Elsevier

Brain Research

Volume 810, Issues 1–2, 9 November 1998, Pages 241-250
Brain Research

Research report
Modulation of GABAA receptor-mediated inhibition by postsynaptic calcium in epileptic hippocampal neurons

https://doi.org/10.1016/S0006-8993(98)00922-6Get rights and content

Abstract

Visualization of neurons during patch clamp recordings from slices provides concurrent neuroanatomical information for physiological studies. Although, the technique becomes increasingly popular in immature brains, it has not been fully utilized in aged/adult and diseased brains including post-surgical human specimen. In the present study, glutamatergic modulation of GABAA receptor-mediated inhibition was investigated by whole-cell patch clamp recordings from visualized hippocampal dentate granule cells (DGCs) in slices that were prepared from surgically-removed human medial temporal lobe specimens and the rat pilocarpine model of temporal lobe epilepsy. GABAA receptor-mediated synaptic inhibition was recorded by isolating inhibitory postsynaptic currents (IPSCs) at a membrane potential of 0 mV where glutamatergic excitatory postsynaptic currents are near equilibrium. Peak amplitude of GABAA IPSC was not different between epileptic DGCs of both human and pilocarpine-treated rat hippocampi and those in the control rat DGCs. However, when high frequency stimulation (30 Hz for 10 s) preceded immediately before the generation of a GABAA IPSC, its peak amplitude was significantly reduced in epileptic DGCs. The application of an NMDA receptor antagonist prevented this decrease indicating that the high frequency stimulation activated the NMDA receptor and that this activation is involved in the induction of response-decrement of GABAA IPSCs in epileptic DGCs. In addition, intracellular application of a calcium chelator, BAPTA through a patch pipette was found effective in preventing the response-decrement of GABAA IPSCs suggesting that postsynaptic calcium-increase is also involved in this process. It is proposed that activation of the NMDA receptor in epileptic DGC may trigger an epileptogenic increase of intracellular free calcium, and this calcium-increase plays a crucial role for the induction of the response-decrement of GABAA IPSCs in epileptic hippocampus, which possibly leads to the initiation of epileptic seizures and ictal events.

Introduction

It has been suggested that GABAA receptor/channel function can be modulated by intracellular calcium (see Ref. [1]for review). Low concentration of intracellular calcium is reported to increase GABAA receptor function 17, 27, while an appreciable increase in the intracellular calcium (>1 μM) was found to decrease GABAA receptor-channel function via phosphorylation-dephosphorylation mechanisms 13, 21, 22. Proposed sources of intracellular calcium increase for this type of modulation are: (1) the voltage-dependent calcium channels [30]and (2) the NMDA subtype glutamate receptor [6]. In epilepsy, there is ample evidence to show that NMDA receptor-mediated synaptic responses are pathologically increased in the hippocampus in temporal lobe epilepsy patients 2, 15, 23and in animal models of chronic epilepsy 16, 26, 40. Increased activities of the NMDA receptor appear to work in favor of inducing glutamate-mediated modulation of GABAA receptor-mediated inhibition in epileptic neurons. Indeed, systemic administration of a NMDA receptor antagonist was reported to restore GABAA receptor-mediated inhibition in epileptic animals [18]. In acute hippocampal slices, epileptogenic responses induced by a bath-application of an NMDA receptor agonist were accompanied by a concurrent reduction of GABAA receptor-mediated inhibition [35].

In the present study, it is hypothesized that epileptic activation of the NMDA receptor triggers an epileptogenic increase of intracellular free calcium, and this calcium-increase is crucial for down-regulating GABAA receptor-mediated inhibition in the epileptic hippocampus. In order to experimentally regulate the increase of intracellular calcium and clamp its concentration, a calcium chelator, BAPTA, was used in an intrapipette solution. Dentate granule cells (DGCs) were visualized prior to the patch clamp recording in slice preparations. Although this visualized patch clamp recording technique is used widely in immature brains, it has not been fully utilized in aged/adult and diseased brains. There is no report to date that the technique has been applied to human epileptic hippocampal specimens in slices.

Section snippets

The pilocarpine model

Sprague–Dawley male rats (100–150 g; N=84) were injected with 320–350 mg/kg pilocarpine (i.p.) according to methods previously described [16]. Sixty-three of 84 rats developed Stage 1 to Stage 5 seizures within 30 min after the injection of pilocarpine, and subsequently experienced status epilepticus. The status epilepticus was controlled by diazepam (4 mg/kg) 3 h after the onset. In the remaining 21 rats, pilocarpine induced no status epilepticus. In the rats that experienced status

Preparation of thin slices from human epileptic hippocampus and from the rat pilocarpine model

In the present study, hippocampal slices were prepared with a thickness of 250 μm in order to visualize individual dentate granule cells (DGCs) prior to the whole-cell patch clamp recording of GABAA receptor-mediated inhibitory postsynaptic currents (GABAA IPSCs). This thickness is 1/2 that of the hippocampal slices which were prepared from the same types of epileptic specimen with the use of the `blind method' for the patch clamp recording from thick slices 14, 15.

With a thickness of 250 μm,

Discussion

The present results with an intracellular BAPTA application suggest that the increase in intracellular calcium concentration, induced by the activation of the NMDA receptor, is directly involved in and responsible for the induction of the response-decrement of GABAA IPSCs in the DGCs of human epileptic hippocampus and the pilocarpine-treated and seizure-experienced rat hippocampus. The present study also demonstrated that the mechanisms responsible for this transient decrement of GABAA

Acknowledgements

I thank Dr. I. Fried for providing human hippocampal specimens, and the members of the UCLA Epilepsy Surgery Program for their clinical expertise. Human study was conducted under the guidance of Declaration of Helsinki and UCLA Human Subject Protection Committee and supported by an NINDS program project grant P01-NS02808. The pilocarpine project was supported by an NINDS FIRST Award R29-NS31180.

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