During the present reporting period, significant progress was made on this research project. The existence of cannabinoid CB2 receptors in the brain has been heretofore controversial. Most evidence has heretofore suggested that only CB1 cannabinoid receptors are found in brain and central nervous system, while cannabinoid CB2 receptors are restricted to the body's periphery - primarily in the immune system. However, this view has been challenged by recent claims that CB2 receptors are present in the central nervous system and by recent claims that CB2 receptors modulate synaptic activity. Therefore, we used highly selective CB2 agonists and antagonists, combined with the use of CB1 and CB2 receptor gene-deleted mice, and molecular neurobiology techniques combined with electrophysiology, to study the existence and function of CB2 receptors in the brain. Firstly, we studied the expression of functional cannabinoid CB2 receptors on dopamine neurons within the ventral tegmental area (VTA) in rats. The rationale for this work was that we had previously reported the expression of functional cannabinoid CB2 receptors in midbrain dopamine neurons in mice. However, little was known as to whether CB2 receptors are similarly expressed in rat brain. We used in situ hybridization and immunohistochemical assays, and detected CB2 gene and receptors in dopamine neurons of the VTA. The CB2 receptors on VTA dopamine neurons were up-regulated by cocaine self-administration. Electrophysiological experiments showed that activation of CB2 receptors by the CB2-selective receptor agonist JWH133 inhibited VTA dopamine neuronal firing in single dissociated neurons. Local administration of JWH133 by micro-injection into the nucleus accumbens inhibited cocaine-enhanced extracellular dopamine and intravenous cocaine self-administration. This effect was blocked by AM630, a selective CB2 receptor antagonist. These data suggest that CB2 receptors are expressed on VTA dopamine neurons and functionally modulate dopaminergic neuronal activity and cocaine self-administration behavior in rats. We then studied species differences in cannabinoid CB2 receptors and receptor responses to cocaine self-administration in rats versus mice. We found that there are significant species differences in CB2 receptor mRNA splicing and expression, protein sequences, and receptor responses to CB2-specific ligands in mice versus rats. Systemic administration of JWH133, a highly selective CB2 receptor agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed ratio schedule of reinforcement in mice, but not in rats. However, under progressive-ratio reinforcement, JWH133 significantly increased the progressive-ratio break-point in cocaine self-administering rats - thus decreasing cocaine's incentive motivational properties. We then examined CB2 receptor gene expression and receptor structure in the brain. We found novel rat-specific CB2c and CB2d mRNA isoforms in addition to CB2a and CB2b mRNA isoforms. Using in situ hybridization RNAscope assays, we found higher levels of CB2 receptor mRNA in different brain regions and cell types in mice versus rats. By comparing CB2 receptor-encoding regions, we found a premature stop codon in the mouse CB2 receptor gene that truncated 13 amino acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2 receptor genes and receptor structures may in part explain the different effects of CB2 receptor-selective ligands on cocaine self-administration in mice versus rats. In addition, we studied CB2 receptor-mediated effects on neuronal plasticity in the hippocampus. The functionality of the endocannabinoid system is primarily ascribed to the well-documented retrograde activation of presynaptic cannabinoid CB1 receptors. However, we found that action potential-driven endocannabinoid release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1 receptors. This hyperpolarization, which is specific to hippocampal CA2 and CA3 pyramidal cells, depends on the activation of CB2 receptors, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to hyperpolarization of the neuron. CB2 receptor activation occurred in a self-regulatory manner, robustly altered the input/output function of CA3 hippocampal pyramidal cells, and modulated gamma oscillations in vivo. Thus, we found - for the first time - a cell-type specific plasticity mechanism in the hippocampus that provides robust evidence for the neuronal expression of CB2 receptors and emphasizes their importance in basic neuronal transmission. Finally, we explored the effects of the novel cannabinoid compound delta-8-tetrahydrocannabivarin (THCV) on nicotine's effects in rodents. We found that THCV inhibits nicotine self-administration in alcohol-preferring (P) rats, inhibits cue-induced nicotine-seeking behavior in P rats tested in the incubation of craving animal model, inhibits nicotine-induced relapse to nicotine-seeking behavior in P rats tested in the reinstatement animal model of relapse, prevents acquisition of nicotine-induced conditioned place preference in mice, significantly attenuates anxiety-like behavior (as measured in the plus maze) in mice placed into nicotine-withdrawal, significantly attenuates somatic signs of withdrawal in mice placed into nicotine-withdrawal, and significantly attenuates the hyperalgesia (as measured using the hot-plate test) in mice placed into nicotine-withdrawal. As THCV is a combined CB1 antagonist and CB2 agonist, such findings are fully congruent with our previous reports of significant anti-addiction actions of cannabinoid CB1 antagonists and CB2 agonists. Further, we propose that the tetrahydrocannabivarins constitute an exciting new target for the development of anti-addiction, anti-craving, and anti-relapse medications.
