Effect of zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures

doi:10.1016/S0169-328X(03)00183-9

Molecular Brain Research

Volume 116, Issues 1–2, 19 August 2003, Pages 1-6

https://doi.org/10.1016/S0169-328X(03)00183-9 Get rights and content

Abstract

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation associated with the neurotransmitter glutamate, or from inadequate inhibitory effects associated with γ-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove amino acids from the synaptic cleft. Excitation initiated by the synaptic release of glutamate is attenuated by the action of glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino-acid carrier-1 (EAAC-1). GABA is removed from synaptic regions by the action of the transporters proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, albino rats with chronic, spontaneous recurrent seizures induced by the amygdalar injection of FeCl₃ were treated for 14 days with zonisamide (ZNS) (40 mg/kg, i.p.). Control animals underwent saline injection into the same amygdalar regions. Treatment control for both groups of intracerebrally injected animals was i.p. injection of equal volumes of saline. Western blotting was used to measure the quantity of glutamate and GABA transporters in hippocampus and frontal cortex. ZNS caused increase in the quantity of EAAC-1 protein in hippocampus and cortex and down regulation of the GABA transporter GAT-1. These changes occurred in both experimental and ZNS treated control animals. These data show that the molecular effect of ZNS, with up-regulation of EAAC-1 and decreased production of GABA transporters, should result in increased tissue and synaptic concentrations of GABA. Although many antiepileptic drugs have effects on ion channels when measured in vitro our study suggests that additional mechanisms of action may be operant. Molecular effects on regulation of transporter proteins may aid in understanding epileptogenesis and inform investigators about future design and development of drugs to treat epilepsy.

Introduction

Zonisamide (ZNS), 1,2-benzisoxazole-3-methanesulfonamide, is an antiepileptic drug known to be effective in treating patients with partial seizures or secondary generalized tonic clonic seizures [6], [9], [25]. Mechanisms for the anticonvulsant effects of ZNS include (a) blocking voltage-sensitive Na⁺ and T-type Ca²⁺ channels [10], [20], [27], [31], (b) modulating the dopaminergic system [17], [18], (c) reducing epileptiform events by inhibiting excitatory glutamatergic transmission [19], (d) inhibiting lipid peroxidation in a rat model of epilepsy [11], and (e) scavenging hydroxyl radical and nitric oxide radicals in a dose-dependent manner by increasing reducing ability in the brain [10], [16], [32].

Prior work has shown that epileptogenesis with collapse of glutamate regulation leading to glutamatergic excitatory synaptic transmission and reduced γ-aminobutyric acid (GABA)-ergic inhibitory synaptic transmission is associated with changes in the molecular expression of amino acid transporters [34].

Some antiepiletic drugs are known to have molecular effects on amino acid transporter proteins. Valproic acid is associated with increased expression [36] or up-regulation [4] of excitatory amino acid transporters in the hippocampus, suggesting that valproic acid may have unique mechanisms of action by affecting the removal of glutamate by up-regulating EAATs of excitatory amino acid transporters.

To study the molecular effects of the antiepileptic drug zonisamide (ZNS), albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl₃ were treated for 14 days with either ZNS, or with saline used as an injection control. Regions of the hippocampus and the frontal cortex were assayed with Western blots for glutamate and GABA transporters.

Section snippets

Materials and methods

Twenty male Wistar rats weighing between 200 and 250 g were used. The animals were kept in hanging cages; they had unlimited access to food and water and were exposed to 12-h light–dark cycles. Surgical procedures were performed with pentobarbital sodium anesthesia (37.5 mg/kg, i.p. ). The experimental design was reviewed and approved by the Committee for the Ethics on Animal Experiments in Miyazaki Medical College. The experiments were carried out under the control of the guideline for Animal

Results

Spontaneous stage 4 kindled behavioral seizures [22] were observed 5 days after FeCl₃ was injected into the amygdala in control rats treated with saline, as previously reported [37]. Seizures were not observed in rats treated with ZNS whether they had an amygdalar FeCl₃ injection or saline injection.

