Distributions of hypothalamic neuron populations co-expressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse

The hypothalamus, which includes the zona incerta, contains a discrete population of catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers may coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons may also express glutamate or GABA. We used Cre/loxP recombination to express enhanced GFP fluorescence in neurons that express the vesicular glutamate (vGLUT2) or GABA transporter (vGAT) and determined TH immunoreactivity in glutamatergic or GABAergic neurons, respectively. EGFP-labeled vGLUT2 neurons were not TH-ir. However, there were discrete TH-ir signals that colocalized with EGFP-positive vGAT neurons, which we validated by in situ hybridization for Vgat mRNA. In order to contextualize the observed pattern of TH+EGFP colocalization in vGAT neurons, we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir vGAT neurons onto atlas templates from the Allen Reference Atlas of the mouse brain. TH-ir vGAT neurons were distributed throughout the rostrocaudal extent of the hypothalamus and were localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a very strong incidence of EGFP fluorescence in TH-ir neurons across all brain regions where it was observed, but the most striking colocalization was found in the zona incerta (ZI), where every TH-ir neuron was EGFP-positive. Neurochemical characterization of these ZI revealed that they display immunoreactivity for dopamine but not dopamine β-hydroxylase. In aggregate, these findings indicate the existence of a novel hypothalamic population that may signal through the release of GABA and/or dopamine.