Patterns of drug-related behavior: role of VTA DA neurons and inhibitory GPCR-dependent signaling

Abstract

Ventral tegmental area (VTA) dopamine (DA) neurons play an important role in modulating activity in the mesocorticolimbic system in response to reward. Output from VTA DA neurons is critical for mediating drug-related behaviors, as inhibition of these neurons blocks drug induced stimulation. Drugs of abuse act on a diverse set of molecular targets, but they share the ability to enhance DA levels throughout the mesocorticolimbic system. Enhanced DA levels engage negative feedback mechanisms on VTA DA neurons by activating inhibitory G protein-dependent signaling pathways, mediated by GABAB receptors (GABABRs) and D2 DA receptors (D2Rs). GABABR- and D2R-dependent feedback in VTA DA neurons is further modulated by Regulator of G Protein Signaling (RGS) proteins, which act to enhance the deactivation of G protein signaling. The R7 family of RGS proteins, including RGS6, is known to modulate neuronal G protein signaling preferentially via Gao, and has been implicated in drug-related behaviors. One goal of the work in this thesis was to assess how changes in VTA DA neuron inhibition may alter drug-related behaviors.In order to assess the relative influence of GABABRs and D2Rs on drug sensitivity, we characterized a neuron-specific CRISPR/Cas9 approach to ablate GABABRs or D2Rs in VTA DA neurons. Our ablation technique prevented VTA DA neuron somatodendritic responses to the GABABR agonist baclofen or the D2R agonist quinpirole in a receptor specific manner. Loss of VTA DA neuron D2R-dependent signaling resulted in enhanced cocaine-induced motor stimulation in both male and female mice, whereas loss of VTA DA neuron GABABR-dependent signaling resulted in enhanced cocaine-induced motor stimulation only in males. Neither GABABR nor D2R ablation had an effect on morphine-induced motor stimulation. These data suggest that VTA DA neuron inhibitory G protein-dependent feedback modulates behaviors in a drug- and sex-specific way. We also wanted to evaluate the role of RGS6 in modulating VTA DA neuron inhibitory feedback and drug-related behaviors. We showed that RGS6 and GB5, the binding partner of R7 RGS proteins, are expressed in the majority of VTA DA neurons. Additionally, both the GABABR and D2R can signal through Gao, the preferred substrate of RGS6, suggesting that RGS6 may modulate both GABABR- and D2R-dependent signaling. Indeed, constitutive RGS6–/– mice exhibited enhanced amplitude of somatodendritic VTA DA neuron D2R-dependent signaling and prolonged deactivation of GABABR-dependent signaling. Next, we utilized the CRISPR/Cas9 approach characterized previously to assess VTA DA neuron specific effects of RGS6–/–. As with constitutive RGS6–/–, VTA DA neuron specific RGS6 ablation enhanced D2R-dependent current amplitude. Further, VTA DA neuron specific RGS6 overexpression reduced D2R-dependent current amplitude. These data suggest a negative regulatory role for RGS6 in VTA DA neuron G protein-dependent signaling. We also report that both male and female constitutive RGS6–/– mice display decreased binge alcohol consumption, but that only female VTA DA neuron specific RGS6–/– display decreased binge alcohol consumption. Together these results suggest that RGS6 modulates VTA DA neuron G protein-dependent inhibition in a receptor-dependent manner, resulting in a sex-dependent influence on alcohol consumption. Most humans using drugs of abuse have co-occurring substance use patterns, so in addition to assessing independent drug mechanisms it is also important to assess how drug-related behavior patterns change during drug co-consumption. As alcohol and nicotine are the most frequently co-used drugs of abuse, the second goal of this work was to assess how abstinence from either nicotine or alcohol, after weeks of concurrent consumption, affects intake of the remaining drug. We did this by utilizing a 3-bottle choice model of concurrent alcohol, nicotine, and water consumption. When we removed the nicotine bottle after 3 weeks of concurrent consumption, we did not observe a change in levels of alcohol consumed, suggesting that mice do not compensate for the absence of nicotine by increasing alcohol consumption. When we instead added the aversive tastant quinine to the alcohol bottle, we saw an acute decrease in alcohol consumption in addition to an increase in the levels of nicotine consumed, suggesting that mice compensate for the absence of alcohol by increasing nicotine consumption. Further, chronically increasing quinine concentration in the alcohol bottle resulted in enhanced nicotine compensation in females but not in males. These results have important implications for treating patients with substance co-use disorders, as they suggest the order of drug abstinence may affect overall treatment outcomes. Collectively, work presented in this thesis provides novel insights about how drug-related behavioral outcomes are affected by drug mechanism, underlying inhibitory architecture, sex of the subject, and drug availability. These insights highlight the importance of gaining a greater understanding of both individual and co-substance use disorders in order to inform patient-specific treatment strategies.