It has been suggested that TH-VGluT2 neurons corelease dopamine and glutamate, however, it is unclear whether these neurons have proteins necessary for the synthesis (aromatic L-amino acid decarboxylase, AADC) or transport (vesicular monoamine transporter, VMAT2 or dopamine transporter, DAT) of dopamine. By in situ hybridization-immunohistochemistry, we found that all TH neurons in the A10 region had AADC, but not all had VMAT2, DAT or D2 receptors (D2R). To determine whether TH-VGluT2 neurons account for TH neurons lacking these dopamine markers, we implemented an anatomical mirror technique, and found that not all TH-VGluT2 neurons lacked VMAT2, DAT or D2R. Next, by quantitative RT-PCR of individual micro-dissected TH neurons, we discovered 2 classes of TH-VGluT2 and 3 classes of TH-only neurons with different latero-medial distribution. Some of the TH-VGluT2 neurons had both VMAT2 and DAT (TH-VGluT2 Class 1) and others lacked both transporters (TH-VGluT2 Class 2). Most of the TH-only neurons contained VMAT2 and DAT (TH-only Class 1), a few had DAT without VMAT2 (TH-only Class 2), and others lacked both VMAT2 and DAT (TH-only Class 3). We concluded that (a) the majority of TH neurons lacking DAT are TH-VGluT2 neurons, (b) very few TH-only neurons express DAT without VMAT2, and (c) TH-VGluT2 neurons lacking DAT also lack VMAT2. Thus we provide evidence that the A10 region contains dopamine neurons with differential compartmentalization and unique signaling properties.
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