Pharmacological upregulation of h-channels reduces the excitability of pyramidal neuron dendrites

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1 Phrmcologicl upregultion of h-chnnels reduces the excitility of pyrmidl neuron dendrites Nichols P. Poolos 1 3, Michele Migliore 4,5 nd Dniel Johnston 1 1 Division of Neuroscience nd 2 Deprtment of Neurology, Bylor College of Medicine, One Bylor Plz, Houston, Texs 77030, USA 3 Current ddress: Deprtment of Neurology nd Regionl Epilepsy Center, University of Wshington, 325 9th Avenue, Settle, Wshington 98104, USA 4 Yle University School of Medicine, Section of Neuroiology, New Hven, Connecticut 06520, USA 5 Permnent ddress: Ntionl Reserch Council, Institute of Advnced Dignostic Methodologies, Plermo 90146, Itly Correspondence should e ddressed to D.J. (dn@ltp.neusc.cm.tmc.edu) Pulished online: 15 July 2002, doi: /nn891 The dendrites of pyrmidl neurons hve mrkedly different electricl properties from those of the som, owing to the non-uniform distriution of voltge-gted ion chnnels in dendrites. It is thus possile tht drugs cting on ion chnnels might preferentilly lter dendritic, ut not somtic, excitility. Using dendritic nd somtic whole-cell nd cell-ttched recordings in rt hippocmpl slices, we found tht the nticonvulsnt lmotrigine selectively reduced ction potentil firing from dendritic depolriztion, while minimlly ffecting firing t the som. This regionl nd inputspecific effect resulted from n increse in the hyperpolriztion-ctivted ction current (I h ), voltge-gted current present predominntly in dendrites. These results demonstrte tht neuronl excitility cn e ltered y drugs cting selectively on dendrites, nd suggest n importnt role for I h in controlling dendritic excitility nd epileptogenesis. Mny centrl nervous system (CNS) neurons hve extensively rorized dendrites on which they receive the mjority of their synptic contcts. Recent dvnces in electrophysiologicl techniques hve shown tht the picl dendrites of hippocmpl nd neocorticl pyrmidl neurons hve mrkedly different electricl properties from those of their corresponding somt, nd these differing properties re due to non-uniform distriutions nd kinetics of voltge-gted chnnels. For exmple, in hippocmpl pyrmidl neurons, the A-type trnsient K + current (I A ) nd the hyperpolriztion-ctivted ction current (I h ; h-chnnel) re present in the dendrites t mny-fold higher densities thn they re in the som 1 5. The nonuniform distriution of voltge-gted chnnels ffect signl processing in the dendrites, ltering the retrogrde propgtion (or ck-propgtion ) of ction potentils (APs), nd the integrtion of synptic potentils. We resoned tht some drugs tht ffect ion chnnels might ct preferentilly on dendritic voltge-gted chnnels, nd thus selectively lter excitility in one region of the neuron. One such clss of drugs consists of nticonvulsnts known to ct on N + chnnels, nd includes phenytoin (PHT), crmzepine (CBZ) nd lmotrigine (LTG). LTG is structurlly novel nticonvulsnt tht is cliniclly effective on oth prtilonset nd primrily generlized seizures, s well s in psychitric illnesses such s ipolr disorder 6,7. Here we consider whether these drugs tht ct on the excitility of CNS neurons s studied with somtic recordings 8 might lso hve some unforeseen effects on dendritic excitility. We found tht LTG hd selective effect on the excitility of the dendrites of hip- pocmpl pyrmidl neurons, mrkedly reducing AP firing when initited from dendritic depolriztion, ut minimlly ffecting APs initited from somtic depolriztion. This effect on dendritic excitility ws not due to ction on N + chnnels, ut rther to n increse in I h, voltge-gted current present in high density in the dendrites. These results show tht drug cn ffect excitility nd AP firing regionlly within neuron, nd provide evidence tht I h is centrlly involved in modulting neuronl excitility nd thus my influence epileptogenesis. RESULTS Actions of nticonvulsnts on dendritic excitility Using somtic nd dendritic recordings in hippocmpl CA1 pyrmidl neurons, we tested the hypothesis tht nticonvulsnt drugs differentilly ffect the excitility of dendrites nd somt. Bth ppliction of LTG during prolonged current injection t the som cused modest reduction of repetitive AP firing (Fig. 1), consistent with previous findings 9,10. When similr rte of AP firing ws elicited from dendritic injection site, however, LTG ppliction mrkedly reduced or olished AP firing (Fig. 1). Injection of rnge of current steps tht produced 5 20 APs in 500 ms under control conditions (Fig. 1c) showed tht LTG olished AP firing for ll ut the highest mplitude current injections in tht series. LTG hd disproportionte effect on AP firing elicited from dendritic depolriztion s compred to tht from the som. The effect of LTG on dendritic AP firing ws reversile with prolonged (>30 min) return to control solution (dt not shown). nture neuroscience volume 5 no 8 ugust

