The opioid epidemic has emerged as one of the leading public health concerns of the 21st century. It has been paralleled by a rise in the popularity of e-cigarettes, which has facilitated increased nicotine use among adolescents. Clinical studies have revealed that the co-use of nicotine and opioids is exceedingly common; most individuals with opioid use disorders (OUDs) also report smoking, and smoking is a strong predictor of opioid misuse. Preclinical studies have explored the biological basis for the clinical phenomenon, revealing that co-exposure to nicotine and opioids may produce cross-sensitization to drug reward. Considered the canonical reward pathway, the mesolimbic dopamine system primarily comprises dopaminergic (DAergic) neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). The VTA also contains a substantial population of y-aminobutyric acid (GABA) neurons, which provide local inhibitory control over DA neurons, but also project distally. Though nicotine and opioids have each separately been shown to alter the activity of VTA DA and GABA neurons, little is known about the way that simultaneous use of these drugs may further modify the mesolimbic system. This dissertation establishes a physiologically relevant model for the co-use of nicotine and opioids, with chronic exposure to nicotine vapor beginning in adolescence, followed by a 6-week co-exposure to both nicotine vapor and morphine via continuous two-bottle choice (C2BC), during adulthood. We use this drug administration paradigm in conjunction with calcium imaging (fiber photometry) to compare how the response of VTA DA and GABA neurons to nicotine vapor changes following exposure to chronic adolescent nicotine and then co-exposure to nicotine + morphine. We report that VTA DA neurons are especially sensitive to the effects of adolescent nicotine exposure, whereas VTA GABA neurons are uniquely altered by co-exposure to nicotine and morphine. In this way, we highlight the feasibility and utility of studying polysubstance use in preclinical models and point to VTA GABA neurons as a potential therapeutic target for the co-treatment of concurrent nicotine and opioid dependencies.