Determining composition and distribution of nicotinic acetylcholine receptors in mouse ventral tegmental area brain region with single-cell sequencing approach.

Introduction: Nicotine affects on the central and peripheral neuron systems by interacting with nicotinic acetylcholine receptors (nAChRs). However, the distribution and composition of these nAChR subunits at single-cell level within ventral tegmental area (VTA) remain unclear.

Methods: Single-cell nuclear suspension was prepared and sequenced from the VTA of nicotine-treated and saline-control C57BL/6 mice. Sequencing data were subjected to quality control and followed by cell type determination. The combination probability of nAChR subunits and acetylcholine metabolism products were estimated with Metacell and single cell Flux Estimation Analysis (scFEA), respectively. The relationships between gene modules and nAChRs were established using high-dimensional weighted gene co-expression network analysis (hdWGCNA).

Results: Sequencing of 53,855 VTA cells from six mice identified eight cell types, with the strongest communication observed between neurons and other cell types. Neuron subtypes included dopamine (DA), gamma-aminobutyric acid (GABA), glutamate (GLU), and GABAergic neurons with glutamate characteristics (GABA+GLU). The co-expression probability of nAChR subunits were determined for each neuron type. scFEA revealed nicotine treatment increased acetylcholine and DA metabolites but decreased phosphatidylcholine. hdWGCNA revealed five modules were significantly correlated with nicotine treatment, nAChR subunits, and DA neurons, indicating a potential role for nAChR subunits on DA neurons.

Conclusions: This study not only revealed differences in the expression of each nAChR subunit but also determined their composition and distribution at the single-neuron level within the VTA region.

Implications: This study not only determined which subunit gene is expressed in which type of neuron, but also predicted potential combinations of different nAChR subunits for different neurons. Such information is critical and useful for researchers to understand the role of each nAChR subunit at the neuronal level.