Neuroscienee Letters, 112 (1990) 43-47 Elsevier Scientific Publishers Ireland Ltd.

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NSL 06798

Anticonvulsant effects of tetronic acid derivatives on picrotoxin induced epileptiform activity in rat hippocampal slices G. Krhr and U. Heinemann Institut.fiir Neurophysiologie, Zentrum Physiologie und Pathophysiologie, Universitiit zu Krln, Cologne (F.R.G.)

(Received 14 November 1989; Revised version received 28 December 1989; Accepted 28 December 1989) Key words." Picrotoxin; Anticonvulsant; Rat; Hippocampus; Tetronic acid derivative; Losigame

We have investigated the effects of a new class of anticonvulsants, the tetronic acid derivatives AO33 (generic name: losigame) and AO78, on field potentials, extracellular calcium concentration changes and intracellular potentials in rat hipppocampal slices treated with the non-competitive GABAA antagonist picrotoxin (PTX). The tetronic acid derivatives reduced and eventually blocked spontaneous epileptiform events, induced by 10 to 30 gM PTX. Stimulus induced burst discharges were shortened in duration, but not blocked. Extracellular calcium concentration changes and associated slow negative field potentials were diminished in a dose dependent manner. Intracellular recordings revealed no effect of AO33 on resting membrane potential, little effect on input resistance, a small increase in the threshold of action potentials and an attenuation of stimulus induced paroxysmal depolarisation shifts (PDSs). Spontaneous PDSs initially decreased in duration until they were no longer observable.

T h e existence o f t h e r a p y resistant f o r m s o f epileptic activity requires the developm e n t o f new a n t i c o n v u l s a n t s . W e have investigated the effects o f the tetronic acid derivatives A O 3 3 ( L o s i g a m e ) a n d A O 7 8 , a new class o f a n t i c o n v u l s a n t s , o n p i c r o t o x i n ( P T X ) i n d u c e d a l t e r a t i o n s in n e u r o n a l activity [3]. In view o f a possible role o f intracellular C a 2 + a c c u m u l a t i o n in epilepsy d e p e n d e n t nerve cell d e g e n e r a t i o n [10] we also s t u d i e d the effects o f the tetronic acid derivatives o n stimulus i n d u c e d changes in [Ca2+]o. T h e e x p e r i m e n t s were p e r f o r m e d on 50 transverse rat h i p p o c a m p a l slices (350-400 p m thick), o b t a i n e d f r o m 150-200 g a d u l t female W i s t a r rats by using p r o c e d u r e s o f p r e p a r a t i o n a n d m a i n t e n a n c e described previously [9]. E x t r a c e l l u l a r recordings were p e r f o r m e d in a r e a CA1 with calcium-selective/reference electrodes (tip diameters: 2-3 g m ) which r e s p o n d e d to a 10-fold c h a n g e in [Ca2+]o with a p o t e n t i a l shift o f 2 6 - 2 9 mV. Electrodes filled with 2 - 4 M p o t a s s i u m acetate (resistances 70-100 M~2) Correspondence: G. Krhr, Institut ffir Neurophysiologie, Zentrum Physiologie und Pathophysiologie, Universitiit zu Krln, Robert-Koch-Strasse 39, 5000 Cologne 41, F.R.G.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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were used for intracellular recordings. The Schaffer collateral/commissural fibers in area CA 1 were stimulated (pulse duration 0.1 ms, interstimulus intervals 50 ms) with bipolar glass-insulated wire electrodes (tip diameters: 50/~m). AO33 and AO78 were dissolved in dimethylsulfoxide (DMSO) and added to the artificial cerebrospinal fluid (ACSF) to give final concentrations of 10-100/~M. The final DMSO concentration in the ACSF was then 2.8-28 mM and showed no effects in control experiments. Both AO33 and AO78 suppressed reversibly spontaneous epileptiform discharges induced by 10-30/zM picrotoxin (PTX). The average frequency of PTX-induced discharges under control conditions was 5.4 + 2.3 per min (n = 15). The effects of AO33 and AO78 on spontaneous transients recorded extracellularly and on spontaneous paroxysmal depolarization shifts (PDSs) recorded intraceUularly are shown in Fig. I A and I B, respectively. The threshold effect of AO33 was obtained with 10/zM and complete suppression was reached with 50/IM after 20-25 min of drug application (n = 7). AO78 was slightly less effective. Complete suppression was seen after 30-40 rain of drug application with 100/tM while 50/~M AO78 only reduced the spontaneous discharges (Fig. 1A; n=3). The tetronic acid derivatives also had a depressant effect on the duration of stimulus induced epileptiform burst discharges but never totally blocked them in the concentration range tested. The depressant ef-

A f.p.

