Effects of Antiepileptic Drugs on GABA Responses and on Reduction of GABA Responses by PTZ and DMCM on Mouse Neurons in Cell Culture

Home , Phensuximide

Epdepsia, 30(1):17-25, 1989 Raven Press, Ltd., New York 0 International League Against Epilepsy Effects of Antiepileptic Drugs on GABA Responses and on Reduction of GABA Responses by PTZ and DMCM on Mouse Neurons in Cell Culture

*?Peter P. De Deyn and ?Robert L. Macdonald

*Department of Neurology, University of Michigan Medical Center, Ann Arbor, Michigan, U.S.A., and ?Research Institute, Born Bunge Foundation, University of Antwerp, Laboratory of Neurochemistry and Neuropharmacology, Antwerp. Belgium

Summary: The mechanisms of action of antiepileptic 98% (2 pM), and ZK 98% (2 pM) did not alter GABA drugs effective against generalized absence seizures responses. DZP enhanced GABA responses in a concen- (antiabsence AEDs) remain uncertain. Antiabsence tration-dependent manner. The inhibition of GABA re- AEDs are generally effective against seizures induced in sponses produced by PTZ 1 mM was unaltered by ESM experimental animals by pentylenetetrazol (PTZ) and (600 pi'@), DMO (6 mM), CGS 98% (1 pkf), or ZK 98% methyl~,7-dimethoxy~-ethyl-~-carboline-3-c~boxy~ate(1 pM). Coapplication of VPA (200 pM) and PTZ (1 mM) (DMCM), drugs which reduce GABAergic inhibition. slightly enhanced the FTZ effect. DZP (>lo nM), how- Thus, antiabsence AEDs have been suggested to enhance ever, reversed the PTZ-induced reduction of GABA re- GABAergic inhibition. We studied the effects of several sponses. The DMCM (250 nM) inhibition of GABA- AEDs on GABA responses recorded from mouse spinal responses was unaltered by ESM (600 pw, DMO cord neurons grown in primary dissociated cell culture. (2 mM), or VPA (200 pM). CGS 98% (2 pM) and ZK Four antiabsence AEDs were included: ethosuximide 912% (2 pM), however, antagonized the DMCM effect. (ESM), dimethadione (DMO), sodium valproate (VPA), DZP (> 10 nM) significantly reversed the DMCM-induced and diazepam (DZP). Two experimental AEDs, CGS inhibition of GABA responses. The lack of effect of VPA, 98% and ZK 912%, with anticonvulsant action against ESM, and DMO on postsynaptic GABA responses sug- PTZ- or DMCM-induced seizures were also included. gests that direct enhancement of postsynaptic GABA ac- Possible effects of the antiabsence and experimental tion is not a common mechanism of action of antiabsence AEDS on PTZ- and DMCM-induced inhibition of GABA AEDs. The AEDs DZP, CGS 9896, and ZK 912% all responses were also evaluated. PTZ and DMCM revers- reversed DMCM, but not PTZ, reduction of GABA re- ibly reduced GABA responses in a concentration- sponses, suggesting that these AEDs blocked DMCM sei- dependent manner. PTZ completely inhibited GABA re- zures by acting at benzodiazepine receptors. However, sponses at 10 mM (ICso of 1.1 mM), whereas DMCM- since only DZP enhanced GABA responses, it is unclear induced inhibition of GABA responses reached a plateau how CGS 98% and ZK 912% blocked PTZ seizures. Key level of 39% of control values at 1 pM (ICso of 33 nM). Words: Anticonvulsants-GABA-Neuron culture-cell ESM (1,200 pm,DMO (6 mM), VPA (200 pM), CGS culture-Spinal cord neurons-Convulsants.

A reduction in central nervous system (CNS) alized absence seizures (antiabsence AEDs) may in- GABAergic inhibition has been suggested to be a volve enhancement of GABAergic inhibition (Mac- cause of epilepsy (Meldrum, 1979; Roberts, 1980; donald and McLean, 1986). Lloyd et al., 1981; Olsen, 1981), and it has been The GABAA receptor is an oligomeric complex suggested that the mechanism of action of antiepi- containing binding sites for GABA, barbiturates, leptic drugs (AEDs) used in the treatment of gener- benzodiazepines, f3-carbolines and picrotoxinlike drugs (Olsen, 1981; Olsen and Leeb-Lundberg, 1981). The structure of the GABA, receptor was Received February 1988; revision accepted June 1988. recently deduced from cDNA clones and is com- Address correspondence and reprint requests to Dr. R. L. posed of two a and two f3 peptide subunits which Macdonald at University of Michigan, Neuroscience Laboratory Building, 1103 East Huron St., Ann Arbor, MI 48104-1687, span the neuronal membrane to form a chloride U.S.A. channel (Schofield et al., 1987). GABA binds to the