2 c We similrly tested the effects of PHT nd CBZ on dendritic excitility. Bth ppliction of PHT (Fig. 2) or CBZ (Fig. 2) did not significntly ffect AP firing elicited from dendritic current injection. We quntified the effect of LTG, PHT nd CBZ on AP firing y verging the numer of APs elicited y series of four 500-ms current injections of progressively grded mplitude producing 5 20 APs under control conditions, then repeting these sme current injections under drug conditions (Fig. 2c). LTG ws found to reduce AP firing from dendritic current injection to 17 ± 5.7% of control vlues (n = 8), compred to much smller reduction of AP firing elicited t the som (83 ± 9.3% of control, n = 6; P < compred to vlue t the dendrites). In contrst, PHT nd CBZ hd no significnt effect on AP firing from dendritic depolriztion Fig. 1. Lmotrigine (LTG) selectively lowered dendritic excitility. () Action potentil (AP) firing elicited y current injection t the som under control conditions (Ctrl) ws modestly reduced y th ppliction of LTG (50 µm). Resting potentil, 68 mv. () AP firing elicited from dendritic current injection under control conditions ws olished y LTG (50 µm). Resting potentil, 64 mv. Dendritic recording distnce, 190 µm. (c) Dendritic current injections ( pa for 500 ms), which under control conditions produced 5 20 APs, led to little or no AP firing in the presence of LTG (100 µm). Resting potentil, 60 mv. Dendritic recording distnce, 180 µm. (PHT, 99 ± 4.5% of control, n = 3, P > 0.05; CBZ, 99 ± 6.2%, n = 3, P > 0.05). In ddition to ffecting AP firing from the dendrites, th ppliction of LTG cused depolriztion of resting potentil which ws concentrtion-dependent. This effect ws similr in the som nd dendrites (50 µm LTG, 3.1 ± 0.26 mv, n = 13; 100 µm LTG, 5.1 ± 0.78 mv, n = 9). In quntifying the effect of LTG on AP firing, resting potentil ws compensted to the originl pre-drug level. When resting potentil ws not compensted, the effect of LTG on AP firing from dendritic current injection ws somewht reduced ut still significntly different from control (Fig. 2d, top trces; AP firing, 53 ± 6.5% of control, n = 6, P < 0.001). In contrst, LTG hd no significnt effect on AP firing from somtic current injection when resting potentil ws uncompensted (Fig. 2d, ottom trces; 99 ± 13% of control, n = 4, P > 0.05). Thus, the stedy-stte depolriztion induced y LTG reduced the mgnitude of the effect of LTG on AP firing y moving resting potentil closer to threshold. However, even when this resting potentil chnge ws uncompensted, LTG continued to selectively ffect AP firing elicited in the dendrites. LTG hs een shown to ffect N + currents in usedependent mnner 9,10. We therefore explored whether the effect of LTG on APs recorded in dendrites might e due to reduction of the mplitudes of ck-propgting APs. Bckpropgting APs ntidromiclly ctivted y 50-Hz trins of stimultion under control conditions showed progressive decline c Fig. 2. LTG, ut not phenytoin or crmzepine, lowered dendritic excitility. () AP firing from dendritic current injection ws minimlly ffected y th ppliction of phenytoin (PHT, 50 µm). Resting potentil, 62 mv. Dendritic recording distnce, 200 µm. () Crmzepine (CBZ, 50 µm) similrly hd miniml effect on AP firing from dendritic current injection. Resting potentil, 67 mv. Dendritic recording distnce, 210 µm. (c) Group dt showing the selective effect of LTG on dendritic AP firing compred to its effect on somtic AP firing, nd the miniml effects of PHT nd CBZ on dendritic AP firing. Drug concentrtions nd numer of oservtions for ech condition: LTG dend, µm, n =8; LTG som, µm, n = 5; PHT dend, µm, n = 3; CBZ dend, µm, n = 3. (d) Dendritic AP firing shown in response to 200-pA current injection under control conditions (top trces) ws reduced y 50% in the presence of LTG (100 µm) when resting potentil ws not compensted to control levels. Resting potentil, 61 mv (Ctrl) nd 58 mv (LTG). Dendritic recording distnce, 180 µm. When the sme experiment ws done using somtic current injection (ottom trces), LTG (100 µm) hd no significnt effect on AP firing. Resting potentil, 66 mv (Ctrl) nd 61 mv (LTG). (e) Bck-propgting APs in the dendrites elicited y 50-Hz ntidromic stimultion under control conditions were not ffected y ddition of LTG (50 µm). Resting potentil, 60 mv. Dendritic recording distnce, 220 µm. d e 768 nture neuroscience volume 5 no 8 ugust 2002

3 Fig. 3. LTG decresed AP firing elicited from the dendrites without ffecting AP ck-propgtion in dendrites. () Dul simultneous whole-cell recordings from som nd dendrites with dendritic current injection. Under control conditions, APs elicited from dendritic current injection initited ner the somtic recording site, then ck-propgted to the dendritic recording site, s shown y expnded trces with the first APs from som nd dendrites (inset). In the presence of LTG (100 µm), AP firing from dendritic current injection ws reduced, ut APs continued to initite ner the somtic recording site nd ck-propgte into the dendrites. Dendritic recording distnce, 165 µm. Resting potentil for ll trces, 64 mv. Clirtion for insets is sme s shown for Fig. 3, ut with voltge 40 mv nd time 12.5 ms. () Sme recording s in (), ut with somtic current injection. LTG (100 µm) decresed AP firing elicited from the som to much lesser extent thn when elicited from the dendrites, nd AP ck-propgtion remined unffected. APs continued to initite ner the somtic recording site (inset). in mplitude ecuse of the slow inctivtion of N + currents 11 (Fig. 2e). LTG (50 µm) did not significntly ffect the mplitudes of ntidromic APs throughout the trin, including the first or lst APs in the trin. The mplitude of the first ck-propgting AP in response to 50-Hz ntidromic ctivtion in the presence of LTG ( µm) ws 97.3 ± 1.9 % (n = 6) of control AP mplitude. Under control conditions, the mplitude of the tenth AP in the trin decremented to 39.2 ± 3.4% (n = 6) of the mplitude of the first AP, while in the presence of LTG the tenth AP ws 40.0 ± 4.5% (n = 6) of the first AP mplitude in LTG (not significntly different from control, P > 0.05). These dt show tht LTG did not ffect the mplitude of ck-propgting APs in the dendrites, nor their decrement due to dendritic N + current entering into slow inctivted stte. Becuse the mplitudes of ck-propgting APs in the dendrites re sensitive to smll chnges in N + currents 11, these results suggest tht the ction of LTG on reducing AP firing elicited in the dendrites ws not due to n effect on AP ck-propgtion per se, nd ws independent of ctions on N + chnnels. To explin how LTG could drmticlly ffect AP initition from dendritic current