Hypothalamic Glutamatergic/Orexinergic Neurons Attenuate Fentanyl-Induced Respiratory Depression via Medullary Pre-BöTzinger Complex Pathways.

Opioid-induced respiratory depression, particularly from fentanyl, is a major contributor to overdose mortality. The hypothalamus plays a modulatory role in breathing, potentially via orexinergic and glutamatergic projections to medullary respiratory centers. Here, we investigated the role of hypothalamic neurons in counteracting fentanyl-induced respiratory depression (FIRD) in male and female mice. We found that orexin (ORX) and glutamatergic (VGlut2+) neurons are distributed in dorsal hypothalamic nuclei, mainly in the lateral hypothalamus (LH) and more sparsely in the paraventricular nucleus (PVN) and send projections to the pre-Bötzinger complex (preBötC) in the medulla. Hypercapnia activated ORX neurons in the LH, but not in the PVN. Pharmacological blockade of ORX signaling with a dual ORX receptor antagonist dose-dependently suppressed respiratory activity and exacerbated FIRD. Using chemogenetics and whole-body plethysmography, we found that activation of dorsal hypothalamic neurons projecting to the preBötC increased respiratory rate and attenuated FIRD. Activation of glutamatergic neurons in either the LH or PVN stimulated ventilation; however, this effect was independent of ORX signaling. Notably, activation of LH-glutamatergic neurons successfully reduced FIRD, whereas activation of PVN glutamatergic neurons failed to attenuate FIRD. Chemogenetic activation of LH neurons projecting to the preBötC increased respiratory rate in an ORX signaling-dependent manner and was sufficient to attenuate FIRD. In contrast, activation of PVN neurons projecting to the preBötC enhanced active movements/arousal of mice but had minimal effects on respiratory parameters. Therefore, these findings suggest that LH-ORX/glutamatergic projections to the medulla drive respiratory activity that can alleviate FIRD in mice of either sex.