Multimodal characterization of transcriptionally defined ventral tegmental area dopamine neurons

Summary Ventral tegmental area (VTA) dopamine (DA) neurons are highly implicated in reward learning, motivated behaviors, and substance use disorders. DA neurons in the VTA are traditionally characterized by expression of genes involved in DA synthesis, release, or reuptake, such as tyrosine hydroxylase (encoded by the Th gene), which is the rate-limiting step in DA synthesis. However, recent transcriptomic studies have revealed substantial cellular heterogeneity within the VTA, including multiple subtypes of VTA DA neurons. Using single nucleus RNA sequencing, we previously identified two transcriptionally distinct Th + subpopulations: a DA/glutamate/GABA Combinatorial neuron marked by Slc26a7 and a DA-only neuron marked by Gch1 . However, the functional properties of these distinct DA neuron classes remain unknown. Here, we developed an AAV-based strategy enabling cell-type-specific access to these populations and performed comparative transcriptional, electrophysiological, and anatomical analyses, providing the first functional characterization of these transcriptionally-defined DA neuron subtypes. Whole-cell recordings revealed similar baseline membrane properties but a divergence in intrinsic excitability and latency to fire action potentials after current input. Anatomical mapping revealed overlapping but biased projection patterns, and Combinatorial neurons, but not DA-only neurons, were selectively recruited following experience with cocaine. Together, these findings reveal functional specialization among transcriptionally-defined VTA DA neuron subpopulations, dissociating DA-specific from multi-neurotransmitter properties and refining our understanding of VTA heterogeneity.