Orexin A activates hypoglossal motoneurons and enhances genioglossus muscle activity in rats.

BJP British Journal of Pharmacology DOI:10.1111/bph.12784 www.brjpharmacol.org RESEARCH PAPER Correspondence Orexin A activates hypoglossal motoneurons and enhances genioglossus muscle activity in rats G H Zhang1,2*, Z L Liu3,4*, B J Zhang3, W Y Geng1, N N Song1, W Zhou1, Y X Cao1, S Q Li4, Z L Huang3 and L L Shen1 1 Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, Shanghai, China, 2Department of Physiology, Basic Medical College of Zhengzhou University, Zhengzhou, China, 3Department of Pharmacology, State Key Laboratory of Medical Neurobiology, Institute of Brain Science, Shanghai Medical College of Fudan University, Shanghai, China, and 4Department of Pulmonary Medicine, Center of Snoring and Sleep Apnea Medicine, Zhongshan Hospital of Fudan University, Shanghai, China Lin-Lin Shen, Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, Shanghai 200032, China. E-mail: ; or Zhi-Li Huang, Department of Pharmacology, State Key Laboratory of Medical Neurobiology, Institute of Brain Science, Shanghai Medical College of Fudan University, Shanghai 200032, China. E-mail: ---------------------------------------------------------------- *These authors contributed equally to this work. ---------------------------------------------------------------- Received 15 December 2013 Revised 28 April 2014 Accepted 1 May 2014 BACKGROUND AND PURPOSE Orexins have been demonstrated to play important roles in many physiological processes. However, it is not known how orexin A affects the activity of the hypoglossal motoneuron (HMN) and genioglossus (GG) muscle. EXPERIMENTAL APPROACH GG muscle electromyograms (GG-EMG) were recorded in anaesthetized adult rats after orexin A or orexin receptor antagonists were applied to the hypoglossal nucleus, and in adult rats in which orexin neurons were lesioned with the neurotoxin orexin-saporin (orexin-SAP). HMN membrane potential and firing were recorded from neonatal rat brain slices using whole-cell patch clamp after an infusion of orexin A or orexin receptor antagonists. KEY RESULTS Unilateral micro-injection of orexin A (50, 100 or 200 μM) into the hypoglossal nucleus significantly enhanced ipsilateral GG activity in adult rats. Orexin A (4, 20, 100 or 500 nM) depolarized the resting membrane potential and increased the firing rate of HMNs in a dose-dependent manner in the medullary slices of neonatal rats. Both SB 334867, a specific OX1 receptor antagonist and TCS OX2 29, a specific OX2 receptor antagonist not only blocked the depolarized membrane potential and the increased firing rate of HMNs by orexin A in the neonatal model but also attenuated GG-EMG in the adult model. A significant decrease in GG-EMG was observed in adult orexin neuron-lesioned rats compared with sham animals. CONCLUSION AND IMPLICATIONS Orexin A activates OX1 and OX2 receptors within the hypoglossal motor pool and promotes GG activity, indicating that orexin A is involved in controlling respiratory motor activity. © 2014 The British Pharmacological Society British Journal of Pharmacology (2014) 171 4233–4246 4233 BJP G H Zhang et al. Abbreviations AP, action potential; GG, genioglossus; GG-EMG, GG muscle electromyogram; HMN, hypoglossal motoneuron; LH, lateral hypothalamus; OSA, obstructive sleep apnoea; orexin-SAP, orexin-saporin; PF, perifornical; REM, rapid eye movement; XIIa, hypoglossal nerve activity Table of Links TARGETS LIGANDS OX1 receptor Orexin A OX2 receptor TCS OX2 29 This Table lists protein targets and ligands which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013). Introduction As peptide transmitters, orexins, including orexin A and B, have been demonstrated to play an important role in many physiological processes, such as feeding behaviour (Willie et al., 2001), energy homeostasis (Kukkonen et al., 2002), sleep/wake cycles (Saper et al., 2005), motivation (Harris and Aston-Jones, 2006), stress responses (Zhang et al., 2006), regulation of the cardiovascular system (Shirasaka et al., 2003) and breathing (Young et al., 2005). Orexin neurons are restricted to the lateral hypothalamus (LH). They project widely throughout the brain, with the exception of the cerebellum. Orexins act on two specific GPCRs, OX1 and OX2 receptors, in target cells (Elias et al., 1998; Peyron et al., 1998; Sakurai et al., 1998; Nambu et al., 1999). The genioglossus (GG) muscle, which is innervated by the medial branch of the hypoglossal nerve, is the largest upper airway dilator muscle. Contraction of the GG muscle promotes anterior tongue movement and oropharyngeal airway widening (Jordan and White, 2008), which prevents obstructive sleep apnoea (OSA) in humans (Remmers et al., 1978). There are two entirely opposite results reporting that plasma orexin A was lower (Nishijima et al., 2003) or higher (Igarashi et al., 2003) in patients with OSA, suggesting that dysfunction of orexin systems might be involved in the pathophysiology of OSA. Importantly, how orexin regulates GG muscle activity is still not clear. Some reports about the effect of orexins on upper airway patency in vivo have been published. Specifically, orexin B micro-injected into the rat pontine Kolliker-Fuse nucleus was reported to enhance the preinspiratory activity of the hypoglossal nerve (Dutschmann et al., 2007). Also orexin A micro-injected into the hypoglossal nucleus was reported to activate the GG muscle in decerebrate cats (Peever et al., 2003). However, the effect of orexin on the hypoglossal motoneuron (HMN), the key role of endogenous orexin in controlling GG activity and the receptor mechanism involved in this process, warrants further investigation. 4234 British Journal of Pharmacology (2014) 171 4233–4246 Thus, in the present study, a GG muscle electromyogram (GG-EMG) was recorded in anaesthetized adult rats before and after the application of orexin A or orexin receptor antagonists to the hypoglossal nucleus, and in adult rats whose orexin neurons were lesioned by the neurotoxin orexin-saporin (orexin-SAP; micro-injected into the bilateral LH 14 days before the recording). To elucidate the receptors involved, a whole-cell patch clamp was used to record the membrane potential and firing of the HMN in medullary slices of neonatal rats. Our findings indicate that endogenous orexin targeting the HMN enhanced GG activity via OX1 and OX2 receptors. Methods Animals Male Sprague-Dawley (SD) rats (250–350 g) and neonatal pups (P6-P10) were obtained from the Sino-British SIPPR/BK Laboratory Animal Limited Company (Shanghai, China). The animals were maintained at an ambient temperature of 22 ± 0.5°C, with a relative humidity of 60 ± 2%. A 12 h light/ dark cycle (lights on at 07:00 h, illumination intensity ≈ 1 (...truncated)