Electrophysiological actions of felbamate on rat striatal neurones

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1 Bridsh Journl of Phrmcology (1995) 116, B 1995 Stockton Press All rights reserved /95 $12.00 A Electrophysiologicl ctions of felmte on rt stritl neurones *Antonio Pisni, *Alessndro Stefni, *Antonio Sinisclchi, *Nicol B. Mercuri, * tgiorgio Bernrdi & l'*polo Clresi *Clinic Neurologic, Dip. Snit e Biologi Cellulre, Universit di Rom 'Tor Vergt', vi 0. Rimondo, Rom, Itly nd tirccs - Clinic Snt Luci, vi Ardetin 306, Rome, Itly Keywords: 1 We hve investigted the effects of the nticonvulsnt drug, felmte (FBM), on stritl neurones, recorded in vitro y using oth intrcellulr nd extrcellulr conventionl recordings in slices nd whole-cell recordings in cutely isolted neurones. 2 FBM, t therpeuticlly relevnt concentrtions ( ym) showed multiple mechnisms of ction. Like other ntiepileptic drugs, FBM ( ym) showed direct inhiitory ction on current-evoked firing dischrge of stritl neurones. A ptch-clmp nlysis of this effect reveled dose-relted reduction of voltge-dependent sodium (N+) currents ( gm), with hlf inhiition dose (IC,0) vlue of 28 pm. 3 We lso tested whether FBM ffected corticostritl glutmtergic trnsmission. In control medium (1.2 mm externl mgnesium), oth extrcellulrly recorded field potentils nd intrcellulrly recorded excittory postsynptic potentils (e.p.s.ps) evoked y corticl stimultion were not ffected y th ppliction of gm FBM. 4 When mgnesium ws removed from the perfusing solution, procedure which revels N-methyl-Dsprtte (NMDA)-medited component in the corticostritl synptic potentil, FBM ( MM) produced dose-dependent reduction of the mplitude of oth the field potentil nd the e.p.s.p. 5 FBM reduced the inwrd currents produced either y th or y focl pplictions of 30 gm NMDA, finding consistent with the hypothesis tht the oserved reduction of the NMDA-medited component of the synptic potentils my e cused t postsynptic level. 6 The reduction of the NMDA-medited component of the synptic trnsmission y FBM nd its depressnt effect on the voltge-dependent N+ chnnels, my ccount for the ntiepileptic ction of this drug. Moreover, the phrmcologicl properties of FBM might render this drug interesting s neuroprotectnt gent. Felmte; ntiepileptic drugs; synptic potentils; ptch-clmp; stritum; excittory mino cids; NMDA; sodium chnnels Introduction FBM (2-phenyl-1,3-propnediol-dicrmte), is new ntiepileptic drug, which hs een shown to e effective in the tretment of prtil onset seizures nd in the Lennox-Gstut syndrome (The felmte study group in Lennox-Gstut syndrome, 1993). FBM hs een demonstrted to e efficcious in lrge spectrum of in vivo epilepsy niml models. In rodents, FBM inhiits seizures induced y mximl electroshock, y picrotoxin, pentylentetrzol nd 4-minopyridine, ut not y icuculline or strychnine (Swinyrd et l., 1986; for review see Burdette & Sckellres, 1994). In Rhesus monkeys, FBM reduced seizures produced y luminium hydroxide injections into corticl regions (Lockrd et l., 1987). Moreover FBM ntgonizes seizures induced y N-methyl-D-sprtte (NMDA) nd kinte-induced sttus epilepticus (White et l., 1992; Chronopoulos et l., 1993). Most common nticonvulsnts ffect N+ chnnels, clcium chnnels, nd/or the GABAA-enzodizepine receptor complex. Investigtions on the mechnism of ction of FBM demonstrted tht this drug hs unique phrmcologicl profile: in fct n ction on voltge-dependent N+ chnnels hs een descried (White et l., 1992). However, its primry ntiepileptic effect ppers to e t the NMDA receptor chnnel complex, where FBM displces 5,7-[3H]-dichlorokynurente from the strychnine-insensitive glycine inding site (McCe et l., 1993). This effect hs een demonstrted lso in humn postmortem rins (Wmsley et l., 1994). In whole-cell recordings from cultured hippocmpl neurones, FBM locked NMDA responses. The sme group reported potentition of GABAA responses y FBM (Rho et l., 1994); however cler ction on GABA receptors is controversil (Ticku et l., 1991; 1 Author for correspondence. Domenici et l., 1994). Furthermore, n ction of FBM on non- NMDA glutmte receptors hs een descried (De Srro et l., 1994; Domenici et l., 1994). Experimentl evidence emerged in fvour of