Modulatory Effects And The Underlying Mechanisms Of Orexin And Histamine On Rat Hypoglossal Motoneurons

[BACKGROUD] Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common disease, characterized by repetitive collapse of the upper airway and chronic intermittent hypoxia (CIH). CIH destroys the normal sleep rhythm and affects many basic physiological processes, leading to repeated arousal from sleep. Pathogenesis of OSAHS is linked to suppression of genioglossal muscle tone in sleep, especially during REM sleep. Genioglossal is the largest upper airway dilator muscle innervated by the medial branch of the hypoglossal nerve, which is controlled by hypoglossal motoneurons (HMs) in hypoglossal motor nucleus (HMN). HMs are influenced by a variety of neuromodulators, some of which are fully studied and believed to alter HMs excitability across sleep-wake states, but others are poorly understood and thus pharmacological strategies designed to treat OSAHS have been largely unsucessful. Orexin and histamine are two important regulatory factors of sleep, which may also play important roles in OSAHSOrexin, mostly cleaved from propro-orexin (PPO) in the lateral hypothalamic area, as endogenous peptide ligand for two orphan G-protein-coupled receptor (OX1R and OX2R), functions in many physiological processes, such as feeding behavior, energy homeostasis, stress response, regulation of cardiovascular system and so on. And orexin has been shown to play a significant role in the promotion and maintenance of arousal. Orexin neurons are mainly located in lateral hypothalamus, however, the axons and terminals containing orexin A distribute widely throughout the brain. Hypothalamic orexin neurons also project to HMN, and both OX1R and OX2R have been identified in the HMN. Our earlier study found chronic intermittent hypoxia significantly upregulated OX1R and OX2R in the medulla. So it is reasonable that orexin A involves in modulating the activition of HMs and plays a key role in the pathogenesis of OSA.Histamine in the central nervous system also functions in many pathophysiological and physiological processes, including the sleep-waking cycle, energy and endocrine homeostasis and motor control. Histaminergic neurons, which are restrictively located in the tuberomammillary nucleus in the posterior hypothalamus, project widely in the brain and also project to HMN. An early report has found HMN expresses abundant H1 receptors (H1R). Using in vivo EMG recording, Horner et al found that microdialysis delivery histamine into HMN significantly increased GG activity. But the role of histamine on signal hypoglossal motoneuron and the underlying mechanism remain unknow.[OBJECTIVES] It is important to verify the effect of CIH on the expression of orexin in the brain and the role of orexin A and histamine in modulating HMs excitability. On one hand, this study would help us understand the real pathogenesis of OSAHS. On the other hand, it might help in the development of pharmacological approaches for the treatment of OSAHS. In the present study, we have focused on the possible role of orexin A and histamine in regulating HMs excitability by self-made CIH animal cabin and performing whole-cell patch clamp recording in brainstem slices. (1) To evaluate the influence of CIH on the expression of PPO in the hypothalamus and orexin receptors in the hypothalamus and the medulla. (2) And then to evaluate the effect and receptor mechanisms of orexin A on HMs under current-clamp. (3) To evaluate the effect of histamine and the underlying mechanisms on HMs, including receptors mechanisms, signal pathway and ionic mechanisms, were studied in the second part under current-clamp and voltage-clamp.