Physical dependence on ethanol may be defined by the appearance of withdrawal symptoms upon cessation of ethanol intake. Changes in the function of three neuronal systems have been implicated in the generation or expression of one of these symptoms, withdrawal seizures: reduction in activity of the GABAA receptor, and increased activity of dihydropyridine-sensitive voltage-dependent calcium channels and NMDA receptor-coupled ion channels. Some evidence for the role of these systems in ethanol withdrawal is derived from lines of mice that have been selectively bred for sensitivity (withdrawal seizure prone, WSP) or resistance (withdrawal seizure resistant, WSR) to ethanol withdrawal seizures. WSP mice have more hippocampal NMDA receptors than WSR mice, both before and after chronic ethanol ingestion, and voltage-sensitive calcium channels are increased more in WSP mice after chronic ethanol treatment. WSP mice are also more sensitive to GABA antagonist-induced seizures than WSR mice, and recent evidence suggests a difference in GABAA receptor structure between these lines. We propose to study mice being generated in a new selection study for sensitivity (HW mice) and resistance (LW mice) to ethanol withdrawal seizures. We will assess the sensitivity of the untreated and ethanol-exposed mice, over several generations, to the convulsant properties of drugs acting at the three neurochemical systems of interest (i.e., picrotoxinin, NMDA, Bay K 8644); the measure used will be handling-induced convulsions, which is the selection measure. We will also characterize the NMDA receptor complex and the voltage-sensitive calcium channel in brain by membrane binding and autoradiography. Replication of earlier receptor binding results in an independent selection study will significantly strengthen the relationship between a particular neurochemical system and the generation of withdrawal seizures. This study also allows concurrent behavioral and neurochemical analyses as the selected lines are separating, strengthening the causal relationship. Studies are also proposed to assess interactions between the NMDA and GABAA receptor complexes that may contribute to ethanol withdrawal seizures. Acutely, ethanol inhibits NMDA receptor function and chronic ethanol in vivo as well as chronic exposure of cells in vitro to the NMDA antagonist MK-801 lower the levels of mRNA for certain subunits of the GABAA receptor. We will use primary cultures of cerebellar granule cells to determine if ethanol and NMDA antagonists similarly affect GABAA receptor function, subunit mRNA levels and subunit proteins, to evaluate the hypothesis that a primary effect of ethanol on NMDA receptor function leads to altered GABAA receptor function. Ethanol withdrawal has been likened to kindling, and an understanding of the mechanisms of ethanol withdrawal seizures may lead to the development of efficacious therapies to reduce these seizures and prevent the development of severe withdrawal symptoms in chronic alcoholics.
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