B

Control

jl

Control

rttl'!l!i 50~M AO 78

I!

t 50;JM AO 33

Wash

I L Ii 12. mv 1rain

Wash 40mY

7 Fig. 1. Effects of tetronic acid derivatives (AO78 and AO33) on spontaneous epileptiform activity induced by 20-30/zM picrotoxin (PTX) in the CAI pyramidal cell layer of rat hippocampal slices. A: 50 pM AO78 reduced reversibly the frequency of extracellularly recorded field potentials. This effect increased after terminating drug perfusion and reached a maximum 15-20 min after onset of drug washout. B: 50 pM AO33 blocked the spontaneous epileptiform activity within 25 min. The first effect (see middle row) was a reduction in the number of action potentials followed by a reduction in the duration of paroxysmal depolarization shifts recorded intracellularly. Resting potential was - 65 mV.

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fects on the duration of both anti- and orthodromic stimulus induced burst discharges were reversible after 40-50 min of washout (Fig. 2; n = 9). Paired-pulse inhibition was observed under control conditions (PTX alone) and this inhibition was not accentuated by 50 MM AO33, AO78 (10(, ~.M) was less effective on stimulus induced burst discharges even after 30-35 min of perfusion (n = 7). It is well known that extracellular free calcium concentration decreases during paroxysmal activity [4]. We therefore studied the effects of AO33 and AO78 on orthoand antidromically induced decreases in extracellular calcium concentration Control

50pM AO 33

Wash

ORTHO

slow f.p. ~ "

f'P' ~

1.6raM ANTI V

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s,ow,.o.

]2mv

-

t

t

t

t

-

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10ms

Fig. 2. Decreases in extracellular calcium concentration [Ca2+]o and associated slow negative field potentials (slow f.p.) produced by repetitive stimulation (20 Hz; 10 s) are reduced by 50 #M AO33. The transient field potentials (f.p.) evoked by paired pulse stimulation were found to be reduced. The calibration bars for calcium concentration ([Ca2+]o) for slow field potentials (slow f.p.) and for transient field potentials on the lower fight apply to all records. The Schaffer collaterai/commissurai fiber system (orthodromic activation of hippocampal pyramidal cells, upper throe rows) was stimulated with 1.6 V (ortbo) the alveus (predominantly antidromic activation of hippocampal pyramidal cells, lower thrce rows) with 3.0 V (anti). The recordings were performed in the stratum pyramidal¢ of the CA 1 region. Stimulus times are indicated by bars and arrows, respectively.

46 ([Ca2+]o). As reported previously PTX augmented stimulus induced calcium concentration changes by 30-50%. AO33 and AO78 reduced both the ortho- and the antidromically induced decreases in [Ca2+]o by up to 65% in a dose-dependent and reversible manner (AO33; n = 9; Fig. 2 and AO78; n = 6). This effect was stronger than that of the N M D A receptor antagonist DL-2-amino-5-phosphonovaleric acid (2-APV) and ketamine [6] and also stronger than the classical calcium entry blockers [5, 7]. The maximal reduction of stimulus induced [Ca2+]o decreases by both classes of drugs was 30%. Intracellular recordings made from CAl pyramidal cells showed that resting membrane potential and input resistance were not affected by AO33. Further, action potential amplitudes induced by either extracellular or intracellular stimulation were not changed. The threshold for inducing action potentials was slightly increased. The most significant effects of AO33 were those on spontaneous PDS in the presence of PTX (Fig. 1B). Initially, the number of action potentials arising from the spontaneous PDSs were reduced. Subsequently, the duration of spontaneous PDSs decreased and with time PDSs could no longer be observed. Evoked PDSs in the presence of PTX were also affected by AO33. Initially, evoked excitatory postsynaptic potentials (EPSP)-like responses did not give rise to action potentials or elicit a reduced number of action potentials in the presence of AO33 (Fig. 3A). Secondly,

