Effect of psychotropic agents on a model of absence epilepsy in rats

Neuropharmacology Vol. 30, No. 6, pp. 651456, Printed in Great Britain. All rights reserved

1991 Copyright

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002%3908/91 $3.00 + 0.00 1991 Pergamon Press plc

EFFECT OF PSYCHOTROPIC AGENTS ON A MODEL OF ABSENCE EPILEPSY IN RATS H.-H. FREYand M. VOITS Department of Pharmacology and Toxicology, School of Veterinary Medicine, Freie Universitiit Berlin, KoserstraBe 20, D-1000 Berlin 33, Germany (Accepted 17 January 1991) Summary-The effect of different groups of psychotropic agents on the spontaneous absence-like paroxysms in the ECoG of rats was studied in order to evaluate the specificity of the model for antiabsence drugs. Morphine-like analgesics increased the number of paroxysms, whereas this was depressed or the discharges were completely abolished by the following drugs: d-amphetamine, tricyclic antidepressants, centrally acting anticholinergics and L-DOPA, NMDA antagonists and memantine. Since the latter drugs have been reported to be effective in petit mal epilepsy, or the NMDA antagonists and memantine, have

a potential anticonvulsant effect, the results are in favour of the usefulness of the model for antiabsence drugs. Key words-absence model in rats, morphine-like analgesics, amphetamine, anti-Parkinson drugs, tricyclic antidepressants, NMDA antagonists, memantine.

Rats showing spontaneously occurring spike-wave paroxysms in the electrocorticogram (ECoG), associated with absence-like behavioural alterations, have been studied by Vergnes, Marescaux, Micheletti, Reis, Depaulis, Rumbach and Warter (1982) and by Vergnes, Marescaux, Depaulis, Micheletti and Warter (1990) and were proposed as a model of absence epilepsy. This proposal seemed justified on the basis of results obtained with single doses of ethosuximide, valproate, antiepileptic drugs: trimethadione and benzodiazepines inhibited or abolished the paroxysms with reasonable doses, whereas carbamazepine and phenytoin, being ineffective against human absences, had no effect in the model in the rat either, or even enhanced the number and duration of discharges (Marescaux, Micheletti, Vergnes, Reis, Depaulis, Rumbach and Warter, 1984). In a recent study (Wahle and Frey, 1990), rats with spike-wave discharges were treated with ethosuximide and valproate for 2 or 4 weeks, respectively. In these experiments, ethosuximide proved effective at concentrations in plasma considered to be therapeutic in human absence epilepsy, but rats treated with valproate showed a temporary effect at very large concentrations only and subsequently developed tolerance to the anticonvulsant effect of the drug. This was explained by increases in concentrations of y-aminobutyric acid (GABA) in the CNS, induced by valproate, because Vergnes, Marescaux, Micheletti, Depaulis, Rumbach and Warter (1984) have shown that GABAergic drugs aggravated the syndrome. Nevertheless, on the basis of these somewhat contradictory results, it seemed necessary to study the effect of a broad spectrum of centrally

acting drugs on the spike-wave discharges, in order to assess the specificity of the model for anti-absence drugs. Such experiments have done before by Micheletti, Warter, Marescaux, Depaulis, Tranchant, Rumbach and Vergnes (1987), with drugs affecting noradrenergic transmission and by Kleinlogel (1985), with similar drugs and neuroleptics. Of these drugs, a,agonists and u,-antagonists reduced the spike-wave discharges, whereas tll -antagonists and a2-agonists increased the duration of discharges. The neuroleptics, having a pronounced CI,-adrenolytic effect, likewise increased the total duration of the spikewave discharges. In the present experiments, the effects of the following groups of drugs on the absence-like spikewave discharges were studied in rats: morphine-like analgesics, d-amphetamine, antidepressent drugs, centrally acting anticholinergics, L-DOPA, memantine and antagonists of N-methyl-n-aspartate (NMDA). METHODS Animals

