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

Abstract

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.

Figure 1

Effects of unilateral micro-injection of orexin A (0.1 μl) into the hypoglossal nucleus on the GG-EMG in adult rats. (A) Location of the hypoglossal nucleus at bregma −13.68 mm in the atlas (left) and micro-injection trace in the hypoglossal nucleus (right) indicated by an arrow. (B) A typical example illustrating GG-EMG increased by orexin A micro-injection at 50 (n = 6), 100 (n = 5) and 200 μM (n = 5). Arrow indicates the time of orexin A injection. The traces of tonic and respiratory-related GG activities induced by different doses of orexin A are shown with smaller time window upon the compressed graphics. (C) Effect of orexin A on the tonic and respiratory-related GG-EMG. Values are means ± SEM. *P < 0.05, vs control. HN, hypoglossal nucleus; CC, central canal.

Figure 2

Orexin A increased the firing rate and decreased the membrane potential of HMNs in a concentration-dependent and reversible manner in neonatal rats. (A) Typical example illustrating the location of the hypoglossal nucleus (a) indicated by a dashed line. (b) Higher magnification of the square area in (a), arrow indicating a hypoglossal motoneuron. The recording electrode is shown entering from the right side of the field. (B) Typical examples illustrating repetitive firing of HMNs before (left), during (middle) and 15 min after (right) perfusion with orexin A at 4 (n = 7), 20 (n = 13), 100 (n = 16) and 500 nM (n = 9), respectively. The evoked depolarizing current steps of these 4 neurons were the same as 300 pA of 1000 ms in duration. (C) Time course of the change in the firing rate induced by orexin A at 4, 20, 100 and 500 nM. (D) Mean peak firing rate (% of control) induced by orexin A. (E) Typical examples illustrating orexin A decreased the membrane potential of HMNs at 4, 20, 100 and 500 nM. (F) Effect of orexin A on the membrane potential of HMNs. Values are means ± SEM. *P < 0.05; **P < 0.01, vs control. 4 V, the fourth ventricle.

Figure 3

Effects of OX receptor antagonists on orexin A-induced firing rate and depolarization of HMNs in neonatal rats. (A) Typical examples indicating that orexin A (100 nM) induced depolarization of HMN in normal ACSF (left), and this effect persisted in TTX-containing ACSF (right) in the same cell. (B) Time course of orexin A (100 nM)-induced firing rate in ACSF containing OX₁ receptor antagonist, SB 334867 (10 μM, n = 7) and OX₂ receptor antagonist, TCS OX2 29 (10 μM, n = 7) alone or together (n = 6). (C) Average peak firing rate (% of control) induced by orexin A in ACSF containing SB 334867 and TCS OX2 29 alone or together. (D) Typical examples illustrating that orexin A (100 nM)-induced depolarization was blocked by SB 334867 (up, n = 7), TCS OX2 29 (middle, n = 7), or together (down, n = 6). (E) Change of orexin A-induced depolarization after addition of SB 334867 (10 μM) and TCS OX2 29 (10 μM) alone or together. Values are means ± SEM. *P < 0.05; **P < 0.01, vs control.

Figure 4

Micro-injection of OX₁ receptor antagonist, SB 334867 (10 mM, 0.1 μl, n = 6) or OX₂ receptor antagonist, TCS OX2 29 (1 mM, 0.1 μl, n = 5) into the hypoglossal nucleus attenuated GG-EMG in adult rats. (A) Typical example illustrating that SB 334867 mainly decreased the respiratory-related GG-EMG compared with control (saline, 0.1 μl). (B) Changes in the tonic and respiratory-related GG-EMG following a micro-injection of SB 334867. (C) Typical example illustrating that TCS OX2 29 decreased both the tonic and the respiratory-related GG-EMG compared with the control (saline, 0.1 μl). (D) Changes in the tonic and respiratory-related GG-EMG following microinjection of TCS OX2 29. Values are means ± SEM. *P < 0.05, **P < 0.01, vs control.

Figure 5

Lesions of orexin neurons in the bilateral LH after orexin-SAP (400 nl per side, 0.43 mg ml⁻¹) micro-injection decreased the respiratory-related GG-EMG in adult rats. (A) Loss of Nissl bodies in the LH after orexin-SAP treatment. (a) Nissl staining showing cells in the saline-treated rats and (c) the cells in the orexin-SAP lesioned rats. Higher magnification of the square area in a (b) and in c (d). (B) Loss of orexin A-immunoreactive neurons in the LH after orexin-SAP treatment. (a) Anti-orexin A antibody immunohistochemistry staining showing cells indicated by an arrow in the saline-treated rats and (c) the cells in the orexin-SAP lesioned rats. Higher magnification of the square area in a (b) and in c (d). (C) Typical example illustrating orexin neurons lesions decreased the respiratory-related GG-EMG. (D) Changes in the tonic and respiratory-related GG-EMG after orexin-SAP treatment (n = 7) compared with the control (n = 6). Values are means ± SEM. **P < 0.01, vs control. 3 V, the third ventricle; orexin-SAP, orexin-saporin.