Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices

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1 Brain Research 885 (2000) lcate/ bres Research reprt Ptassium-induced enhancement f persistent inward current in hippcampal neurns in islatin and in tissue slices G.G. Smjen *, M. Muller Department f Cell Bilgy, Bx 3709, Duke University Medical Center, Durham, NC 2770, USA Received 9 May 2000; accepted 2 September 2000 Abstract Previus wrk suggested a rle fr the vltage-dependent persistent sdium current, I Na,P, in the generatin f seizures and spreading depressin (SD). Ordinarily, INa,P is small in hippcampal neurns. We investigated the effect f raising external K cncentratin, [K ], n whle-cell persistent inward current in freshly islated hippcampal CA pyramidal neurns. INa,P was identified by TTX-sensitivity and dependence n external Na cncentratin. When nne f the in channels were blcked, INa,P was nt usually detectable, prbably because cmpeting K current masked it, but after raising [K ] INa,P appeared, while K currents diminished. With K channels blcked, INa,P culd usually be evked in cntrl slutin and raising [K ] caused its reversible increase in mst cells. The 2 increase did nt depend n external calcium [Ca ]. In CA pyramidal neurns in hippcampal slices a TTX-sensitive persistent inward current was always recrded and when [K ] was raised, it was reversibly enhanced. Strng deplarizatin evked irregular current fluctuatins, which were als augmented in high [K ]. The findings supprt a rle f ptassium-mediated psitive feedback in the generatin f seizures and spreading depressin Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmissin Tpic: Sdium channels Keywrds: Persistent sdium current; Sdium channel; External ptassium; Intracellular calcium; Seizure; Spreading depressin. Intrductin cmpnent f the extracellular ptential shift during ictal seizures revealed a lng-lasting prminent current sink Accumulatin f K ins in cerebral interstitial spaces limited t cell bdy layers, fr which the surce was has been blamed fr the eruptin f spreading depressin distributed ver the dendritic fields [45]. By cntrast, (SD) by Grafstein [7] and fr the generatin f seizure vltage shifts and [K ] increase assciated with SD start discharges by Green [8] and by Fertziger and Ranck [2]. in the dendrite layers [24] where intrinsic ptical signals Glr et al. [5,6] identified a sustained extracellular are als maximal [,34] and CSD shws pwerful inward negative ptential shift during epileptifrm seizures that currents [45]. In spite f the bvius differences between was limited t the cell bdy layers in dentate gyrus and seizures and SD, there are als imprtant similarities. Bth, hippcampus. We have cnfirmed this bservatin [40] and seizures and SD can be triggered by similar insults, and demnstrated that the accumulatin f K ins was als bth are inhibited by similar physical r pharmaclgical maximal in the interstitium f cell bdy layers [4]. interventins, such as cling, hypertnicity, acidsis and Current surce density (CSD) analysis f the sustained certain depressant drugs [4,32]. An event that begins with an epileptifrm discharge can smetimes terminate in SD [39] and a seizure that starts in a fcus can then spread *Crrespnding authr. Tel.: ; fax: ver a large area with a velcity resembling that f SD 548. [4,44]. address: g.smjen@cellbi.duke.edu (G.G. Smjen). Frm these and similar bservatins it has been clear Present address: Zentrum fur Physilgie und Pathphysilgie, Abthat bth tnic seizures and spreading depressin require teilung Neur- und Sinnesphysilgie Humbldtallee 23, D Gttingen, Germany. inward currents that inactivate very slwly r nt at all / 00/ $ see frnt matter 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 G.G. Smjen, M. Muller / Brain Research 885 (2000) The nature f these currents was, hwever nt knwn. A micrscpe bjective with patch pipettes; tight seal was vltage-dependent persistent sdium current was demn- established, and the whle-cell recrding cnditin created strated, amngst thers, in hippcampal neurns [3,26] by suctin. T recrd Na as well as K currents the pipettes (reviewed by Crill [7] and by Taylr [43]). It is nt knwn were filled with a slutin cntaining (in mml/ l): KF whether INa,P flws thrugh distinct classes f Na chan- 29, NaCl 4, EGTA 0, CaCl2 0.5, MgCl2 2, Hepes 