,~

B

Control

20~JM PTX

45min 20JJM PTX

+lOmV 30rain 50~M AO 3;3

15min lOOgM AO 33

Wash

Wash

40mY 50ms

50m~

40mV

Fig. 3. The effectof 20/aM picrotoxin (PTX) on stimulus induced EPSP-IPSP-sequencesat different membrane potentials (A; - 9 mV hyperpolarized from rest and + I 1 mV depolarized from rest; and B: + l0 mV depolarized from rest) is shown in the upper row. A: the control recordings are superimposed. In the presence of 100/aM AO33 the number of action potentials were reduced. The stimulation intensity was 2.0 V, the resting membranepotential (RMP) -66 mV. B: hoar-thresholdstimulation (1.6 V) in a different CA1 cell (RMP -65 mV) induced an EPSP whichwas followedby a paroxysmaldepolarisation shift after some delay. Fifty/aM AO33increased this delay in a reversible manner. Stimulation indicated by stars.

47 at threshold stimulation, the delay by which P D S followed the stimulus induced E P S P was increased (Fig. 3B). I P S P antagonized by P T X did not recover in AO33. Lastly, A O 3 3 decreased the n u m b e r o f action potentials evoked by a given depolarizing current injection. We conclude that AO33 and AO78 have the capability to prevent spontaneous epileptiform discharges induced by the non-competitive G A B A A antagonist P T X [2]. We believe that AO33 acts in a m a n n e r that is different from phenytoin and carbamazepine. These drugs produce a continuous decline o f action potential amplitudes due likely to a use-dependent block o f inward sodium currents [8]. Similarly, the tetronic acid derivatives and valproic acid [1] enhance spike frequency a c c o m m o d a t i o n , i.e. they reduce the n u m b e r o f action potentials evoked by antidromic stimulation in low Ca 2+ medium. Since the input resistance o f CA1 cells and IPSPs evoked in control medium are negligibly affected by AO33 ( K 6 h r and Heinemann, in preparation), we think that the drugs do not act via a G A B A mimetic action. We suggest that AO33 and A O 7 8 m a y act by enhancing potassium channel function or by reducing a depolarizing inward current such as the slow Ca or N a currents. This research was supported by a grant from Dr. W. Schwabe, Karlsruhe, and by the D F G (He 1128/2-4). We are grateful to M. G r o e n e n w a l d and G. Heske for assistance in the experiments and the preparation o f the manuscript. 1 Franceschetti, S., Hamon, B. and Heinemann, U., The action of valproate on spontaneous epileptiform activity in absence of synaptic transmission and on evoked changes in [Ca2+]oin the hippocampal slice, Brain Res., 386 (1986) 1-11. 2 Galindo, A., GABA picrotoxin interaction in the mammalian central nervous system, Brain Res., 14 (1969) 763-767. 3 Hablitz, J.J., Picrotoxin induced epileptiform activity in hippocampus: role of endogenous versus synaptic factors, J. Neurophysiol., 51 (1984) 1011- 1027. 4 Heinemann, U., Lux, H.D. and Gutnick, M.J., Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain Res., 27 (1977) 237-243. 5 Jones, R.S.G. and Heinemann, U., Differential effects of calcium entry blockers on pre- and postsynaptic influx of calcium in the rat hippocampus in vitro, Brain Res., 416 (1987) 257-266. 6 K6hr, G. and Heinemann, U., Effects of NMDA-antagonists on picrotoxin-, low Mg2+ and low Ca2+induced epileptogenesis and on evoked changes in extracellular Na +- and Ca2+-concentrations in rat hippocampal slices, Epilepsy Res., in press. 7 Louvel, J., Abbes, S., Godfraind, J.M. and Pumain, R., The action of various organic calcium channel blockers on epileptic phenomena in hippocampal slices. In E.-J. Speckmann, H. Schulze and J. Walden (Eds.), Epilepsy and Calcium, Urban & Schwarzenberg, Munich, 1986, pp. 277-299. 8 Macdonald, R.L. and McLean, M.J., Anticonvulsant drugs: mechanisms of action. In A.V. DelgadoEscueta, A.A. Ward, D.M. Woodbury and R.J. Porter (Eds.), Advances in Neurology/Basic mechanisms of Epilepsies, Raven, New York, 1986, pp. 713-736. 9 Mody, I., Stanton, P.K. and Heinemann, U., Activation of N-methyl-v-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling. J. Neurophysiol., 59 (1988) 1033-1054. 10 Siesj6, B.K., Cell damage in the brain: A speculative synthesis, J. Cereb. Blood Flow Metab., 1 (1981) 155-185.