17 18 P. P. DE DEYN AND R. L. MACDONALD receptor to open the chloride channel, resulting in tions. We also determined the concentration- membrane hyperpolarization and inhibition. dependent effects of the convulsant drugs PTZ and Several convulsants, including bicuculline, picro- DMCM on GABA responses and the effects of the toxin, penicillin, D-tubocurarine, pentylenetetrazol AEDS on PTZ- and DMCM-induced reduction of (PTZ) and a-keto-6-guanidino-valeric acid have GABA responses. been shown to be GABA antagonists, blocking the inhibitory action of GABA by interacting with ei- MATERIALS AND METHODS ther GABA or picrotoxin binding sites (Curtis et al., 1971; Curtis et al., 1972; Hill et al., 1972; Hill and Primary dissociated cell culture Simmonds, 1973; Macdonald and Barker, 1978~; Cultures of spinal cord neurons were prepared Johnston and Willow 1981; Olsen and Leeb- from dissected spinal cords and attached dorsal root Lundberg, 1981; De Deyn et al., 1988). Benzodiaz- ganglia from 12-14-day-old fetal mice as described epine receptor ligands which were convulsant or previously (Ransom et al., 1977). Experiments con- proconvulsant, such as the P-carbolines PCCM, formed to the policy of the American Physiological PCCE, and DMCM and the pyrazoloquinoline CGS Society. The tissue was minced and then mechani- 82 16 reduced GABAergic inhibition through an al- cally dissociated by trituration in Ca2+- and losteric interaction with the GABAA receptor M$+-free balanced salt solution to a suspension of (Braestrup et al., 1983; Peterson, 1983). single cells and small clumps. The dissociated cells AED reduction of seizures induced in experimen- were suspended in culture medium [W% Eagle’s tal animals by either intravenous (i.v.) or subcuta- miminal essential medium (MEM) supplemented neous (s.c.) administration of PTZ has been used to with 5.5 g glucose and 1.5 g NaHCO,/L, 5% heat- predict antiabsence efficacy of drugs such as diaz- inactivated horse serum, and 5% Nu-Serum I1 (Col- epam (DZP), trimethadione (TMO), sodium val- laborative Research) 325 mOsm] and then plated on proate (VPA), and ethosuximide (ESM) (Swinyard, sterile collagen-coated 35-mm dishes. The cultures 1969). The antiabsence AEDs were also effective were maintained in an incubator with an atmo- against DMCM-induced seizures (Petersen, 1983). sphere of 93% room air and 7% CO, at 35°C. The Experimental drugs also have an anticonvulsant ac- bicarbonate/CO, buffer maintained pH at 7.4. tion in experimental animals against PTZ or DMCM 5-Fluoro-2’deoxyuridine was added to the cultures seizures. The pyrazoloquinoline, CGS 9896, was on days 4-6 to suppress the growth of rapidly di- anticonvulsant in the PTZ test (Petrack et al., 1983), viding non-neuronal cells. Medium was changed and the p-carboline, ZK 91296, was protective twice weekly. Cultures were maintained for 4-9 against PTZ- and DMCM-induced seizures (Pe- weeks before electrophysiologic experiments were tersen et al., 1984; Klockgether et al., 1985). made. Enhancement of GABA-mediated inhibition has Experimental procedures been proposed as the mechanism of action of several AEDs used extensively in human therapy (Ec- Solutions cles et al., 1963; Nicoll, 1972; Macdonald and All recordings were made in a high magnesium Barker, 19786, 1979; Gent and Phillips, 1980; Shultz ion concentration of Dulbecco’s phosphate- and Macdonald, 1981). Anticonvulsant benzodiaz- buffered saline (PBS) after removal of growth me- epines such as DZP directly enhance GABA re- dium. The elevated magnesium ion concentration in sponses (Choi et al., 1977; Macdonald and Barker, the recording solution suppressed spontaneous syn- 19786), whereas VPA increases brain and synapto- aptic and action potentials. The recording solution soma1 GABA concentration (Godin et al., 1969; contained (in mM): NaCl 137, Na2HP04,8.06, KCl Simler et al., 1973; Iadarola and Gale, 1979). How- 2.68, KH2P041.47, CaCl, 1, MgCI, 10, and glucose ever, although DZP has been shown to enhance 5.6 (pH 7.4). Heavy paraffin oil was applied to the GABA responses, no clear action of TMO [or its surface of the bathing solution to retard evapora- active metabolite, dimethadione (DMO)] VPA, or tion. ESM on GABAergic inhibition at clinically relevant Solutions of drugs were always prepared on the concentrations has been reported. day of the experiment in the following manner: In this study, we determined whether or not the DMCM, DZP, and VPA were dissolved in dimeth- antiabsence (ESM, DMO, VPA, and DZP) and two ylsulfoxide (DMSO) to form lo-, 1-, and 60-mM experimental (CGS 9896 and ZK 91296) AEDs had stocks, respectively. DMO, ESM, and PTZ were an action on postsynaptic GABA responses on dissolved in recording solution to form 12-, 6-, and mouse spinal cord neurons grown in primary disso- 10-mM stocks, respectively. Aliquots were re- ciated cell culture at clinically re’cvant concentra- moved and diluted in bathing medium to obtain the

