Evidence of widespread neuronal expression of CB2 cannabinoid receptor MRNA and proteins in rat central nervous system.

Leonardo Guasti, Gregory Michael, Maurice Elphick. Queen Mary, University of London, London, United Kingdom.

Several recent studies have reported evidence that the CB2 cannabinoid receptor is expressed in the central nervous system (CNS) (Van Sickle et al., 2005; Gong et al., 2006). Here we have used a variety of techniques to analyse in detail the expression and localisation of CB2 receptors in the rat CNS and spleen.

Using RT-PCR with primers complementary to sequences in exons 1 and 3 of the rat cnr2 gene, mRNA expression was detected in the spleen and in all brain regions examined. Using two non-overlapping DIG-labelled cRNA probes and two 35S-labeled oligonucleotides for mRNA in situ hybridisation revealed CB2 receptor expression predominantly in the marginal zone of spleen white pulp, whilst in brain widespread neuronal expression was observed. To investigate CB2 receptor protein expression, novel antibodies were generated against a peptide antigen corresponding to the C-terminal 14 amino-acid residues of the rat CB2 receptor. Western blot analysis of HEK-293 cells expressing HA-tagged-CB2 revealed the same pattern of immunostained bands when probing with anti-CB2 or anti-HA antibodies. Moreover, the most intensely stained band corresponded to the expected molecular mass for CB2 (∼45 kDa). This band was also detected in both spleen and brain homogenates, but with ∼10 fold lower abundance in brain than in spleen. Immunohistochemistry of spleen sections revealed staining in the marginal zone, consistent with CB2 mRNA expression. In the brain, the distribution of CB2-immunoreactivity corresponded with CB2 mRNA expression, showing low-level but widespread neuronal localisation. For example, both CB2 mRNA and CB2 protein were detected in the cell body and/or dendrites of neurons in the olfactory bulb (mitral cells), cerebellum (Purkinje cells) hippocampus and neocortex (pyramidal cells).

These data suggest that CB2 receptors may mediate postsynaptic effects of endocannabinoids in normal brain function. Furthermore, the methods reported here will be useful for future analysis of changes in CB2 receptor expression in the CNS that are thought to occur in association with a variety of neuropathological conditions (e.g. neuropathic pain).

Gong et al. (2006) Brain Res. 1071:10-23

Van Sickle et al. (2005) Science, 310:329-32.

Supported by Medical Research Council grant G0401647.