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J Appl Physiol 102: 241-248, 2007. First published September 7, 2006; doi:10.1152/japplphysiol.00679.2006
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Vigilance state-dependent attenuation of hypercapnic chemoreflex and exaggerated sleep apnea in orexin knockout mice

Akira Nakamura,1 Wei Zhang,2 Masashi Yanagisawa,3,4 Yasuichiro Fukuda,1 and Tomoyuki Kuwaki1,2

Departments of 1Autonomic Physiology and 2Molecular and Integrative Physiology, Chiba University Graduate School of Medicine, Chiba-city, Chiba, Japan; 3Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas; and 4Exploratory Research for Advance Technology Yanagisawa Orphan Project, Japan Science and Technology Corporation, Tokyo, Japan

Submitted 16 June 2006 ; accepted in final form 1 September 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Exogenous administration of orexin can promote wakefulness and respiration. Here we examined whether intrinsic orexin participates in the control of breathing in a vigilance state-dependent manner. Ventilation was recorded together with electroencephalography and electromyography for 6 h during the daytime in prepro-orexin knockout mice (ORX-KO) and wild-type (WT) littermates. Respiratory parameters were separately determined during quiet wakefulness (QW), slow-wave sleep (SWS), or rapid eye movement (REM) sleep. Basal ventilation was normal in ORX-KO, irrespective of vigilance states. The hypercapnic ventilatory response during QW in ORX-KO (0.19 ± 0.01 ml·min–1·g–1·%CO2–1) was significantly smaller than that in WT mice (0.38 ± 0.04 ml·min–1·g–1·%CO2–1), whereas the responses during SWS and REM in ORX-KO were comparable to those in WT mice. Hypoxic responses during wake and sleep periods were not different between the genotypes. Spontaneous but not postsigh sleep apneas were more frequent in ORX-KO than in WT littermates during both SWS and REM sleep. Our findings suggest that orexin plays a crucial role both in CO2 sensitivity during wakefulness and in preserving ventilation stability during sleep.

chemostimulation; control of breathing; behavioral state control; hypothalamus

OREXIN-CONTAINING NEURONS have a critical role for multiple physiological functions, such as energy homeostasis, thermogenesis, sleep-wake architectures, nociception, and cardiovascular and respiratory functions (29, 33, 38).

The prepro-orexin knockout (ORX-KO) mouse is a unique and intriguing animal model. Because the two subtypes of the orexin peptides (orexin A and B) are cleaved from a common precursor molecule, prepro-orexin, by undergoing proteolytic processing, ORX-KO mice completely lack both of the orexins (4). The ORX-KO mouse is one of the model animals of human narcolepsy, showing cataplexy attacks and sleep-onset rapid eye movement (REM) sleep that normally occurs exclusively after non-REM sleep (4). These are very similar to the clinical features of human narcolepsy. In addition, ORX-KO mice showed low basal blood pressure, probably through low sympathetic vasoconstrictor tone, attenuated fight-or-flight responses, and a tendency to be obese (17). However, the respiratory phenotype of ORX-KO has not fully been examined.

We have focused on a possible role of orexin for respiratory regulation in a vigilance state-dependent manner for the following reasons. First, the axons of orexin-containing neurons project to some respiration-related sites, such as the nucleus tractus solitarius, the pre-Bötzinger complex, and the hypoglossal and phrenic nuclei (11, 19, 35). Second, intracerebroventricular administration of orexin promoted both wakefulness (10) and ventilation (36). Because basal respiration and respiratory reflex regulations are much different between awake and sleep states (7, 18), orexin may be a missing link between vigilance state and vigilance state-dependent respiratory control. Third, orexin-deficient mice facing a stressor presented an attenuated fight-or-flight response including a rise in respiration and blood pressure, although basal ventilation was comparable to that in the control mice (17, 37). This observation indicated that orexin might be a master switch to elicit integrated behavioral and autonomic outputs and that orexin might modulate respiration only in a specific situation. Fourth, human narcolepsy is thought to be caused by degeneration of orexin-containing neurons (31) and narcolepsy patients had frequent sleep apneas compared with healthy controls (5). Patients with Guillain-Barré syndrome also had low levels of orexin in their cerebrospinal fluid (27), and the syndrome is sometimes associated with respiratory paralysis. From these backgrounds, we hypothesized that the orexin system might modulate breathing, in particular, vigilance state-dependent regulation of breathing.

