Effects of neurosteroids on spike-wave discharges in the genetic epileptic WAG/Rij rat

doi:10.1016/S0920-1211(98)00067-9

Epilepsy Research

Volume 33, Issue 1, 1 January 1999, Pages 23-29

https://doi.org/10.1016/S0920-1211(98)00067-9 Get rights and content

Abstract

Effects of i.p. administration of the neurosteroids, allopregnanolone and pregnenolone sulfate, were studied in WAG/Rij rats, a genetic model for generalized absence epilepsy. EEG recordings showed that allopregnanolone, a positive modulator of the GABA_A receptor, in doses ranging from 5 to 20 mg/kg, increased dose-dependently the number and total duration of spike-wave discharges. Pregnenolone sulfate, a positive modulator of NMDA receptors, also increased those parameters, though only at the highest dose used (100 mg/kg). Significant changes in spike-wave discharges occurred during the first hour post-injection and were not accompanied with behavioral alterations. The obtained data indicate that both these neurosteroids aggravate the spike-wave activity. This finding contrasts with the anti-convulsant effects of some neurosteroids and they point to a different pharmacological profile of epilepsy with convulsive or non-convulsive seizures.

Introduction

In recent times considerable interest has focused on neurosteroids as endogenous modulators of excitability of the central nervous system. Neurosteroids influence the excitability of the brain by non-genomic mechanisms, e.g. by positive allosteric modulation of GABA_A and, possibly, excitatory amino acid receptors (McEwen, 1991, Wu et al., 1991, Paul and Purdy, 1992). Neurosteroids such as allopregnanolone, which are positive modulators of the GABA_A receptor, show potent anti-convulsant effects. These effects are observed in various models of convulsive seizures, such as pentylenetetrazol-, bicuculline-, penicillin-, picrotoxin-, cocaine-, nicotine- and excitatory amino acid seizure models (Landgren et al., 1987, Belelli et al., 1990, Luntz-Leybman et al., 1990, Maione et al., 1992, Kokate et al., 1994, Gasior et al., 1997, Budziszewska et al., 1998). Moreover, neurosteroids inhibit the development of kindling, which points to their role in a control mechanism in genesis of this model of epilepsy (Holmes et al., 1984, Carter et al., 1997).

In contrast to a growing body of evidence for the involvement of neurosteroids in regulation of convulsive seizures, no data have been available on the role of these compounds in a non-convulsive absence epilepsy. It has been proposed that a particular strain of rats, WAG/Rij, is a useful model for a generalized absence epilepsy in man (Van Luijtelaar and Coenen, 1986, Coenen et al., 1992). All rats older than 6 months of this strain show 7–10 Hz spike-wave discharges in the cortical EEG, together with concomitant behavioral episodes of twitching of the vibrissae and accelerated breathing (Van Luijtelaar and Coenen, 1986). This genetic model has been amply validated by pharmacological and behavioral studies (Peeters et al., 1988, Peeters et al., 1989a, Coenen et al., 1991, Van Luijtelaar et al., 1991a, Van Luijtelaar et al., 1991b, Coenen et al., 1992, Van Luijtelaar et al., 1995, Van Luijtelaar et al., 1996). In this type of epilepsy, both GABAergic and glutamatergic mechanisms are crucially involved (Peeters et al., 1989b, Peeters et al., 1990, Ramakers et al., 1991, Peeters et al., 1992, Peeters et al., 1994).

The purpose of the present study was to find out whether neurosteroids were involved in controlling the number and duration of spike-wave discharges in that model of epilepsy. Therefore the role of allopregnanolone and pregnenolone was investigated on EEG of WAG/Rij rats. Among neurosteroids, allopregnanolone is the most potent positive modulator of the GABA_A receptor (Majewska, 1991, Hawkinson et al., 1994), whereas pregnenolone acts as both a GABA antagonist and a NMDA agonist (Majewska, 1991, Wu et al., 1991).

