Single-Cell Profiling Identifies Reward Behavior-Related Neurons and Alterations in the Ventral Tegmental Area Based on Arvcf-Knockout Mouse Model

The ventral tegmental area (VTA) is a crucial brain region for dopamine synthesis and reward processing, yet the molecular diversity and functional roles of nondopaminergic VTA neurons remain poorly characterized. While ARVCF (a member of ARVCF delta catenin family) has been implicated in dopamine release and reward-related behaviors (e.g., nicotine/alcohol and natural rewards), its influence on VTA neuronal subpopulations at single-cell resolution is unknown. Based on the Arvcf-knockout (Arvcf-KO) mouse model and nicotine exposure, we constructed a reward behavior-related profile at the single-cell transcriptome level and explored the cell subpopulations associated with reward behavior in VTA, as well as how these populations communicate with dopaminergic neurons. Following single-nucleus RNA Sequencing (snRNA-seq) from VTA, we obtained 96,240 cells of wild-type and Arvcf-KO mice with and without nicotine treatment. Subsequent cell-type abundance analysis revealed a significant reduction in the abundance of neuronal populations upon Arvcf-KO (FDR < 0.05). By integrating single-cell transcriptomics, neurology proteomics, and multiplex immunofluorescence imaging, we discovered a subpopulation of glutamatergic-dopaminergic combinatorial neurons, which is significantly associated with reward. Further cell communication analysis indicated that Arvcf-KO reduced the signal output from these neurons to dopaminergic neurons, represented by Wnt signaling. Finally, integrative analyses of metabolite detection and 2-way analysis of variance based on snRNA-seq indicated that the glutamatergic properties represent a key regulator of reward learning behaviors induced by nicotine and related stimuli. Taken together, our single-cell analysis identified that VTA combinatorial neurons are crucial for nicotine-induced reward through cellular signaling and glutamatergic properties, implying potential new therapeutic targets for addiction treatment.