[METHODS] 1. Forty rats were randomly divided into five groups of eight rats each. Two groups were exposed to intermittent air (IA), served as controls. Two more groups were exposed to intermittent hypoxia (IH). The last group was exposed to IH for 5 weeks followed by re-oxygenation (ROX) for 5 weks. Groups IH and LA underwenttreatment 8 hours daily for 1 week or 5 weeks,7 or 35 consecutive days. IA and IH animals were placed in identical intermittent hypoxia chambers of our own design filled with nitrogen and oxygen or with air. Oxygen concentration in the chamber flctuated between 6-21% in the IH cycle and was stable at about 21% in the IA cycle. Every cycle was 1 minute. Expression of PPO mRNA in the hypothalamus and orexin receptors mRNA in the hypothalamus and the medulla was detected by real-time PCR (RT-PCR).2. HMs membrane potential and firing were reorded from neonatal rats (P6-10) brain slices using whole-cell patch clamp after an infusion of orexin A or OXR antagonists.3. The effects of histamine on HMs and the underlying mechanisms including recoptors mechanisms, signal pathway and ionic mechanisms were studied under curent-clamp and voltage-clamp using neonatal rats (P10-16) brain slices.[RESULTS] 1. CIH upregulated PPO mRNA levels in the hypothalamusRats exposed to one week of IH showed less PPO mRNA expression in the hypothalamus than IA rats (relative OD 0.49±0.11 vs 1.00±0.15, respectively; P<0.05). Unlike 1 weeks of IH,5 weeks of IH was associated with PPO mRNA levels 2.10-fold higher than in control (P<0.05). Eight CIH rats from the ROX group were exposed to air for 5 weks of re-oxygenation, the level of expression of PPO mRNA showed no significant difference from the 5-week IA group (relative OD 1.32±0.09 vs 1.00±0.12, respectively; P>0.05). However, significant differences were observed between the 5-week re-oxygenation group and the 5-week IH group.2.CIH increases orexin receptor expression in the hypothalamus and medullaTwo regions of the brain were evaluated. The first was the hypothalamus because it is the most important area related to arousal, and the second was the medulla, which is linked to the development of OSA. RT-PCR showed that 1 week of IH affected OX1R expression in neither the hypothalamus nor the medulla. Rather, it upregulated OX2R expression in the medulla, relative to IA controls (P<0.05). When the duration of exposure to IH extended to 5 weeks, both OX1R and OX2R mRNA were found to have been upregulated significantly in both regions. Average OX2R mRNA in the medulla increased to 4.5-fold control levels and average OX1R mRNA increasd to 2.41-fold control levels. After 5 weeks of ROX, the upregulation of OX1R and OX2R mRNA was completely reversed in the hypothalamus (P>0.05), and the rats showed levels similar to those of ROX rats subjected to 5 weeks of IA. However, the expression of OX2R mRNA in the ROX group was still higher in the medulla than control group (P<0.05) but significantly lower than the CIH group (P<0.05).3. Orexin A increased the firing rate and decreased the membrane potential of the HMs.The recorded HMs exhibited a concentration-dependent and reversible excitatory response to orexin A at 4,20,100 and 500 nM, with an increase in the peak firing rate by 108.75±0.42%(P>0.05, n=7),148.45±6.01%(P<0.05, n=13),203.95±9.18% (P<0.05, n=16), and 310.91±18.19%(P<0.05, n=9), and decreased membrane potential at 1.00±0.13 mV (n=7),3.98±0.46 mV (n=13),7.27±0.72 mV (n=16) and16.72±1.49 mV (n=9), respectively.4. OXR antagonists blocked orexin A-induced increases in firing rate and depolarization of HMsIn the presence of TTX (1 μM), orexin A still depolarized the HMs (n=6), suggesting that orexin A-induced depolarization in HMs was a direct postsynaptic effect. In the presence of SB 334867, an OX1R specitic antagonist, the peak firing rate increased by orexin A (100 nM) was lower than that induced by the neuropeptide alone (174.08±4.72% vs.203.95±9.18%, P<0.05, n=7), and the orexin A-induced depolarization was also blocked. The same phenomenon was seen in the presence of TCS OX229, an OX2R specitic antagonist. Only in the presence of both SB 334867 and TCS OX229, orexin A failed to excite HMs (n=6). These results indicat that orexin