The orexin receptor 1 (OX₁R) in the rostral medullary raphe contributes to the hypercapnic chemoreflex in wakefulness, during the active period of the diurnal cycle

Abstract

It has been shown that orexin plays an important role in the hypercapnic chemoreflex during wakefulness, and OX₁Rs in the retrotrapezoid nucleus (RTN) participate in this mechanism. We hypothesized that OX₁R in the rostral medullary raphe (MR) also contributes to the hypercapnic chemoreflex. We studied the effects on ventilation in air and in 7% CO₂ of focal antagonism of OX₁R in the rostral MR by microdialysis of SB-334867 in rats during wakefulness and NREM sleep, under dark and light periods. During wakefulness in the dark period, but not in the light period, SB-334867 caused a 16% reduction of the hyperventilation induced by 7% CO₂ compared with vehicle. There was no significant effect in sleep. The basal ventilation, body temperature and ${\dot{V}}_{{\text{O}}_2}$ were not affected. No effect was observed in a separate group of animals which had the microdialysis probe misplaced (peri-raphe). We conclude that OX₁R in the rostral medullary raphe contribute to the hypercapnic chemoreflex in wakefulness, during the dark period in rats.

Introduction

The orexin-containing neurons have been linked to the control of breathing, and there is a strong suggestion that these neurons are critical in the coordination between breathing and arousal states (Nakamura et al., 2007, Williams and Burdakov, 2008). The orexins, also known as hypocretins, include 2 subtypes of neuropeptides, the orexin-A and orexin-B (hypocretin-1 and hypocretin-2, respectively), which are derived from the same precursor (prepro-orexin), and bind to two G-protein coupled receptors: orexin receptor-1 (OX₁R), selective for orexin-A, and orexin receptor-2 (OX₂R), non-selective for both orexin-A and -B (de Lecea et al., 1998, Sakurai et al., 1998). The expression of orexin-containing neurons is restricted to the dorsal and lateral hypothalamus, but they have widespread projections to the whole brain (Peyron et al., 1998, Nambu et al., 1999), which explains the multiplicity of functions that are modulated by orexin, such as control of energy homeostasis, feeding behavior, reward processes, sleep–wake states, stress response, nociception, cardiovascular and respiratory control. Some evidence, indeed, supports the role of orexin on the control of breathing. Prepo-orexin knockout mice have a 50% attenuation of the hypercapnic chemoreflex during wakefulness, and this effect is partially restored with the administration of orexin-A and orexin-B (Deng et al., 2007, Nakamura et al., 2007). This is consistent with recent findings showing that orexin neurons are activated by pH and CO₂ in vitro and in vivo (Williams et al., 2007, Sunanaga et al., 2009). Moreover, the intracerebroventricular injection of an OX₁R-selective antagonist (SB-334867) decreased the respiratory chemoreflex by 24% in mice (Deng et al., 2007), and recently we have showed that the antagonism of OX₁R in the RTN caused a 30% reduction of the ventilatory response to CO₂ during wakefulness, and a 9% reduction during NREM sleep in rats (Dias et al., 2009).

Axonal processes immunoreactive to orexin have been demonstrated within the brainstem, including the medullary raphe (Nambu et al., 1999, Nixon and Smale, 2007). Similarly, OX₁R expression was found in the medullary raphe nuclei (Hervieu et al., 2001, Marcus et al., 2001). Moreover, the administration of orexin in the fourth ventricle induces c-Fos expression in the raphe pallidus (Zheng et al., 2005). It is unknown, however, whether the OX₁R in the medullary raphe contribute to hypercapnic chemoreflex.

We therefore investigate if OX₁R in the rostral medullary raphe are involved with hypercapnic chemoreflex. We also asked if their role varies whether the animal is in the dark-active period or in the light-inactive period of the diurnal cycle. This question was due to the fact that there is a diurnal variation of the orexin-A levels in rat cerebrospinal fluid with higher values at the dark period, compared with the light period (Desarnaud et al., 2004). Thus, the role of orexin on control of breathing may be related to the diurnal cycle.

To address our questions, we submitted adult freely moving rats to focal microdialysis of vehicle and then SB-334867 (OX₁R antagonist) into the rostral medullary raphe to focally inhibit OX₁R, and studied the effects of both treatments on breathing in room air and in 7% CO₂ during wakefulness and NREM sleep, in the dark and light periods of the diurnal cycle.