The Journal of Neurophysiology Vol. 77 No. 4 April 1997, pp. 2139-2152
Copyright ©1997 The American Physiological Society

Physiological and Pharmacological Alterations in Postsynaptic GABA_A Receptor Function in a Hippocampal Culture Model of Chronic Spontaneous Seizures

John W. Gibbs III⁴, Sompong Sombati^{1, 5}, Robert J. Delorenzo^{1, 2, 3, 5}, and Douglas A. Coulter^{1, 3, 4, 5}

¹ Department of Neurology, ² Department of Biochemistry and Molecular Biophysics, ³ Department of Pharmacology and Toxicology, and ⁴ Department of Anatomy, Medical College of Virginia; and ⁵ Medical College of Virginia Comprehensive Epilepsy Institute of Virginia Commonwealth University, Richmond, Virginia 23298-0599

Gibbs, John W., III, Sompong Sombati, Robert J. DeLorenzo, and Douglas A. Coulter. Physiological and pharmacological alterations in postsynaptic GABA_A receptor function in a hippocampal culture model of chronic spontaneous seizures. J. Neurophysiol. 77: 2139-2152, 1997. Cultured rat hippocampal neurons previously exposed to a media containing no added Mg²⁺ for 3 h begin to spontaneously trigger recurrent epileptiform discharges following return to normal medium, and this altered population epileptiform activity persisted for the life of the neurons in culture (>2 wk). Neurons in "epileptic" cultures appeared similar in somatic and dendritic morphology and cellular density to control, untreated cultures. In patch-clamp recordings from hippocampal pyramidal cells from "epileptic," low Mg²⁺ pretreated hippocampal cultures, a rapid (within 2 h of treatment), permanent (lasting 8 days) and statistically significant 50-65% reduction in the current density of functional -aminobutyric acid-A (GABA_A) receptors was evident when the GABA responses of these cells were compared with control neurons. Functional GABA receptor current density was calculated by determining the maximal response of a cell to GABA 1 mM application and normalizing this response to cellular capacitance. Despite the marked GABA efficacy differences noted above, the potency of GABA in activating chloride currents was not significantly different when the responses to control and "epileptic" pyramidal cells to multiple concentrations of GABA were compared. The EC₅₀ for GABA was 4.5 ± 0.2 (mean ± SE) for control neurons and 3.5 ± 0.4 µM, 5.2 ± 0.5 µM, 3.7 ± 0.3 µM, and 4.6 ± 0.3 µM for epileptic neurons2 h, 2 days, 3 days, and 8 days after low Mg²⁺ pretreatment, respectively. Modulation of GABA responses by the benzodiazepine, clonazepam, was significantly reduced in epileptic neurons compared with controls. The kinetically determined clonazepam 100 nM GABA augmentation efficacy decreased from 44.1% in control neurons to 9.3% augmentation in neurons recorded from cultures 10 days posttreatment. The kinetics of GABA current block by the noncompetitive antagonist picrotoxin were determined in hippocampal cultured neurons, and an IC₅₀ of 14 µM determined. Bath application of picrotoxin at half of the IC₅₀ concentration (7 µM) induced epileptiform activity in control cultures and this activity appeared very similar to the epileptiform activity induced by prior low Mg²⁺ treatment. This concentration of picrotoxin was determined experimentally to block 30% of the GABA_A-mediated receptor responses in these cultures, and this level of block was sufficient to trigger spontaneous epileptiform activity. The 50% reduction of GABA responses induced as a permanent consequence of low Mg²⁺ treatment therefore was determined to be sufficient in and of itself to induce the spontaneous epileptiform activity, which was also a consequence of this treatment.

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