Abstract
In the lateral hypothalamic area (LHA), some neurons, express the neuropeptide neurotensin (Nts) and regulate feeding, drinking and physical activity. Many LHA Nts neurons also contain the inhibitory neurotransmitter GABA, but some modulate glutamate (glut) signaling. This suggests that there are different subtypes of LHA Nts neurons. LHA Nts neuronal terminals have been seen within both the Ventra Tegmental Area (VTA) and Substantia Nigra Compacta (SNc) implying that subpopulations of LHA Nts neurons have distinct midbrain projection targets and mechanisms of action. Furthermore, some LHA Nts neurons co‐express the long form of the leptin receptor (LepRb) and are activated by the anorectic hormone leptin (NtsLepRb neurons) while others are activated by dehydration (NtsDehy neurons). I examined the hypothesis that NtsLepRb and NtsDehy neurons are distinguishable from each other based on their expression of GABA or glut and whether they project preferentially to the VTA or SNc. Until now, the lack of reagents to simultaneously identify Nts, GABA and glutamate prevented this analysis. To overcome this, we designed a dual genetic recombinase approach in order to simultaneously label Nts and GABA or glutamate‐containing neurons. We first generated mice that express FlpO recombinase specifically in Nts neurons (NtsFlpO mice) which we crossed with mice that express Cre recombinase in GABA or glutamate neurons. The resulting offspring expressed FlpO recombinase in Nts neurons and Cre in GABA/Glutamate Neurons; NtsFlpO/vGatCre or NtsFlpO/vGlutCre mice respectively. I injected vectors for FlpO‐inducible green fluorescent protein (GFP) and Cre‐inducible red fluorescent protein (RFP) in the LHA these mice with causes GFP expression only within LHA Nts neurons and RFP expression only in GABA/Glut neurons enabling the simultaneous detection of Nts and GABA and/or glutamate containing neurons via fluorescent microscopy. To examine the midbrain projections of each subtype of LHA Nts neurons, Fluorogold (FG) retrograde tract tracer was injected into the VTA or SNc of mice that express GFP only in Nts neurons (NtsGFP mice) and followed with leptin, or dehydration treatment. Brains were analyzed via immunostaining to label NtsLepRb, or NtsDehy neurons and FG. Our data shows that many LHA Nts neurons contain GABA and some express glutamate. Additionally, both LHA NtsLepRb neurons project to the VTA to the SNc but LHA NtsDehy neurons do not project to either midbrain region. The neurotransmitter content of these cue‐distinct subpopulations will be determined using Leptin and Dehydration treatment too.Support or Funding InformationThis work is supported by NIH awards to JAB (1F31 DK107081‐01A1, 5 T32 ES007255‐27) and GML (RC105025)
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