The overall goal of this proposal is to provide mentored career development training for a young physician scientist. The primary vehicle of this mentored training involves the development of research to elucidate the molecular mechanisms that underlie alterations in gamma-aminobutyric acid (GABA)A receptor adaptations that influence the development of ethanol dependence. The candidate will receive broad-based training in alcoholism research as well as several methods that will be required to address his research plan including confocal microscopy, transgenic mouse technology, intracerebroventricular (ICV) administration of drugs and measurement of seizure thresholds. In addition, he will be guided to develop new methods to investigate the phosphorylation of GABAA receptors. The research plan will focus on GABAA receptor adaptations since ethanol dependence produces marked increases in CNS excitability and anxiety that involve adaptations in the pharmacological properties of GABAA receptors. Dr. Kumar proposes to focus on the role of receptor trafficking, phosphorylation and PKC activity in mediating GABAA receptor adaptations. He will test the hypothesis that PKC interactions with GABAA receptors may determine receptor subunit adaptations throughout brain.
Specific Aim 1 will determine if ethanol dependence alters the association of GABAA receptors with PKC isozymes in cerebral cortex, hypothalamus and amygdala. Next, he will determine if genetic deletion of PKCgamma or PKCepsilon isozymes alter ethanol-induced adaptations in GABAA receptor function and seizure susceptibility.
Specific Aim 2 will utilize these mice to determine if PKCy and PKCe differentially alter the effects of ethanol on membrane expression and internalization of specific GABAA receptors.
The final aim will investigate the role of PKC'y and PKCe in the phosphorylation state of GABA! receptors, again using mutant mouse models. ICV administration of PKC isoform specific inhibitor/activator will be used to establish a cause and effect relationship between the alterations in PKC and subsequent effects on receptor membrane expression, internalization and function. We predict that these experiments will delineate specific GABAA receptor adaptations involved in ethanol dependence-induced enhancement of seizure susceptibility (bicuculline seizure threshold). These studies will provide important mechanistic information on the molecular basis of ethanol-induced adaptations in GABAA receptors that influence the development of ethanol tolerance and dependence.
