Research reportChanges in extracellular glutamate and GABA levels in the hippocampal CA3 and CA1 areas and the induction of glutamic acid decarboxylase-67 in dentate granule cells of rats treated with kainic acid
Introduction
It has been proposed that various types of seizures are induced by an imbalance between glutamatergic excitation and GABAergic inhibition [6]. In order to assess synaptic activities, extracellular levels of glutamate and GABA have been investigated using brain microdialysis techniques during chemically induced seizures in animals and seizures in human epileptics 10, 14. Limbic seizures in rats or rabbits have been observed to be accompanied by an increase 21, 23, 36, 43, 48, 49or no change 7, 21, 26, 32, 46in extracellular glutamate. Changes in extracellular GABA levels are also inconsistent 7, 21, 22, 44, 48, 49. The discrepancies among these findings are probably due to the following factors: (1) the seizures were induced by a variety of chemical agents 27, 44, 47, 48, and their mechanisms of action may well not be uniform; (2) the microdialysis was performed in the hippocampus 7, 21, striatum [8], entorhinal cortex [46]and piriform cortex [48]; (3) the microdialysis was not necessarily performed throughout a series of behavioral changes. Sometimes the analyses were made only for the first 1–2 h after an injection of kainic acid 22, 36.
In the hippocampus, basic differences in structure and function exist between the CA1 and CA3 areas. Limbic seizure induced by the systemic administration of kainic acid is an established model of temporal lobe epilepsy 4, 19, and different roles are played by these two hippocampal areas during such seizures [13]. Regarding the action of kainate, the binding sites or receptors for kainate in the CA3 areas are of the high-affinity type, whereas those in CA1 are of the low-affinity type 5, 16. An intraventricular or intrahippocampal injection of kainic acid results in neuronal loss in the CA3 pyramidal layer; CA1 pyramidal cells, in contrast, are vulnerable to the systemic administration of kainate 4, 29, 31, 40. Nevertheless, most microdialysis studies in the hippocampus have been carried out without making a distinction between the CA1 and CA37, 21, 23, 26, 49.
In the present study, we investigated the effect of a systemic administration of kainic acid on the extracellular glutamate and GABA levels in the CA1 and CA3 areas of freely moving rats over a 10-h period. Furthermore, to ascribe the long-lasting increase in extracellular GABA levels to any change in gene expression, we examined the expression of the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, and GABA immunoreactivity in the rat hippocampus by in situ hybridization and immunohistochemistry, respectively. The changes in the extracellular glutamate and GABA levels and in GAD67 expression were found to differ between the hippocampal CA1 and CA3 areas.
Part of this work was published in an abstract [12].
Section snippets
Brain microdialysis
Male Sprague–Dawley rats (180–240 g) were anesthetized with Nembutal (50 mg/kg, i.p.) and mounted on a stereotaxic frame (Narishige, Tokyo, Japan). A guide cannula (CUP 11, BAS, Tokyo, Japan) was implanted in CA3 or CA1 according to the atlas of Paxinos and Waston [33]. The coordinates used were A (from bregma) −3.8, L 1.8, V 0.8 and A −4.3, L 4.8, V 5.8 mm for the CA1 and CA3 areas, respectively. After 3 days, the rats were individually transferred to a plexiglas bowl allowing free movement,
Animal behavior
After receiving a subcutaneous administration of kainic acid (10 mg/kg), all animals experienced behavioral changes characteristic of limbic seizures as described by Lothman and Collins [24]. Within 10–20 min after the kainate injection, staring posture alternating with hyperactivity was present. These symptoms were gradually replaced by trains of repetitive `wet dog shake'. At 1–1.5 h after the injection, `wet dog shakes' were succeeded by automatisms and mild to severe intermittent seizures
Discussion
In the present investigation, the rat hippocampal CA3 and CA1 regions were separately dialyzed through the course of seizures after a systemic administration of kainic acid. The extracellular glutamate and GABA levels were changed differently in these two areas. Glutamate increased temporarily in CA3, then decreased slightly and fluctuated. In CA1, the glutamate level decreased consistently. The extracellular GABA levels were increased with different time courses in CA3 and CA1. In CA3, the
Acknowledgements
We would like to thank Ms. Hiroko Kuzume for providing technical assistance. This work was supported by Research Grant-in-Aid 07279107 for Scientific Research on Priority Areas on "Functional Developments of Neural Circuits", the Ministry of Education, Science, Sports and Culture. R.G. Ding was a Sasakawa Foundation Fellow.
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