Identification of cannabinoid type 1 receptor expressing cocaine amphetamine-regulated transcript neurons in the rat hypothalamus and brainstem using in situ hybridization and immunohistochemistry

Recent data have indicated that the neuropeptide cocaine amphetamine-regulated transcript (CART) may be a downstream mediator of the effect of CB1 receptor antagonist on appetite regulation. In order to identify possible interactions between CART and central CB1R expressing neurons, a detailed mapping of CART and CB1R expression and immunoreactivity in the brain was initiated. Single radioactive in situ hybridizations revealed a predominant overlap between CART and CB1R mRNA in hypothalamic and lower brainstem nuclei. Using double in situ hybridization, co-localization between CART and CB1R mRNA expressing neurons was observed to be most pronounced in the retrochiasmatic and lateral hypothalamic areas, as well as in all parts of the dorsal vagal complex. Further attempts to immunohistochemically characterize the distribution of CB1R were, however, deemed impossible as any of eight commercially available antibodies/antisera gave rise to non-specific staining patterns. Furthermore, the staining pattern obtained was not discriminate between CB1R knockout mice and wild type mice. Collectively, we demonstrate at the messenger level that CB1R expressing perikarya colocalize with CART expressing neurons in hypothalamic and brainstem areas known to be important in appetite control, whereas interactions at the protein level necessitate a demand for cautious interpretations of immunohistochemical results.