The experiments were done in WAG/Ola/Hsd rats of both sexes (Hartan Olac, Bichester, U.K.), weighing 250-300g at the time of the study. They were housed singly in plastic cages at constant temperature (2426°C) and humidity (SO%), under a 12 hr light cycle (7 a.m.-7 p.m.). They were fed on a standard diet (Altromin 1324, Altromin, Lage, Germany) and tap water was available for 24 hr a day. 651

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Surgery

The rats were provided with ECoG electrodes under chloral hydrate anesthesia (360mg/kg intraperitoneally). After fixation in a stereotactic instrument, the skull was exposed and 4 holes (0.7 mm diameter) were drilled with a dental drill for the epicortical electrodes. Coordinates were, with the bregma as reference, 3.0 anterior, 3.0 lateral and 5.0 posterior, 4.0 lateral. Stainless steel screws served as electrodes and were connected to a teflon-coated stainless steel wire and a female connector. The assembly was held in place by dental acrylic cement. The animals were allowed to recover for 7 days before recording of the ECoG commenced.

diluted with isotonic saline to an injection volume of 2 ml/kg: morphine.HCl (E. Merck, Darmstadt), fentanyl (Fentanyl-Janssen, Janssen GmbH, Neuss), meperidine (Dolantin, Farbwerke Hoechst), pentazocine (Fortral, Withrop), buprenorphine (Temgesic, Boehringer Mannheim), d-amphetamine sulphate (E. Merck, Darmstadt), imipramine (Tofranil, Geigy), amitryptiline (Saroten, Roche), biperidine (Akineton, Nordmark), trihexyphenidyl . HCl (Lederle). LDOPA (Serva Feinchemikalien), ketamine (Ketanest, Parke-Davis), CPP (3-(2-carboxypiperazin-4-yl)propylI-phosphonic acid (by courtesy of Dr Turski, Schering AG), memantine (Akatinol Memantine, Merz & Co.) and naloxone.HCl (DuPont). The doses refer to the free base or acid, respectively.

Recording

The ECoG was recorded on an electroencephalograph (San-ei rectilinear thermal-writing oscillograph 8 K 22, San-ei Instrument Co., Tokyo, Japan) from 6 rats placed in individual glass boxes. The glass boxes were in a grounded Faraday cage. From each rat, the ECoG was derived from 2 ipsilateral electrodes from the right or left cortex, respectively. Typical spike-wave paroxysms lasted 2.5-5.5 set on the average and showed spikes at a rate of 8-9 set with average amplitudes of 0.24.4mV (Fig. 2), accompanied by rhythmic twitching of the vibrissae and behavioural arrest in the position occupied at the start of the complex. Sometimes the eyes are closed for the duration of the paroxysm. Paroxysms of at least 1 set duration were counted. For the experiments, rats were selected having at least 30 spike-wave paroxysms during the 30min period recorded, with a reasonable variation in the number of paroxysms from day to day. E.xperimental procedure

RESULTS

Morphine-like analgesics

Morphine was tested in doses of 0.05, 0.1, 1.O and 2.0 mg/kg. With doses of 0.1 mg/kg and greater, there was a tendency to increases in the number of paroxysms and total duration, from 15 min-2 hr

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After a control period of 30min, the respective drug was injected subcutaneously and further ECoG records of 30min duration were taken from 1545 min, 60-90 min, 120-l 50 min and 240-270 min after the injection. Deviations from this protocol are given under Results. For each single recording period, the number of spike-wave paroxysms, their duration and the total duration of paroxysms during 30min were counted and the average duration of the individual paroxysms was calculated. These values were related to the control period of the individual rats on the same day, which was set to 1.O. Rats were only used once every 2 weeks. Groups of 4-6 rats were used per dose.

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The following drugs were used, mostly in the form of commercial ampoules, the contents of which were

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Fig. 1. Effect of buprenorphine, 0.1 mg/kg (a) and 0.2 mg/kg (b) (SC), on the number (N) and average duration of single paroxysms (D) and total duration of paroxysms, during 30 min (S). The ordinate gives the values relative to the control period (= 1.0) and the abscissa the time after injection. The median and the range of observations is marked.

