The dopaminergic (DA) neurons in the ventral tegmental area (VTA) of the middle brain play an important role in emotion-related behaviour, and the alteration of excitability of VTA DA neurons are believed to be the key determinants in behaviours of depression and drug addictions. The excitability of VTA DA neurons controls the release of DA in the projection fiber terminals thus controls the function of VTA DA neurons. After many years of hard work, we begin to understand how the excitability of VTA DA neurons is regulated, but these achievements are far from satisfactory. Furthermore, with recent progress and realization that property of VTA DA neurons, against the classical view that VTA DA neurons are homogeneous population, are distinctly different, thus the accumulated limited knowledge on the mechanism of excitability modulation of VTA DA neurons is especially short of expectation. The most outstanding indications of the heterogeneity of VTA DA neurons are represented by the distinctly different electrophysiological properties among VTA DA neurons projecting to different brain regions. However, the underlying mechanism is not clear. In this application, we plan to investigate the underlying mechanism determining the distinct excitability of VTA DA neurons projecting to the cortical and the limbic regions of the brain by single cell RNA sequencing (scRNA-seq). The main focus of the study will be on the intrinsic ion channels and the related modulation mechanism of VTA DA neuron excitability. Based on this, we will further study the mechanism which underlies the alteration of excitability of VTA DA neurons in the condition of a depression state, and still further the related depression behaviour.

After retrobeads injection, following brain slice preparation and recording, the recorded and retrobeads-labelled neurons were aspirated into the patch pipette and were then broken into the PCR tube containing 1 μl lysis buffer. For mRNA in individual cells, mRNA was amplified by SMARTer Ultra Low Input RNA for Illumina Kit, which was qualified and reversely transcribed to cDNA by Qubit and Agilent Bioanalyzer 2100 electrophoresis. After fragmentation of cDNA (300 bp) by ultrasound, sequencing libraries (end repair, addition of poly(A), and ligation of sequencing connectors) were built using the Ovation Ultralow Library System V2. After that, the constructed libraries were sequenced using Illumina HiseqXten.