Major depression and addiction are mental health problems associated with stressful events in life with high relapse and recurrence even after treatment. Many laboratories were not able to detect the presence of CB2 cannabinoid receptors (CB2-Rs) in healthy brains, but CB2-R expression has been demonstrated in rat microglial cells and other brain-associated cells during inflammation. Thus, neuronal expression of CB2-Rs has been ambiguous and controversial, and its role in depression and substance abuse is unknown. In this study we tested the hypothesis that genetic variants of the CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances, including opiates, cocaine, and ethanol, in rodents. Here we demonstrate that a high incidence of Q63R but not H316Y polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated after exposure to stressors and administration of abused drugs. Mice that developed an alcohol preference had reduced CB2 gene expression, and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 antisense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. Using electron microscopy we report the subcellular localization of CB2-Rs that are mainly on postsynaptic elements in rodent brain. Our data demonstrate the functional expression of CB2-Rs in the brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuroimmunocannabinoid activity.

BackgroundAddiction and major depression are mental health problems associated with stressful events in life with high relapse and reoccurrence even after treatment. Many laboratories were not able to detect the presence of cannabinoid CB2 receptors (CB2-Rs) in healthy brains, but there has been demonstration of CB2-R expression in rat microglial cells and other brain associated cells during inflammation. Therefore, neuronal expression of CB2-Rs had been ambiguous and controversial and its role in depression and substance abuse is unknown.Methodology/Principal FindingsIn this study we tested the hypothesis that genetic variants of CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances including opiates, cocaine and ethanol in rodents. Here we demonstrate that a high incidence of (Q63R) but not (H316Y) polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated following exposure to stressors and administration of abused drugs. Mice that developed alcohol preference had reduced CB2 gene expression and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 anti-sense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. We report for the using electron microscopy the sub cellular localization of CB2-Rs that are mainly on post-synaptic elements in rodent brain.Conclusions/SignificanceOur data demonstrate the functional expression of CB2-Rs in brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuro-immunocannabinoid activity.

Two well-characterized cannabinoid receptors (CBrs), CB1 and CB2, mediate the effects of cannabinoids and marijuana use, with functional evidence for other CBrs. CB1 receptors are expressed primarily in brain and peripheral tissues. For over a decade several laboratories were unable to detect CB2 receptors in brain and were known to be intensely expressed in peripheral and immune tissues and have traditionally been referred to as peripheral CB2 CBrs. We have reported the discovery and functional presence of CB2 cannabinoid receptors in mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. We used RT-PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays to determine the functional expression of CB2 CBrs in rat brain and mice brain exposed to chronic mild stress (CMS) or those treated with abused drugs. RT-PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild-type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in brain and CB2 expression was detected in neuron-specific enolase (NSE) positive hippocampal cell cultures. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus-maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system (CNS). Thus, contrary to the prevailing view that CB2 CBrs are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 CBrs and their gene transcripts are widely distributed in the brain. This multifocal expression of CB2 immunoreactivity in brain suggests that CB2 receptors may play broader roles in the brain than previously anticipated and may be exploited as new targets in the treatment of depression and substance abuse.

Recent aggregation of evidence for the roles of endogenous agonist and receptor systems that are mimicked or activated by cannabanoid ligands has provided a focus for work that has elucidated details of some of the multiple physiological roles and pharmacological functions that these systems play in brain and peripheral tissues. This chapter reviews some of the approaches to improved elucidation of these systems, with special focus on the human genes that encode cannabanoid receptors and the variants in these receptors that appear likely to contribute to human addiction vulnerabilities.

Confocal laser microscopy of DiI‐labeled slices of postnatal rat cerebellum (postnatal Day 4–10; P4–10) was compared to infrared microscopy and the rapid Golgi method (P0–14) to investigate postnatal migration of granule neurons. Vertical migration of the granule neurons occurred already at birth (P0). Surprisingly, mossy fibers often reached the external granule cell layer and were in close contact with the external granule cells. These mossy fibers may play a role in initiating granule cell migration. At this age, cell bodies of the immature neurons were attached to the external basal lamina by a process and extended down toward the presumptive internal granule cell layer. At P14, some granule cells remained attached to the surface, although their cell bodies exhibited the typical morphology of mature granule neurons and were located deep in the internal granule cell layer. These cells extended their endfeet‐like processes all the way to the surface of the brain. These results indicate that the vertical pathways of granule cell migration form early and persist throughout the period of granule cell migration. Confocal infrared microscopy of DiI‐labeled sections and the rapid Golgi method also allowed demonstration of tangentially migrating neurons that made one or more turns on the way to the internal granule cell layer. The rapid Golgi method confirmed that many Bergmann glial processes end at the level of the tangentially migrating granule cells whereas others project to the surface. These observations show that migratory granule cells take several different routes to their final destination, which cannot be explained by so‐called radial glial guidance. The only mode of migration in evidence is consistent with process elongation and translocation of the nucleus within the preformed processes. © 2003 Wiley‐Liss, Inc.

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.