0, nels, r whether it represents failure f inactivatin f the Na2ATP 4, ph 7. r 7.3, tip resistance MV. T cnventinal, fast, transient Na current. Recently the blck K currents, KF was substituted by 09 mm CsF weight f evidence seemed t be shifting tward the latter and 20 mm TEA-Cl. interpretatin [2,3]. An Axpatch D amplifier in vltage clamp cnfigura- The physilgical rle attributed t INa,P is the regula- tin and the pclamp-6 (Axn Instruments) suite f prtin f the resting excitability f neurns. Its usual grams was used t recrd whle-cell currents. Pipette and cnductance is t weak t explain the deplarizatin seen cell capacitances were cmpensated in the custmary in such severely pathlgical cnditins as seizures r SD. manner. Series resistance was cmpensated t 70%. The INa,P greatly increases, hwever, under pathlgical cn- hlding ptential was 270 mv pipette vltage. Current ditins. In heart and skeletal muscle a persistent Na vltage (I V ) curves were recrded usually at ne, sme- current is enhanced by elevated [K ] [9] and hypxia times at 2-min intervals. Tw different prtcls were causes an increase in heart muscle [5,28] as well as in used: either eight sweeps, each beginning with a pre-pulse neurns [9]. In cmputer-based simulatins we recently f 00 ms t 290 mv t remve inactivatin, fllwed by demnstrated that Na -mediated, vltage-dependent, slw- 200-ms deplarizing steps at 2-s intervals in 5-mV ly inactivating inward current can generate an SD-like increments, taking the pipette vltage frm 270 t 35 deplarized state [29]. We have therefre asked whether mv; r 2 sweeps f a 200-ms hyperplarizing pre-pulse elevatin f [K ] wuld als cause an increase in INa,P in fllwed by 400-ms deplarizing steps in 0-mV incre- hippcampal neurns. This indeed turned ut t be the ments, taking the pipette frm 270 t 40 mv. case. Sme f the findings have been reprted in an The current recrds were read with Clampfit (Axn abstract [38]. Instruments) sftware. After subtractin f linear leak and hlding currents, the data were further prcessed with the Excel (Micrsft) prgram. Junctin ptentials were calcu- 2. Methds lated with the JPCalc prgram [3]. 2.. Islatin f neurns 2.3. Flurescence imaging Hippcampal CA pyramidal cells were islated accrd- The nn-permeant frms f the flurescent calcium ing t the methd f Kay and Wng [30]. Briefly: rats f indicatr dyes flu-3 (0 mm) and fura-red (30 mm) g bdy weight were decapitated under ether (Mlecular Prbes) were added t the pipette slutin [22]. anesthesia. Brains were remved and 500-mm thick slices The 488 nm excitatin light was used and emissin was were cut frm hippcampus. The CA regin was cut int recrded at 520 nm (flu-3) as well as 640 nm (fura-red) smaller pieces and these tissue fragments were digested fr with COMOS (Birad) sftware. Flurescence intensities 75 min. The digestin medium cntained (in mml/ l): were recrded frm tw intersecting elngated rectangular NaCl 25, KCl 5, CaCl2, MgCl2 2, D-glucse 25, areas f interest at 0- r 20-s intervals; images were [2-hydrxyethyl]piperazine-[2-ethanesulfnic acid] recrded at 60-s intervals. Backgrund-crrected flures- (Hepes) 0, ph 7.0, with trypsin 0.75 mg/ ml, at rm cence ratis (flu-3/ fura-red) were cmputed subsequently temperature. After digestin the tissue pieces were washed using Excel (Micrsft) sftware. Flu-3 flurescence and then incubated in trypsin-free xygenated medium at increases while fura-red flurescence decreases with rising rm temperature. Tissue fragments were dispersed by 2 [Ca ] i. trituratin with a graded series f fire plished Pasteur pipettes Hippcampal slices 2.2. Recrding f vltage-dependent currents Tissue slices f 400 mm thickness were prepared as Cell suspensins were placed in a chamber f abut 0.7 described abve, and placed in an Osl style interface ml capacity n the stage f a Zeiss Axiskp and main- chamber in flwing artificial cerebrspinal fluid (ACSF) f tained in flwing Hepes-buffered medium f the fllwing the fllwing cmpsitin (in mml/ l): 30 NaCl, 3.5 cmpsitin: (in mml/l:) NaCl 30, KCl 3.5, CaCl2.2, KCl,.25 NaH2PO 4, 24 NaHCO 3,.2 CaCl 2,.2 MgSO 4, MgCl.0, glucse 25, Hepes 0, ph 7.3 r 7.35, at 0 glucse, ph 7.4, saturated with 95% O, 5% CO, C. When K cncentratin was raised, Na was temperature 368C. When K cncentratin was raised, Na equivalently reduced. Cells were apprached under the was equivalently reduced.