Specificity of an absence model in rats

after injection. The difference was, however, not significant. Fentanyl (0.01, 0.03 and 0.05 mg/kg) led to significant increases in number and total duration of spikewave paroxysms. This was observed at 0.01 mg/kg after 15 min only, after the larger doses also after 1 hr. Meperidine (2.0 and 4.0mg/kg). Only the larger dose increased the number and total duration of paroxysms significantly at 15 min and 1 hr. Buprenorphine (0.1 and 0.2 mg/kg) induced a highly significant and long-lasting increase in number and total duration of spike-wave paroxysms (Figs 1 and 2). Pentazocine (1.0, 2.0 and 4.0 mg/kg) led to an increase in the number and total duration, which was short-lasting (15 min-1 hr) and seemed most pronounced with the medium dose. Naloxone (0.3 and 1.0 mg/kg) had a short-lasting tendency to increase number and total duration of the paroxysms. The effect of morphine, 2 mg/kg, was apparently not reduced after 1 mg/kg naloxone at 15 min and 1 hr; however, the paroxysms increased considerably after 2 hr, at which time the action of naloxone should have terminated. d-Amphetamine

A dose of 0.5 mg/kg suppressed the spike-wave paroxysms nearly completely at 15 min and there was

still a significant reduction of the number and total duration 1 hr after the injection. Also, the duration of the single paroxysms tended to be reduced up to 4 hr. After a dose of 1 mg/kg, the spike-wave discharges were completely abolished for 2 hr and still reduced after 4 hr (Fig. 3). Antidepressants

Imipramine (5.0 and 10.0 mg/kg); the smaller dose significantly reduced the number and total duration of paroxysms, beginning from 1 hr after injection. With the larger dose, a depression of these parameters was already evident after 15 min. Amitryptiline (5 mg/kg i.p. and 10 mg/kg s.c.), led to a significant reduction in the number and total duration of the paroxysms, for the whole duration of the experiment. The duration of the individual paroxysms tended also to be reduced for the first hour after injection (Fig. 4). The smaller dose was given intraperitoneally because there was a local irritation at the site of the subcutaneous injection of lOmg/kg. Central anticholinergics and L-DOPA

Biperidine (0.5 and 1.0 mg/kg) led, dose-dependently, to a pronounced suppression of the number, duration and total duration of the spike-wave paroxysms (Fig. 5) and the same effect was obtained with trihexyphenidyl at the same dose.

Fig. 2. The ECoG of 2 rats during the control period (a) and 2 hr after the subcutaneous injection of 0.1 mg/kg buprenorphine (b). NP M/6-H

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In the case of L-DOPA (200 mg/kg orally), the first recording period was from 30-60 min. During the first two periods, the number of paroxysms and the total duration were significantly reduced. N-Methyl-o-aspartate-antagonists

Ketamine (2.0, 4.0 and 5.0 mg/kg); with a dose of 4 mg/kg and especially at 5 mg/kg, the number and total duration of spike-wave paroxysms were significantly reduced. This effect lasted for the whole duration of the experiment with the 5 mg/kg dose. At 2 mg/kg there was no apparent effect. With CPP (2.0 and 5.0 mg/kg), there was a dosedependent reduction in the number and total duration of the spike-wave paroxysms and also the duration of the individual complexes tended to be shortened (Fig. 6).

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With this drug (5.0 and lO.Omg/kgi.p.), the smaller dose reduced the spike-wave paroxysms and with the larger dose there was a complete suppression of discharges for 1 hr and a strong depression still after 2 hr (Fig. 7). DISCUSSION

The aim of the present study was to obtain some insight into the specificity of the spontaneous spike-

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Fig. 4. Effect of amitryptiline 5.0 mg/kg (i.p.) (a) and 10.0 mg/kg (s.c.) (b), on the number (N), average duration of single paroxysms (D) and total duration of paroxysms during 30 min (S). For further explanations see Fig. I.

