Chemical flavorants in vaping products alter neurobiology in a sex-dependent manner to promote vaping-related behaviors.

Electronic nicotine delivery systems (ENDS) are distinctly different from combustible cigarettes due to the availability of flavor options. Subjective measures have been employed to demonstrate that adults and adolescents 'prefer' flavors for various reasons: 1) they are pleasing and 2) they mask the harshness of nicotine. Despite this, there have been few investigations into the molecular interactions that connect chemical flavorants to smoking or vaping-related behaviors. Here we investigated the effects of three chemical flavorants (hexyl acetate, ethyl acetate, and methylbutyl acetate) that are found in green apple (GA) ENDS e-liquids; but are also found in other flavor categories. We used a translationally-relevant vapor self-administration mouse model and observed that adult male and female mice self-administered GA flavorants in the absence of nicotine. Using α4-mCherryα6-GFP nicotinic acetylcholine receptor (nAChR) mice, we observed that mice exposed to GA flavorants exhibited a sex-specific increase (upregulation) of nAChRs that was also brain region-specific. Electrophysiology revealed that mice exposed to GA flavorants exhibited enhanced firing of ventral tegmental area dopamine neurons. Fast-scan cyclic voltammetry revealed that electrically-stimulated dopamine release in the nucleus accumbens core is increased in mice that are exposed to GA flavorants. These effects were similarly observed in the medial habenula. Overall, these findings demonstrate that ENDS flavors alone change neurobiology and may promote vaping-dependent behaviors in the absence of nicotine. Furthermore, the flavorant-induced changes in neurobiology parallel those caused by nicotine, which highlights the fact that non-menthol flavorants may contribute to or enhance nicotine reward and reinforcement. SIGNIFICANCE STATEMENT: The impact of flavors on vaping is a hotly debated topic; however, few investigations have examined this in a model that is relevant to vaping. Although a full understanding of the exact mechanism remains undetermined, our observations reveal that chemical flavorants in the absence of nicotine alter brain circuits relevant to vaping-related behavior. The fact that the flavorants investigated here exist in multiple flavor categories of vaping products highlights the fact that a multitude of flavored vaping products may pose a risk toward vaping-dependent behaviors even without the impact of nicotine. Furthermore, as the neurobiological changes impact neurons of the reward system, there exists the possibility that non-menthol flavorants may enhance nicotine reward and reinforcement.