By examining the respiratory phenotype of ORX-KO, we attempted to determine possible roles of endogenous orexin in respiratory regulation. Our hypothesis was that the orexin system might contribute to respiratory regulation in a vigilance state- dependent manner. The aim of this study was, therefore, to test our hypothesis by 1) assessing baseline characteristics of ventilation and chemoreceptor reflex in response to hypoxia and hypercapnia during sleep/awake periods and by 2) examining sleep apneas using our previously established method for simultaneous measurements of vigilance states and ventilation in mice (22).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

ORX-KO mice with mixed genetic background of 129/Sv and C57BL/6 were generated as reported previously (4) and back-crossed to C57BL/6 for five times. They were maintained in heterozygotes and crossed to obtain null mutants and wild-type (WT) littermates. Genotype of ORX-KO mice was identified by PCR on DNA extracted from the tail as has been reported (17). Mice used in this study were 24- to 36-wk-old male ORX-KO homozygotes (n = 13, body weight 38 ± 2 g) and WT (n = 10, body weight 35 ± 1 g) mice. Body weight of ORX-KO tended to be higher than WT but there was no statistical difference between the two. All mice were housed in plastic cages in a room maintained at 23–25°C with lights on at 7:00 AM and off at 7:00 PM. Mice had food and water available ad libitum. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use Committee of Chiba University Graduate School of Medicine.

Simultaneous Measurement of Respiration and Vigilance States

Methods of surgery, recording of electroencephalography (EEG), electromyography (EMG), and respiration, and definition of vigilance states and of apneas were the same as our previous report dealing with normal mice (22). In brief, mice were surgically implanted with electrodes for EEG and EMG under isoflurane anesthesia at least 7 days before the experiments. On the experimental day, the mouse was put into a whole body plethysmography chamber and the electrode codes were connected to a slip ring so that the animal moved freely in the chamber. All the recordings were 6 h in length and were performed from 1000 to 1600. The chamber was continuously flushed with a gas mixture at rate of 500 ml/min for the entire 6-h period with the use of either room air, hypoxic (15% O2-balance N2), moderate hypercapnic (5% CO2-21% O2-N2 balance), or severe hypercapnic (10% CO2-21% O2-N2 balance) gas mixtures. Chamber PCO2 and PO2 were continuously monitored (Respina IH26, NEC-San-Ei-Instrument, Tokyo, Japan) at the outlet of the chamber. Each animal was tested under one of the four gas conditions per day at random order during 4–7 days.

EEG, EMG, and pressure signals were amplified and fed into a personal computer after analog-to-digital conversion (Power Lab, ADInstrument, Castle Hill, NSW, Australia). Sleep-wake architecture was determined by visual inspection of neck EMG and digitally filtered EEG (0.25–4, 4–8, and 8–30 Hz). Vigilance state was classified into quiet wakefulness (QW), active wakefulness (AW), slow wave sleep (SWS), or REM sleep. Respiratory frequency (fR) and amplitude of the plethysmography signals [representing tidal volume (VT)] were calculated using signal-analysis software, Chart (ADInstrument). The ambient temperature and pressure and the chamber temperature were measured intermittently (about every 2 h) during the 6 h of recording, and the average values were used for later calculation of VT. Rectal temperature of each mouse was recorded immediately after the plethysmographic recording while the animal was awake. Individual measurements of rectal temperature revealed a mean value of 36.5 ± 0.1°C in ORX-KO and 36.1 ± 0.1°C in WT mice. There was no difference between ORX-KO and WT mice. In each genotype, there was no difference in rectal temperature among the four gas conditions. Based on these values, VT was calculated according to the formula used by Epstein et al. (9). Minute volume was defined as the product of inspiratory VT and fR. Respiratory parameters were not calculated for AW because the animal's movement might distort the plethysmographic signals.

Apnea was defined as cessation of plethysmographic signals for at least two respiratory cycles (22). Apneas were classified as postsigh if the preceding breath was at least 100% above the average amplitude during the preceding 10 s. Apnea without a preceding sigh was defined as spontaneous.

We also calculated the ratio of {partial}-band (0.25–4 Hz) power to beta-band (8–30 Hz) power in EEG using fast Fourier transform (FFT) to examine sleep intensity. REM latency was defined as the time from beginning of sleep to beginning of REM sleep. We calculated REM latency to evaluate whether ORX-KO mice have a tendency to begin REM sooner than the WT mice after they fell into asleep because sleep-onset REM in ORX-KO mice has been observed in the previous report (4). When SWS terminated without consecutive REM sleep, such an episode was excluded from calculation of REM latency. Arousal frequency was defined as the counts per hour of the transition from sleep to awake.

Blood Gas Analysis

In a separate group of the animals (n = 5 for each genotype), a catheter was implanted in the right femoral artery under aseptic procedure under isoflurane anesthesia. Blood gas analysis was conducted with blood taken from the arterial catheter during conscious state.

