Spatial Distribution of Neurons Expressing Single, Double, and Triple Molecular Characteristics of Glutamatergic, Dopaminergic, or GABAergic Neurons in the Mouse Ventral Tegmental Area

The ventral tegmental area (VTA) is a heterogeneous midbrain area that plays a significant role in diverse neural processes such as reward, aversion, and motivation. The VTA contains three main neuronal populations, namely, dopamine (DA), γ-aminobutyric acid (GABA), and glutamate neurons, but some neurons exhibit combinatorial molecular characteristics of dopaminergic, GABAergic, or glutamatergic neurons. However, little information is available regarding detailed distribution of neurons with single, double, and triple molecular characteristics of glutamatergic, dopaminergic, or GABAergic neurons in mice. We present a topographical distribution map of three main neuronal populations expressing a single molecular characteristic of dopaminergic, GABAergic, or glutamatergic neurons, and four neuronal populations co-expressing double or triple molecular characteristics in combinatorial manners, in the mouse VTA, following analysis of triple fluorescent in situ hybridization for the simultaneous detection of tyrosine hydroxylase (TH, marker for dopaminergic neurons), vesicular glutamate transporter 2 (VGLUT2, marker for glutamatergic neurons), and glutamic acid decarboxylase 2 (GAD2, marker for GABAergic neurons) mRNA. We found that the vast majority of neurons expressed a single type of mRNA, and these neurons were intermingled with neurons co-expressing double or triple combinations of VGLUT2, TH, or GAD2 in the VTA. These seven neuronal populations were differentially distributed in the VTA sub-nuclei across the rostro-caudal and latero-medial axes. This histochemical study will lead to a deeper understanding of the complexity of neuronal molecular characteristics in different VTA sub-nuclei, and potentially facilitate clarification of diverse functions of the VTA.