Distributions of hypothalamic neuron populations coexpressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse

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 EGFPVgat-fluorescence, respectively. EGFP(Vglut2) neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFPVgat 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 EGFPVgat 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 EGFPVgat 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 EGFPVgat. Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine beta-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.