neuroprotectnt ction of this drug. In in vivo models, FBM reduced the re of infrction following ilterl crotid ligtion (Wsterlin et l., 1992). When tested in vitro ginst hypoxic insults in the CAl region of the hippocmpus, FBM showed mrked neuroprotection (Wllis et l., 1992). In n ttempt to elucidte further the mechnism of ction of FBM, we studied the electrophysiologicl effects of this drug on stritl neurones, recorded either from corticostritl rin slice preprtion or from cutely isolted cells. Corticostritl fires represent the mjor excittory input to the neostritum (Reui & Cuenod, 1979). Overctivity of excittory pthwys within the sl gngli hs een thought to ply n importnt role in the pthogenesis of neurodegenertive disorders such s Huntington's disese nd Prkinson's disese (Turski & Turski, 1993). The phrmcologicl modultion of excittory mino cid trnsmission in the sl gngli might e of interest for the tretment of such disorders. Our findings suggest tht FBM might deserve evlution for its phrmcologicl properties s neuroprotectnt gent. Methods Preprtion nd mintennce of the slices nd of isolted cells Adult mle Wistr rts ( g) were used for ll the experiments. Detils of the preprtion nd mintennce of the

2 2054 slices hve een previously descried (Clresi et l., 1991). Briefly, rts were killed y hevy low to the chest under ether nesthesi, which severed mjor lood vessels. The rin ws quickly removed nd coronl slices ( um thick) including cortex nd neostritum were prepred from tissue locks with the use of virtome. A single slice ws then trnsferred to recording chmer nd continuously perfused with solution (360C, 2-3 ml min-) contining (in mm): sodium chloride (NCl) 126, potssium chloride (KCl) 2.5, monosodium cid phosphte (NH2PO4) 1.2, mgnesium chloride (MgCl2) 1.2; clcium chloride (CCl) 2.4, glucose 10 nd sodium cronte (NHCO3) 26; the solution ws gssed with mixture of 02 (95%) nd CO2, (5%). In some experiments MgCl2 ws omitted from the solution. For whole-cell recordings, isolted stritl cells were used. Preprtion nd mintennce of cutely isolted stritl cells hve een descried in detil y Stefni et l. (1994). In rief, coronl slices, 450 pm thick, were incuted in HEPESuffered Hnk's lnced slt solution (HBSS), uled with 100% 02 nd wrmed t 34WC. One hour lter one slice ws A. Pisni et l Felmts In the Wtum trnsferred to HBSS medium supplemented with 1.5 mg ml-' protese XIV. After enzymtic tretment, the tissue ws rinsed nd mechniclly triturted. The cell suspension ws finlly plced in Petri dish mounted on stge of n inverted microscope (Nikon). Electrophysiologicl studies Extrcellulr recording electrodes were filled with 2 M NCl; intrcellulr recording electrodes were filled with either 2 M KCl or 2 M K-cette (30-60 MCI). For synptic stimultion, ipolr electrodes were used. These were plced in the cortex, close to the recording electrode, or in the white mtter etween the cortex nd the stritum. The field potentil mplitude ws defined s the verge of the mplitude from the pek of the erly positivity to the pek negtivity, nd the mplitude from the negtivity to the pek lte positivity (Alger & Teyler, 1976). During voltge-clmp experiments hedstge voltge ws monitored continuously. An Axoclmp 2A mplifier ws used for recordings. Trces were displced on n oscilloscope nd / (V 2 ftk"po 20 mv.ft L na 80 ms 1.0r x E E mv -50 mv 400 pa c._. 0.6 :3 c 'O ms EE- 0.2 o z log dose FBM (M) Figure 1 Felmte (FBM) reduces repetitive firing dischrge nd inhiits N+ currents in stritl neurones (). A depolrizing current-pulse (+1 na intensity) induced tonic firing dischrge in stritl neurone recorded from corticostritl slice preprtion (RMP -80mV) (). In the presence of FBM 100Mm, the firing frequency evoked y the depolrizing step ws reduced. Note tht the ction potentils were prtilly truncted y digitl plotting (). Effect of FBM on the IN of stritl neurone. On the left, the currents were ctivted y depolrizing step pulses to -2OmV (from holding potentil of -70mV): control () nd FBM 5OMM (). The FBM-medited inhiition ws 60%. On the right, dose-response curve for the inhiitory ction of FBM on the pek mplitude of IN. Ech point represents the men ± s.e.men reltive mplitude of 3 experiments. The IC50 for FBM ction ws 28 AM.