3 04 G.G. Smjen, M. Muller / Brain Research 885 (2000) 02 0 Slices were left undisturbed fr 90 min befre recrding slwly inactivating inward current was detectable in all but began. tw f a ppulatin f 42 islated CA hippcampal neurns. The mean amplitude (nrmalized t cell capaci Whle-cell recrding in tissue slices tance) was pa/ pf (mean6st.d.), with a range frm 0 t pa/pf in cells with average capacitance Patch pipettes were pulled frm thick-walled glass tubes f pf. In 0 trials the bath K cncentratin was and filled with the fllwing slutin (in mml/ l): 20 raised frm 3 t 20 mm; the mean persistent inward K-glucnate, 8 KCl, 0 Hepes, EGTA, 0.5 CaCl 2, 2 current in this sample in cntrl slutin was MgCl 2, 4 ATP-Na 2, ph 7.35, smlarity 290, pipette tip pa/pf (mean6s.e.m.) and it increased t pa/ resistance 3 5 MV. Cells in CA stratum pyramidale were pf (P,0.03 by paired t-test). Fig. illustrates an example fund by blind search. Seal resistances f $0.5 GV, f the reversible enhancement f the persistent inward mre usually $.0 GV were accepted. Pipette capacitance current in ne f these cells. In this case the maximal was cmpensated. Whle-cell recrding cnditin was persistent inward current increased during elevatin f established by gentle suctin. Cell capacitance and series [K ] by a factr f almst 4 (Fig. A,C) while the resistance were nt cmpensated. Hlding ptential was calculated maximal cnductance dubled (Fig. D). The 265 mv. Series f test pulses were delivered at 90-s sample currents f Fig. B shw an increase nt nly f intervals. Each prtcl cnsisted f a pre-pulse f 400 ms the slwly inactivating inward current, but als f an t 290 mv fllwed by a series f either eight r 0 inward tail current that fllws replarizatin. As lng as deplarizing steps f 600 ms in 0-mV increments, either the persistent inward current was small, tail currents were frm 270 t 0, r frm 270 t 20 mv. Data prcessing usually small r absent in recrdings made with CsF was similar t that fr islated cells. pipettes. Marked tail currents were seen whenever the Each cell was examined first fr 5 min in nrmal persistent inward current grew large, but there was n slutin, then fr 5 min in elevated K (0 mml/ l) simple crrelatin between the amplitudes f the tw. slutin. If the seal held, then either recvery was b- In the absence f channel blcking agents, when KFserved during washing with nrmal ACSF fr 5 30 min, filled pipettes were used, the persistent current was a r the cell was expsed t high K slutin with tet- mixture mainly cmpsed f the delayed rectifier K rdtxin (TTX).0 mm added. Slices were expsed t current, I K,DR [36] and I Na,P. As lng as the cells were high K nly nce. bathed in nrmal slutin, a persistent inward current was detected in nly tw ut f cells. When the K 2.6. Statistics cncentratin in the bath was raised, such an inward current appeared in nine f the cells. Its amplitude was Except when therwise nted, numerical data are given 2. pa/pf (n53) at 0 mm, 23.6 pa/pf (n57) at 20 as the mean6s.e.m. Significance was calculated by paired mm and 26.9 pa/pf (n53) at 40 mm [K ]. Apparent- tw-tailed t-test. ly in cntrl slutin the pwerful IK,DR masked the weak I Na,P, but as [K ] increased, INa,P strengthened sufficiently t cmpete. Fig. 2B illustrates the emergence f INa,P in 3. Results high [K ] slutin. The transient Na current, I Na,T, usually increased in the 3.. The effect f elevated ptassium cncentratin, first few minutes f recrding frm a cell, and then its [K ],n islated neurns amplitude drifted slwly ver time, either increasing r decreasing. Raising [K ] did nt alter this curse. In Fig. Whle-cell currents were evked by deplarizing vlt- 2 the I V curves and the nrmalized cnductances f I Na,T age steps in patch-clamped neurns freshly islated frm and INa,P f ne cell are cmpared. CA regin f rat hippcampus. The recrding pipettes were filled