A modulates the excitability of HMs via both OX1R and OX2R.5. Histamine depolarized HMs via the activation of an inward current, and exhibit desensitizationCurrent-clamp recording showed that histamine depolarized HMs in a dose-dependent manner, with elevated membrane potential 1.13±0.09 mV (n=9)、2.96 ±0.24mV (n=10),7.26±0.33 mV (n=13),12.29±1.10 mV (n=9) at concentration of 3 μM,10 μM,30 μM and 100 μM, respectively. The depolarization evoked by the second application of 30 μM histamine was significantly small than the former (3.17 ±0.43 mV vs.7.60±0.35 mV, P<0.05, n=9), indicating that histamine-activated response had desensitization. In voltage-clamp experiments, histamine elicited stable inward whole-cell currents on the HMs, suggesting that histamine depolarized HMs via the activation of an inward current.6. H1 receptor mediates the histamine-induced postsynaptic excitation on HMsWhen perfusing the slices with ACSF containing 0.5 μM TTX,30 μM histamine still evoked a strong depolarization on the HMs. TTX, coupled with bicuculline, APV and CNQX couldn’t block the inward current induced by histamine. These data suggested a marked postsynaptic excitatory effect induced by histimine. In ACSF with pyrilamine (30 μM), a highly selective histamine H1 receptor antagonist,30 μM histamine failed to depolarize the motoneurons. However, in ACSF with cimetidine, a highly selective histamine H2 receptor antagonist,30 μM histamine excited 7 motoneurons (7.10±0.22 mV, n=7 vs.7.26±0.33 mV, P>0.05, compared with histamine alone).2-PyEA (10-100 μm), a highly selective histamine H1 receptor agonist, mimicked the excitatory effect of histamine on HMs in a concentration-dependent manner. But dimaprit, a highly selective histamine H2 receptor agonist, had no effect on HMs at 10,30 or 100 μM. R-a-methylhistamine, a selective histamine H3 receptor agonist, also had no effect on HMs. All these results strongly suggested that only histamine H1 receptors mediate the histamine-induced excitatory effect on single HM.7. The signal pathway underlying excitatory action of histamine on HMs.In the presence of a PKA inhibitor H-89 (10μM) or a PKC inhibitor chelerythrine (10 μM),2-PyEA (30 μM) still elicited a stable inward current of HMs (P>0.05, compared with 2-PyEA alone). However, application of PLC-IP3 inhibitor U-73122(10 μM) completely blocked the excitatory effects of histamine on HMs. These data indicated that the pathway underlying excitatory action of histamine on HMs was H1R/Gq/11/PLC/IP3/Ca2+.8. The inward current produced by histamine and 2-PyEA is possibly mediated by membrane exchange of Na+and Ca2+Ba2+(4 mM BaCl2), a broad spectrum blocker of K+channels, or ZD 7288 (50 μM), the blocker of HCN, could not block the effect of 2-PyEA (30 μM). However, in the Tris-ACSF, the inward current induced by 2-PyEA (30μM) was smaller than 2-PyEA (30 μM) in the normal ACSF. And in the no Ca2+ pipette solution,2-PyEA (30 μM) induced inward current was 36.93±5.49 pA(n=6), also smaller than 2-PyEA induced in the normal pipette solution. These data suggested that the effect of histamine and 2-PyEA depended extracellular Na+ and intracellular Ca2+, might via activing Na+/Ca2+exchanger(NCX).[CONCLUSION] Considering the fact that th expression of orexin and its receptors were here found to be upregulated in the hypothalamus and medulla of CIH rats and this upregulation was reversed by re-oxygenation, and orexin A regulated the excitability of HMs via both OX1R and OX2R, we came to the conclusion that orexin may be one of the important factors in sleep fragmentation and may play an important role in the pathophysiology of OSAHS. Histamine, coupled to H1R, transmits the signal via Gq/11/PLC/IP3/Ca2+ andactivates Na+/Ca2+ exchanger, induces an inward current and depolarizes HMs. We hoped our work would establish the initial basis for the further study of the role of the orexin and histamine system in the pathophysiology and clinical application of OSAHS.