Orexin Facilitates the Hypoxic Ventilatory Response: Influence of Estrus Cycle

Abstract

Orexin neurons project to nuclei participating in the hypoxic ventilatory response (HVR), including to the nucleus tractus solitarius (nTS) and paraventricular nucleus of the hypothalamus (PVN), nuclei that also express orexin receptors (OxRs). Many nTS‐projecting PVN neurons that are activated by acute hypoxia (Hx) are also immunoreactive (IR) for corticotropin‐releasing hormone (CRH). Orexin neurons are active in the dark (active) phase and silent in the light (inactive) phase. Orexin contributes to the HVR in males, especially in the dark phase. The possibility that orexin neurons facilitate the HVR through pathways that involve the PVN and/or nTS has not been investigated. Whether orexin influences the HVR in females is also unknown. This is a relevant issue because recent data suggest that orexin neurons are potently inhibited by estrogen. Here we hypothesized that orexin: 1) facilitates the HVR in females, especially during diestrus (i.e., low estrogen) in the dark phase, and 2) contributes to the HVR by facilitating the activation of CRH neurons in the PVN and/or neurons in the nTS. We tested these hypotheses using adult female and male Sprague Dawley rats (age 3‐4 months) and whole‐body plethysmography to measure ventilation (VE) during normoxia and hypoxia. Daily vaginal smears were performed to determine the stage of estrus cycle. Female rats (n=4) were exposed to graded hypoxia (FIO2from 0.21 to 0.09) over 3‐5 minutes to avoid the effects of hypoxia on body temperature and metabolic rate. Each rat was tested after vehicle and again following suvorexant (a dual OxR antagonist; 20 mg/kg, i.p.). Each rat was tested during proestrus/estrus and again in diestrus. The same rats were tested in the dark phase and then approximately 2 weeks later in the light phase. We measured the effects OxR blockade on VE during normoxia and at FIO2=0.15, 0.13, 0.11 and 0.09 (at least five breaths at each level), as well as on metabolic CO2 production (VCO2) in normoxia. Male rats were exposed to hypoxia (FIO2=0.11; n=4) or normoxia (n=4) for 2 hrs in the dark phase, after either suvorexant or vehicle, in order to induce c‐Fos expression. Immunofluorescence was performed to quantify the activation (i.e., Fos‐IR) of the PVN (particularly CRH neurons) and nTS neurons. During estrus, the HVR (determined by the increase in VE/VCO2) was slightly reduced following OxR blockade in the dark phase (drug: p=0.022). The inhibitory effect of OxR blockade on the HVR in the dark phase was much stronger during diestrus (by ~50% at FIO2=0.11; O2 level x drug: p=0.006). In the light phase there was no significant effect of OxR blockade on the HVR, regardless of estrus cycle stage. In male rats, OxR blockade significantly reduced the number of activated CRH PVN neurons (by ~50%; drug: p=0.012), and nTS neurons (by ~50%; drug x O2 level: p=0.027). Thus, orexin contributes to the HVR in females, especially during diestrus in the dark phase, when orexin neurons presumably have their highest activity. Orexin may facilitate the peripheral chemoreflex via pathway(s) that involve both the nTS and CRH neurons in the PVN.