The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFP^Vglut2 - or EGFP^Vgat -fluorescence, respectively. EGFP^Vglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFP^Vgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFP^Vgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFP^Vgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFP^Vgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFP^Vgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFP^Vgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine β-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.

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