The goal of this study is to define the effect of delta-9-tetrahydrocannabinol (THC), the major psychoactive and immunosuppressive component in marijuana, on microglia within the brain. Microglia are a resident population of macrophages, which constitute a major element of the immune system of the brain. These cells respond to trauma and infection by migrating to sites of injury, produce proinflammatory cytokines and cytotoxic substances, and phagocytize infectious agents and damaged tissue. Microglia, also, have been implicated in neuropathological processes, such as AIDS dementia and multiple sclerosis as a consequence of chronic activation. We have demonstrated that microglia in vitro express CB1 and CB2 cannabinoid receptors, the latter of which are present at high levels when these cells are in an inflammatory state. These receptors have been shown to play a role in cannabinoid-mediated modulation of immune cell functions at non-neuronal sites and our preliminary data suggest that they play a similar role in microglia. We propose to assess for functional linkages for CB1 and CB2 receptors in cannabinoid-mediated modulation of microglial functions. These studies are highly significant since recognition that specified activities are linked functionally to cannabinoid receptors would provide insight regarding therapeutic regimens for ablating inflammatory processes which contribute to AIDS-associated and other forms of neuropathogenesis. The hypothesis to be tested is that THC and other cannabinoids alter select functional activities of microglia and do so by a cannabinoid receptor-mediated process. In order to test this hypothesis, the following Specific Aims are proposed as guidelines to the research. First, we will define the pattern of cannabinoid receptor expression by microglia in the brain. We have demonstrated that microglia differentially express CB1 and CB2 receptors in vitro in relation to cell activation and will establish whether these events occur in vivo. Organotypic brain slice experiments as well as in vivo infectivity studies will be performed. Cannabinoid receptor expression will be assessed using histological, immunocytochemical, pharmacological, and molecular approaches. Second, we will define the functional relevance of cannabinoid receptors on microglia. We have demonstrated that microglia in vitro, when in """"""""responsive"""""""" and """"""""primed"""""""" inflammatory states, express high levels of the CB2 receptor. We will assess for cannabinoid-mediated effects through this receptor on functions associated with these activation states including chemotaxis, chemokinesis, phagocytosis, and antigen processing. Functional assays will employ paired cannabinoid enantiomers, receptor subtype-specific antagonists, and cannabinoids exhibiting differential ligand binding affinities to establish structure-activity relationships.
