Defining the caudal hypothalamic arcuate nucleus with a focus on anorexic excitatory neurons.

KEY POINTS: Excitatory glutamate neurons are sparse in the rostral hypothalamic arcuate nucleus (ARC), the subregion that has received the most attention in the past. In striking contrast, excitatory neurons are far more common (by a factor of 10) in the caudal ARC, an area which has received relatively little attention. These glutamate cells may play a negative role in energy balance and food intake. They can show an increase in phosphorylated Stat-3 in the presence of leptin, are electrically excited by the anorectic neuromodulator cholecystokinin, and inhibited by orexigenic neuromodulators neuropeptide Y, met-enkephalin, dynorphin and the catecholamine dopamine. The neurons project local axonal connections that excite other ARC neurons including proopiomelanocortin neurons that can play an important role in obesity. These data are consistent with models suggesting that the ARC glutamatergic neurons may play both a rapid and a slower role in acting as anorectic neurons in CNS control of food intake and energy homeostasis. ABSTRACT: Here we interrogate a unique class of excitatory neurons in the hypothalamic arcuate nucleus (ARC) that utilizes glutamate as a fast neurotransmitter using mice expressing GFP under control of the vesicular glutamate transporter 2 (vGluT2) promoter. These neurons show a unique distribution, synaptic characterization, cellular physiology and response to neuropeptides involved in energy homeostasis. Although apparently not previously appreciated, the caudal ARC showed a far greater density of vGluT2 cells than the rostral ARC, as seen in transgenic vGluT2-GFP mice and mRNA analysis. After food deprivation, leptin induced an increase in phosphorylated Stat-3 in vGluT2-positive neurons, indicating a response to hormonal cues of energy state. Based on whole-cell recording electrophysiology in brain slices, vGluT2 neurons were spontaneously active with a spike frequency around 2 Hz. vGluT2 cells were responsive to a number of neuropeptides related to energy homeostasis; they were excited by the anorectic peptide cholecystokinin, but inhibited by orexigenic neuropeptide Y, dynorphin and met-enkephalin, consistent with an anorexic role in energy homeostasis. Dopamine, associated with the hedonic aspect of enhancing food intake, inhibited vGluT2 neurons. Optogenetic excitation of vGluT2 cells evoked EPSCs in neighbouring neurons, indicating local synaptic excitation of other ARC neurons. Microdrop excitation of ARC glutamate cells in brain slices rapidly increased excitatory synaptic activity in anorexigenic proopiomelanocortin neurons. Together these data support the perspective that vGluT2 cells may be more prevalent in the ARC than previously appreciated, and play predominantly an anorectic role in energy metabolism.