Molecular and Functional Characterization of Transcriptionally Defined Subpopulations of VTA Dopamine Neurons - PROJECT SUMMARY Substance use disorder is a complex neurobiological disease characterized by a loss of control over drug-taking and drug-seeking behaviors. Further exacerbated by the COVID-19 pandemic, the rate of drug-related overdoses and subsequent deaths has increased dramatically over the past decade, surpassing 107,000 in 2022. All drugs of abuse increase dopamine (DA) transmission within the nucleus accumbens (NAc) from DA projections from the ventral tegmental area (VTA). While the presence of tyrosine hydroxylase (Th) has long been used to identify DA neurons, more recent studies have revealed remarkable heterogeneity among VTA DA neurons, with some neurons co-expressing markers for both DA and glutamate (Glut) transmission that are similarly Th+. However, the role of DA-only compared to combinatorial cells in substance use disorder is currently unknown. Using single nucleus RNA sequencing to comprehensively profile the VTA, we previously identified unique markers for these two subpopulations of DA neurons. Slc26a7, a gene that encodes an anion transporter, serves as a selective marker for combinatorial neurons that harbor expression of genes implicated in both Glut and DA synthesis and neurotransmission. Likewise, the GTP cyclohydrolase Gch1 was identified as a marker for DA-only neurons. Using a fluorescent in situ hybridization protocol, I validated these findings showing the Slc26a7 marks DA+/Glut+ cells while Gch1 marks DA+/Glut- cells. I have shown unique induction of the neuronal activity marker Fos in Slc26a7+ cells in the VTA 1 hour following cocaine, but not fentanyl, experience; this same response was not observed in Gch1+ cells, suggesting a difference in response to cocaine between these two distinct DA neuron populations. These results suggest that two subpopulations of DAergic cells in the VTA respond to cocaine in unique ways and may in turn drive distinct downstream effects and behavioral responses to cocaine. Following these findings, I hypothesize that differences in cellular targets and neurophysiology confer distinct behavioral roles of DA subpopulations. Using these selective markers, I have designed and generated novel adeno-associated viruses (AAVs) to both express distinct fluorophores and manipulate the neurons in a cell-type specific way. Using these AAVs, this project aims to take a multidisciplinary approach to rigourously investigate and determine any differences in cell types through the following aims: (1) Characterize anatomical and cellular localization of projections, (2) Determine neurophysiological differences, and (3) Determine the role of combinatorial cells in behavior. The proposed studies will deepen our understanding of the role of these combinatorial cells in SUD, providing avenues for therapeutic exploration for a disease largely lacking treatment options. Under this award, I will master behavioral paradigms and electrophysiology, techniques that will aid my success as a physician-scientist.
