Marijuana's active constituent, delta9-THC, produces psychoactive by activating G-protein-coupled CB1 cannabinoid receptors in the CNS. Although previous work has characterized the role of adaptations in CB1 receptor-mediated G-protein activation in cannabinoid tolerance and dependence, little is known about the involvement of downstream components in the signaling pathway. The proposed studies will address the potential involvement of downstream components in the signaling pathway. The proposed studies will address the potential role of novel regulators of CB1 receptor signaling (RGS) are GTPase activating proteins for the G(i/o) and G(q) subfamilies of G-proteins. They accelerate the G-protein inactivation rate, thus providing a form of desensitization that occurs downstream of G-protein activation but upstream of effector modulation. Thus, RGS proteins may be involved in the homeostatic control of effector regulation. The proposed studies will investigate the potential roles of RGS proteins in modulating the acute and chronic effects of cannabinoids. First, to determine whether tolerance of delta9-THC in mice is associated with changes in effector regulation, the effects of chronic delta9-THC administration on adenylyl cyclase activity will be examined in various brain regions, and compared to immunoreactive levels of RGS proteins in modulating the acute and chronic effects of cannabinoids. First, to determine whether tolerance to delta9-THC in mice is associated with changes in effector regulation, the effects of chronic delta9-THC administration on adenylyl cyclase activity will be examined in various brain regions, and compared to immunoreactive levels of RGS proteins. In parallel studies, the effect of acute and chronic delta9-THC on transcriptional regulation of RGS expression will be determined by in situ hybridization, and compared to autoradiograms of CB1 receptor-activated G-proteins in the same brains These studies will be performed in time-course fashion, and compared to the development of delta9-THC tolerance at the behavioral level. Similar studies will the be performed before and after antagonist, precipitated withdrawal, to determine the contribution of RGS proteins to cannabinoid dependence, little is known about the involvement of downstream components in the signaling pathway. The proposed studies will address the potential role of novel regulators of CB1 receptor signaling in the mechanisms of cannabinoid tolerance and dependence. The newly discovered regulators of G-protein signaling (RGS) are GTPase activating proteins for the G(i/o) and G(q) subfamilies of G-proteins. They accelerate the G-protein inactivation rate, thus providing a form of desensitization that occurs downstream of G-protein activation but upstream of effector modulation. The proposed studies will investigate the potential roles of RGS proteins in modulating the acute and chronic effects of cannabinoids. First, to determine whether tolerance of delta9- THC in mice is associated with changes in effector regulation, the effects of chronic delta9-THC administration on adenylyl cyclase activity will be examined in various brain regions, and compared to immunoreactive levels of RGS protein. In parallel studies, the effects of acute and chronic delta9- on transcriptional regulation of RGS expression will be determined by in situ hybridization, and compared to autoradiograms of CB1 receptor-activated G-proteins in the same brains. These studies will be performed before and after antagonist-THC tolerance at the behavioral level. Similar studies will then be performed before and after antagonist- precipitated withdrawal, to determine the contribution of RGS proteins to cannabinoid dependence. Next, transgenic approaches will be used to investigate the relationship between CB1 receptors and RGS proteins. To determine whether expression of particular RGS isoforms is associated with the expression of CB1 receptors, RGS mRNA will be examined in CB1 knockout mice by in situ hybridization. To determine whether expression of particular RGS isoforms is associated with the expression of CB1 receptors, RGS mRNA will be examined in CB1 knockout mice by in situ hybridization. To determine whether expression of particular GS isoforms is associated with the expression of particular RGS isoforms is associated with the expression of CB1 receptors, RGS mRNA will be examined in CB1 knockout mice by in situ hybridization. To determine whether RGS9-2, a novel RGS isoform that is highly expressed in striatum, modulates CB1 receptor function, the effects of RGS9 knockout on the acute and chronic effects of delta9-THC will be examined in both behavioral and biochemical studies. Co-transfection with effector modulation in defined cellular population. These studies will run concurrently with those above to use information gained from these cells in studying the roles of RGS proteins in the CNS. This combined biochemical and molecular biological approach in both animals and isolated cells will lead to a greater understanding of the roles of RGS proteins in modulating CB1 receptor function in both the normal and delta9-THC tolerant/dependent state.
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