Evidence has emerged during the past few years that demonstrates the existence of an endogenous cannabinoid system in the brain. This system consists of a G-protein coupled receptor (CB1), several potential effector systems, and the endogenous ligand anandamide. An additional cannabinoid receptor (CB2) is found primarily in macrophages. The goal of the proposed research is to identify additional receptors and/or mechanisms through which cannabinoids produce their behavioral effects and establish the physiological role of the endogenous system in brain. Our current findings suggest that selective analogs can be developed that do not produce the full array of cannabinoid effects. We will continue developing highly potent novel agonists to fully explore the structure-activity relationships, as well as to produce selectivity for behavioral effects and for CB1 and CB2 receptors. Efforts will be made to develop additional competitive antagonists as well as irreversible antagonists for both CB1 and CB2 receptors. These analogs will be evaluated in CB1 and CB2 receptor binding assays, several mouse pharmacological assays, rat drug discrimination, and for inhibition of macrophage function. A major objective is to determine what role the endogenous cannabinoid system plays in the development of tolerance and dependence. It has been shown that the development of tolerance is associated with receptor down-regulation and elevated CB1 mRNA levels in cerebellum of mice. Studies are proposed for determining whether similar changes occur in cerebral cortex, hippocampus and striatum. Further evaluation of tolerance in selected brain areas will include measurement of receptor levels, determination of receptor sequestration, MAP kinase activity and levels of krox-24 and CB1 mRNA. The recent discovery of a specific cannabinoid antagonist allowed us to develop for the first time a model of cannabinoid dependence in both mice and rats. We will fully characterize this model regarding the dose of delta9-THC and the frequency of administration that is required for development of dependence. The time required for recovery from dependence will be established and efforts will be made to detect abrupt withdrawal. The biochemical changes observed in the development of tolerance will also be examined during the development of and recovery from dependence. Additionally, CREB levels will be measured in dependent animals, since it has been implicated in the development of dependence for several classes of compounds.
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