Attenuated phrenic long-term facilitation in orexin neuron-ablated mice

https://doi.org/10.1016/j.resp.2009.07.025Get rights and content

Abstract

We examined phrenic long-term facilitation (LTF) in urethane-anesthetized, vagotomized, paralyzed, and artificially ventilated orexin neuron-ablated mice and their wild-type littermates. Effect of isocapnic single hypoxic episode (SHE, for 45 s) and intermittent hypoxia (IH, 5 times of SHE separated by 5 min) on phrenic nerve activity (PNA) was measured for 1–2 h. In wild-type mice, amplitude of PNA gradually increased after cessation of IH and reached 55 ± 15% above the baseline (n = 7, p < 0.05) whereas the burst rate of PNA did not change. Qualitatively similar but significantly attenuated response (16 ± 8%) was observed in orexin neuron-ablated mice. SHE did not affect amplitude nor frequency in both animals. We conclude that orexin contributes to eliciting phrenic LTF at least in part in mice. This study also showed, for the first time, phrenic LTF following IH in WT mice. Characteristics of phrenic and ventilatory LTF in mice were similar to those in rats.

Introduction

Respiratory long-term facilitation (LTF) is a long-lasting (>1 h) augmentation of respiratory motor output even after cessation of intermittent hypoxic stimuli (Millhorn et al., 1980a). LTF following intermittent hypoxia (IH) has been extensively studied by measuring phrenic nerve activity in anesthetized, paralyzed, vagotomized, and artificially ventilated rats and cats (Hayashi et al., 1993, Fregosi and Mitchell, 1994, Morris et al., 2000, Peng et al., 2003). LTF is a pattern sensitive phenomenon since it is not observed after sustained hypoxia (Baker and Mitchell, 2000). Although involvement of medullary raphe serotonergic neurons in LTF has been disclosed (Millhorn, 1986, Bach and Mitchell, 1996), how IH affect these neurons and the mechanism of pattern sensitivity remains largely unknown.

IH has been considered a model of hypoxia during sleep apnea. LTF may compensate for factors that predispose to sleep-disordered breathing, particularly during obstructive sleep apnea (OSA) by keeping patency of the respiratory tract (Mahamed and Mitchell, 2007). However, the relationships between LTF and OSA have not been reliably proved (Mahamed and Mitchell, 2007, Mateika and Narwani, 2009).

Orexin is a hypothalamic neuropeptide regulating sleep/wake states (Sakurai, 2007). Deficiency of orexin results in narcolepsy in human, dog, and mice (Chemelli et al., 1999, Lin et al., 1999, Thannickal et al., 2000). In addition to sleep/wake states, orexin also regulates food intake (Willie et al., 2001) and autonomic functions including respiration (see (Gestreau et al., 2008, Kuwaki, 2008, Williams and Burdakov, 2008) for review). Orexinergic neurons innervate respiratory nuclei such as preBötzinger complex, nucleus tractus solitarius, raphe pallidus, and phrenic and hypoglossal motoneurons (Peyron et al., 1998, Fung et al., 2001, Berthoud et al., 2005, Young et al., 2005, Rosin et al., 2006). The exogenous administration of orexin induces phrenic and hypoglossal activation (Young et al., 2005, Dutschmann et al., 2007) and hence increases ventilation (Zhang et al., 2005, Deng et al., 2007). Orexin deficient mice showed exaggerated sleep apnea (Nakamura et al., 2007) and lack of ventilatory LTF during both sleep and wake sates (Terada et al., 2008). Stress-induced ventilatory activation was also attenuated in orexin deficient mice (Kayaba et al., 2003, Zhang et al., 2006a). In addition, narcolepsy patients have a high incidence of sleep apnea (Chokroverty, 1986) and chronic IH sensitizes stress response (Ma et al., 2008).

Although these results indicated possible involvement of orexin in LTF and we actually observed lack of ventilatory LTF in orexin deficient mice, confirmation by observing phrenic LTF seemed to be indispensable because some characteristics of ventilatory LTF and phrenic LTF are not identical at least in rats. Ventilatory LTF is predominantly frequency dependent (Turner and Mitchell, 1997, Mitchell et al., 2001b, Olson et al., 2001, Kline et al., 2002, McGuire et al., 2002, Terada et al., 2008) whereas phrenic LTF is mainly amplitude dependent (Mitchell et al., 2001a). Magnitude of ventilatory LTF (20–25%) is much smaller than that of phrenic LTF (>50%) (Mitchell et al., 2001a, Feldman et al., 2003). Before measuring phrenic LTF in orexin deficient mice, we first tried to establish mice model of phrenic LTF because there is no such report. Once such model is established, it would be readily applicable to other mutant mice and thus facilitate molecular examination of LTF.

At present, there are two mouse models of orexin deficiency. One is a conventional knockout (Chemelli et al., 1999) and the other is the orexin neuron-ablated mouse (Hara et al., 2001). The orexin neuron-ablated mouse was developed using a transgenic technique by introducing a truncated Machado–Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch under the control of the orexin promoter. In these orexin/ataxin-3 transgenic (ORX/ATX-Tg) mice, the orexinergic neurons are selectively and postnataly degenerated: degeneration begins 1–2 weeks after birth and reaches >99% loss at the age of 4 months. Both orexin knockout mice and ORX/ATX-Tg mice showed narcolepsy and similar phenotypes so far tested (Willie et al., 2001, Zhang et al., 2006b, Sakurai, 2007, Kuwaki et al., 2008). We used orexin neuron-ablated mice in this study because they are free from possible in utero effects of orexin deficiency.

The specific aim of this study was threefold. First, we examined whether phrenic LTF could be observed in anesthetized, vagotomized, paralyzed, and artificially ventilated mice under isocapnic conditions. Second, we examined whether there was any difference between phrenic LTF and ventilatory LTF in WT mice by comparing our present and former results (Terada et al., 2008). Finally, we examined whether phrenic LTF was attenuated in orexin deficient mice as was the case for ventilatory LTF.

Section snippets

Methods

All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Use Committee of Chiba University Graduate School of Medicine.

Results

Apneic threshold before hypoxic stimulation as expressed as expired CO2 was ∼2% for both WT and ORX/ATX-Tg mice (Table 1). Ventilator settings, PNA (amplitude and burst frequency), and heart rate during basal period before hypoxic stimulation were not different between WT and mutant mice (Table 1). In a detailed inspection of the PNA, we found that the burst intervals were irregular in ORX/ATX-Tg whereas they were highly regular in WT mice (Fig. 1). As a result, CV of burst frequency was

Discussion

This study demonstrated phrenic LTF following IH in mice. To the best of our knowledge, this is the first report showing phrenic LTF in mice. This study also supported our hypothesis that orexin is necessary for eliciting phrenic LTF at least in mice.

Our first aim was to examine whether phrenic LTF could be observed in mice. In WT mice, we observed a considerable phrenic LTF following IH but not after SHE. Though we could not examine possible effect of sustained hypoxia in the present study

Acknowledgement

Part of the study was supported by a Grant-in-Aid for Scientific Research (17590183, 20590226) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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