Statistical Procedure

All data are expressed as means ± SE. Possible effects of inspired gas composition on the sleep state and respiratory parameters were assessed by two-way (between factor was the genotype, and within factor was the gas composition) ANOVA with repeated measures design, in which room air was treated as the control. Differences in respiratory parameters among vigilance states (QW, SWS, and REM sleep) were also analyzed using two-way (between factor was the genotype, and within factor was the vigilance states) ANOVA with repeated measures design. A contrast procedure was used to compare among within-factors. Post hoc comparisons of Student-Newman-Keuls procedure or unpaired t-test were used to compare between genotypes. A P value <0.05 denoted the presence of a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Basic Parameters of Sleep-Wake State and Ventilation in ORX-KO mice

Time-related changes in sleep-wake state and ventilation.   When the mice breathed normal room air, total sleep time (SWS plus REM sleep) varied between 40 and 80% except for the initial 30-min period (Fig. 1A). There was no apparent time-related trend or cyclic change after 30 min from the beginning of the experiment. The same was true when the mice breathed hypoxic (15% O2; Fig. 1A) and mild hypercapnic (5% CO2; Fig. 1B) gas mixtures. When the mice breathed severe hypercapnic gas mixture (10% CO2; Fig. 1B), on the other hand, longer time (2–3 h) was needed compared with the other gas conditions before a relatively stable value of 40–80% was reached. Total sleep time did not differ between ORX-KO and WT mice in a specific gas condition.


Figure 1
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  		Fig. 1. Time-related changes in sleep-wake state and ventilation. A and B: total sleep time [sum of slow-wave sleep (SWS) and rapid eye movement (REM) sleep] was averaged for every 30-min period and plotted as the dependent variables on the observation time. Mice breathed room air (solid line in A), hypoxic gas mixture (15% O2, dashed line in A), mild hypercapnic gas mixture (5% CO2, solid line in B), or severe hypercapnic gas mixture (10% CO2, dashed line in B). C–F: respiratory minute volume was separately calculated during quiet wake (solid line), SWS (broken line), and REM sleep (dashed line) and averaged for every 30-min period. Values are means ± SE of 5 wild-type (WT) mice and 5 prepro-orexin knockout (ORX-KO) mice. Note that there are missing variables for respiratory minute ventilation during REM sleep because of the brief and highly varying nature of mouse REM periods.

 
Respiratory minute volume stayed at a narrow range over the 6 h in QW, SWS, or REM sleep states when the mice breathed room air (Fig. 1C). When the mice breathed hypoxic or hypercapnic gas mixtures, the largest respiratory minute volume was observed during the first or second 30-min period, indicating some acclimatization to the stimuli (Fig. 1, D–F). Nevertheless, respiratory minute volume in the hypercapnic conditions kept a higher level than that in the normal gas condition over the entire observation period. In addition, rank order of the respiratory minute volume (QW > SWS > REM) did not change through the time in a given gas condition.

Together with the brief and highly varying nature of mouse REM periods (as evidenced by missing variables in Fig. 1, C–F), these results support the validity of calculating a mean value for 6 h.

Sleep-wake state.   Mild hypercapnia and hypoxia did not affect sleep-wake state in both WT and ORX-KO mice (Fig. 1, A, and B, Table 1), as was the case in our previous report using WT mice of 129/Sv strain (22). Severe hypercapnia (10% CO2), on the other hand, significantly increased total time and endurance of QW periods in the WT mice. A similar tendency was observed in ORX-KO mice. In both genotypes, total sleep time tended to be reduced by severe hypercapnia, although the difference did not reach statistical significance (P = 0.11 for WT and P = 0.10 for ORX-KO). Severe hypercapnia increased EEG power {partial}/beta ratio during SWS in WT mice and during all vigilance states in ORX-KO mice, indicating a compensatory increase in sleep intensity for a reduced sleep time.


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  		Table 1. Effects inspired gas composition on sleep architecture

 
The difference between sleep-wake architecture in ORX-KO mice and that in WT mice was characterized by the following two points. First, sleep fragmentation was observed in ORX-KO mice, as evidenced by significantly shorter SWS duration and by larger arousal frequencies than those in WT mice. Total times spend in SWS and REM sleeps were not different between ORX-KO and WT mice. Second, REM latency was significantly shorter in ORX-KO mice than in WT mice. Any episode of sleep-onset REM (i.e., REM latency = 0) was not found in the present experimental setup (daytime observation). These observations were consistent with the data for light period in a previous report (4) using ORX-KO mice of mixed genetic background (129/Sv x C57/BL6). Hypoxic or hypercapnic environment did not affect these characteristics in ORX-KO mice.