Epilepsia, Vof. 30, No. 1, 1989 AEDs: MECHANISMS OF ACTION 19 applied concentrations. The final solutions con- Direct effects of convulsant drugs and AEDs on tained GO.1% DMSO. GABA responses were accepted only if the GABA DMCM was applied at concentrations between 1 responses returned to control amplitude within 5-10 nM and 10 pM. PTZ concentrations ranged be- min of removal of the drug-containing micropipette. tween 62.5 pM and 10 mM. In the experiments test- In the studies evaluating effects of coapplication of ing the effects of AEDs on DMCM- and PTZ- the AEDs and the convulsant drugs on GABA re- induced reduction of GABA responses, DMCM sponses, data were accepted only when the original was used at a concentration of 250 nM and PTZ was direct effect of the convulsant drug could again be used at a concentration of 1 mM. The ranges of evoked after recovery of the GABA response from AED concentrations tested were determined from the combination effect. human clinical pharmacologic data when available. Drug application Concentrations equivalent to the clinically useful For evaluation of drug effects on GABA re- therapeutic range in CSF or comparable to the free sponses, all drugs were applied by a perfusion mi- levels in serum were used (Gugler et al., 1977; cropipette. A blunt-tipped pm) micropipette, Eadie and Tyrer, Sherwin, Wilder and (10-15 1980; 1982; filed with the recording solution containing convul- Karas, 1982). sant or convulsant and AED, was positioned 15-30 Experimental apparatus pm from the soma of the cell under study. The open For experiments, the culture dish containing end of the perfusion micropipette was connected to bathing solution was placed on a microscope stage a pressure regulator, set between 0.4 and 0.8 psi, by heated by a Pellitier device with temperature regu- tight-fitting polyethylene tubing. Pressure-pulse du- lated at 3435°C. The stage was mounted on a Leitz ration, regulated by a voltage-activated three-way inverted microscope fitted with phase-contrast op- valve, was 10 s. Under these conditions, local per- tics to facilitate micropipette placement (using Leitz fusion produced no artifacts, and application of re- micromanipulators) and to penetrate cells under di- cording solution (with or without vehicle) was vir- rect visual control. tually free of effects. When the effect of coapplication of convulsant drugs and AEDS was studied, Electrophysiologic recordings the drugs were applied through one perfusion mi- Intracellular recordings were made from the so- cropipette to avoid flow artifacts. The concentra- mata of spinal cord (>20 pm) neurons using glass tions reported in this study are those contained in micropipettes (25-50 Ma) filled with 3M KCI. Use the perfusion micropipettes. Although some reduc- of an active bridge circuit (Model 8100,Dagan, Min- tion in the concentration of drug at the neuron may neapolis, MN, U.S.A.) allowed simultaneous re- have occurred due to dilution in the surrounding cording of membrane potential and injection of medium, increasing durations of application or al- current (for steady-state polarization or periodic tering the position of the perfusion micropipette did stimulation) using a single micropipette. The pream- not increase the effects of the drugs. Thus, although plifier output signal was led to a six-channel poly- there may be some small inaccuracy in the drug graph (Model 2600, Gould Instruments, Cleveland, concentrations, the reported concentrations are OH, U.S.A.) for continuous recording. probably close to those at the surface of the neuron. The perfusion micropipettes and recording mi- GABA responses cropipettes were held by Leitz micromanipulators. GABA (0.5 M, pH 3.4) was applied iontophoret- To decrease leakage of drugs into the bathing me- ically using 500-ms duration rectangular current dium, the tips of the perfusion micropipettes were pulses at 5-s intervals. Tips of iontophoretic pi- kept in the oil phase between drug application trials. pettes were positioned to within 2 pm of neuronal They were lowered in the aqueous phase only when somata. The use of 3M KC1-filled micropipettes re- drug application was desired. sulted in elevation of intracellular chloride ion concentration and a shift in the chloride equilibrium mgs potential from about -65 mV to about -20 mV. CGS 9896 [2-(p-chlorophenyl)-2,5-dihydro- Under these conditions, an increase in chloride con- pyrazolo)-4,3-C)quinolin-3(5H)-11 was obtained ductance resulted in an outward chloride current from Ciba-Geigy (Summit, NJ, U.S.A.). DZP was giving depolarizing GABA responses (Nowak et al., obtained from Hoffman-LaRoche (Nutley, NJ, 1982). Responses of -10-15 mV in amplitude were U.S.A.). ZK 912% (ethyl 5-benzyloxy4methoxy- evoked following membrane hyperpolarization (to methyl-p-carboline-3-~-carboxylate)and DMCM between -70 and -90 mV) to avoid saturation at (methyl-6,7-dimethoxy4ethyl-~-carboline-3-~arb0x- or near the chloride equilibrium potential. ylate) were obtained from A/S Ferrosan (Denmark).

