Marijuana has been used for many centuries as a psychoactive agent and also for its medicinal applications. For example, in humans, it has been used as an analgesic, anti-inflammatory, immunosuppressant and anticonvulsant. Binding studies with analogs of delta-9- tetrahydrocannabinol, the active component of marijuana (i.e., CP 55,940 etc.) have revealed that receptors for these ligands exist not only in the brain, but also in peripheral tissues (e.g., spleen, testis). To date, two cannabinoid receptors have been cloned, the central and peripheral cannabinoid receptor. We have used in situ hybridization to map the distribution of the peripheral cannabinoid receptor (CB2). This receptor was first discovered by Munro et in HL60 cells upon their differentiation into macrophages. Several in situ hybridization studies by different groups have suggested that mRNA for CB2 was present in the marginal zones of the spleen, but the cell types have not been identified. To further characterize the cell types in the spleen which may be responsible for binding cannabinoids, we have used specific immunocytochemistry in combination with in situ hybridization. We have also detected the mRNA for CB2 in testis, and are studying the hormonal regulation of mRNA expression in testosterone-treated young rats. In addition, we want to determine if there is a correlation between the mRNA level and a particular stage in spermatogenesis. Thus, we will synchronize spermatogenesis in rats using a vitamin A-deficient diet. It is known that vitamin A deprivation leads to the complete cessation of spermatogenesis in mammals, and that it is reversible upon supplementation with vitamin A. We have postulated that in the event that this receptor is missing, we could elucidate its function in a living organism. Thus, to study the significance of the presence of such a receptor, we will produce transgenic mice lacking the peripheral cannabinoid receptor. This gene will be targeted and mutated by homologous recombination. As a first step, we have successfully cloned the mouse CB2 receptor and characterized it by restriction endonuclease mapping. We have subcloned the fragments containing the coding region and those immediately flanking this region. We are presently mapping and sequencing these fragments to mutate the CB2 mouse gene in the plasmid construct and prepare it for homologous recombination.
