Repeated cocaine administration upregulates CB2 receptor expression in striatal medium-spiny neurons that express dopamine D1 receptors in mice

Cannabinoid CB₂ receptors (CB₂R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB₂R action remain unclear. We have previously reported that cocaine self-administration upregulates CB₂R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB₂R expression in striatal medium-spiny neurons that express dopamine D₁ or D₂ receptors (D₁-MSNs, D₂-MSNs) and microglia. Due to the concern of CB₂R antibody specificity, we developed three mouse CB₂-specific probes to detect CB₂R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB₂R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration of heroin produced a dose-dependent reduction in striatal CB₂ mRNA expression. RNAscope ISH assays detected CB₂R mRNA in striatal D₁- and D₂-MSNs, not in microglia. We then used transgenic CX3CR1^eGFP/+ microglia reporter mice and D₁- or D₂-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1^eGFP/+, D₁-MSNs, or D₂-MSNs, respectively. We found that CB₂R upregulation occurred mainly in D₁-MSNs, not in D₂-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB₂R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB₂R upregulation occurs mainly in D₁-MSNs in the nucleus accumbens. Given the important role of D₁-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB₂Rs modulate cocaine action.