Epilepsia, VOI.30. NO.I, 1969 20 P. P. DE DEYN AND R. L. MACD0NAL.D

DMO (5,5-dimethyl-2,4-oxazolidine-dione),PTZ tained at 10 mM. The DMCM effect was also con- (6,7,8,9-tetrahydro-SH-tetrazoIo-[1,5-a]azepine), centration dependent (Fig. 2). A significant 9.9 * and GABA were purchased from Sigma Chemical 2.3% decrease of GABA responses (p < 0.001) was (St. Louis, MO, U.S.A.). VPA and ESM were ob- found at 10 nM. The inhibition of GABA responses tained from Parke-DavidWarner-Lambert Re- reached a maximum of 39% at 1 FM.Although both search Laboratories (Ann Arbor, MI, U.S.A.). PTZ and DMCM reduced GABA responses, DMCM was more potent than PTZ. The estimated Algebraic and statistical methods ICs0 values for the inhibition of GABA responses Mean values and SD were calculated for GABA were mM for PTZ and nM for DMCM. response amplitudes before, during, and after drug 1.1 33 application. Mean values and SD for the effects of Direct effect of the AEDs on GABA responses the coapplied convulsant drugs and AEDs on ESM (600 and 1,200 wICI), DMO (2 and 6 mM), GABA responses were also calculated. CGS 98% (2 pV), and ZK 912% (2 plf) did not All effects were expressed as percentage of alter GABA responses significantly (Table 1). VPA change in GABA response from control GABA re- (200 produced a small (2.1%) but significant sponse. The statistical significance of differences reduction of GABA responses. DZP applied at con- between GABA responses with and without drug centrations between 1 and 500 nM enhanced GABA application was calculated using the two-tailed Stu- responses in a concentration-dependent manner. dent's t test; p < 0.05 was considered statistically For DZP, a significant 12.6 f 3.4% enhancement of significant. GABA responses (p < 0.001) was obtained at 10 nM, and a peak enhancement of 82.4 k 5.7% was RESULTS obtained at 500 nM (Table 1). Direct effects of convulsant drugs PTZ and DMCM Effects of AEDs on FTZ-induced antagonism of on GABA responses GABA responses Application of PTZ or DMCM to spinal cord neu- The reduction of GABA responses produced by rons did not alter resting membrane potential or PTZ (1 mM) was unaltered by ESM (600 pM) and conductance. Application of recording solution DMO (2 and 6 mM) (Table 2). Similarly, the benzo- (with or without vehicle) (n = 10) did not signifi- diazepine receptor antagonists, CGS 9896 (1 pV) cantly alter GABA responses (100.8 k 1.9%). and ZK 91296 (1 phi), did not alter PTZ (1 mM) PTZ and DMCM rapidly and reversibly reduced reduction of GABA responses Vable 2 and Fig. 4A GABA responses. The PTZ effect was concentra- and B). VPA (200 FM), however, coapplied with tion-dependent (Fig. 1). A si&icant 6.2 -+ 2.1% PTZ (1 mM), slightly enhanced (p < 0.05) the in- decrease of GABA responses (p < 0.001) was ob- hibition of GABA responses induced by PTZ (Ta- tained at 125 ptf, and complete inhibition was ob- ble 2).

flG. 1. Concentration-dependent -10 - effects of pentylenetetrazol (PTZ) on GABA responses on spinal cord - neurons. GABA responses (bot- -20 tom) show the effect on one cell of - the respective concentrations of 6 -30 applied PTZ. Initial control (CTL) % -40 - GABA response shows a stable response before drug application. ,$ - GABA responses returned to con- 9 trol values within 2 min after re- --60 - moval of PTZ. lontophoretic GABA application is indicated by a dash. 0 --70 - Effects are expressed as percent- 6 age of change of the original GABA 6 response. Data are means plus SD N of responses from 3 to 13 neurons. SD shown in one direction only. "p < 0.001 from control GABA responses. Drug concentrations shown on abscissa are logarithm molar. I I I -4 -3 -2 Drug Conc Log (M)

Epilepsia, Vol. 30, NO, 1, 1989 AEDs: MECHANISMS OF ACTION 21

FIG. 2. Concentration-dependent effects of methyl-6,7- dimethoxy-4-ethyl-p-carboline- 3-carboxylate (DMCM) on GABA responses on spinal cord neu- -10 - rons. GABA responses (bottom) X show the effect on one cell of 5 the respective concentrations 0" of applied DMCM. Initial control ? -20 - (CTL) shows a stable GABA re- 2 sponse before drug application. 9 GABA responses returned to control values within 10 min af- O ter removal of the DMCM- % -30- containing micropipette. lonto- E phoretic GABA application is in- 5 dicated by a dash. Effects are f: expressed as percentage of change of the original GABA response. Data are means plus SD of responses from 5 to 15 neurons. SD shown in one direction only. "p < 0.001 from control ,I I I I GABA responses. Drug concen- -9 -8 -7 -6 -5 trations shown on abscissa are Drug Con< log (M) logarithm molar.

In contrast, DZP with a threshold concentration significantly antagonized the DMCM-induced inhi- of 10 nM (p < 0.05) significantly reversed the FTZ- bition of GABA responses (Table 3 and Fig. 4C and induced reduction of GABA responses. Coapplica- D). DZP, with a threshold concentration of 10 nM tion of PTZ (1 mM) and DZP (100 nM)resulted in a (p < 0.05), significantly reversed the DMCM- signifcant 7.4 & 5.3% enhancement of the GABA induced inhibition of GABA responses (Fig. 3B). response (p < 0.001) (Table 2 and Fig. 3A). Simultaneous application of DMCM 250 nM and DZP 500 nM resulted in a small increase (5.5%) in Effects of AEDs on DMCM-induced decreases of GABA responses compared to control responses GABA responses (Table 3). DMCM (250 nM) reduction of GABA-responses pM), mM), was unaltered by ESM (600 DMO (2 or TABLE 2. Efiecrs of AEDS on PTZ-induced VPA (200 CLM) (Table 3). Coapplication of DZP, antagonism of GABA responses CGS 9896 or ZK 91296, however, influenced the No. of GABA DMCM-induced inhibition of GABA-responses cells response (Table 3). CGS 9896 (2 pM) and ZK 91296 (2 CLM) studied (% control AED (n) rt SEM) FTZlmM 60.2 2 7.4 TABLE 1. Direct eflects of AEDS on GABA responses 7 PTZImM+ESM600@ 61.3 rt 5.8 FTZlmM 61.9 2 3.8 GABA response 4 No. of cells (% control PTZ 1 mM + DM 2 mM 62.4 2 5.4 FTZlmM 58.0 2 5.6 AED studied (n) f SEM) 3 PTZ 1 mM + DM06mM 59.1 rt 11.1 ESM 6OOJLM 8 102.3 2 3.3 FTZlmM 4 66.3 2 8.9 ESM 1,200 JLM 5 100.4 2 2.1 PTZ 1 mM + CGS 98% 1 )LM 67.3 2 8.4 PTZlmM 62.3 rt 7.2 DMO 4 2mM 3 99.3 ? 1.7 FTZ 1 mM + ZK912% 1 )LM 61.0 rt 11.6 6mM 4 99.3 2 1.4 FTZlmM 7 60.0 2 6.2 VPA 200 )LM 8 97.9 2 1.7" FTZl mM + VPA200uM 57.8 2 5.4" FTZlmM 63.2 rt 7.2 CGS 98% 2 pM 10 100.2 ? 4.6 5 ZK 912% 2 pM 6 100.3 2 2.7 FTZ 1 mM + DZP 1 nM 61.5 2 8.5 FTZImM 67.6 5 6.2 DZP 6 1nM 5 100.1 2 1.2 FTZ 1 mM + DZP 1OnM 70.6 2 4.7' FTZImM 6.3 10 nM 5 112.6 ? 3.4' 8 64.0 t 100 nM 38 143.7 ? 17.2' PTZ I mM + DZP 100nM 107.4 2 5.3' PTZlmM 57.6 9.0, 500 nM 5 182.4 2 5.7' 2 f FTZ 1 mM + DZP500nM 126.8 rt 8.1 a p < 0.01, 'p < 0.001, from control, Student's two-tailed f test. Abbreviations as in Table I. AED, antiepileptic drug; ESM, ethosuximide; DMO, p < 0.05, 'p < 0.001 from FTZ 1 mM, Student's 2-tailed r dimethadione; VPA, sodium valproate; DZP, diazepam. test, paired samples.

