Endocannabinoids and the Control Of Behavior and Cardiovascular Function

National Institute On Alcohol Abuse And Alcoholism

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Abstract

The vagal gut-brain axis has been recognized as a vital regulator of the metabolic, motivational, and emotional states. These include anxiety, depression, reinforcement, food and alcohol craving, and involve the endocannabinoid system. Our previous observations indicated that the brain non-penetrant CB1 receptor (CB1R) antagonist, JD5037, suppresses alcohol preference in mice by blocking CB1R in ghrelin-producing cells and hampering the permissive role of ghrelin to drink alcohol (Cell Metab 2019; 29: 1320-1333). During the current reporting period, we have evaluated the possible contribution of CB1R located on sensory afferents of the gastrointestinal (GI) tract in the voluntary intake of alcohol by C57Bl/6J mice, using two drinking paradigms: the two-bottle/free choice and the drinking in the dark paradigms. We found that selective deletion of CB1R from ghrelin-producing cells only partially occluded the inhibitory effect of JD5037 in alcohol drinking transgenic (Ghrl-Cre CB1flox/flox) mice. We then created transgenic mouse lines to delete CB1R in dorsal root ganglia (Wnt-Cre CB1flox/flox) and nodose ganglia (Phox2b-Cre CB1flox/flox) and found the inhibitory effect of JD5037 to be occluded only in the latter. Likewise, the deletion of GHSR1A in nodose ganglia in the transgenic (Phox2b-Cre Ghsr1aflox/flox) mouse line occluded the inhibition of alcohol drinking by the GHSR1A antagonist PF-52960457 in alcohol preferring mice. RNA sequencing of nodose ganglia revealed the existence of multiple clusters of neurons, which differ with respect to genetic markers, function, and innervation (Cell Rep., 2019; 27: 2508-2523). We found that the deletion of CB1R from the subpopulation Trpv1 positive neurons only marginally affected alcohol drinking (Trpv1-Cre CB1flox/flox), whereas the deletion of CB1R from advillin positive neurons (Avil-Cre/ERT2 CB1flox/flox) abolished the modulatory effect of JD5037 on alcohol drinking. Interestingly, Cnr1 and Avil expression were found to overlap in the same subset of nodose neurons, whereas there was minimal overlap between Cnr1 and Trpv1 expression in the various subsets. Additionally, to achieve localized and side-specific deletion of specific genes, we have developed a technique of microinjection of the neurotoxin saporin coupled to an antibody against specific receptors, such as Cnr1, that allows the internalization of saporin exclusively in neurons expressing the specific receptor targeted by its antibody. Using this technique, we will test whether there is any preference for left of right nodose neurons that express CB1 receptors in projecting to central pathways involved in the control of alcohol drinking behavior. In conclusion, CB1 receptor on sensory afferents of the GI tract may contribute to alcohol seeking behavior in mice. Ongoing studies further dissect the roles of two major subpopulations of nodose ganglion nerve terminals that project to the muscular vs. mucosal layers of the GI tract by testing the effects of CB1 ligands in Glp1r-Cre CB1flox/flox and GPR65-Cre CB1flox/flox mice, respectively, and examining the nature of interaction between CB1 and Ghsr1a on sensory afferent neurons.