Differential effects mediated by GABAA receptors in thalamic nuclei in lh/lh model of absence seizures

doi:10.1016/S0920-1211(97)01023-1

Epilepsy Research

Volume 27, Issue 1, April 1997, Pages 55-65

https://doi.org/10.1016/S0920-1211(97)01023-1 Get rights and content

Abstract

Absence seizures represent synchronized burst-firing of thalamocortical neurons, which are driven by tonic GABAergic output of nucleus reticularis thalami (NRT). Activation of GABA_A receptors on NRT neurons reduces NRT output and retards thalamocortical burst-firing. Although this mechanism in NRT may underlie antiabsence effects of benzodiazepines, it does not explain observations that barbiturates can worsen absence seizures. In this study we tested the hypothesis that clonazepam and phenobarbital produce differential effects on GABA_A receptors in the lh/lh genetic model of absence seizures after microinjection into NRT compared to VLa, a prototypic relay nucleus containing thalamocortical neurons. In NRT, phenobarbital (16–1600 nmol/cannula), clonazepam (160–2200 pmol/cannula) and muscimol (8.8–263 pmol/cannula) significantly suppressed absence seizure frequency. In VLa, phenobarbital (1.6 nmol) and muscimol (0.88 pmol) increased seizure frequency, whereas higher doses (160 nmol and 88 pmol, respectively) significantly suppressed seizure frequency. In contrast, clonazepam produced no effect on seizure frequency even at a dose of 2.2 nmol; this same dose significantly suppressed absence seizures after microinjection into NRT. These findings suggest that activation of GABA_A receptors in NRT may suppress absence seizures, and that phenobarbital may worsen absence seizures through actions on GABA_A receptors in thalamocortical cells (VLa). Region-specific GABA_A receptor isoforms may underlie the contrasting effects of clonazepam after microinjection into NRT and VLa.

Introduction

The search for more effective antiabsence compounds should be facilitated by studying the mechanisms of action of antiabsence AEDs in the neuronal populations which generate the synchronized burst-firing of absence seizures. Three neuronal populations comprise a thalamocortical circuit that appears to generate absence seizures: neocortical pyramidal neurons, thalamic relay neurons, and neurons within the nucleus reticularis thalami (NRT) 44, 45. Studies of this thalamocortical circuit have led to the following theoretical mechanistic framework for absence seizures. Activation of T-type calcium channels 14, 33within thalamic relay neurons appears to be a critical step within the cascade of events that leads first to synchronized burst-firing of these neurons 8, 10and then to oscillatory burst-firing between relay cells and other neuronal populations within the circuitry 9, 22, 44. T-channels rapidly inactivate after being activated and a relatively lengthy hyperpolarization is required for their deinactivation 7, 10, 49. Hence, their continued participation in synchronized burst-firing requires the presence of a tonic hyperpolarizing mechanism. This mechanism appears to be provided by tonically active GABAergic afferents from NRT neurons 2, 22, 27, 43, 44. Studies from our and other labs have shown that these afferents activate GABA_B receptors as a critical process in the generation of synchronized burst-firing 16, 24, 41.

GABA_A receptors on NRT neurons also appear to have a regulatory role within this thalamocortical circuit. NRT neurons, which are exclusively GABAergic [20], have recurrent inhibitory connections with neighboring NRT neurons 11, 12. By activating GABA_A receptors, these recurrent connections regulate GABAergic output from NRT neurons to thalamic relay neurons. The net effect of increased GABA_Aergic synaptic transmission within NRT is thus to reduce synchronized burst-firing of thalamic relay neurons 15, 22, 23, 48. In principle, therefore, this type of GABA_A receptor-mediated inhibition of NRT outflow should decrease the likelihood of absence seizures.

A clinical observation which has thusfar complicated efforts to produce a comprehensive mechanistic framework for absence seizures is the differential efficacy of two classes of compounds that act at different binding sites on the GABA_A receptor complex: benzodiazepines (e.g. clonazepam), which suppress absence seizures; and anticonvulsant barbiturates (e.g. phenobarbital), which lack efficacy or even worsen them 4, 5, 26. If clonazepam exerts its antiabsence actions by activating GABA_A receptors in NRT 15, 21, then why does phenobarbital lack efficacy against absence seizures 4, 5, 26? One possible explanation may be the existence of different pentameric isoforms of the GABA_A receptor, each with unique pharmacological properties 1, 6, 13, 25, 30, 32, 34, 39. Localization of different GABA_A receptor isoforms in different neuronal populations could, in principle, account for the discrepant clinical effects of phenobarbital and clonazepam.

To begin seeking evidence of functionally different effects produced by GABA_A receptors in the neuronal populations which generate absence seizures, we used the lethargic (lh/lh) genetic model 16, 17, 28, 29, 36to analyze the effects of compounds acting at different regulatory sites of GABA_A receptors within two thalamic nuclei: NRT and VLa, a prototypic relay nucleus. By microinjecting these compounds into NRT or VLa and analyzing their effects on the frequency and duration of spontaneous absence seizures in these mice, we were able to test two alternative hypotheses. The first hypothesis was that activation of NRT GABA_A receptors by GABA_A receptor agonists or benzodiazepines but not by anticonvulsant barbiturates would suppress absence seizures in lh/lh mice. This hypothesis was formulated based upon two observations: (i) that benzodiazepines ameliorate and anticonvulsant barbiturates may worsen absence seizures 4, 5, 26; and (ii) that activation of GABA_A receptors in NRT should have an antiabsence effect, an idea supported by clonazepam's effects on GABA_A receptors in NRT 15, 21. The second hypothesis was that activation of VLa GABA_A receptors by phenobarbital but not by muscimol or clonazepam would worsen absence seizures. This hypothesis was formulated as the logical corollary of the first hypothesis, to account for phenobarbital's worsening of absence seizures. If phenobarbital does not produce this effect by acting on GABA_A receptors in NRT, then it may produce this effect by activating GABA_A receptors in thalamic relay neurons (e.g. VLa). If so, then it should follow that GABA_A receptors in VLa respond differently to muscimol and clonazepam than to phenobarbital. Preliminary accounts of this work were reported in abstract 18, 19.