injection while minimlly ffecting APs elicited from the som, we simultneously mde whole-cell recordings from the som nd distl dendrites. When current ws injected into the dendrites under control conditions, APs initited ner the somtic recording site (s determined y compring the ltency to onset of dendritic nd somtic APs; Fig. 3, inset), nd then ck-propgted into the dendrites (Fig. 3, Ctrl), consistent with prior studies 12,13. In the presence of LTG, c d Fig. 4. LTG decresed dendritic excitility y incresing ctivtion of I h. () Whole-cell dendritic recordings showing suthreshold response to oth hyperpolrizing nd depolrizing current injection. Under control conditions (Ctrl), slow relxtion of memrne potentil ( sg ) in response to oth hyperpolrizing nd depolrizing current injection demonstrted the presence of I h. In the presence of LTG (50 µm), the sg ws incresed in response to oth depolrizing nd hyperpolrizing current injection. Resting potentil, 64 mv. Dendritic recording distnce, 190 µm. () Appliction of ZD-7288 (ZD, 10 µm), specific locker of I h, locked the sg in memrne potentil nd mrkedly incresed input resistnce in response to current injection under control conditions, producing n opposing effect on dendritic excitility compred to LTG. Resting potentil, 70 mv. Dendritic recording distnce, 240 µm. (c) Appliction of ZD-7288 (10 µm) cused n initilly suthreshold dendritic current injection (left trces, Ctrl) to produce repetitive AP firing (left trces, ZD). Susequent ppliction of LTG (50 µm) produced miniml effect on dendritic AP firing in the presence of I h lockde y ZD (left trces, ZD/LTG). Similrly, the voltge sg produced y I h with suthreshold hyperpolrizing nd depolrizing current injections under control conditions (right trces, Ctrl) ws locked y ZD-7288 (right trces, ZD). Adding LTG (50 µm) hd no effect on the suthreshold response in the presence of ZD (right trces, ZD/LTG). Resting potentil, 68 mv. Dendritic recording distnce, 180 µm. (d) Group dt showing tht the decrese in dendritic AP firing cused y LTG ws lrgely locked y previous ppliction of ZD-7288 (ZD/LTG). Drug concentrtions nd numer of oservtions for ech condition: LTG, µm, n = 8; ZD/LTG, 50 µm LTG nd 10 µm ZD-7288, n =5. nture neuroscience volume 5 no 8 ugust

4 Fig. 5. LTG cuses depolrizing shift in I h ctivtion. () Dendritic cell-ttched ptch recordings of I h. Hyperpolrizing voltge commnds (V c, leled t right of current trces) from 45 mv holding potentil produced slowly ctivting inwrd currents chrcteristic of I h. Dendritic recording distnce, 210 µm. () I h recorded from dendrites ws mrkedly lrger thn tht recorded t the som. Current trces shown re with holding potentil of 45 mv nd voltge commnd of 115 mv, nd re from different neurons; dendritic recording distnce, 200 µm. (c) I h ctivtion from dendritic cell-ttched ptch recordings. Addition of LTG (100 µm) to the pipette solution produced n 11 mv depolrizing shift in I h ctivtion (LTG) compred to control conditions (Ctrl; V 1/2 roughly 83 mv; V 1/2 in LTG, roughly 72 mv). Numer of oservtions for ech point in ctivtion curves: control, n =7; LTG, n =6. (d) Representtive I h from voltge commnds ner rest in neuron under control conditions (Ctrl) nd seprte neuron with LTG (100 µm) in the recording pipette; to fcilitte comprison, control current trce ws scled such tht mximl I h ws the sme in oth control nd LTG conditions. AP firing ws significntly reduced; however, APs still initited ner the somtic recording site nd ck-propgted into the dendrites (Fig. 3, LTG). When current ws insted injected t the som (Fig. 3), APs continued to initite ner the som (Fig. 3, inset) ut LTG hd much smller effect on AP firing rte, nd did not ffect the ck-propgtion of APs into the dendrites. (Note tht the initil dendritic AP in the trin ws of similr mplitude in oth control nd drug conditions, ut tht the mplitude of susequent APs vried with firing frequency, reflecting decrement due to frequency-dependent slow inctivtion of N + chnnels 11.) These results confirmed tht LTG ws c d not ltering the ck-propgtion of APs into the dendrites, ut insted ws decresing the trnsmission of current injected from the dendrites. LTG ffects suthreshold response of dendrites Prolonged hyperpolrizing nd depolrizing suthreshold current pulses injected into the dendrites under control conditions produced voltge wveforms with slowly ctivting relxtion (or sg ) tht indicted the presence of the I h current 14,15. I h is slowly ctivting, non-inctivting inwrd current which is ctive t resting potentil nd increses its ctivtion with hyperpo- Fig. 6. I h ctivtion decresed AP firing in computtionl model. () Simulted AP firing in the dendrites (upper trces) nd t the som (lower trces) in response to dendritic current injection. Responses re shown under control conditions with resting potentil (rp) set t 70 mv, nd during incresed I h ctivtion (10-mV shift) with rp set t 70 mv or 65 mv to simulte the depolrizing ction of 10-mV shift in I h ctivtion. A simulted increse in I h ctivtion olished AP firing from dendritic current injection when resting potentil ws held t control levels, nd produced significnt decrese in AP firing when resting potentil ws llowed to depolrize to 65 mv. () Sme conditions s in (), ut with somtic current injection. A simulted increse in I h ctivtion produced much smller effect on AP firing from somtic current injection thn with dendritic current injection when resting potentil ws held t 70 mv. When resting potentil ws held t 65 mv, incresed I h ctivtion no longer reduced AP firing from somtic current injection. Dendritic distnce, 200 µm. (c) Simulted decy of voltge trnsients in response to 700-pA step current injection t 350 µm in the dendrites. When resting potentil ws held t 70 mv, the stedy-stte