Epilepsia. Vol. 30, No. 1. 1989 22 P. P. DE DEYN AND R. L. MACDONALD

CTL PTZ lmhf PTZ 1mM PTZ 1mM PTZ 1mM CTL + DZ 10nM + DZ lOOnlI + DZ 500nU FIO. 3. Blockade of pentylenetetrazol (PTZ 1 mM) (panel A) and DMCM (250 nM) (panel B) -induced reduction of GABA responses on spinal cord neurons by DZP (10, 100, and nM). Column 2 shows PTZ and 500 - methyl 6,7-dimethoxy-4-ethyl-p- CABb carboline-3carboxylate (DMCM) effects. Columns 3-5 show effects of CTL DMChl 250nM DMCM 250nM DMCM 250nXI DUCM 250nJf CTL simultaneous convulsant and diaze- + DZ 10nU + DZ 100nM + DZ 500r~Xf pam (DZP) applications. Columns 1 and 6 show control (CTL) GABA responses before and after drug applications, respectively. Control responses between drug .applications are not shown. lontophoretic GABA B. application is indicated by a dash. nn1 2 3 4 5 6

DISCUSSION CTL PTL ImU CTL PTZ ImY CTL + ZK 91296 To understand the mechanism(s) of action of antiabsence AEDs better, we investigated the effects of some clinically and/or experimentally proven AEDs on GABA responses recorded from mouse spinal cord neurons in cell culture. The ef- L*HA fects of two convulsant drugs used in chemical an- fTL PTZ ImM CTL PTZ lmbl CTL imal models of epilepsy, PTZ and DMCM, were studied as well. Furthermore, the possible antagonistic effect of the AEDS on PTZ- and DMCM- induced reduction of GABA responses was determined. The convulsant drugs PTZ and DMCM reduced GABA responses in a concentration-dependent CTL DMUCY 250nY CTL DYCY 250nY CTL manner, consistent with previous observations t ZK 91296 (Pellmar and Wilson, 1977; Macdonald and Barker, 1978~;Jensen and Lambert, 1983; Skerritt and Macdonald, 1984). This reduction of postsynaptic GABAergic inhibition might underlie the convulsant activity of these drugs. Our results also con- 25OnM firm that DMCM exerts its effect on GABA re- CTL DYCM CTL DYCY 250nM CTL sponses through an interaction with the benzodiaz- t CGS 9896 epine receptor. Indeed, the benzodiazepine receptor antagonists CGS 9896 and ZK 91296 (De Deyn and Macdonald, 1987) completely antagonized the DMCM effect. DMCM is an inverse ago-

nist at the benzodiazepine receptor (Jensen and I 2 3 I 5 Lambert, 1983; Skerritt and Macdonald, 1984), FIG. 4. Lack of antagonism of pentylenetetrazol (PTZ 1 mM) which probably explains the protective activity of and antagonism of methyl-6,7-dimethoxy-4-ethyl-p-car- ZK 912% against DMCM-induced seizures (Klock- boline-3-carboxylate (DMCM 250 nM)-induced reduction of gether et al., 1985). PTZ, however, was previously GABA-responses on spinal cord neurons by ZK 91296 (2 pM, panels A and C) and CGS 9896 (2 pM, panels B and D). Col- demonstrated to inhibit GABA responses through umn 2 shows the PTZ and DMCM effect. Column 4 shows the an interaction with a different site on the GABA effect of simultaneous convulsant and experimental antiepi- receptor, possibly the picrotoxin receptor (Macdon- leptic drug (AED) applications. Columns 1, 3, and 5 show control (CTL) responses, respectively, before, between, and ald and Barker, 1977, 1978~). after drug applications. lontophoretic GABA application is The anticonvulsants ESM, DMO, and VPA ap- indicated by a dash.