3 stored on digitl system. Vlues given in the text nd in the figures re expressed s men ± s.e.men. The sttisticl significnce of the experiments ws evluted with the use of Student's t test. Drugs were th-pplied y switching the superfusing solution to one contining known concentrtions of drugs. In some experiments NMDA ws pplied y ejecting few nnolitres of 500 ym solution from the tip of lunt pipette eneth the surfce of the superfusing solution nd just ove the tissue slice. Ptch-clmp recordings in the whole-cell configurtion were performed with fire-polished pipettes (Corning 7052), with impednce from 3 to 8 MQ. Bth s well s dilysing solutions were designed to seprte clerly the voltge-dependent tetrodotoxin (TTX)-sensitive N+ current (IN). Pipettes were filled with n internl solution consisting of (in mm): EGTA 10, TrisPO4 72, Tris se 41, mgnesium 4, phosphocretine 20, ATP 2-4, GTP 0-0.2, leupeptin 0.2; ph ws djusted to A. Pisni et l Felmte In the shitum 7.3 with phosphoric cid; the osmolrity ws mosm 1-1. After otining the whole-cell configurtion, cells were thed in medium composed of (in mm): HEPES 10, NCl 30-60, TEA , clcium 2, mgnesium 1, cdmium 0.4. s well s drug solutions were pplied with liner rry of six, grvity-fed cpillries positioned within 500 pm of the ptched neurone. Recordings were mde with n Axoptch ID t room temperture. Series resistnce compenstion (70-80%) ws employed. Dt were low-pss filtered (corner frequency = 5 khz). For dt quisition nd nlysis pclmp 55.1 running on PC486 ws used. Sources nd hndling of compounds 2055 Compounds were otined from Sigm (St. Louis, U.S.A.) with the exception of FBM (2-phenyl-1,3-propneidol-dicrmte) which ws gift from Schering-Plough (Milno, I 400 pa 5 ms 1.0 _ _ x _E Prepulse voltge (mv) Figure 2 Effect of felmte (FBM) on the stedy-stte inctivtion curve of N+ chnnels. () Trces represent currents ctivted y step depolriztions to -40 mv from progressively less polrized potentils (from -110 to -20 mv), oth in control condition () nd in the presence of 30 sm M FBM (). () The dt points were collected s normlized current mplitude (verticl xis) t -20 mv following the corresponding conditioning voltge step (sciss scle): (0) ; (-) FBM 30 ym; ([l) wsh. The curves were well fitted ccording to the Boltzmnn eqution I= 1/1 +exp [V conditioning-vip2/vhj where V1/2 is the conditioning voltge t which IN is hlf mximl. The slope fctor ws 7.29 in control condition, 7.22 in the presence of FBM, 7.4 fter FBM wsh.