Ventilation.   When the mice breathed normal room air, fR in ORX-KO tended to be higher and VT tended to be smaller than those in WT mice during both awake and sleep periods (Table 2). In other words, ventilation in ORX-KO tended to be shallow and fast. However, respiratory minute volume was almost identical between the ORX-KO and WT mice during every vigilance state (Fig. 2). In both genotypes, fR (QW > SWS, REM) and VT (QW > SWS > REM) showed a clear sleep-wake dependency (Table 2).


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  		Table 2. Respiratory parameters in each vigilance state

 

Figure 2
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  		Fig. 2. Hypercapnic (left) and hypoxic (right) responses of respiratory minute volume in WT and ORX-KO mice during quiet awake (top) and sleep (bottom) periods. Values are means ± SE of 5 WT mice and 5 ORX-KO mice. Same data under room air breathing are presented in the left and right panels for the sake of better comparison.

 
Chemoreflex

Hypercapnic ventilatory responses.   Mild (5% CO2-21% O2) and severe (10% CO2-21% O2) hypercapnic gas challenges significantly and dose-dependently increased ventilation in both ORX-KO and WT mice (Table 2, Fig. 2). In the WT mice, slopes of the hypercapnic response were greatest during the QW period (0.38 ± 0.04 ml·min–1·g–1·%CO2–1) and decreased during SWS (0.30 ± 0.05 ml·min–1·g–1·%CO2–1) and REM (0.15 ± 0.04 ml·min–1·g–1·%CO2–1) periods in this order (Fig. 2). In ORX-KO mice, on the other hand, the slope of the hypercapnic response during QW periods (0.19 ± 0.01) was comparable to or even smaller than those for SWS (0.25 ± 0.03) and REM sleep (0.08 ± 0.02) periods. During sleep periods, there was no difference between ORX-KO and WT mice in all parameters (fR, VT, minute volume, and slope of the hypercapnic response). Thus, ORX-KO mice showed a vigilance state-dependent abnormality of hypercapnic ventilatory responses.

Hypoxic ventilatory responses.   In the WT mice, hypoxia induced significant increases in minute volume only during QW periods but not during sleep periods (Fig. 2). The same was true for ORX-KO mice, and there was no difference between the two genotypes.

Blood gas analysis.   There was no difference in arterial PO2 and arterial PCO2 between ORX-KO and WT mice even when the mice breathed hypoxic or hypercapnic gas mixtures (Table 3). These results showed that the gas exchange in the lung was normal in ORX-KO. These results are also consistent with the preserved minute volume in ORX-KO mice when breathing normal room air.


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  		Table 3. Effects inspired gas composition on blood gas

 
Sleep Apnea

In the WT littermates, similar results were obtained to our previous report using WT mice of 129/Sv strain (22). Characteristics were summarized as three points (Fig. 3). First, postsigh apneas were observed exclusively in SWS, whereas sighs and spontaneous apneas were observed during both SWS and REM sleep periods. Second, hypoxia increased postsigh occurrence index during SWS, whereas hypercapnia decreased the index. Severe hypercapnia (10% CO2-21% O2) completely abolished postsigh apnea. Third, frequencies of sighs and spontaneous apneas were not affected by the difference in gas mixtures during both SWS and REM sleep periods.


Figure 3
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  		Fig. 3. Sigh and apnea occurrence index in WT and prepro-orexin knockout (KO) mice. Top: results during SWS. Bottom: results during REM sleep. Values are means ± SE of WT mice (open bars, n = 5) and KO mice (solid bars, n = 5). ND, not detected; RA, under room air; 15O2, under 15% O2; 5CO2, under 5% CO2: 10CO2, under 10% CO2. {dagger}P < 0.05 compared with room air. *P < 0.05 compared with WT mice.

 
In ORX-KO mice, qualitatively similar results to those in WT mice were obtained (Fig. 3). In a quantitative point of view, however, there was a significant difference between the genotypes. The occurrence index of spontaneous apneas was much greater in ORX-KO than in WT mice during both SWS and REM sleep. Such difference was preserved when the breathing gas mixtures were changed to hypoxic or hypercapnic gases. Frequencies of sighs and postsigh apneas were comparable between the ORX-KO and WT mice. No postsigh apnea was observed during the REM period, suggesting that the state specificity of postsigh apnea was preserved in ORX-KO mice as in the WT mice.

