Neuronal versus Glial CB2 Receptors: Insights from a Novel CB2-KO-eGFP Reporter Mouse Line.

The cannabinoid CB2 receptor (CB2R) has emerged as a promising therapeutic target for pain and central nervous system disorders, yet its brain expression has remained controversial due to low basal levels and the lack of reliable antibodies. Previous green fluorescent protein (GFP) reporter mouse lines have produced conflicting findings, possibly because GFP was either randomly inserted into the genome or placed in the 3'-untranslated region of the CB2R gene (Cnr2), complicating interpretation. Here, we report a new CB2-KO-eGFP mouse line in which the endogenous Cnr2 coding region was precisely replaced with enhanced GFP through targeted knock-in, generating a combined CB2R knockout and GFP reporter. Loss of CB2R expression was confirmed by qRT-PCR, RNAscope in situ hybridization, and cannabinoid pharmacological assays. GFP-immunostaining was detected across multiple brain regions, including cingulate cortex, hippocampus, red nucleus, and cerebellum, and in several cell types such as microglia, astrocytes, and neurons. Flow cytometry revealed strong GFP signals in spleen and blood cells and quantifiable GFP expression in brain tissue. Notably, ~ 70% of microglia and ~ 4% of neurons in cortex and hippocampus expressed GFP under normal physiological conditions. These findings demonstrate that CB2R is indeed expressed in healthy brain tissue and across multiple neural and glial cell types, resolving long-standing uncertainty regarding CB2R localization. Functionally, CB2R deletion reduced cannabinoid-induced analgesia, hypothermia, and catalepsy, confirming the receptor's physiological relevance. This new mouse line provides a reliable and highly informative tool for defining CB2R expression and function in both the brain and peripheral immune system.