4 2056 Itly). FBM ws dissolved in dimethylsulphoxide (DMSO) in 100 mm stock solution, nd diluted in perfusion medium to the finl concentrtion immedited prior to use. The highest concentrtion of DMSO in finl working solution ws out 0.3%. Experiments with this concentrtion of the vehicle were performed in ll the configurtions descried. In none of these did DMSO produce significnt effects on intrinsic memrne properties or on synptic potentils mplitude (dt not shown). The limited soluility of FBM precluded the possiility of testing higher concentrtions of this drug. In fct, thppliction of 1 mm FBM, or the corresponding volume of vehicle, induced n irreversile memrne depolrizing. During intrcellulr recordings, performed in the voltge-clmp mode, 1TX 1 um ws pplied throughout the recording in A. Pisni et l Fdm in the stratm order to void contmintion of the inwrd current y voltgectivted N' conductnces. Results Electrophysiologicl chrcteristics of the recorded cells nd ction of FBM on firing ctivity nd N+ current Conventionl current- nd voltge-clmp recordings were otined from 56 electrophysiologiclly identified 'principl' stritl neurones. The chrcteristics of these cells hve een descried in detil previously (Clresi et l., 1990). These neurones showed high resting memrne potentil (RMP, 120 Mg 1.2 mm 100 E 80 o 60._ I Time (min) Mgnesium 1.2 mm Wsh (iii) 2 mv 2 ms (mi (iv) (v) Figure 3 In mgnesium-free solution the reduction in the field potentil mplitude produced y dding 1.2mM mgnesium to the thing solution is mimicked y 100pM felmte (FBM). In () the grph shows the time-course of single experiment. Bthppliction of 1.2mM mgnesium contining solution produced reduction of the field potentil mplitude. After wsh-out of mgnesium, ppliction of 100 pm FBM induced n inhiition which mimicked the effect of mgnesium. In () the trces represent the different phses of the experiment: control in mgnesium-free solution, in the presence of 1.2mM mgnesium, wsh in the sence of mgnesium (iii), (iv), 100pM FBM (v), wsh FBM (vi).

5 -80±5 mv), sence of spontneous ction potentil dischrge, tonic firing ctivity during current-induced memrne depolriztion. Morphologicl studies suggest tht these cells re medium-sized spiny neurones which re the lrge mjority of neuronl popultion in the stritum (Groves, 1983) nd represent the GABAergic output drive towrds glous pllidus nd sustnti nigr. As shown in Figure l, depolrizing current pulses ( + 1 na intensity, 200 ms durtion) evoked sustined ction potentil dischrge with no detectle ccomodtion (Figure l). As shown in Figure l, th ppliction of 100 gim FBM reduced the current-evoked firing ctivity of stritl neurones (n = 13). Interestingly, in most of the recorded neurones, the first ction potentils occurring in the initil phse of the current-induced memrne depolriztion were not inhiited. In 3 out of 13 cells only prtil recovery ws oserved. These findings suggested tht FBM, s lredy descried for other nticonvulsnts, modultes the voltge-dependent IN. A. Pisni et l Felmte In the strium In order to test this possiility, we isolted IN in whole-cell recordings from cutely dissocited medium-sized spiny neurones. This current ws completely locked y nnomolr concentrtions ( JlM) of TTX (n= 10, dt not shown). Figure l shows tht FBM modultes the lek-sutrcted IN (-60%, Figure l). In ll the neurones tested (n=30), FBM ( um) reduced the pek IN. As reveled y the dose-response curve (l), the ICm for this effect ws 28 gm. The ction of FBM on IN ws further investigted y stedy-stte inctivtion protocols (Figure 2). The neurones ws clmped t progressively less polrized potentil (from to -20 mv, s indicted in Figure 2) nd finlly tested with the test pulse t -40 mv. IN ws normlized to the current induced y the prepulse to -110 mv. As shown in Figure 