Epilepsia, Vol. 30, NO,I, 1989 AEDs: MECHANISMS OF ACTION 23

TABLE 3. Effects of AEDS on DMCM-induced of GABA responses. This is consistent with behav- antagonism of GABA responses ioral findings for this benzodiazepine receptor ago- No. of GABA nist (Haefely et al., 1981). cells response Neither VPA, DMO, nor ESM antagonized studied (% control AED (n) t SEM) DMCM-induced reduction of GABA responses. DZP, however, antagonized the DMCM-induced in- DMCM 250 nM 62.4 t 5.8 DMCM 250 nM + ESM 600 FM 64.5 t 7.0 hibition of GABA responses. This is probably due DMCM-. .-. . -250 . nM.- . 60.6 t 3.6 to its high-affinity binding at the benzodiazepine re- DMCM 250 nM + DMO 2 mM 57.8 t 3.3 DMCM 250 nM 62.3 2 4.4 ceptor in competition with DMCM (Haefely et al., DMCM 250 nM + VPA 200 61.7 t 6.4 1981). CGS 98% and ZK 91296, although devoid of DMCM 250 nM 58.7 t 4.0 DMCM 250 nM + DZP 10 nM 63.3 t 4.1" any intrinsic effect, completely antagonized the DMCM 250 nM 60.5 t 5.5 DMCM-induced reduction of GABA responses. DMCM 250 nM + DZP 100 nM 71.7 t 13.4" DMCM 250 nM 58.7 t 2.6 This is explained by the previously demonstrated DMCM 250 nM + DZP 500 nM 105.5 t 9.96 antagonistic effect of these compounds at the ben- DMCM 250 nM 63.0 2 6.5 DMCM 250 nM + CGS 98% 2 phi 99.0 2 1.3' zodiazepine receptor (De Deyn and Macdonald, DMCM 250 nM 59.2 2 5.5 1987). The demonstrated antagonism of the DMCM DMCM 250 nM + ZK 912% 2 phi 99.2 k 3.6' effect might be the underlying mechanism for the Abbreviations as in Table 1. protective action against DMCM-induced seizures p c 0.05, bp < 0.001 from DMCM 250 nM, Student's two-tailed f test, paired samples. for ZK 91296 (Klockgether et al., 1985). The lack of effects of VPA, ESM, and DMO on plied at concentrations equivalent to the clinically postsynaptic GABA action contrasts with their clin- useful therapeutic range in cerebrospinal fluid ical efficacy against generalized absence seizures, (CSF), did not alter GABA responses. Moreover, and suggests that direct postsynaptic enhancement these drugs did not alter neuronal membrane poten- of GABAergic inhibition is not a common mecha- tial or input resistance. These results confirm pre- nism of action of antiabsence AEDs. These results vious findings for ESM and VPA (Barnes and Dich- also suggest that the disturbance responsible for ter, 1984; Macdonald et al., 1984; Macdonald et al., generalized absence seizures may not be localized 1985; McLean and Macdonald, 1986; Buchalter and to the GABA receptor. Alternatively, the therapeu- Dichter, 1986). At higher concentrations, however, tic efficacy of these AEDs may be due to the stim- previous studies demonstrated an enhancement of ulation of GABA synthesis andor the inhibition of GABA responses by VPA (Macdonald and Bergey, GABA metabolism as has been shown for VPA 1979; Harrison and Simmonds, 1982). The observed (Volzke and Doose, 1973; Hammond and Wilder, concentration-dependent effect of DZP on GABA 1985). Finally, there may be alterations in patholog- responses with a maximal direct effect of 82.4% is ic neurons, and their interactions may not have consistent with previous findings (Choi et al., 1977; been present in our neurons in culture. Macdonald and Barker, 19786; Skemtt et al., 1984). Our results suggest that DZP most probably ex- The two experimental AEDs CGS 9896 and ZK erts its antiabsence effect by enhancing GABA- 91296, however, applied at a concentration of 2 pM mediated inhibition through an interaction with the remained inactive in this paradigm. This finding is benzodiazepine receptor. CGS 98% and ZK 912%, consistent with previous work describing CGS 98% two experimentally proved AEDs, were shown to and ZK 91296 to be pure antagonists at the benzo- be pure antagonists at the benzodiazepine receptor. diazepine receptor (De Deyn and Macdonald, Their anticonvulsant effect against DMCM-induced 1987). seizures can be explained by their benzodiazepine With the exception of DZP, none of the tested receptor antagonistic effect. Their mechanism AEDs antagonized or reversed PTZ-induced inhibi- against PTZ-induced seizures is less clear. The ex- tion of GABA responses. The applied PTZ concen- istence of an endogenous benzodiazepine receptor tration (1 mM) was comparable to the critical brain inverse agonist has been proposed (Guidotti et al., PTZ level for onset of seizures in mice (Yonekawa 1982). CGS 9896 and ZK 91296 could enhance et al., 1980). Behavioral studies, however, demon- GABAergic inhibition by displacing this endoge- strated the protective action of VPA, DMO, ESM, nous compound. Regardless of mechanism of ac- CGS 98%, and ZK 91296 against PTZ-induced sei- tion, CGS 98% and ZK 912% may be clinically use- zures (Chen et al., 1963; Krall et al., 1978; Petrack ful AEDs. et al., 1983; Petersen et al., 1984). DZP (100 nM) coapplied with PTZ (1 mM) reversed the inhibition Acknowledgment: We thank Nancy Fox for technical

