedite (middle) nd strong (right) stimuli. Note tht the strongest stimulus evoked spike in the presene of I h, ut not in its sene. () I h hd n exittory effet on the reltionship etween V pek nd stimulus strength with M urrent loked y XE991. Dt re shown in the sene (red) or presene (lue) of ZD7288. The ma dt (dshes) denote the RMP. () Similr results were otined in seond ell s in. 29 Nture Ameri, In. All rights reserved. XE XE + ZD Stimulus strength (µa) Stimulus strength (µa) P o.1, pired t test), similr to its effet in the sene of XE991. Of prtiulr interest, lokde of the M urrent olished the inhiitory effet of I h on pek EPSP voltge. In the present of XE991, I h exerted purely exittory effet on V pek for oth wek nd strong stimuli (Fig. 6 ; ompre with Fig. 2, with norml M urrent). This result ws seen in ll of the ells tested (n ¼ 7). In ddition, when M urrent ws loked, the presene of I h now inresed the exitility of the ell, s evidened y spike firing t lower stimulus strengths in the sene of ZD7288 thn in its presene (Fig. 6). These findings onfirm our modeling results nd further support the ide tht I h lone exerts n exittory influene on neuronl tivity nd tht the inhiitory effets of I h require n intertion with voltge-gted K + urrents. DISCUSSION We found tht I h exerted dul depolrizing/hyperpolrizing effets on the pek voltge of suthreshold EPSPs s funtion of synpti strength. For wek EPSPs, I h exerted depolrizing effet on V pek. In ontrst, for strong EPSPs, I h exerted n inhiitory, hyperpolrizing effet. Although previous studies hve desried n inhiitory influene of I h on EPSP mplitude (DV EPSP ) nd firing of oth N + nd C 2+ spikes 1,15,17,19,23,26, our results provide, to the est of our knowledge, the first demonstrtion tht I h n lso exert n inhiitory effet on suthreshold pek EPSP voltge. This is n importnt distintion euse pek EPSP voltge, rther thn EPSP mplitude, determines the effet of the EPSP on neuronl firing 25. Our results lso provide insight into the mehnism of the prdoxil effet y whih the depolrizing inwrd I h n produe net hyperpolrizing effet on pek EPSP voltge. First, using simple omputtionl model, we found tht I h ting in the sene of other voltge-gted hnnels exerted purely depolrizing effet on the pek memrne potentil hieved y suthreshold EPSPs. This indites tht the diret exittory effet of I h to depolrize the memrne predomintes over its inhiitory effet to inrese resting memrne ondutne. This diret exittory effet of I h lso underlies its lssil ontriution to the pemker depolriztion tht genertes rhythmi firing in oth rdi myoytes nd ertin CNS neurons, suh s thlmi rely neurons 12,13. In ontrst with the results of our simple model in whih I h is purely exittory, we found tht I h produed n inhiitory effet on V pek of lrge, suthreshold EPSPs in models ontining delyed-retifier 7 voltge-gted K + hnnels. The exittory-to-inhiitory rossover effet of I h on pek EPSP voltge tht ourred in suh models provides n exmple of the nonliner interply of voltge-dependent urrents. The depolriztion of the resting memrne y I h enhned the resting voltge-gted K + ondutne eyond tht ttined in the sene of I h. At the pek of wek EPSP, the outwrd driving fore on K + ws quite smll ompred with the lrge inwrd driving fore on urrent through I h hnnels, using the diret depolrizing effet of I h to e dominnt. In ontrst, t the pek of lrge EPSPs, the outwrd driving fore on K + ws inresed nd the inwrd driving fore on I h ws deresed. As result, the inhiitory effet of the K + urrent ws dominnt. Our omputtionl results further suggest tht the inhiitory effet of I h requires tht the K + urrent kinetis e reltively slow ompred with the time ourse of the EPSP. This llows the I h -medited inrese in resting K + ondutne to persist throughout the EPSP nd thus inhiit EPSP pek voltge. Both our experimentl nd omputtionl findings implite the M-type K + urrent s eing the prole meditor of the inhiitory effets of I h in CA1 pyrmidl neurons. The reltively negtive voltge rnge of tivtion of the M urrent llows it to respond to the smll hnges in resting potentil medited y I h ; the slow tivtion kinetis of the M urrent ensure tht suh hnges in resting tivtion influene the pek EPSP voltge 3,38,4,41. Moreover, in experiments in whih we loked the M urrent with XE991, I h exerted purely exittory effet, onfirming tht M urrent is neessry for the inhiitory effet of I h. One other importnt omputtionl result is tht the inhiitory effet on somti pek EPSP voltge used y the intertion of I h nd M urrent does not depend on hnnel distriutions in the somtodendriti omprtments. This is importnt, s I h is known to e present in grdient of inresing density in pil dendrites, wheres it is unler whether M urrent is restrited to xosomti omprtments or is present in dendrites 38,4,41. In ontrst