voltge recorded in the dendrites under control conditions (Ctrl; solid red line) ws mrkedly reduced y 10-mV shift in I h ctivtion (LTG; solid lue line). The corresponding responses t the som (dshed lines) show n even lrger proportionl reduction in the stedy-stte voltge. (d) Sme responses s in (c), ut with resting potentil held t 65 mv to simulte the depolriztion occurring with 10-mV shift in I h ctivtion. Under these conditions, the stedy-stte memrne potentil t the som resulting from current injection in the dendrites ws still reduced compred to control. (e) Plot shows the stedy-stte depolriztion from resting potentil occurring long the dendrite produced y c d e 650-pA current injection t 350 µm in the dendrites. Solid lines show depolriztion under either control (Ctrl) or incresed I h ctivtion conditions (LTG; 10-mV shift) normlized to the pek depolriztion t the site of current injection under control conditions. Dshed line shows depolriztion during incresed I h ctivtion normlized to pek depolriztion in tht condition. Resting potentil, 70 mv in ll trces. 770 nture neuroscience volume 5 no 8 ugust 2002

5 Fig. 7. LTG decresed synptic ctivtion of pyrmidl neurons y reducing temporl summtion in frequency-dependent mnner. () Simulted somtic AP firing in response to synptic stimultion in the dendrites. At low stimulus frequencies (<5 Hz), AP firing rte ws modestly ffected y incresed I h ctivtion (10-mV shift; LTG). As the stimulus rte incresed, AP firing rte under control conditions incresed rpidly due to temporl summtion, ut incresed t much slower rte with incresed I h ctivtion, with the gretest effect of incresed I h occurring etween 10 nd 30 Hz. At higher frequencies (>30 Hz), the effect of incresed I h diminished. Trces t right show smple AP firing in control nd incresed I h conditions t 4-, 10- nd 40-Hz stimultion rtes. Resting potentil, 65 mv for ll trces. Clirtion is 20 mv, 100 ms. () Experimentl dt showing superimposed dendritic responses to current injection of lph wveforms (upper trces) under control (red trces) nd LTG (100 µm; lue trces) conditions. LTG hd miniml effect on 5-Hz trin of stimuli, ut decresed temporl summtion for 20-Hz trin (rrowhed). Dendritic recording distnce: 5 Hz, 240 µm; 20 Hz, 210 µm. Resting potentil, 60 mv for oth. Somtic recordings of EPSPs resulting from stimultion of Schffer collterls (lower trces) showed tht LTG hd similr frequencydependent effect on temporl summtion s for lph wveforms, minimlly ffecting low frequency EPSPs (5 Hz) ut significntly reducing the postsynptic response to 20-Hz stimultion (rrowhed). Resting potentil: 5 Hz, 61 mv; 20 Hz, 60 mv. Time clirtion, 200 nd 100 ms for 5 Hz nd 20 Hz, respectively. (c) Plot of the reduction in temporl summtion c cused y LTG (100 µm) s mesured with lph wveform dendritic current injection or synptic stimultion. LTG significntly decresed temporl summtion t 10 nd 20 Hz, ut not t 5 or 40 Hz. (Sttisticl significnce compred to control, *P < 0.05 or **P < ) lriztion. Becuse of these unique properties, I h tends to stilize memrne potentil towrds rest, producing either depolrizing sg in memrne potentil with prolonged hyperpolrizing current injection (reflecting ctivtion of n inwrd current), or hyperpolrizing sg in response to prolonged depolrizing current injection (due to dectivtion of n inwrd current; Fig. 4 nd, Ctrl). This voltge sg thus reduced stedy-stte input resistnce in response to prolonged current injection. I h is distriuted in the hippocmpl pyrmidl neuron in non-uniform grdient pttern, with low density t the som, nd density tht increses up to sevenfold with incresing distnce in the picl dendrites 16. The high dendritic density of I h contriutes significntly to locl integrtion properties, reducing the input resistnce, time constnt nd temporl summtion of EPSPs in the dendrites 17,18. In the presence of LTG, suthreshold current injection in the dendrites produced responses with greter voltge sg, prticulrly in response to depolriztion, suggestive of lrger I h t rest (Fig. 4). LTG thus reduced input resistnce in the dendrites: when mesured with 500-ms current injections producing <15 mv voltge excursions from rest, LTG ( µm) lowered stedy-stte input resistnce y 23.3 ± 5.2% compred to control (n = 7, P < 0.005) in response to depolrizing current injections, nd y 13.6 ± 6.7% in response to hyperpolrizing current injections (not sttisticlly different from control, n = 5, P > 0.05). Consistent with the smller density of I h t the som, however, LTG hd much smller effect t the som on the response to suthreshold current injection (dt not shown). Appliction of the specific I h locker ZD-7288 t ner-sturting doses (10 µm) 19 hd the opposite effect (Fig. 4). ZD olished the slowly ctivting voltge relxtion present under control conditions in response to oth hyperpolrizing nd depolrizing current injection, nd mrkedly incresed input resistnce. When ZD-7288 ws pre-pplied efore LTG, it lrgely locked the effect of LTG on dendritic excitility: suthreshold current injections in the dendrites under control conditions ecme suprthreshold with repetitive AP firing in the presence of ZD-7288; when LTG ws dded, there ws miniml effect on AP firing (Fig. 4c). Similrly, ZD-7288 olished the effect of LTG on suthreshold current injection in the dendrites (Fig. 4c). Group dt (Fig. 4d) shows tht I h lockde y ZD-7288 prevented the effect of LTG on dendritic AP firing (94 ± 14% of control, n = 5, P >0.05). These results suggest tht LTG cted vi I h, incresing its stedy-stte ctivtion t memrne potentils ner rest. This would hve the effect of diminishing the dendritic input resistnce, time constnt nd temporl summtion of excittory inputs. LTG increses ctivtion of I h We exmined the mechnism of ction of LTG on I h using cellttched ptch