Section snippets

lh/lh mice

Colonies of lh/lh and +/+ (coisogenic non-epileptic strain) mice were initiated with stocks obtained from Jackson Labs and were bred and housed in an immunoprotected environment in the Duke University Vivarium. The +/+ strain comprises all F1 progeny of C57Bl/6JEi females X C3H/HeSnJ males. Male lh/+ (heterozygote) mice were bred with female lh/+ mice to produce 25% lh/lh (about 30 male lh/lh/month) in the F2 generation. By 3 weeks of age, lh/lh mice were easily distinguished from their

Intra-NRT microinjections: muscimol, phenobarbital and clonazepam

Bilateral intra-NRT injection (n=6) of the GABA_A agonist muscimol (8.8–263 pmol/cannula) produced a dose-dependent and significant (P<0.025) suppression of absence seizures in lh/lh mice (Fig. 1). The effect was apparent within 15 min of injection and was maximal (53%) by 45 min after injection. The effect persisted for the entire 150 min period after injection. The IC₅₀ of muscimol was 64 pmol/cannula.

Bilateral intra-NRT injection (n=5) of the anticonvulsant barbiturate phenobarbital (16–1600

Principal findings

Three principal findings emerged from this study. First, intra-NRT microinjection of muscimol, phenobarbital and clonazepam produced significant suppression of absence seizure frequency in lh/lh mice. Second, intra-VLa microinjection of muscimol and phenobarbital enhanced absence seizure frequency at lower doses and significantly suppressed seizure frequency at higher doses; however, muscimol had an efficacious (97%) seizure-suppressant effect in contrast to the weak effect of phenobarbital

Acknowledgements

We thank Mrs Betty Worrell and Mrs Sarah Sneed for providing administrative assistance during the study. Alex Huin, Eric Akawie and Yin Yin provided technical assistance during initial parts of the study. Funding was provided by grants to D.A.H. from NINDS (RO1 NS30977) and from the Veterans Administration (Merit Review).

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The GABA neurotransmitter is widely expressed in the central nervous system, and the involvement of GABA and its receptors (GABAA and GABAB) in the pathophysiological mechanisms of absence seizures is well known and has been widely investigated in rodent models [2]. It is well known that the GABAergic neurons of the reticular thalamic nuclei (RTN) and their axonal connections to the relay nuclei of the thalamus contribute to the development and regulation of absence seizures [38]. Intraperitoneally administered GABA mimetics such as vigabatrin and tiagabine result in contraindications in absence seizures [39-42].
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Absence Epilepsy (AE) is a prototypic epileptic syndrome that develops during brain maturation but cannot be fully explored in human patients. Genetic animal models, especially rats with spike-and-wave discharges recorded on the electroencephalogram, the hallmark of absence seizures, offer strong face validity with the human pathology that allows precise exploration of the pathophysiology of this form of epilepsy. Using an array of different methods, recent studies have demonstrated that spike-and-wave discharges are initiated in the primary somatosensory cortex and then rapidly propagate to motor cortices and thalamic nuclei. More specifically, in vivo electrophysiological intracellular recordings showed that the pyramidal neurons of the deep layers of this cortex exhibit fast activation, hyperexcitability and hypersynchronizing characteristics in favor of their role as ictogenic neurons in absence seizures. Furthermore, longitudinal studies during brain maturation suggest the progressive development of these features. Exploration of the different key players in the maturation of the primary somatosensory cortex should determine the anomalies that lead to the development of the cortical generator of absence seizures.
Genetic Models of Absence Epilepsy in Rats and Mice
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Several genetic models of absence epilepsy have been developed, since the 1960s, in both mice and rats, based on the observation that some animals display spontaneous spike-and-wave discharges during cortical EEG recording, concomitant with behavioral arrests. For most of these models, the electrophysiological, behavioral, and pharmacological features are quite reminiscent of those observed in human patients with typical absence epilepsy. Here, we describe the different models, how they have been discovered and developed, how seizures occur, which comorbidities are associated, and how they can be used to better understand the physiopathology of absence epilepsy and its genesis. In particular, these models offer the opportunity to explore the neural circuits involved in the generation of seizures at different levels of integration, from unitary recording to functional magnetic resonance imaging. In addition, they allow performance of proof of concept experiments for innovative therapeutic strategies, and identification of early biomarkers.
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GABA-mediated mechanisms have been extensively studied within the CTC system, and there is evidence that both GABAA and GABAB receptors are involved in the generation of SWDs (Snead, 1995; van Luijtelaar and Sitnikova, 2006). GABAergic neurons in the NRT and their projections to the specific relay nuclei of the thalamus are involved in the development/regulation of absence seizures (Hosford et al., 1997; Snead, 1992; Snead et al., 1992). Interestingly, it has been suggested that the balance between GABAA(Cl−) and GABAB(K+)-mediated conductances is essential for the control of SWDs (Kaminski et al., 2001; Marescaux et al., 1992b; Vergnes et al., 1984).
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