The overall goal of this project is to investigate biochemical mechanism of how tolerance to and physical dependence upon barbiturates develop. The objective of this renewal application will focus on the role of GABAA and GABAB receptors in barbiturate tolerance and dependence. We will continue to utilize a rat model (intracerebroventricular, icv, infusion of pentobarbital) as an experimental model useful for determining how different binding sites [for GABA, benzodiazepines (BZ) and convulsants, e.g., picrotoxin and t-butylbicyclophosphorothionate (TBPS)] of the GABAA receptor complex and GABAB receptors in different regions of the brain are involved in chronic effects of pentobarbital.
The specific aims are designed to test the hypothesis formulated.
Specific Aim I. Characterization of GABAA receptors in different regions of the brain during the development of tolerance to and physical dependence upon pentobarbital. Studies on GABAA receptors in discrete areas of the brain will be the first step in gaining insight into the biochemical mechanisms of barbiturate actions. The investigations will be conducted at different time intervals during the development of tolerance to and physical dependence upon pentobarbital. The results obtained will be compared with those from acutely treated animals. The specific studies are as follows: 1. Biochemical characterization of different binding sites (GABA, BZ, TBPS) of the GABAA receptors; 2. Autoradiographic characterizations of different binding sites (GABA, BZ, TBPS) of the GABAA receptors; 3. In situ hybridization characterization of GABAA receptor subunit mRNA (alpha, beta, gamma, delta); and, 4. Cl- influx - functional characterization of GABAA receptors.
Specific Aim II. Characterization of GABAB receptors in different regions of the brain during the development of tolerance to and physical dependence upon pentobarbital. The specific studies are: 1. Biochemical characterization of ligand binding to GABAB receptors; and 2. Autoradiographic characterization of ligand binding to GABAB receptors.
Specific Aim III. Investigation of GABA release mechanisms in different regions of the brain during the development of tolerance to and physical dependence upon pentobarbital. The release of GABA affected by pentobarbital is intimately related to postsynaptic GABAA receptors and presynaptic GABAB receptors (autoreceptors).
Specific Aim I V. Systematic assessments of the degree of the development of tolerance to and physical dependence upon pentobarbital. The results obtained from these experiments will be correlated with the results derived from those of Specific Aims I-III.
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