recordings in the dendrites. This technique llowed us to study I h from loclized region of the dendrites, while leving intct the modultion of this current y the intrcellulr milieu 20. Using pipette solution tht isolted I h, hyperpolrizing voltge commnds from holding potentil of 45 mv produced slowly ctivting inwrd currents which were non-inctivting (Fig. 5). The mgnitude of these currents ws smll for voltge commnds ner rest ( 75 mv) nd progressively incresed with lrger hyperpolrizing commnds, s is typicl for I h. The currents t the distl dendrites were fr lrger in pek mplitude thn those t the som (Fig. 5). We plotted the voltge-dependent ctivtion of I h y mesuring the pek inwrd til current upon return to holding potentil fter 1-s voltge commnd. I h ctivtion ner resting potentil ( 60 to 70 mv) ws smll under control conditions, nd hd V 1/2 of roughly 83 mv (Fig. 5c). Addition of LTG to the pipette solution produced n 11-mV depolrizing shift in the I h ctivtion curve, with V 1/2 of roughly 72 mv. This shift in I h ctivtion cused sustntil increse in the mount of I h ctive round resting potentil (Fig. 5d). LTG did not increse mximl I h mesured t the most hyperpolrized potentils (I h t 115 mv in control, 30.0 ± 5.3 pa, n =6; in LTG, 34.0 ± 6.3 pa, n = 8, P > 0.05). LTG nture neuroscience volume 5 no 8 ugust

6 lso depolrized the resting potentil, with n verge depolriztion of 5 mv upon rek-in t the end of the experiment, compred to control neurons. (This pprent shift in resting potentil is included in the 11-mV shift of I h ctivtion; tht is, without ccounting for the resting potentil chnge, there ws roughly 6-mV ctivtion shift.) We ttriute this effect to prole LTG entry into the dendritic memrne, ffecting locl I h nd thus locl mesurements of resting potentil. LTG reduces synptic ctivtion of pyrmidl neurons How does incresed I h ctivtion ffect AP firing in response to repetitive synptic stimultion, s would occur during epileptiform firing? To study how modultion of I h would lter not only the postsynptic response to trins of EPSPs in the dendrites ut lso the trnsmission of voltge trnsients in the dendrites, we used computtionl model (Methods) of the CA1 hippocmpl pyrmidl neuron. We first simulted AP firing from current injection in control conditions nd then with I h ctivtion shifted in depolrized direction y 10 mv. This simulted increse in I h ctivtion significntly lowered the rte of AP firing from dendritic current injection, oth when resting potentil ws held t its control level (Fig. 6, middle trces) nd when resting potentil ws set 5 mv more depolrized, simulting the effects of LTG on resting potentil (Fig. 6, right trces). Conversely, simulted current injection t the som showed only modest reduction of AP firing when I h ctivtion ws incresed nd resting potentil ws fixed t control levels (Fig. 6, middle trces); nd when resting potentil ws held depolrized y 5 mv, incresed I h ctivtion cused no reduction (or smll increse) in AP firing t the som (Fig. 6, right trces). This model replicted our experimentl findings of selective effect of incresed I h ctivtion on dendritic excitility. We then used the model to ddress questions more difficult to study experimentlly. Incresed I h ctivtion seemed to hve competing influences on somtic AP firing in response to dendritic inputs: reduction in dendritic input resistnce reduced the depolriztion seen t the som from n excittory dendritic input, wheres the depolrizing chnge in resting potentil rought the som closer to threshold. We simulted suthreshold current injections in the dendrites nd found tht, s expected, when resting potentil ws held t the control level, incresed I h ctivtion cused significnt decrese in the stedy-stte dendritic potentil produced y dendritic current injection (Fig. 6c). However, the decrese in stedy-stte voltge seen t the som ws proportiontely lrger thn in the dendrites, suggesting tht the voltge trnsient hd more decy during its trnsmission from injection site to som. When resting potentil ws set t depolrized level during incresed I h ctivtion, the resting potentil chnge opposed some of the effects of I h on dendritic input ttenution y moving closer to threshold. Even under these conditions, the stedystte memrne potentil reched t the som in response to dendritic input remined lower thn control (Fig. 6d). This finding showed tht the effect of incresed I h on dendritic voltge ttenution outweighed the effect of the resting potentil chnge t the som, nd thus inhiited somtic AP firing from dendritic inputs. A simultion of the decy of voltge trnsients long the dendrite clrified how I h preferentilly ttenuted dendritic inputs. We simulted depolrizing current injection t distl dendritic loction (350 µm), nd mesured the stedy-stte voltge long the length of the dendrite (Fig. 6e). A 10-mV shift in I h ctivtion cused stedy-stte voltge t the site of current injection to e reduced to 45% of control, ut t the som, the voltge trnsient hd decyed to 15% of the control vlue. A plot of the normlized stedy-stte voltge (Fig. 6e, dshed line) showed tht voltge trnsients hd n incresed decy with distnce under conditions of incresed I h, demonstrting tht incresed I h hd lowered the length constnt of the dendrite. This suggested tht the effects of LTG on somtic initition of AP firing from dendritic current injection were due to decrese in input resistnce t the site of current injection s well s to greter ttenution of voltge trnsients long the length of the dendrite. We then used the model to study the effects of LTG on synpticlly driven AP firing, using series of simulted rndom EPSP trins delivered in the dendrites. Under control conditions, s stimultion frequency incresed up to 20 Hz, the numer of APs elicited y EPSPs tended to increse, s temporl summtion