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wave paroxysms in rats, as a model of absence epilepsy. For that purpose, a number of centrally acting drugs, not being specific anticonvulsants, were tested for their ability to increase or decrease number and duration of spike-wave paroxysms. Morphine-like analgesics, as a group, increased number and total duration of paroxysms, though the increase was not always significant. The most pronounced effect was seen with buprenorphine. Though no direct antagonism by naloxone could be shown, the ‘release’ of the effect of morphine after the effect of naloxone was over, points to a participation of opiate receptors. An inhibitory, ‘anticonvulsant’, effect was seen with d-amphetamine, antidepressant drugs, centrallyacting anticholinergics and L-DOPA, as well as with NMDA antagonists and memantine. d-Amphetamine was mentioned by Livingston (1966) as effective against petit ma1 epilepsy but because ethosuximide and valproic acid are by far superior, it is no longer used in this indication. Fromm and Glass (1977) and Fromm, Wessel, Glass, Alvin and van Horn (1978) have reported on tricyclic antidepressants being active in a model of petit mal, corticofugal inhibition in the cat, and also clinically, in patients with absences and myoclonicastatic seizures. The known inhibitory effect of these

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Fig. 3. Effect of d-amphetamine, 0.5 mg/kg (a) and 1.Omg/kg (b) (SC), on the number (N), average duration of the single paroxysms (D) and total duration of paroxysms, during 30 min (S). For further explanations see Fig, I.

Specificity of an absence model in rats

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Fig. 5. Effect of biperidine, 0.5 mg/kg (a) and 1.0mg/kg (b) (s.c.), on the number (N), average duration of single paroxysms (D) and total duration of paroxysms, during 30 min (S). For further explanation see Fig. 1.

Fig. 6. Effect of CPP, 2.0 mg/kg (a) and 5.0 mg/kg (b) (so.), on the number (N), average duration of single paroxysms (D) and total duration of paroxysms, during 30 min (S). For further explanation see Fig. 1.

drugs on the uptake of monoamines might play a role in this anticonvulsant effect. There is only one report of an anticonvulsant activity of centrally acting anticholinergics (Millichap, Pitchford and Millichap, 1968). These authors found procyclidine to be active in the pentetrazole seizure threshold test in mice, which is often used as a model of petit ma1 epilepsy, but the ED,, (83 mg/kg) was disproportionately large, and larger still than the ED,, against maximal electroconvulsions (16 mg/kg for procyclidine, 37 mg/kg for trihexyphenidyl). Trihexyphenidyl was not studied in the pentetrazole seizure threshold test. Procyclidine was also tried in some clinical patients with petit mal and it was partially effective in some, which were suffering from refractory myoclonic seizures. The effect of L-DOPA against spontaneous spikewave discharges in the rat, might be a consequence of the increase of cerebral concentrations of dopamine and noradrenaline which, according to previous experiments (Kilian and Frey, 1973; Przegalinski, 1985), can decrease the susceptibility of rodents to pentetrazole-induced seizures. Since NMDA antagonists, a property also discussed for memantine (Kornhuber, Bormann, Retz, Hiibers and Riederer, 1989), have an inherent anticonvulsant effect and the role of excitatory amino

acids in absences has not been studied, the ‘anticonvulsant’ effect in the present study cannot be termed nonspecific until further studies reported. Memantine has a near chemical relationship to adamantine, which is known to liberate dopamine and might also, in this way, influence the spike-wave paroxysms. In summary, the results of the present experiments are more in favour than against a specificity of the

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Fig. 7. Effect of 10 mg/kg memantine (i.p.) on the number (TV),average duration of single paroxysms (D) and total duration of paroxysms during 30min (S). For further explanation see Fig. 1.