either with a KF-based slutin that des nt 3.2. Elevated ptassium cncentratin and flurescence impede any f the knwn majr in currents, r a slutin with CsF as the majr electrlyte and 20 mm TEA, t Laser-induced flurescence in cells filled with calcium blck mst K -mediated utward currents. The hlding indicatr dyes causes a pwerful, slwly reversing ptenptential was 270 mv. Pipette and cell capacitances were tiatin f I Na,P [37,38]. T see whether the effects f neutralized and series resistance was 70% cmpensated. elevated [K ] and f flurescence interact, dye-filled cells Linear leak and hlding current were subtracted ff line. were intermittently expsed t laser light while vltage- T cnstruct current vltage (I V ) curves, cells were dependent currents were als tested, first in nrmal slu- stimulated by series f deplarizing steps, preceded by tin and then in elevated [K ]. Currents were recrded hyperplarizatin t 290 mv. Sets f test pulses were with KF filled micrpipettes, s that the persistent inward applied at - r 2-min intervals. currents were, initially, quite small r absent. Fig. 3 When K-currents were blcked, a vltage dependent, illustrates the changes f the maximal persistent inward

4 G.G. Smjen, M. Muller / Brain Research 885 (2000) Fig.. Reversible enhancement f persistent inward sdium current, INa,P in an islated neurn during expsure t elevated external ptassium cncentratin. (A D) Data frm the same cell, recrded with a pipette filled with a slutin based n CsF, cntaining 20 mm TEA, t blck mst K currents. N flurescent dyes were used. Current amplitudes fr (A), (C) and (D) were measured as the average during the last 5 ms f deplarizatin. (A) The maximal amplitude f the persistent inward current recrded befre, during and after expsure t 20 mm [K ] in the bath. The brken vertical lines indicate change f bath slutin. The hrizntal bar indicates intermittent illuminatin by laser but, in the absence f dyes, the illuminatin had n effect n I Na,P. (B) Sample recrdings f currents evked by vltage step t 20 mv pipette ptential, crrespnding t membrane ptential (V m) f26.7 mv after crrectin fr junctin ptential. Superimpsed recrdings btained befre raising [K ], 8 min after switching t high K slutin, and after 20 min f washing with nrmal slutin. Sampling rate 2000 Hz, filtered ff-line at 00 Hz. (C) Current vltage (I V ) curves f the slwly inactivating current btained just befre switching t high K slutin, after 8 min f expsure t 20 mm [K ], after 8 min f intermittent laser illuminatin while expsed t high [K ], and after 20 min washing with nrmal ACSF. (D) Whle-cell cnductance in nansiemens as functin f V m, calculated by dividing the currents shwn in part (C) by the driving ptential, defined as the difference between V and the reversal ptential f the current. m currents recrded at ne min intervals frm 0 such dye- f influx f Ca thrugh vltage-dependent channels. filled cells. The rapid increase f the inward current after These increases dissipated slwly, creating a sawtth high-k slutin was intrduced int the bath, strngly pattern when the flurescence rati was pltted against suggests that raising [K ] accelerated the laser-induced time (Fig. 4A). The amplitude f these vltage-dependent 2 increase. Of the 0 cells three were lst during the high K Ca respnses gradually decreased with repetitin, prba- treatment. When the seven remaining cells were washed in bly due t rundwn. Raising [K ] caused a small increase nrmal slutin, the inward current decreased fr a shrt in the baseline flurescence rati in mst cells, indicating 2 perid, but in five f the cells it then resumed t increase an increase in resting [Ca ] i. The magnitude f the under the influence f the cntinuing laser pulses. enhancement f INa,P and f the change in flurescence Laser illuminatin f cells that were nt filled with dye rati baseline were, hwever, nt crrelated. had n effect n the persistent inward current, regardless f The effect f calcium n INa,P was further tested in three 2 whether the bath K cncentratin was nrmal, r elevated cells by remving Ca frm the bath while raising [K ]. 