Epilepsia, Vol. 30, No. I. 1989 24 P. P. DE DEYN AND R. L. MACDONALD assistance and Liliane Van den Eynde and Marjorie Mills Johnston GAR, Willow M. Barbiturates and GABA receptors. for secretarial assistance. Dr. Peter De Deyn is the recip- Costa E, Di Chiara G, Gessa GL, eds. GABA and benzodi- ient of a Rotary International Foundation Award. azepine receptors. New York: Raven Press, 1981:1914 Klockgether T, Schwarz M, Turski L, Sontag K-H. ZK 912%, an anticonvulsant p-carboline which lacks muscle relaxant REFERENCES properties. Eur J Pharmacol 1985;110:309-15. Krall RL, Penry JK, White BG, et al. Antiepileptic drug devel- Barnes DM, Dichter MA. Effects of ethosuximide and tetra- opment: 11. Anticonvulsant drug screening. Epilepsia methyl-succinimideon cultured cortical neurons. Neurology 1978;1940%28. 1984;25:620-5. Lloyd KG, Munari C, Bossi L, et al. 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tor ligands with opposing pharmacologic actions. Psycho- 98% (1 pM). La co-aplicacion de VPA (200 pM) y el PTZ (1 mM) pharmacology 1983;38:129-37. aument6 ligeramente 10s efectos del PTZ. Sin embargo el DZP Ransom BR, Neale E, Henkart M, et al. Mouse spinal cord in (>lo nM) revirti6 la reducci6n de las respuestas GABA induci- cell culture. I. Morphology and intrinsic neuronal electro- das por el PTZ. La reducci6n de las respuestas GABA producida physiologic properties. J Neurophysiol 1977;40:1132-50. por el DMCM (250 nM) no fue alterada por la ESM (600 pM), la Roberts E. Epilepsy and antiepileptic drugs: a speculative syn- DMO (2 mM) o el VPA (200 m. Sin embargo el CGS 98% thesis. In: Glaser GH, Penry JK, Woodbury DM, eds. Anti- (2 pM), y el ZK 912% (2 pM), antagonizaron el efecto del epileptic drugs: mechanisms of action. New York: Raven DMCM. El DZP (>I0 nM) revirti6 significativamente la inhibi- Press, 1980:667-7 13. ci6n de las respuestas GABA inducidas por el DMCM. La falta Schofield PR, Darlison MG, Fujita N, et al. Sequence and func- de efectos de CPA, ESM y DMO sobre las respuestas GABA tional expression of the GABA, receptor shows a ligand- post-sinlpticas sugiere que el incremento de la acci6n GABA gated receptor super-family. Nature 1987;328:221-7. post-sinlptica no es un mecanismo comdn de actuaci6n de las Schulz DW, Macdonald RL. Barbiturate enhancement of AEDs antiausencia. Todas las AEDs DZP, CGS 98% y ZK 912% GABA-mediated inhibition and activation of chloride ion revirtieron la reducci6n de las respuestas GABA producidas por conductance: correlation with anticonvulsant and anesthetic el DMCM per0 no las inducidas por el PTZ lo que sugiere que actions. Brain Res 1981;109:177-88. estos AEDs bloquean 10s ataques DMCM actuando sobre 10s Sherwin AL. Ethosuximide. Relation of plasma concentration to receptores de la benzodiazepina. Sin embargo, puesto que el seizure control. In: Woodbury DM, Penry JK, Pippenger CE, incremento de las respuestas GABA s6lo se produce por el DZP, eds. Antiepileptic drugs. 2nd Ed. New York: Raven Press, permanece todavia sin aclarar el por qut el CGS 98% y el ZK 1982:637-45. 912% bloquean 10s ataques producidos por el PTZ. Simler S, Ziesielski L, Maitre M, et al. Effect of sodium di- (A. Portera-Slnchez, Madrid) n-propylacetate on audiogenic seizures and brain gamma- aminobutyric acid level. Biochem Pharmacol 1973;22: 1701-8. Skenitt JH, Macdonald RL. Benzodiazepine receptor ligand ac- ZUSAMMENFASSUNG tions on GABA-responses. p-carbolines, purines. Eur J Phar- Der Wirkmechansimusvon Antiepileptika gegen generalisierte macol 1984;lOl:135-41. Absencen ist unklar. Antiabsencemittel sind generell wukungs- Skenitt JH, Werz MA, McLean MJ, Macdonald RL. Diazepam voll gegen PTZ- und Methyl-6,7-Dimethoxy-4-Athyl- and its anomalous p-chloro-derivative Ro 5-4864: Compara- p-Carbolin-3-Carboxylat (DMCM) induzierte tierexpenmentelle tive effects on mouse neurons in cell culture. Brain Res Anfdle, also von Medikamenten, die die GABA-erge Inhibition 1984;3 10:99-105. reduzieren. Es wurde vermutet, dap Antiabsencemittel die Swinyard EA. Laboratory evaluation of antiepileptic drugs. Ep- GABA-erge Inhibition verstarken. Wir untersuchten die ilepsia 1%9;10:107-19. Wirkung von verschiedenen Antiepileptika auf GABA- Volzke E, Doose H. Dipropylacetate (Depakine, Ergenyl) in the Antworten in spinalen