Molecular heterogeneity of midbrain dopaminergic neurons--Moving toward single cell resolution

Abstract

Since their discovery, midbrain dopamine (DA) neurons have been researched extensively, in part because of their diverse functions and involvement in various neuropsychiatric disorders. Over the last few decades, reports have emerged that midbrain DA neurons were not a homogeneous group, but that DA neurons located in distinct anatomical locations within the midbrain had distinctive properties in terms of physiology, function, and vulnerability. Accordingly, several studies focused on identifying heterogeneous gene expression across DA neuron clusters. Here we review the importance of understanding DA neuron heterogeneity at the molecular level, previous studies detailing heterogeneous gene expression in DA neurons, and finally recent work which brings together previous heterogeneous gene expression profiles in a coordinated manner, at single cell resolution.

Keywords: Dopamine neuron subtypes; Midbrain; Molecular diversity; Selective vulnerability; Single cell expression profiling.

Figure 1. Overview of the midbrain DA system

A, Schematic of the pathway of DA production and reuptake. B, Rostral and caudal coronal sections from the Allen Reference Atlas, depicting key DA neuron containing regions in the midbrain. C, Sagittal section, depicting the midbrain DA neuron clusters A8-10, and their key projections to the Amygdala (AMY), Caudate Putamen (CP), Accumbens (ACB) and cortex (CTX).

Figure 2. Differential expression of various genes across DA neuron subtypes at single cell resolution

Dissociated and FACS sorted midbrain cells from postnatal day 4 Slc6a3/Dat::Cre;Ai9 mice were analyzed for the expression of 96 genes using a microfluidic multiplexed qRT-PCR platform, with cycle threshold (Ct) values as output(84). Following an unbiased, hierarchical clustering analysis, 153 individual neurons were assigned to one of six DA neuron subtypes: 1A (red), 1B (orange), 2A (green), 2B (light blue), 2C (dark blue), 2D (purple). The expression profile of 15 key genes is illustrated across these subtypes, without normalization. The value for each cell (i.e. the y axis) is depicted as 20-Ct.

Figure 3. Single cell comparison of Sox6 and Otx2 within each subpopulation

Expression of Sox6 (purple) and Otx2 (orange), shown for each individual neuron within the six DA neuron subtypes. Relative expression values are depicted as 24-Ct. Sox6 is expressed in the vast majority of Cluster 1 cells (DA^{1A, B}) and to a lesser extent in Cluster 2 (most notably in subtype DA^2C). In contrast, Otx2 is mainly detected in Cluster 2 subtypes DA^2B and DA^2C. Occasional cells show expression of both these transcription factor mRNAs.