2 2 (Fig. ). Lack f [Ca ] was cmpensated by raising Mg cncentratin frm.0 t 2.2 mm. Fig. 4B,C illustrates 3.3. K -induced augmentatin f INa,P is nt calcium ne such trial, recrded with a CsF-filled pipette. As 2 dependent expected, remving Ca suppressed the sawtth scillatins and depressed the baseline flurescence (Fig. 4A). The deplarizing pulses that were applied t btain I V 2 Mbilizatin f Ca frm internal stres, if any, culd nt curves caused the flurescence rati t increase, as a result cmpensate fr the reductin f the external surce f the 2

5 06 G.G. Smjen, M. Muller / Brain Research 885 (2000) 02 0 N-methyl-D-glucamine (NMDG ) fr 50 mm Na in the bath, in the presence f 20 mm [K ] in fur flurescent cells, depressed INa,P frm t pa/pf (P,0.04). TTX-sensitivity, reversal ptential, and Na dependence cnfirmed that this is a slwly inactivating sdium current, I Na,P Neurns in tissue slices Since islated neurns are truncated and are in an abnrmal envirnment, we asked whether raising [K ] wuld als enhance INa,P in intact neurns in their natural habitat. T answer this questin, whle-cell recrdings were made frm pyramidal cells in CA regin f rat hippcampal tissue slices. The slices were maintained in an interface tissue slice chamber perfused with bicarbnate-buffered artificial cerebrspinal fluid (ACSF) at 368C. Glucse in the bath was 0 mm, instead f the 25 mm used fr islated cells. The pipettes were filled with K-glucnate-based slutin and n in channel blcking drugs were used, except at the end f sme trials when mm TTX was added t the bath t verify the Na dependence f the inward currents. The vltage f cells in situ cannt perfectly be cntrlled by the whle-cell patch clamp methd, because the membrane ptential f the axn and f the dendritic tree cannt be cntrlled by feedback applied t the cell bdy. Nnetheless, with this technique changes in the relative magnitude f the vltage-induced currents can reliably be assessed. Since the membrane ptential was presumably nt unifrm ver the entire cell surface, in this sectin we reprt pipette vltages withut crrectin fr series resistance r junctin ptential, because this was the nly reliably knwn vltage. The capacitance f the cells in tissue was nt neutralized, but this des nt intrduce a serius errr in the measurement f currents that change Fig. 2. Cmparing the persistent and the transient sdium currents. Frm nly slwly. The hlding ptential was 265 mv; t btain ne cell, recrded by KF filled micrpipette. (A) I V curves f the I V curves the pipette was first stepped t 290 mv fr 400 maximal amplitude f the fast transient inward current, I Na,T, in cntrl slutin and in 20 mm bath [K ]. (B) I V curves f the persistent current ms and then a series f 600-ms deplarizing pulses were measured during the last 5 ms f deplarizatin frm the same traces as applied in 0-mV increments, either frm 270 t 0 mv r thse used fr (A). In the absence f channel blcking drugs the persistent frm 270 t 20 mv. Deplarizatin evked trains f current is a mixture cnsisting mainly f INa,P and I K,DR. (C) Vltage current spikes presumably drawn by actin ptentials fired dependence f the nrmalized cnductances f INa,T and f the mixed in the axn, utside the vltage clamped regin, as well as persistent current in elevated [K ]. Cnductance was calculated by dividing current by driving vltage, nrmalized t the maximal amplitude. slwer transient inward current surges prbably reflecting calcium actin ptentials generated in the dendritic tree [8,27]. Persistent currents were measured within segments in. During the same bath change, [K ] was raised t 0 f traces tward the end f the deplarizing vltage steps mm. INa,P was augmented, demnstrating the independence where firing ceased, r else between spikes. 