The dorsomedial hypothalamus (DMH) receives inputs from the preoptic area (POA), where ambient temperature mediates physiological adaptations of energy expenditure and food intake. Warm-activated POA neurons suppress energy expenditure via brown adipose tissue (BAT) projecting neurons in the dorsomedial hypothalamus/dorsal hypothalamic area (dDMH/DHA). Our earlier work identified leptin receptor (Lepr)-expressing, BAT-projecting dDMH/DHA neurons that mediate metabolic leptin effects. Yet, the neurotransmitter (glutamate or GABA) used by dDMH/DHAneurons remains unexplored and was investigated in this study using mice. We report that dDMH/DHAneurons represent equally glutamatergic and GABAergic neurons. Surprisingly, chemogenetic activation of glutamatergic and/or GABAergic dDMH/DHA neurons were capable to increase energy expenditure and locomotion, but neither reproduced the beneficial metabolic effects observed after chemogenetic activation of dDMH/DHAneurons. We clarify that BAT-projecting dDMH/DHA neurons that innervate the raphe pallidus (RPa) are exclusively glutamatergic Lepr neurons. In contrast, projections of GABAergic or dDMH/DHAneurons overlapped in the ventromedial arcuate nucleus (vmARC), suggesting distinct energy expenditure pathways. Brain slice patch clamp recordings further demonstrate a considerable proportion of leptin-inhibited dDMH/DHAneurons, while removal of pre-synaptic (indirect) effects with synaptic blocker increased the proportion of leptin-activated dDMH/DHAneurons, suggesting that pre-synaptic Lepr neurons inhibit dDMH/DHAneurons. We conclude that stimulation of BAT-related, GABA- and glutamatergic dDMH/DHAneurons in combination mediate the beneficial metabolic effects. Our data support the idea that dDMH/DHAneurons integrate upstream Lepr neurons (e.g., originating from POA and ARC). We speculate that these neurons manage dynamic adaptations to a variety of environmental changes including ambient temperature and energy state. SIGNIFICANCE STATEMENT: Our earlier work identified leptin receptor expressing neurons in the dDMH/DHA as an important thermoregulatory site. Dorsomedial hypothalamus (DMH) Lepr neurons participate in processing and integration of environmental exteroceptive signals like ambient temperature and circadian rhythm, as well as interoceptive signals including leptin and the gut hormone glucagon-like-peptide-1 (GLP1). The present work further characterizes dDMH/DHAneurons as a mixed glutamatergic and GABAergic population, but with distinct axonal projection sites. Surprisingly, select activation of glutamatergic and/or GABAergic populations are all able to increase energy expenditure, but are unable to replicate the beneficial metabolic effects observed by Lepr activation. These findings highlighting dDMH/DHA Lepr neurons as a distinct subgroup of glutamatergic and GABAergic neurons that are under indirect and direct influence of the interoceptive hormone leptin and if stimulated are uniquely capable to mediate beneficial metabolic effects. Our work significantly expands our knowledge of thermoregulatory circuits and puts a spotlight onto DMH-Lepr neurons for the integration into whole body energy and body weight homeostasis. Lepr Lepr Lepr Lepr Lepr Lepr Lepr Lepr Lepr Lepr
