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

Cannabinoid CB receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB receptors (CBRs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CBRs in the brain. However, this finding is disputed because of the specificity of CBR antibody signals. We used two strains of currently available partial CB-knockout (CB-KO) mice as controls, four anti-rat or anti-mouse CBR antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CBR-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 CB-like immunostaining in mouse midbrain dopamine neurons. CBR deletion in CB-KO mice may reduce or leave CBR-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CBR band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB-KO mice. Quantitative RT-PCR assays detected CB 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 CB-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CBR-specific. Non-specific binding may be related to the expression of mutant or truncated CBR-like proteins in partial CB-KO mice and the use of anti-rat CB antibodies because the epitopes are different between rat and mouse CBRs.