Distributions of hypothalamic neuron populations co-expressing 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 glutamate or GABA. We used Cre/loxP recombination to express enhanced GFP fluorescence (EGFP) in neurons that express the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined TH immunoreactivity in glutamatergic or GABAergic neurons, respectively. EGFP-positive vGLUT2 neurons were not TH-ir. However, discrete TH-ir signals 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 (ARA) of the mouse brain. TH-ir 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 very strong presence of EGFP fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in the zona incerta (ZI) – a region assigned to the hypothalamus in the ARA – where every TH-ir neuron was EGFP-positive. Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine β-hydroxylase. In aggregate, these findings indicate the existence of a novel hypothalamic population in the mouse that may signal through the release of GABA and/or dopamine.