Section snippets

Methods

Six-month-old WAG/Rij male rats weighing 330–360 g, kept in an animal house on a reversed 12/12 light-dark cycle (light on at 20:00), were used as experimental subjects. They had free access to standard laboratory food and water throughout the experimental period. The rats were implanted with cortical electrodes (Plastics One, MS 333/2-A) under pentobarbital anaesthesia (Narcovet, 60 mg/kg). The electrodes were placed on the surface of the cortex: one in the frontal region (coordinates with the

Effects of allopregnanolone on the number, total and mean duration of spike-wave discharges

The results are presented in Table 1. The ANOVA showed that i.p. injection of allopregnanolone had a significant effect on the number (F=14.34, df 3,30, P<0.0001) and total duration (F=4.80, df 3,30, P<0.01) of spike-wave discharges. An enhancing effect of allopregnanolone on spike-wave discharges was found only during the first hour post-injection. A dose-dependent effect is suggested by the data shown in Table 1, but also by differences between groups. Multiple range tests (P<0.05) revealed

Discussion

The present study shows that allopregnanolone, a positive modulator of the GABA_A receptor (Majewska, 1991, Hawkinson et al., 1994), aggravates the spontaneously occurring spike-wave discharges in WAG/Rij rats. These data are in line with some previous findings that stimulation of GABA_A receptors, e.g. with muscimol, increases the number of spike-wave discharges both in WAG/Rij rats and in GAERS, another important Wistar model of generalized absence epilepsy (Vergnes et al., 1984, Peeters et

Acknowledgements

This study was supported by grant 4 PO5A 032 09, obtained from the State Committee for Scientific Research (KBN), and by a CISP grant from the University of Nijmegen.