2, 30.M FBM shifted the inctivtion curve towrds more negtive vlues y 10.4 mv, without cler modifiction of the slope. A full recovery of the FBM response ws otined (Figure 2). Anlogous findings were oserved in 5 neurones (-11±3 mv). Effects of FMB on corticlly-evoked synptic potentils In control medium, synptic potentils evoked y corticl stimultion hve een shown to e completely locked y - mino-3-hydroxy-5-methyl-4-isoxzolepropionic cid (AM- PA) receptor ntgonists (Clresi et l., 1992). However, when mgnesium is omitted from the externl solution, NMDA-medited component is unmsked y the removl of the voltge-dependent lockde of this ion on the NMDA receptor-chnnel complex (Clresi et l., 1992). In this condition, re-ppliction of 1.2 mm mgnesium in the medium significntly reduced (-39±3%, n=4; P<0.005) the mplitude of extrcellulrly recorded field potentil (Figure 3 compre with ). This effect ws fully reversile (Figure 3(iii)) nd ws mimicked y the NMDA ntgonist, APV (50 gm, n = 4; dt not shown). In control conditions (1.2 mm mgnesium), FBM ( gm) did not ffect the mplitude of oth field potentils (Figure 4) nd e.p.s.ps (Figure 5). However, in the sence of mgnesium, FBM decresed the mplitude of field potentil (Figure 4) nd e.p.s.p. (Figure 5) in dose-dependent fshion. This inhiition ws fully reversile in most of the experiments fter period of wsh lsting t lest 15 min (Figure 4c(iii)). In some experiments only prtil recovery ws oserved fter 30 min wsh (Figure 5c(iii)). The mximl inhiition of the synptic potentils ws reched with 100 Mm FBM. These concentrtions of FMB cused neither chnges of the memrne potentil, nor modifictions of the current/voltge reltionship t suthreshold levels (dt not shown). As shown in Figure 3, sturting concentrtion of FBM (100 MM) produced the sme percentge inhiition in the field potentil mplitude s ws cused y 1.2 mm mgnesium. In the sence of mgnesium n increse in the synptic potentil mplitude ws oserved. For this reson in most of the experiments, the intensity of the stimultion ws reduced, in order to hve suthreshold e.p.s.p. efore the ppliction of FBM. In mgnesium-free solution, 'epileptic' dischrge ctivity ws not detected from stritl slices. This my e prtilly explined y the effectiveness of hyperpolrizing potssium conductnces in stritl neurones (Clresi et l., 1987). Effects of FBM on NMDA-induced responses in voltgeclmped stritl neurones In order to chrcterize the effect of FBM t the postsynptic level, we tested whether FBM ffected the postsynptic sensitivity to NMDA of voltge-clmped stritl neurones recorded from slices. These experiments were performed in the presence of mgnesium nd of 1 JlM TTX in the thing solution. NMDA ws either th-pplied or loclly dministered y locl pressure ejection. As shown in Figure 6, in the presence of mgnesium, in ll the tested neurones (6 out of 6), 100 JIM FBM cused significnt reduction (-24±3%; P<0.001) of.0 c0 c %I- 0 ' E 0-0. ~0._ 60r 50H 40o 30 H F- C log dose FBM (lgm) Mgnesium 1.2 mm (i Mgnesium-free Wsh ms 2 mv 2057 Figure 4 Effects of felmte (FBM) on the mplitude of extrcellulry recorded field potentils. The grph in () shows tht, in the sence of mgnesium, incresing doses (10-300,uM) of FBM produced dose-dependent decrese in the mplitude of field potentils, reching plteu t 100pM. Ech point represents the men (±s.e.men) of severl experiments (1OpM, n=4; 30M, n=5; 100pM, n=7; 300pM, n=4). In () nd (c) the trces represent the field potentils (ech trce is the verge of four sweeps) recorded in the presence of mgnesium () nd when mgnesium ws omitted (c). Note tht 100pM FBM hd no effect on field potentil mplitude in mgnesium-contining solution (,), while when mgnesium ws omitted cler decrese in the field potentil mplitude ws oserved (-(iii)). Clirtions pply for oth () nd (c).