2 f the high [K ] effect frm Ca influx (Fig. 4B). After subtracting linear leak and hlding current ff line, a slwly inactivating inward current was evident in 3.4. TTX and lw [Na ] suppress high K -augmented recrdings frm all bserved cells in nrmal slutin (Figs. persistent inward current 5 and 6) (als nted in an earlier study, see Ref. [27]). I V curves were recrded at 90-s intervals ver a perid f Expsing cells t 0.5 mm tetrdtxin (TTX) sup min in cntrl slutin, fllwed by a similar pressed the laser- and high [K ] -enhanced persistent length f time during perfusin with slutin cntaining 0 inward current (n53) (see als Ref. [37]). Substituting mm K. Equilibratin f in cncentratins between the

6 G.G. Smjen, M. Muller / Brain Research 885 (2000) Fig. 3. Raising [K ] hastens flurescence-induced ptentiatin f I Na,P. The maximal persistent inward currents measured in 0 flurescent islated cells befre, during and after raising bath [K ] t 20 mm. All recrdings were made with pipettes filled with KF-based slutin, with the flurescent dyes flu-3 and fura red added. During the entire perid, the cells were illuminated at 0- r 20-s intervals by -s pulses f laser light. The initial cntrl perid varied frm 5 t 4 min, expsure t 20 mm [K ] frm 6 t 4 min. interstitial fluid f the slice and the bath in an interface 2509 pa and after washing fr 5 20 min with nrmal chamber is usually cmpleted in abut 30 min [6,0]. Even slutin it decreased t 2344 pa (Fig. 5). thugh tissue [K ] prbably did nt reach the bath level, At the end f high [K ] treatment mm tetrdtxin persistent inward currents were enhanced in all trials (Figs. (TTX) was added t the bath in fur trials. Persistent 5 and 6). The maximal amplitude and the reversal ptential inward currents were substantially reduced by TTX, even f the current shifted t a mre deplarized vltage range. thugh the lss f the seal between pipette and cell When bath [K ] was raised, the maximal persistent current membrane 0 5 min after the start f TTX administratin increased frm pa (mean6s.e.m.; n56) t precluded recrding the full effect f the drug. Fig. 6A pa at 230 mv pipette vltage, while at 220 shws average I V curves frm all fur TTX-treated cells. mv it increased frm pa t pa. The The depressin by TTX affected the persistent inward difference was significant at bth vltages (P,0.02 and currents ver the entire vltage range. The curve f Fig. P,0.000, respectively, by tw-tailed paired t-test). An 6B was cnstructed by subtracting the cmpsite I V curve even mre striking change ccurred at a pipette ptential btained in the presence f TTX and high [K ] frm that f 0 mv, at which level in nrmal slutin the current flw taken in high [K ] withut TTX, and it represents the was utward, pa, but in 0 mm [K ] it turned TTX-sensitive part f the persistent inward current. Exinward, t pa (P,0.000). traplatin indicates that the TTX-sensitive current re- With bath [K ] elevated, spntaneus fluctuatins f verses clse t 42 mv, similarly t the TTX-sensitive the hlding current were nticed in several f the cells. INa,P f islated neurns [37,38]). Strngly deplarized ptentials evked irregular current fluctuatins even in nrmal slutin, which culd be described as vltage-dependent nise. The deplarizatin- 4. Discussin induced fluctuatins became mre marked when [K ] was raised (Fig. 5A). These irregular currents, which need nt The data shw that elevatin f extracellular K cn- be related t I Na,P, added t the persistent current am- centratin, [K ], causes a reversible increase f persistent plitude, which was measured as an average ver ms vltage-gated inward current in hippcampal CA pyramisegments f trace. This additin was mst marked at the dal cells. In islated cells this current was shwn t depend mre deplarized vltages (Fig. 6). n external Na cncentratin, while bth in neurns in Recvery during washing with nrmal slutin fllw- slices and in islatin it was suppressed by TTX, identify- ing high [K ] expsure was recrded in fur cells. In this ing it as persistent sdium current, I Na,P. sub-grup the cntrl recrding f the mean persistent Our gal was t investigate hw this current might inward current was, at 230 mv deplarizatin, 2408 pa, behave under clinically relevant cnditins, and fr this while after 5 6 min in 0 mm [K ] it increased t reasn in many f the trials we avided the use f in