of EPSPs incresed (Fig. 7). With simulted increse in I h ctivtion, t low stimultion frequencies (<5 Hz) there ws little chnge in AP firing compred to control. However, t frequencies etween 10 nd 30 Hz, higher I h cused significnt reduction in synpticlly evoked AP firing compred to control, presumly due to decrese in temporl summtion of EPSPs. At still higher frequencies, there ws decline in temporl summtion under control conditions, nd similr decrese in the effect of incresed I h. These results suggest tht LTG might decrese the pyrmidl neuron response to synptic stimultion in frequencydependent mnner, with pek effect etween 10 nd 30 Hz. To investigte these computtionl phenomen experimentlly, we mde dendritic whole-cell recordings nd used short trins of lph wveform current injections to simulte EPSPs. When trins of stimuli were delivered t low (5 Hz) frequency, there ws little temporl summtion, nd ppliction of LTG hd little effect on the extent of summtion (Fig. 7, top trces). At higher frequencies (10 20 Hz), there ws more temporl summtion, nd here LTG cused decline in temporl summtion during the trin. This effect declined t higher frequencies (40 Hz; Fig. 7c). When trins of synpticlly evoked responses were sustituted for lph wveform current injections, nd recordings mde t the som, LTG hd little effect on low frequency stimultion (5 Hz), ut mrkedly decresed temporl summtion t higher frequencies (20 Hz; Fig. 7, ottom trces). (Note tht with short trins of EPSPs, temporl summtion ws superimposed on synptic fcilittion, unlike the cse with lph wveform current injections.) Incresed I h cused frequency-dependent decrese in temporl summtion which ws of gretest mgnitude round 20 Hz, nd hd little effect on low-frequency synptic ctivity (Fig. 7c). These results were in greement with those otined y computtionl modeling, nd were similr whether experimentlly otined with postsynptic current injection or synptic stimultion, suggesting tht the frequency-dependent effects of incresed I h on synptic ctivtion of pyrmidl neurons were lrgely medited postsynpticlly. DISCUSSION These results demonstrte new mode of ction for CNS drugs. LTG exerts n inhiitory effect on AP firing in the hippocmpl pyrmidl neuron y selectively ltering the excitility of the picl dendrites while minimlly ffecting the excitility of the som. This regionl selectivity is direct consequence of the drug s ction on I h, which is distriuted in non-uniform grdient long the neuron, with the distl dendrites contining much higher density of I h thn the som. Increses in dendritic I h reduce input resistnce, length constnt nd temporl summtion, suppressing the effect of dendritic excittory synptic inputs on perisomtic AP initition in the pyrmidl neuron, thus cusing n overll reduction in excitility. These regionl effects of LTG within single neuron demonstrte tht drugs my hve unex- 772 nture neuroscience volume 5 no 8 ugust 2002

7 pected nd differing effects on different comprtments of the neuron, in prticulr on the dendritic tree (which y some estimtes ccounts for >90% of the surfce re of pyrmidl neurons 21 ), underscoring the importnce of studying dendritic physiology when evluting phrmceuticl mechnisms of ction. LTG seems to ct y shifting the ctivtion of I h in depolrizing direction. Becuse there is little I h ctive t the typicl neuronl resting potentil, smll increses in I h cn led to significnt modultion of suthreshold neuronl ehvior. The contriutions of I h to the integrtive properties of dendrites include reduction in input resistnce, dendritic length constnt, nd temporl summtion, ll of which ct to reduce dendritic excitility The reduction of dendritic input resistnce nd length constnt cuses given excittory input in the dendrites to produce less of locl depolriztion which then decys with distnce long the dendrites. This produces smller voltge chnge t the som, ultimtely reducing AP firing in response to dendritic depolriztion. Increses in I h lso reduce temporl summtion, diminishing AP firing from repetitive synptic inputs in the dendrites. This proly results from the grdul uildup of I h dectivtion during trin of EPSPs, producing net hyperpolrizing current 18. The frequency-dependence of this effect my derive from the reltively slow ctivtion nd dectivtion kinetics of I h (16 20 ms ner rest 16 ). Thus, low-frequency EPSPs do not chieve significnt stedy-stte I h dectivtion, wheres higher frequencies ( 20 Hz) will led to incresed I h dectivtion nd therefore decresed temporl summtion. (At still higher frequencies the I h effect my e overcome y incresing EPSP summtion.) Increses in I h lso depolrize resting potentil, moving it towrds threshold for AP firing, thus countercting some of the inhiitory effect on excittory inputs. Our experiments nd simultions showed tht the lnce of these two influences is different in the two comprtments of the neuron. In response to somtic inputs, when the chnge in resting potentil ws experimentlly uncompensted (s would occur in vivo), LTG hd little or no effect on AP firing, suggesting tht the smll chnge in locl input resistnce ws counterlnced y the depolriztion of resting potentil (which ws more uniform throughout the neuron). Incresed I h hd more drmtic influence on dendritic inputs nd their ility to drive somtic firing, nd this remined inhiitory even when the resting potentil chnge ws uncompensted. This is ecuse incresed I h not only hd much lrger effect on the locl depolriztion cused y dendritic input (owing to its incresed dendritic density), ut lso further ttenuted tht potentil during its spred from the dendrites to the som. These two effects comined to sustntilly reduce the effects of dendritic inputs on somtic AP firing, while minimlly ffecting somtic inputs. Thus it ppers tht incresed ctivtion