6 2058 the inwrd current cused y pressure ejection of NMDA (500 Mm, Figure 6). This effect ws completely reversed fter 15 min FBM wsh (Figure 6(iii)). Figure 6 shows tht, in the presence of mgnesium in the perfusing medium, the inwrd current induced y th ppliction of NMDA (30 MM, n=8) ws lso reduced y 100 M FBM (-29±4%, n=5; P<0.001) (Figure 6). In some of the recorded neurones (4 out of 8) only prtil recovery ws oserved fter 15 min wsh (Figure 6(iii)). Experiments were performed in order to evlute the effect of FBM (100 pm) on the current-voltge A. Pisni et l Felmte In te strtum reltionship of the recorded neurones. In none of the tested cells (n=4), did FBM significntly ffect the current-voltge plot (dt not shown). Discussion The present study provides evidence tht FBM limits the firing ctivity of stritl neurones, y cting directly on voltge-dependent N' currents. In ddition, FBM locks the NMDA c 0 '4-._ 0._ G) 0. 6i E ci so 0- di log dose FBM (gm) 1000 Mgnesium 1.2 mm o up ~. c / Mgnesium-free 4 4 Wsh 4 20 msec 20 mv Actions of felmte (FBM) on the mplitude of corticlly-evoked excittory postsynptic potentils (e.p.s.ps). The grph Figure 5 in () shows the dose-dependent reduction of the mplitude of intrcellulry recorded e.p.s.p. in mgnesium-free thing medium. The effect incresed in dose-dependent fshion, reching plteu t 1001M. Ech point represents the men (+s.e.men) of severl experiments (1O AM, n=4; 30 pm, n =4; 100pM, n= 7; 300pM, n = 3). The trces in the lower prt represent the e.p.s.ps (ech trce is the verge of four sweeps) recorded in the presence of 1.2mm mgnesium () nd when mgnesium ws omitted (c). In control conditions no effect of FBM ws oserved on e.p.s.p. mplitude (,). When mgnesium ws removed from the thing solution, 100pM FBM reduced the e.p.s.p. mplitude, nd only prtil wsh-out ws oserved (c-(iii)). Clirtions pply for oth () nd (c).

7 A. Pisni et l Femte In the stritum 2069 NMDA30gM In NMDA 30 gm, NMDA 30 gm 200 pa 1 min NMDA30 gm In NMDA330gM NMDA 30 gm OD (iii) 200 pa FIgure 6 Effects of felmte (FBM) on NMDA-induced inwrd current in voltge-clmped stritl neurones recorded from slice preprtion in the presence of mgnesium in the medium. () Focl ppliction of 301AM NMDA induced rpid nd trnsient inwrd current (), which ws reduced in the presence of 100AM FBM (). After FBM wsh-out, recovery of the NMDAinduced inwrd current ws oserved ((iii), RMP -85mV) (). Bth-ppliction (30s) of 30AM NMDA induced slow inwrd current. In the presence of 100AM FBM, the NMDA-induced inwrd current ws reduced. After wsh-out of FBM, prtil recovery of the NMDA-induced inwrd current ws recorded. 1 min component of oth extrcellulrly nd intrcellulrly recorded synptic potentils evoked y corticl stimultion nd reduces NMDA-induced inwrd current mesured in voltge-clmped stritl neurones. Although n interction with the NMDA receptor chnnel complex hs een lredy suggested for FBM (McCe et l., 1993; Rho et l., 1994; Domenici et l., 1994), to our knowledge this study represents the first investigtion of the electrophysiologicl ctions of FBM on n identified glutmtergic pthwy in the mmmlin rin. It is worth noting tht oth limittion of firing ctivity of stritl neurones nd modultion of corticostritl synptic potentils occurred t concentrtions very similr to those chieved either in the rin nd plsm of FBM-treted nimls nd in plsm of humn sujects during clinicl trils (McCe et l., 1993; Wgner et l., 1990), or in surgicl smples from ptients receiving therpeutic dosges of this drug (Adusumlli et l., 1994; Tylor et l., 1995). The suppression of IN hs een indicted s common mechnism of ction of mny nticonvulsnts: the interction with the N' chnnels hs in fct een demonstrted lredy for different ntiepileptic drugs, like phenytoin (Yri et l., 1986), crmzepine (McLen & McDonld, 1986), oxcrzepine (Clresi et l., 1995), gpentin (Wmil & McLen, 1994), lmotrigine (Cheung et l., 1992). In line with those evidences, FBM ws lso found to interfere with the voltge-dependent TTX-sensitive IN of stritl neurones. In prticulr, we hve shown cler effect on the stedy-stte inctivtion properties of IN.. This ction my well ccount for the FBM-medited decrese of the current-induced ction potentil dischrge of stritl neurones, intrcellulry recorded from corticostritl slices. Limittion of high frequency repetitive firing ctivity y FBM ws use-dependent, s reveled y the evidence tht the ction potentils occurring in the lte phse of the current-induced depolriztion were preferentilly inhiited. The