7 08 G.G. Smjen, M. Muller / Brain Research 885 (2000) 02 0 Fig. 5. Enhancement f persistent inward current by elevated [K ] in a pyramidal neurn in a hippcampal tissue slice. (A) Sample whle-cell currents evked by deplarizing steps t pipette vltage f 230 mv, in nrmal ACSF, 6 min after raising [K ] in the bath t 0 mm, and after 5 min f washing with nrmal ACSF. (B) I V curves f the same cell as in (A). The current was measured during the last 50 ms f the Fig. 4. External calcium is nt required fr the enhancement f I Na,P.(A) deplarizing vltage step. Rati f flurescences f flu-3/ fura red recrded at 20-s intervals frm 2 an islated neurn. The sawtth pattern is created by Ca influx 2 thrugh vltage-gated Ca currents due t deplarizing vltage steps Patch clamp recrdings made frm islated cells and applied at -min intervals. At the brken vertical line the bath slutin frm cells in tissue slices each have their limitatins, was changed t ne cntaining 0 mm K, and n added calcium. (B) theretical and practical, and the tw methds cmplement I V curves f the slwly inactivating currents f the same cell as in (A), 2 ne anther. The islated neurns had lst mst f their btained just befre and 4 min after switching t high K, lw Ca dendritic tree and were kept at rm temperature in a bath slutin. The recrding pipette cntained CsF-based slutin. buffered by Hepes. Vltage clamping neurns in slices was less perfect than in islated cells, but the cnditins were channel blcking drugs. When all in channels were mre nearly nrmal, because cells retained mrphlgical available fr activatin, INa,P was detectable nly in a integrity, they were at a temperature that is nrmal fr rats, minrity f the islated cells in nrmal slutin. When in CO 2 / bicarbnate-buffered slutin. In additin, the [K ] was raised, INa,P made its appearance even in the recrding pipettes were filled with K-glucnate-based cells where its presence was nt evident befre. It seems slutin, instead f the mre alien KF r CsF. The reliable that in islated neurns in cntrl slutin INa,P was augmentatin f the persistent inward current in the cells usually hidden by the strng utward K currents. Its in slices thus validates the electrphysilgically mre enhancement in elevated [K ] was, in part, due t perfect data btained frm islated cells. weakening f the utward K currents resulting frm the In neurns in slices a persistent inward current was psitive shift f the K equilibrium ptential. That this is always detected even thugh K currents were nt supnt the whle explanatin is apparent frm the fact that pressed. The reasn culd be the presence f persistent elevated [K ] did enhance INa,P in the majrity f cells inward current in the dendrites that are lst during the even when K currents were pharmaclgically sup- dissciatin f islated cells [33]. In many cells the high pressed. K effect was mst cnspicuus at the mst deplarized