GLAST protein immunoreactivity in the hippocampus of FS animals was reduced by 30% of that of the control group (SS; see Fig. 1). Expression of GLAST was not effected by ZNS treatment (SZ, FZ).

Discussion

Treatment of animals with ZNS prevented seizures and resulted in increased production of EAAC-1 in both the hippocampal region and in frontal cortex. Further, ZNS-treated animals had down-regulation of GABA transporter (GAT-1) production in hippocampus and frontal cortex. EAAC-1, a neuronal glutamate transporter, provides re-uptake of glutamate for GABA synthesis [14], [28] as opposed to acting as a protective mechanism for attenuating glutamate excitotoxicity [7]. As reported by Sepkuty et al.

Acknowledgements

This study was partially supported by a Grant-in-Aid for Encouragement of Young Scientists (08770777 and 10770490) from the Ministry of Education, Science, Sport and Culture, Japan (to Y.U.).

References (37)

N. Ikegaki et al.
Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry
Mol. Brain Res.
(1994)
Y. Kanai
Family of neural and acidic amino acid transporters: molecular biology, physiology and medical implications
Curr. Opin. Cell Biol.
(1997)
K. Kitani et al.
Increasing anticonvulsant effect of AD-810 (zonisamide) in aging BDF1 mice
Life Sci.
(1987)
M. Kito et al.
Mechanisms of T-type calcium channel blockade by zonisamide
Seizure
(1996)
I.E. Leppik et al.
Efficacy and safety of zonisamide: results of a multicenter study
Epilepsy Res.
(1993)
E.W. Lothman et al.
Alterations in neurotransmitter amino acids in hippocampal kindled seizures
Epilepsy Res.
(1987)
A. Mori et al.
The anticonvulsant zonisamide scavenges free radicals
Epilepsy Res.
(1998)
M. Okada et al.
Effects of zonisamide on extracellular levels of monoamine and its metabolite, and on Ca2+ dependent dopamine release
Epilepsy Res.
(1992)
M. Okada et al.
Effects of zonisamide on dopaminergic system
Epilepsy Res.
(1995)
R.J. Racine
Modification of seizure activity by electrical stimulation. II. Motor seizure
Electroencephalogr. Clin. Neurophysiol.
(1972)

D.M. Rock et al.

Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD 810, CI 912), a novel anticonvulsant

Epilepsy Res.

(1989)

J.D. Rothstein et al.

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate

Neuron

(1996)

H. Schagger et al.

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

Anal. Biochem.

(1987)

C.L. Schauf

Zonisamide enhances slow sodium inactivation in Myxicola

Brain Res.

(1987)

S. Suzuki et al.

Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex

Epilepsy Res.

(1992)

Y. Ueda et al.

Simultaneous monitoring of the seizure-related changes in extracellular glutamate and gamma-aminobutyric acid concentration in bilateral hippocampi following development of amygdaloid kindling

Epilepsy Res.

(1995)

Y. Ueda et al.

Hippocampal gamma-aminobutyric acid transporter alterations following focal epileptogenesis induced in rat amygdala [In Process Citation]

Brain Res. Bull.

(2000)

Y. Ueda et al.

Amygdalar injection of FeCl3 causes spontaneous recurrent seizures

Exp. Neurol.

(1998)

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Research reportEffect of zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Mol. Brain Res.

Curr. Opin. Cell Biol.

Life Sci.

Seizure

Epilepsy Res.

Epilepsy Res.

Epilepsy Res.

Epilepsy Res.

Epilepsy Res.

Electroencephalogr. Clin. Neurophysiol.

Epilepsy Res.

Neuron

Anal. Biochem.

Brain Res.

Epilepsy Res.

Epilepsy Res.

Brain Res. Bull.

Exp. Neurol.

Research report
Effect of zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures