Despite the fact that marijuana is one of the major drugs of abuse, it has been only recently that major advances have been made in understanding the actions of the cannabinoids on the central nervous system. The development of potent cannabinoid agonists led to the discovery of cannabinoid receptors in brain. However, the precise role of these receptors in brain remains to be fully established. It is our hypothesis that the behavioral effects of delta9-THC does not arise from its actions on a single biochemical system but rather represent the actions of cannabinoids on several biochemical systems. the goal of the proposed research is to identify additional receptors and/or mechanisms through which cannabinoids produce their behavioral effects and determine their exact role in the brain. The emphasis will be directed toward characterizing the pharmacological profile of newly synthesized cannabinoids in order to determine whether there is a common pharmacophore for all cannabinoid behavioral effects. Synthetic analogs will be evaluated for their ability to produce motor hypoactivity, hypothermia, catalepsy and antinociception in mice, and stimulus generalization to delta9-THC in delta9-THC-trained rats and monkeys. In addition, attempts will be made to develop specific cannabinoid antagonists by evaluating analogs that exhibit little or no agonistic activity in the above mentioned behavioral tests as potential antagonists of delta9-THC. Potential irreversible antagonists include nitrogen mustard, isothiocynate, and photoaffinity label analogs. In order to ascertain that novel analogs which have been used to label the cannabinoid receptor share all of the properties of delta9-THC, cross tolerance studies will be conducted to determine whether delta9-THC- tolerant mice are completely tolerant to all of the effects of the novel analogs CP 55,950 and WIN 55,212. Delta9-THC and related analogs will also be evaluated in rats trained to discriminate CP 55,940 from vehicle to determine whether there is complete generalization. An additional strategy will be to identify the central sites which are responsible for specific actions of the cannabinoids. Potent cannabinoid agonists and putative antagonists will be administered via indwelling cannula into rat brain regions. Identification of specific brain sites will allow us to conduct subsequent studies to determine whether the effects associated with this region are mediated through the adenylate cyclase system. In addition to evaluating receptor affinity of new analogs in the 3H-CP 55,940 receptor assay, studies will be conducted to determine whether alterations in cannabinoid receptor density and KD account for development of tolerance. Autoradiography will be used to determine whether the nitrogen mustards eliminate cannabinoid receptor binding uniformly throughout the brain. Finally, characterization of cannabinoid action on adenylate cyclase will include evaluation of novel agonists and antagonists in brain membranes and in primary cell culture. These in vitro studies will be complemented with in vivo studies which will be carried out to determine whether tolerance results in alterations in adenylate cyclase activity.
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