8 G.G. Smjen, M. Muller / Brain Research 885 (2000) During epileptifrm ictal events [K ] rises in hippcam- pal frmatin and necrtex t 8 2 mm, and during spreading depressin (SD) several times higher [2,23,4]. It is likely that during seizures, hypxia and SD the enhancement f the persistent inward currents is even mre marked than it was in these trials. Elevatin f [K ] caused less f an increase in INa,P than did intracellular flurescence f islated neurns [37,38], but the effect f high [K ] was cmparable t that seen in similar islated hippcampal neurns deprived f xygen, as reprted by Hammarstrm and Gage [9]. In intact brains, as als in brain tissue slices, hypxia causes [K ] t increase, and this increase begins sn after withdrawal f xygen [20,35]. Simultaneus decrease f xygen tensin and increase f [K ] prbably reinfrce each ther s effect n I Na,P. It may be, that elevated [K ], hypxia, and intracellular flurescence mdulate the persistent sdium current thrugh a cmmn intermediate step r steps. Regardless f mechanism, enhancement f persistent inward current prbably plays an imprtant part in the generatin f tnic seizures, SD, and SD-like hypxic deplarizatin. In cmputer-based simulatin either INa,P r an NMDA receptr-cntrlled current culd generate SDlike deplarizatin. When bth currents were available, as they are in live brains, the threshld was lwer and the nset faster [29]. Fig. 6. I V curves averaged frm fur cells, demnstrating TTX sensitivity f persistent inward current. (A) I V curves f the persistent cmpnent f currents. Pints are the averages frm fur cells, in nrmal ACSF, in elevated bath [K ], and in elevated bath [K ] with.0 mm TTX added. (B) The TTX-sensitive cmpnent f the persistent current, calculated frm the data f (A), by subtracting data btained in the presence f TTX and high [K ] frm thse in high [K ] withut TTX. Acknwledgements Supprted by NIH grant NS References vltages, in the range frm 0 t 20 mv (Fig. 6). It must be remembered, hwever, that these psitive pipette vltprpagatin f SD and hypxic SD-like deplarizatin in rat [] P.G. Aitken, G.C. Tmbaugh, D.A. Turner, G.G. Smjen, Similar ages are translated t mre mderate deplarizatin at hippcampus recrded ptically and electrically, J. Neurphysil. 80 sme distance frm the cell sma. The disprprtinate (998) grwth f the current evked by the mst psitive pipette [2] C. Alzheimer, P.C. Schwindt, W.E. Crill, Mdal gating f Na vltage may thus signal the increasing cntributin by the channels as a mechanism f persistent Na current in pyramidal mre distal dendrites. The irregular current fluctuatins neurns frm rat and cat sensrimtr crtex, J. Neursci. 3 (993) was als mst cnspicuusly bsted at strngly deplar- [3] P.H. Barry, JPCalc, a sftware package fr calculating liquid ized pipette vltage. Cnceivably these fluctuatins repre- junctin ptential crrectins in patch-clamp, intracellular, epithelial sent dendritic NMDA receptr-cntrlled currents. Vltage and bilayer measurements and fr crrecting junctin ptential 2 dependence mediated by Mg is a well-dcumented measurements, J. Neursci. Methds 5 (994) feature f NMDA-cntrlled currents [25]. High [K ] [4] J. Bures, ˇ O. Buresva, ˇ J. Krivanek, ˇ in: The Mechanism and Applicatins f Lea s induces irregular firing in afferent neurns and thus release Spreading Depressin f Electrencephalg- raphic Activity, Academia, Prague, 974. glutamate at synaptic sites. Such firing is suppressed by [5] E. Carmeliet, Cardiac inic currents and acute ischemia: frm TTX, but elevated [K ] is als expected t cause nn- channels t arrhythmias, Physil. Rev. 79 (999) synaptic glutamate release [,4,42] that is nt affected [6] S.R. Chebab, M.A. Hester, J. Jing, P.G. Aitken, G.G. Smjen, by TTX. These cnjectures, althugh plausible, require Interstitial space, electrical resistance and in cncentratins during further study. hyptnia f hippcampal slices f rats, J. Physil. 487 (995) After nly 5 6 min f raising bath [K ], the cn- [7] W.E. Crill, Persistent sdium current in mammalian central neurns, centratin f K in interstitial fluid f the tissue slice is Annu. Rev. Physil. 58 (996) almst certainly much lwer than it is in the bath [6,0]. [8] G. Czeh, P.G. Aitken, G.G. Smjen, Membrane currents in CA

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