Mauseneuronen, die in Zellkulturen gew- treatment of epilepsy. Epilepsia 197334:185-93. achsen waren. Es wurden 4 Absencemittel untersucht: Ethosux- Wilder BJ and Karas BJ. Valproate-relation of plasma concen- imid (ESM), Dimethadion (DMD), Sodium Valproat (VPA) und tration to seizure control. In: Woodbury DM, Penry JK, Pip- Diazepam (DZP). Zusatzlich wurden 2 experimentelle Antiepi- ueneer CE. eds. Antieoileotic drum. 2nd Ed.. New York: leptika, CGS 98% und ZK 912%, die gegen PTZO oder DMCM- Rav& Press, 1982:59118. induzierte Anfalle wirkungsvoll sind, eingeschlossen. Mogliche Yonekawa WD. Kupferberg HJ, Woodbury DM. Relationship Wirkungen der Antiabsence- und experimentellen Antiepileptika between pentylenetetrazol-inducedseizures and brain pentyl- auf PTZ- und DMCM-induzierte Hemmung der GABA- enetetrazol levels in mice. J Pharmacol Exp Ther 1980; Antworten wurden ebenfalls ausgewertet. PTZ und DMCM 2 14:58%93. zeigten eine konzentrationsabhlngige reversible Reduktion der GABA-Antworten. PTZ zeigte eine komplette Hemmung der RESUMEN GABA-Antworten bei 10 mM (IC501,l mM), DMCM-Hemmung der GABA-Antworten zeigte ein Plateau von 39% der Kontroll- Los mecanismos de acci6n de las medicaciones antiepiltpticas werte bei 1 pM(IC, von 33 mM). ESM (1200 M,DMD (6 mM), eficaces contra 10s ataques generalizados de ausencia (AEDs VPA (200 pM), CGS 98% (2 pM) und ZK 98% (2 pM) anderten antiausencia) permanecen inciertos. Los AEDs antiausencia nicht die GABA-Antworten. DZP verstarkte die GABA- son, generalmente, eficaces contra ataques experimentales indu- Antworten konzentrationsabhangig. Die durch PTZ (1 mM) cidos por el pentilentetrazol (PTZ) y el metil-6,7-dimetoxy-4-etil- hervorgerufene Hemmung der GABA-Antworten war bei ESM pcarbolina-3-carboxilato (DMCM) en animales, medicaciones (600 pM), DMD (6 mM), CGS 98% (1 mM) und ZK 3836 (1 mM) que reducen la inhibicibn GABAfrgica. Hemos estudiado 10s unveriindert. Zusatliche Anwendung von VPA (200 mM) und efectos de varios AEDs sobre respuestas-GABA registradas en PTZ (1 mM) verstarkten geringfiigig den PTZ-Effekt. DZP las neuronas de la mfdula espinal de ratones que habian crecido (10 nM) kehrte die durch FTZ hervorgerufene Reduktion der en cultivos de celulas primarieas disociadas. Cuatro AEDs an- GABA-Antworten um. Die durch DMCM (250 nM) hervorg- tiausencia fueron incluidos: etoxusimida (ESM), dimetadiona erufene Hemmung der GABA-Antworten war durch ESM (DMO), valproato s6dico (VPA) y diazepan (DZP). Tambitn se (600 pM), DMD (2 mM) und VPA (200 pkf) unbeeinflusst. CGS incluyeron dos AEDs experimentales, CGS 98% y ZK 912%, 98% (2 phf) und ZK 912% (2 fl antagonisierten die DMCM- con acci6n anticonvulsiva contra 10s ataques inducidos por PTZ Wirkung. DZP (>lo nM) kehrte die durch DMCM-induzierte o DMCM. Tambifn se valoraron 10s posibles efectos de 10s Hemmung der GABA-Antworten um. Das Fehlen einer Wirkung AEDs antiausencia y experimentales sobre el PTZ y la inhibici6n von VPA. ESM und DMD auf die postsynaptischen GABA- de las respuestas-GABA inducidas por el DMCM. El PTZ y el Antworten legen nahe, dap eine direkte Verstarkung der DMCM redujeron las respuestas-GABA de modo reversible y postsynaptischen GABA-Aktion kein gemeinsamer Mechanis- dependiendo de sus concentraciones. El PTZ inhibi6 cmpleta- mus der Antiabsencemittel darstellt. Die Antiepileptika DZP, mente las respuestas-GABA a 10 mM (ICs0 de 1.1 mM) mientras CGS 98% und ZK 912% kehrten die DMCM-Wirkung auf die que la inhibici6n de las respuestas GABA inducida por el DMCM GABA-Antworten urn, jedoch nicht die von PTZ, was vermuten alcanz6 un nivel estable del39% de 10s valores control con I pM Iapt, dap diese Antiepileptika die DMCM-Anfalle iiber die (lC50de 33 mM). La ESM (1200 pM), la DMO (6 mM), el VPA Wirkung an den Benzodiazipin-Rezeptren verhinderte. Da je- (200 pM), el CGS 98% (2 pM) y el ZK 98% (2 pM) no alteraron doch nur DZP GABA-Antworten verstarkte, ist unklar, in las respuestas-GABA. El DZP aument6 las respuestas GABA de welcher Weise CGS 98% und ZK 912% die PTZ-Anfiitle ver- una manera concentraci6n-dependiente.La inhibici6n de las re- hinderten. spuestas-GABA producidas por el PTZ (1 mM), no se alter6 con las ESM (600 pM), la DMO (6 mM), el CGS 98% (1 pW o el ZK (C. Benninger, Heidelberg) Epilepsia, Vol. 30, No. I, 1989