of I h y LTG reduces the overll excitility of hippocmpl pyrmidl neurons y ttenuting their response to dendritic inputs. The ction of LTG on I h my constitute n importnt new nticonvulsnt mechnism. The frequency-dependent effect of LTG on temporl summtion of EPSPs llows for the selective reduction of AP firing tht results from pthologiclly high levels of excittory synptic ctivity in the dendrites, while preserving AP firing from lower frequency inputs. This fulfills criterion for idel nticonvulsnt ction: tht norml rin function e unffected while excessive firing is suppressed. Other mechnisms of ction hve een scried to LTG tht my contriute to its nticonvulsnt effect, including reduction of N + currents, C 2+ currents nd glutmte receptor ctivtion 9,10,22,23. However, the ction of LTG on I h, nd the role of I h in modulting oscilltory ehvior in other neurons 15 my provide unique explntion of its efficcy ginst primrily generlized epilepsy (such s sence epilepsy), feture not shred y other nticonvulsnts with ctions on N + chnnels such s PHT or CBZ 24. A further impliction of these results is tht I h my hve n importnt role in epileptogenesis, s is supported y recent evidence demonstrting ltertions in I h in n niml model of ferile seizures 25, nd the involvement of I h in mintining the spontneous ctivity of CA1 hippocmpl interneurons 26 nd modulting presynptic excitility 27. These multiple ctions of I h, some of which my increse rther thn decrese neuronl excitility, imply tht its effects on epileptogenesis my e complex. Nonetheless, our results, long with the growing pprecition for the importnt role of I h in regulting neuronl excitility, suggest tht I h my e useful trget for further CNS drug development. METHODS Electrophysiology. Hippocmpl slices (400 µm) were prepred from 6 10 week-old mle Sprgue-Dwley rts using stndrd procedures 4. Animl protocols were pproved y the Animl Reserch Committee t Bylor College of Medicine. Neurons were visulized with differentil interference contrst microscopy using Zeiss Axioskop (Oerkochen, Germny). Recordings were mde t C. The extrcellulr recording solution contined 125 mm NCl, 25 mm NHCO 3, 10 mm dextrose, 2.5 mm KCl, 2 mm CCl 2, 2 mm MgCl 2 nd 1.25 mm NH 2 PO 4. CNQX (10 µm) nd icuculline methiodide (20 µm) were dded to the extrcellulr solution except when synptic stimultion ws used. The wholecell recording pipette solution contined 120 mm potssium gluconte, 20 mm KCl, 10 mm HEPES, 4 mm N 2 -ATP, 2 mm MgCl 2, 0.3 mm Tris- GTP nd 0.2 mm EGTA (ph 7.3 with KOH). For cell-ttched ptch recordings, the pipette solution contined 120 mm KCl, 20 mm TEA-Cl, 10 mm HEPES, 5 mm 4-minopyridine, 2 mm CCl 2, 1 mm MgCl 2 nd 1 mm BCl 2 (ph 7.3 with KOH). Recorded neurons hd resting potentils etween 60 nd 72 mv (men 64 ± 0.7 mv, n = 36). Whole-cell current-clmp recordings were mde using Dgn BVC- 700 mplifier (Minnepolis, Minnesot), were smpled t 10 KHz, nd filtered t 2 KHz. Ptch recordings used n Axon Instruments AxoPtch 1C mplifier (Foster City, Cliforni), nd were smpled t 2 KHz nd filtered t 500 Hz. Dt cquisition used custom softwre written for the Igor Pro nlysis environment (Wvemetrics, Lke Oswego, Oregon). Extrcellulr stimultion in the lveus with tungsten electrode (A-M Systems, Everett, Wshington) ws used to ntidromiclly ctivte APs, nd stimultion in the strtum rditum ws used to elicit EPSPs. Dendritic current injections simulting EPSPs were modeled y n lph function of the form: I = I mx (t/α)e αt where α = 0.1. Drugs were pplied in the th mde either from queous stock solutions (LTG, ZD-7288) or dissolved in DMSO such tht the finl DMSO concentrtion ws <1% (PHT, CBZ). All drugs were otined from Sigm (St. Louis, Missouri) except s noted. LTG ws gift from GlxoSmithKline (Reserch Tringle Prk, North Crolin); ZD-7288 ws otined from Tocris (Blwin, Missouri). Concentrtion rnges of nticonvulsnts used in this study were chosen with reference to the estlished pek therpeutic free cererospinl fluid concentrtions in humns 28 : 39 µm LTG, 7 µm PHT nd 13 µm CBZ. Synptic stimultion experiments were performed with ddition to the th of 50 µm APV, 20 µm MK-801, 100 µm CGP (Tocris) nd 20 µm icuculline methiodide (to lock NMDA nd GABAergic currents). Are CA3 ws isolted from the rest of the slice y knife cut to diminish repetitive firing from reduced GABAergic inhiition. In ddition, EGTA (10 mm) ws dded to the pipette solution to reduce potentition of postsynptic responses from repetitive synptic stimultion. Temporl summtion ws mesured s the rtio of fifth response mplitude (mesured from seline) to the first response mplitude. Group dt re expressed s men ± s.e.m. Sttisticl significnce ws clculted using Student s t-test. Modeling. All simultions were crried out using the NEURON simultion progrm 29. The relistic model of hippocmpl CA1 pyrmidl neuron ws sed on tht used in previous work 30 nd included N +, DR- nd A-type K + voltge-gted conductnces. A non-inctivting, non- nture neuroscience volume 5 no 8 ugust