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Hormones have an extremely large impact on seizures and epilepsy. Stress and stress hormones are known to reinforce seizure expression, and gonadal hormones affect the number of seizures and even the seizure type. Moreover, hormonal concentrations change drastically over an individual's lifetime, especially in women. The prevailing view on steroid hormones is that estrogen and corticosteroid hormones are proconvulsant, whereas progesterone is anticonvulsant, but recent studies in various absence models show that this is not the case in absence epilepsy. Progesterone facilitates the occurrence of absences, most likely through its conversion to the neurosteroid allopregnanolone and the non-genomic action of this neurosteroid on classical GABA_A receptors. On the other hand, during pregnancy, when progesterone is enhanced, spike-wave discharges, typical for absence seizures, are diminished, suggesting an opposite relation between absence seizures and progesterone. How progesterone affects absence seizures differently during pregnancy, and during the various phases of the ovarian cycle is a challenging question. Most likely local neuroplastic changes of δ subunit containing GABA_A receptors are interesting candidates to be involved in how and in which way progesterone exerts its effects on absence seizures and how this may be different across puberty, various phases of the ovulation cycle, pregnancy, and menopause. Estradiol fails to have an effect on absence seizures, while testosterone probably exerts its effects via its metabolites 5α-dihydrotestosterone and later to the GABA_A receptor agonist 3α-androstanediol. Stressors also modulate absence seizures, it is tentatively proposed that this may occur also via modulation of GABA_A receptors.
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While epidemiological data suggest a female prevalence in human childhood- and adolescence-onset typical absence epilepsy syndromes, the sex difference is less clear in adult-onset syndromes. In addition, although there are more females than males diagnosed with typical absence epilepsy syndromes, there is a paucity of studies on sex differences in seizure frequency and semiology in patients diagnosed with any absence epilepsy syndrome. Moreover, it is unknown if there are sex differences in the prevalence or expression of atypical absence epilepsy syndromes. Surprisingly, most studies of animal models of absence epilepsy either did not investigate sex differences, or failed to find sex-dependent effects. However, various rodent models for atypical syndromes such as the AY9944 model (prepubertal females show a higher incidence than prepubertal males), BN model (also with a higher prevalence in males) and the Gabra1 deletion mouse in the C57BL/6J strain offer unique possibilities for the investigation of the mechanisms involved in sex differences. Although the mechanistic bases for the sex differences in humans or these three models are not yet known, studies of the effects of sex hormones on seizures have offered some possibilities. The sex hormones progesterone, estradiol and testosterone exert diametrically opposite effects in genetic absence epilepsy and pharmacologically-evoked convulsive types of epilepsy models. In addition, acute pharmacological effects of progesterone on absence seizures during proestrus are opposite to those seen during pregnancy. 17β-Estradiol has anti-absence seizure effects, but it is only active in atypical absence models. It is speculated that the pro-absence action of progesterone, and perhaps also the delayed pro-absence action of testosterone, are mediated through the neurosteroid allopregnanolone and its structural and functional homolog, androstanediol. These two steroids increase extrasynaptic thalamic tonic GABAergic inhibition by selectively targeting neurosteroid-selective subunits of GABA_A receptors (GABA_ARs). Neurosteroids also modulate the expression of GABA_AR containing the γ2, α4, and δ subunits. It is hypothesized that differences in subunit expression during pregnancy and ovarian cycle contribute to the opposite effects of progesterone in these two hormonal states.
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Agonists and antagonists of GABAB promote and suppress generalized absence seizures in genetic animal models of this disorder, respectively. Peripherally administered GABA enhancers in a majority of cases suppress convulsive seizures in both animals and humans, but promote the occurrence of spike and wave discharges in thalamo-cortical circuits that are typical of generalized absence epilepsy [22, 32]. The GABA receptor-mediated hyperpolarization of relay nuclei of the thalamus de-inactivates low-threshold calcium currents and facilitates rhythmic activity in thalamocortical circuit [32].
Epilepsy is a common neurological disease but the mechanism of seizure generation has been only partially unraveled. Furthermore, almost 30% of epileptic patients are resistant to pharmacological treatment. Therefore, elucidation of the basic mechanism of seizures and search for new antiepileptics in order to treat the drug-resistant form of epilepsy and to improve the efficacy of current therapies seem justified. The aim of this overview is a brief presentation of some new concepts and research directions in pathogenesis and pharmacotherapy of epilepsy. Development of ideas on the mechanisms of seizures and antiepileptic drugs reflects the progress in our understanding of the central nervous system physiology, particularly of neurotransmission. Hyperactivity of excitatory amino acid systems, insufficient GABA_A receptor-mediated neurotransmission, and disturbances in intrinsic properties of neuronal membranes are still regarded as the most important mechanisms of seizures. New data add to the complexity of GABA-glutamate interaction showing both excitatory and inhibitory role of GABA and glutamatergic neurons in the central nervous system. Moreover, besides synaptic NMDA and GABA_A receptors, also extrasynaptic receptors for the amino acid transmitters have been recently implicated in the pathomechanism of epilepsy. Changes in expression, polymorphisms, lost- or gain-function mutations as well as cellular energetic imbalance can contribute to the disturbed function of the ligand- and voltage-dependent sodium, potassium, chloride and calcium channels, resulting in epileptiform activity. Voltage-dependent sodium and calcium channel blockers, and GABA mimetics are the most clinically useful groups of antiepileptic drugs and the newest research in this field is focused on more selective and subtle regulations of their molecular targets. Of interest is an emerging role of extrasynaptic GABAA receptors, various kinds of potassium ion channels, hyperpolarization-activated cyclic nucleotide gated (HCN) channels, acid-sensing ion channels, and gap junctions in the regulation of neuronal excitability and seizures. Iono- and metabotropic glutamate receptors used to be viewed as an attractive target for new anticonvulsants, however, opinions are now less enthusiastic, since their competitive and non-competitive antagonists possess undesired side effects. Positive or negative allosteric modulators of glutamate receptors with fewer side-effects can be more promising. The introduction of new compounds acting through novel pharmacological mechanisms gives hope that the proportion of patients with uncontrolled epilepsy will substantially decrease. However, this may be possible if molecular background of the pharmacoresistance in epilepsy is deciphered.
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Data on the role of sex hormones on genetic animal models of absence epilepsy are limited. Progesterone and allopregnanolone have been shown to enhance SWDs.6,7 On the other hand, despite its proconvulsant effect in convulsive types of epilepsy, 17β-estradiol was reported to have no effects on the occurrence of SWDs in WAG/Rij rats.7
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Effects of neurosteroids on spike-wave discharges in the genetic epileptic WAG/Rij rat

Abstract

Introduction

Section snippets

Methods

Effects of allopregnanolone on the number, total and mean duration of spike-wave discharges

Discussion

Acknowledgements

Neurosci. Biobehav. Rev.

Eur. Neuropsychopharmacol.

Pharmacol. Biochem. Behav.

Neurosci. Biobehav. Rev.

Epilepsy Res.

Epilepsy Res.

Eur. J. Pharmacol.

Brain Res.

Neuropharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Epilepsy Res.

Life Sci.

Life Sci.

Brain Res. Bull.

Brain Res. Bull.

Epilepsy Res.

Neurosci. Lett.

Electroenceph. Clin. Neurophysiol.