pprent discrepncy etween the FBM-induced reduction of the N+ oserved in the whole-cell recordings nd the lck of effect of this drug on the ction potentils evoked t the eginning of the long-lsting depolrizing pulse my hve different explntions. First, the recording conditions were different: in the whole-cell mode, the extrcellulr N+ concentrtion ws mintined rther low (< 60 mm, in comprison with the N+ concentrtion during conventionl recordings from slices, which ws mm) thus llowing us to void the ctivtion of very lrge currents nd the genertion of spce-clmp prolems. Moreover, it should e noted tht, in the isolted cell preprtion, N+ currents minly depend on the ctivtion of somtic chnnels since most of the dendritic roriztion is lost. On the other hnd, in slice preprtions the firing dischrge evoked y depolrizing pulse represents the integrtion etween somtic nd dendritic ctive conductnces. Thus, it is conceivle tht the ction of FBM on N+-dependent ction potentils is less evident in the ltter experimentl condition which reflects oth proximl nd distl neuronl ctivity. However, in some experiments, ws oserved tht FBM ws lso le to prevent the genertion of the first ction potentil when it ws induced y the miniml suprthreshold current (dt not shown). A reduction in the efficcy of the glutmtergic trnsmission hs een climed s prt of the mechnism of ction of some nticonvulsnts (McDonld & Kelly, 1993; Wlker & Snder, 1994). Besides the 'locl nesthetic'-like properties, shred with most common nticonvulsnts, here we hve shown tht FBM ffects corticostritl glutmtergic trnsmission. In prticulr, in the sence of mgnesium from the externl medium, cler reduction of the synptic potentil mplitude induced y FBM ws oserved, suggesting rther selective

8 2060 A. Pisni et l Felmte In the strtum ction on the NMDA-medited component of the glutmtergic potentils. We found tht FBM lters the postsynptic sensitivity to NMDA, in fct it reduces the NMDA-induced responses in voltge-clmped stritl neurones. This evidence suggests tht the FBM-induced decrese in the NMDAmedited component of synptic potentils my lso e due to n interction t postsynptic site. Recently, it hs een hypothesized tht some 'new' nticonvulsnt drugs ct presynpticlly ffecting glutmte relese, presumly through modultory ction on voltgedependent clcium chnnels regulting trnsmitter relese (Cheung et l., 1992; Clresi et l., 1995; Stefni et l., 1995). The evidence tht FBM does not lter the mplitude of synptic potentils recorded under physiologicl conditions (plus 1.2 mm mgnesium), seems to suggest tht the presynptic mechnisms regulting relese from corticostritl terminls re not ffected y this drug. A further indiction for lck of effect on presynptic terminls comes from recent report which suggests tht conotoxin-sensitive, N-type voltge-dependent clcium chnnels, which hve een shown to ply mjor role in the control of the corticostritl trnsmission (Clresi et l., 1994) re not ffected y FBM (Stefni et l., in preprtion). However, our experimentl model does not llow us to exclude n effect t the presynptic level. Incresed glutmtergic trnsmission within the sl gngli hs een implicted in the pthophysiology of epileptogenesis nd of neurodegenertion disorders, such s Huntington's disese nd Prkinson's disese (Nefsey et l., 1979; Turski et l., 1986; Bel, 1992). Mny groups reported tht ntgonists of NMDA nd non-nmda receptors s well s clcium ntgonists were effective s neuroprotective gents in wide vriety of in vitro nd in vivo models (Simon et l., 1986; Choi et l., 1988). Recent evidence suggests tht FBM my hve n nti-prkinsonin potentil, since it reversed dopmine D2 receptor ntgonist-induced kinesi (Kretschmer, 1994). Yet, competitive nd non-competitive NMDA receptor ntgonists hve een found efficcious in experimentl models of Huntington's disese (Bel et l.,1993; Greene et l., 1993). Unfortuntely, most of the competitive ntgonists cting on the glutmte recognition site of the NMDA receptor pper to e ssocited with severe cognitive side effects, similr to those cused y the noncompetitive NMDA chnnel lockers (Sveinjornsdottir et l., 1993). FBM competes with glycine or D-serine t the cogonists recognition site of the NMDA receptor chnnel complex (McCe et l., 1993). Its ntiepileptic efficcy my well e explined y the multiple effects descried. Recent clinicl experience suggests tht the use of FBM my e ssocited with severe dverse effects, such s plstic nemi, (Nightingle, 1994). 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