8 specific ction current I h = g h *n*(v E rev ), with E rev = 30 mv ws inserted in the som nd the picl dendrites. Its dendritic distriution nd ctivtion kinetics were consistent with the ville experimentl dt on CA1 neurons 16. Thus, the voltge dependence of the ctivtion gte vrile ws modeled s n = 1/(1 + exp(0.151(v V 1/2 ))), with V 1/2 = 82 mv in the som nd proximl dendrites (<100 µm), nd V 1/2 = 90 mv for loctions >100 µm from the som; its time constnt ws pproximted s α n = exp(0.033(v V t ))/(0.011(1 + exp(0.083(v V t )))), with V t = 75 mv. A pek conductnce density of g h = 3 ps/µm 2 ws used t the som, nd linerly incresed with distnce, d (µm), s g h *( d/100). LTG ppliction ws modeled with 10-mV depolrizing shift of the ctivtion curve. The resting potentil chnge resulting from this shift in I h ctivtion ws modeled s fixed 5-mV depolriztion. Synptic conductnces were represented y doule exponentil function with rise nd decy time constnts of 3 nd 30 ms, respectively, pek conductnce of 16 ns, nd reversl potentil of 0 mv. For the simultions of Fig. 7, five independent synpses were rndomly plced nd rndomly (poisson) ctivted on dendritic comprtments µm from the som. The verge AP firing rte t ech verge frequency ws clculted from 50 simultions, ech 1 s long. The model nd simultion files re pulicly ville on the ModelDB dtse of Sensel ( Acknowledgments We thnk R. Gry for writing the dt cquisition softwre nd providing help in ll phses of experimenttion nd dt nlysis. We lso thnk R. Chitwood, C. Bernrd nd A. Frick for reding erlier versions of the mnuscript. Reserch ws supported y the Ntionl Institutes of Helth (N.P.P. nd D.J.), the Ntionl Institute of Defness nd Other Communiction Disorders Humn Brin Project (M.M.), the Ntionl Epifellows Foundtion (N.P.P.) nd the Hnkmer Foundtion (D.J.). Competing interests sttement Authors declre competing finncil interests: see the Nture Neuroscience wesite ( version for detils. RECEIVED 12 FEBRUARY; ACCEPTED 25 JUNE Husser, M., Spruston, N. & Sturt, G. J. Diversity nd dynmics of dendritic signling. Science 290, (2000). 2. Johnston, D., Mgee, J. C., Colert, C. M. & Christie, B. R. Active properties of neuronl dendrites. Annu. Rev. Neurosci. 19, (1996). 3. Mgee, J., Hoffmn, D., Colert, C. & Johnston, D. Electricl nd clcium signling in dendrites of hippocmpl pyrmidl neurons. Annu. Rev. Physiol. 60, (1998). 4. Poolos, N. P. & Johnston, D. Clcium-ctivted potssium conductnces contriute to ction potentil repolriztion t the som ut not the dendrites of hippocmpl CA1 pyrmidl neurons. J. Neurosci. 19, (1999). 5. Johnston, D. et l. Dendritic potssium chnnels in hippocmpl pyrmidl neurons. J. Physiol. (Lond.) 525, (2000). 6. Messenheimer, J. in The Tretment of Epilepsy: Principles nd Prctice (ed. Wyllie, E.) (Willims nd Wilkins, Bltimore, 1996). 7. Clrese, J. R. Lmotrigine nd the tretment of ipolr disorder. Introduction. Eur Neuropsychophrmcol. 9 (Suppl. 4), S (1999). 8. White, H. S. in The Epilepsies 2 (eds. Porter, R. J. & Chdwick, D.) 1 30 (Butterworth-Heinemnn, Boston, 1997). 9. Kuo, C.-C. & Lu, L. Chrcteriztion of lmotrigine inhiition of N + chnnels in rt hippocmpl neurons. Br. J. Phrmcol. 121, (1997). 10. Xie, X., Lncster, B., Pekmn, T. & Grthwite, J. Interction of the ntiepileptic drug lmotrigine with recominnt rt rin type IIA N + chnnels nd with ntive N + chnnels in rt hippocmpl neurons. Pflugers Arch. 430, (1995). 11. Colert, C. M., Mgee, J. C., Hoffmn, D. A. & Johnston, D. Slow recovery from inctivtion of N + chnnels underlies the ctivity-dependent ttenution of dendritic ction potentils in hippocmpl CA1 pyrmidl neurons. J. Neurosci. 17, (1997). 12. Golding, N. L. & Spruston, N. Dendritic sodium spikes re vrile triggers of xonl ction potentils in hippocmpl CA1 pyrmidl neurons. Neuron 21, (1998). 13. Sturt, G., Spruston, N., Skmnn, B. & Husser, M. Action potentil initition nd ckpropgtion in neurons of the mmmlin CNS. Trends Neurosci. 20, (1997). 14. Sntoro, B. et l. Moleculr nd functionl heterogeneity of hyperpolriztion-ctivted pcemker chnnels in the mouse CNS. J. Neurosci. 20, (2000). 15. Ppe, H.-C. Queer current nd pcemker: the hyperpolriztion-ctivted ction current in neurons. Annu. Rev. Physiol. 58, (1996). 16. Mgee, J. C. Dendritic hyperpolriztion-ctivted currents modify the integrtive properties of hippocmpl CA1 pyrmidl neurons. J. Neurosci. 18, (1998). 17. Sturt, G. & Spruston, N. Determinnts of voltge ttenution in neocorticl pyrmidl neuron dendrites. J. Neurosci. 18, (1998). 18. Mgee, J. C. Dendritic I h normlizes temporl summtion in hippocmpl CA1 neurons. Nt. Neurosci. 2, (1999). 19. Hrris, N. C. & Constnti, A. Mechnism of lock y ZD 7288 of the hyperpolriztion-ctivted inwrd rectifying current in guine pig sustnti nigr neurons in vitro. J. Neurophysiol. 74, (1995). 20. Chen, S., Wng, J. & Siegelum, S. A. Properties of hyperpolriztionctivted pcemker current defined y cossemly of HCN1 nd HCN2 suunits nd sl modultion y cyclic nucleotide. J. Gen. Physiol. 117, (2001). 21. Fil, J. C. & Hrris, K. M. in Dendrites (eds. Sturt, G., Spruston, N. & Husser, M.) (Oxford University Press, Oxford, 1999). 22. Stefni, A., Spdoni, F., Sinisclchi, A. & Bernrdi, G. Lmotrigine inhiits C 2+ currents in corticl neurons: functionl implictions. Eur. J. Phrmcol. 307, (1996). 23. Wng, S. J., Sihr, T. S. & Gen, P. W. Lmotrigine inhiition of glutmte relese from isolted cererocorticl nerve terminls (synptosomes) y suppression of voltge-ctivted clcium chnnel ctivity. Neuroreport 12, (2001). 24. Coulter, D. A. Antiepileptic drug cellulr mechnisms of ction: where does lmotrigine fit in? J. Child Neurol. 12 (Suppl.1), 2 9 (1997). 25. Chen, K. et l. Persistently modified h-chnnels fter complex ferile seizures convert the seizure-induced enhncement of inhiition to hyperexcitility. Nt. Med. 7, (2001). 26. Lupic, C. R., Bell, J. A., Hoffmn, A. F. & Wtson, P. L. Contriution of the hyperpolriztion-ctivted current (I h ) to memrne potentil nd GABA relese in hippocmpl interneurons. J. Neurophysiol. 86, (2001). 27. Beumont, V. & Zucker, R. S. Enhncement of synptic trnsmission y cyclic AMP modultion of presynptic I h chnnels. Nt. Neurosci. 3, (2000). 28. Shorvon, S. D. Hndook of Epilepsy Tretment (Blckwell, Oxford, 2000). 29. Hines, M. L. & Crnevle, N. T. The NEURON simultion environment. Neurl Comput. 9, (1997). 30. Migliore, M., Hoffmn, D. A., Mgee, J. C. & Johnston, D. Role of n A-type K + conductnce in the ck-propgtion of ction potentils in the dendrites of hippocmpl pyrmidl neurons. J. Comput. Neurosci. 7, 5 15 (1999). 774 nture neuroscience volume 5 no 8 ugust 2002