to the dul depolrizing/hyperpolrizing effets of I h on V pek, ll mnipultions tht inresed the M urrent, whether in the sene or presene of I h, hyperpolrized V pek with no rossover effet. This is onsistent with lrge numer of previous studies tht hve shown n inhiitory influene of the M urrent 4,41.The purely inhiitory nture of the M urrent rises euse its two tions to enhne memrne ondutne nd to generte hyperpolrizing outwrd urrent oth t in the sme diretion to inhiit pek EPSP voltge nd neuronl firing. This is in ontrst to I h, whose diret depolrizing nd shunting effets hve opposing influenes. The ft tht the M urrent n e modulted y neurotrnsmitters nd seond messenger sdes rises the possiility tht the mode of tion of I h on dendriti integrtion n e tuned from inhiition to exittion. For exmple, the loss of M urrent tht ours on musrini reeptor stimultion 45 nd phosphtidylinositol (4,5)- isphosphte depletion 46 should drive I h into purely exittory 582 VOLUME 12 [ NUMBER 5 [ MAY 29 NATURE NEUROSCIENCE

7 29 Nture Ameri, In. All rights reserved. mode of tion on V pek. In ontrst, the lrge inrese in M urrent tht ours in response to n inrese in AMP 32 should use I h to exert predominntly inhiitory effet on V pek (Fig. 5). As hnges in oth the M urrent nd I h re thought to our in epilepti diseses 24,29,31,32, n understnding of how these two urrents intert to regulte exitility my e importnt for the development of new therpeuti pprohes. The dul depolrizing/hyperpolrizing effets of I h on pek EPSP voltge hve interesting implitions for how this urrent my differentilly regulte neuronl firing depending on the stte of exitility of neuron. Under onditions in whih spike threshold is low nd negtive to the I h rossover voltge for inhiition, mnipultions tht enhne I h will inrese V pek for suthreshold EPSPs nd will thus hve n exittory effet on the ility of n EPSP to trigger n tion potentil. In ontrst, when spike threshold is high nd positive to the rossover voltge, mnipultions tht enhne I h will derese V pek for EPSPs ner threshold nd will thus inhiit the ility of n EPSP to eliit n output. Therefore, the polrity of the effet tht hnge in I h exerts on neuronl firing will depend on the overll exitility of the ell. Even if I h nd the M urrent remin onstnt, the effet of their intertion on neuronl output n shift from exittory to inhiitory s result of modultory hnges in other voltge-gted ondutnes tht lter spike threshold. Suh nonliner suthreshold intertions mong voltge-gted hnnels provide rih vriety of mehnisms for fine-tuning the reltionship etween exittory synpti input nd neuronl output. METHODS Slie eletrophysiology. Whole-ell reordings were otined from hippompl CA1 pyrmidl ells in sumerged horizontl rin slies from postntl dy 28 4 mie. Reordings were rried out t C with inhiitory trnsmission eing loked y GABA A (2 mm gzine) nd GABA B reeptor ntgonists (1 mm CGP-55845). Stimulting urrent pulses (.1.2 ms) were pplied through fol extrellulr eletrodes with onstnt urrent genertor one every 15 s. For grded stimultion, urrent mplitude ws djusted to evoke n EPSP in ontrol onditions nd then inremented until spike threshold ws rehed. Identil urrent pulses were repplied fter ddition of 1 mm ZD7288tolokI h. All proedures onformed to US Ntionl Institutes of Helth regultions nd were pproved y the Institutionl Animl Cre nd Use Committees of Columi University nd the New York Stte Psyhitri Institute. See Supplementry Methods online for full detils. Sttistil nlysis. The verge sg rtio ws expressed s [(1 DV ss / DV min ) 1%], where DV ss ¼ RMP V ss, DV min ¼ RMP V min, V ss is the stedy-stte potentil nd V min is the initil minimum potentil. Comprisons were mde using pired t tests where pproprite. An unpired t test ws used to ompre ontrol RMP with the RMP in the presene of XE991. P o.5 ws onsidered to e sttistilly signifint. Results re expressed s men ± s.e.m. Computtionl modeling. Computtionl models were implemented nd run in NEURON 47 (version 5.9; The I h 1,17 nd M-type K + ondutne 4,42,43 models were sed on experimentl studies. See Supplementry Methods for full detils. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk J. Dudmn for helpful dvie nd for providing ustom dtquisition routines written in Igor. This work ws prtilly supported y grnt MH8745 from the US Ntionl Institutes of Helth to S.A.S. L.A. nd M.S.G. were supported in prt y US Ntionl Institutes of Helth Diretor s Pioneer Awrd, prt of the US Ntionl Institutes of Helth Rodmp for Medil Reserh, through grnt numer 5-DP1-OD M.S.G. ws supported in prt y Columi University s Medil Sientist Trining Progrm. AUTHOR CONTRIBUTIONS M.S.G. performed ll experiments, wrote the omputer progrms, rried out the nlysis nd wrote the initil drft of the mnusript. 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