CB₂ receptor antibody signal specificity: correlations with the use of partial CB₂-knockout mice and anti-rat CB₂ receptor antibodies

Cannabinoid CB₁ receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB₂ receptors (CB₂Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB₂Rs in the brain. However, this finding is disputed because of the specificity of CB₂R antibody signals. We used two strains of currently available partial CB₂-knockout (CB₂-KO) mice as controls, four anti-rat or anti-mouse CB₂R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB₂R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB₂-like immunostaining in mouse midbrain dopamine neurons. CB₂R deletion in CB₂-KO mice may reduce or leave CB₂R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB₂R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB₂-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB₂-KO mice. Quantitative RT-PCR assays detected CB₂ mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB₂-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB₂R-specific. Non-specific binding may be related to the expression of mutant or truncated CB₂R-like proteins in partial CB₂-KO mice and the use of anti-rat CB₂ antibodies because the epitopes are different between rat and mouse CB₂Rs.