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spontaneous spike-wave paroxysms in the rat, as a model of absence epilepsy. This model should be studied further in this respect because it has obvious advantages over the methods now used as models of petit ma1 epilepsy. Especially, it is excellently suited for long-term studies of anti-absence drugs, since rats that display the spontaneous spike-wave discharges, do so for life and without any provocation. Acknowledgements-The

experiments were performed with the excellent technical assistance of Mrs U. Augustin and Mrs P. Layer. The study was supported by Deutsche Forschungsgemeinschaft (Fr. 348/7-l). REFERENCES Fromm G. H. and Glass J. D. (1977) The effect of tricyclic antidepressants on corticofugal inhibition of the spinal trigeminal nucleus. Electroencephalogr. din. Neurophysiol. 43: 637645.

Fromm G. H., Wessel H. B., Glass J. D., Alvin J. D. and van Horn G. (1978) Imipramine in absence and myoclonic-astatic seizures. Neurology 28: 953-957. Kilian M. and Frey H.-H. (1973) Central monoamines and convulsive thresholds in mice and rats. Neuropharmacology 12: 681-692. Kleinlogel H. (1985) Spontaneous EEG paroxysms in the rat: Effects of psychotropic and alpha-adrenergic agents. Neuropsychobiology 13: 206-213.

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Kornhuber J.. Bormann J.. Retz W.. Hiibers M. and Riederer P. ‘(1989) Memantine displaces [‘H]MK-801 at therapeutic concentrations in postmortem human frontal cortex. Eur. J. Pharmac. 166: 589-590. Livingston S. (1966) Drug Therapy for Epilepsy, pp. 104107. C. C. Thomas, Springfield, IL. Marescaux C., Micheletti G., Vergnes M., Reis J., Depaulis

A., Rumbach L. and Warter J. M. (1984) A model of chronic spontaneous petit ma/-like seizures in the rat: Comparisons with pentylenetetrazol-induced seizures. Epilepsia 25: 326-33 1. Micheletti G., Warter J.-M., Marescaux C., Depaulis A., Tranchant C., Rumbach L. and Vergnes M. (1987) Effects of drugs affecting noradrenergic neurotransmission in rats with spontaneous petit-mal-like seizures. Eur. J. Pharmat. 135: 397402.

Millichap J. G., Pitchford G. L. and Millichap M. G. (1968) Anticonvulsant activity of antiparkinsonism agents. Proc. Sot. exp. Biol., N. Y. 127: 1187-l 190. Przegalinski E. (1985) Monoamines and the pathophysiology of seizure disorders. In: Antiepileptic Drugs. Vol. 74 of Handbook ofexp. Pharmacol. (Frey H.-H. and Jam D., I&), pp. 1011137. Springer, BerlinVeranes M.. Marescaux C.. Micheletti G.. Reis J.. Denaulis AT, Rumbach L. and Warter J.-M. (1982) Spontaneous paroxysmal electroclinical patterns in the rat: a model of generalized non-convulsive epilepsy. Neurosci. Lett. 33: 97-101.

Vergnes M., Marescaux C., Micheletti G., Depaulis A., Rumbach L. and Warter J.-M. (1984) Enhancement of spike and wave discharges by GABAmimetic drugs in rats with spontaneous petit ma/-like epilepsy. Neurosci. Lett. 44% 91-94.

Vergnes M., Marescaux C., Depaulis A., Micheletti G. and Warter J.-M. (1990) Spontaneous spike-and-wave discharges in Wistar rats: A model of genetic generalized nonconvulsive epilepsy. In: Generalized Epilepsy. Neurobiological Approaches (Avoli M., Gloor P., Kostopoulos G. and Naquet R., Eds), pp. 238-253. Birkhluser, Boston. Wahle H. and Frey H.-H. (1990) Development of tolerance to the anticonvulsant effect of valproate but not to ethosuximide in a rat model of absence epilepsy. Eur. J. Pharmac. 181: l-8.

Wilcoxon F. (1949) Some Rapid Approximate Statistical Procedures. Amer. Cyanamid Co., New York.