Because ORX-KO mice tended to be obese and obesity is sometimes associated with obstructive sleep apnea (6), we examined whether the observed apneas were obstructive or not. For this purpose, we recorded intercostal EMG in additional three ORX-KO mice under normal room air condition. All observed postsigh and spontaneous apneas in ORX-KO mice were central type, as was the case in normal mice in our pervious report (22).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present study, we used the same method as our previous report (22). In comparison between WT mice in the previous study (129/Sv strain) and the present study (mainly C57/BL6 strain), we found general consistency in vigilance state architecture, vigilance state dependency of ventilation, and hypoxia and hypercapnia dependency of the occurrence of postsigh and spontaneous sleep apneas. A notable exception was that the basal fR during QW was larger and the hypoxic response was smaller in the present study than in the previous study. These results are in agreement with the reports showing strain difference in respiratory regulation (23, 30) and thus support the validity of our methodology.

Our hypothesis was that the orexin system might contribute to respiratory regulation in a vigilance state-dependent manner. The present study supported our hypothesis. The ORX-KO mice showed attenuated hypercapnic ventilatory responses, not during sleeping periods but specifically during QW, whereas the basal respiratory minute volume was comparable between ORX-KO mice and WT littermates. Moreover, sleep apneas (spontaneous apneas) were more frequent in ORX-KO than in WT mice. The latter result indicated that orexin might also contribute to preserving ventilatory stability during sleep. Thus, orexin seems to be indispensable during both wakefulness and sleep periods for respiratory integrity, although its roles in respiratory regulation are different between the vigilance states.

Our findings suggest that orexin contributes to ventilatory responses to hypercapnia specifically during wakefulness but not during sleeping periods. This notion may provide insights into the well-known fact that the hypercapnic ventilatory response is greater during wakefulness compared with that during sleep (7). We propose that the hypercapnic response during sleep periods relies on unknown mechanisms that are independent of orexin and the response is strengthened by orexin during wake periods. This proposal is consistent with the report that the spontaneous activity of orexin-containing neurons was increased during wake periods and decreased or even negligible during sleep periods (20, 21). Orexin-containing fibers innervated some of CO2-chemosensitive areas such as the median raphe and retrotrapezoid nuclei in the lower brain stem (11, 19, 35). Wakefulness drive to ventilation has been proposed to increase the hypercapnic responsiveness (24) and administration of orexin may increase ventilation through its awakening effect. However, the present results using knockout mice clearly showed that waking per se could not augment hypercapnic responsiveness without orexin.

Orexin seemed not to be involved in the hypoxic response even during the wake periods. This indicates that intrinsic orexin is not a general respiratory stimulant but an activator for hypercapnic responses. However, this notion is tentative because the hypoxic response in the WT littermates was much smaller than the hypercapnic response (note the difference of the vertical axes in Fig. 2). This made it difficult to compare hypoxic responses between the genotypes.

Although precise mechanisms of sleep apneas are not fully understood even in the WT mice, the mechanism underlying spontaneous sleep apnea seems largely different from that for postsigh sleep apnea for two reasons. First, postsigh apneas were observed exclusively during SWS whereas spontaneous apneas were seen during both SWS and REM sleep. Second, postsigh apneas during SWS were increased by hypoxia and decreased by hypercapnia. No such dependency on the gas composition was observed for spontaneous apneas. Our present results provided the third evidence for discrimination of spontaneous and postsigh sleep apneas. In ORX-KO mice, we observed higher frequency of spontaneous but not postsigh sleep apneas than in WT mice. Therefore, orexin may exert an inhibitory effect on the genesis of spontaneous sleep apneas. Our observation is in line with the previous finding that narcolepsy patients had high incidence of sleep apneas (5), although the author did not classify sleep apneas into spontaneous and postsigh. The present study is, to the best of our knowledge, the first attempt to elucidate mechanisms of sleep apneas by using KO mice.

We do not think that a decreased influence of orexin can explain all of human sleep apneas because most of human apneas are reported to be obstructive type. Nevertheless, there are good reasons to suspect possible involvement of orexin-deficiency in some cases of human apneas. First, orexin neurons were activated by hypoglycemia (3) and are inhibited by glucose (1, 34). Hyperglycemia is a powerful predictor of impaired breathing during sleep in both humans and animals (25, 26). Second, some obstructive sleep apnea patients showed low levels of plasma orexin (2, 28), although an opposite result (15) and no change in cerebrospinal orexin (16) have also been reported. Detailed examination awaits this issue.

Some previous findings suggested that the hypothalamus could modulate ventilation (8, 13, 14, 32). However, the described sketch was confusing. For example, Horn and Waldrop (14) demonstrated that the posterior hypothalamus exerted an excitatory effect on both hypoxic and hypercapnic ventilatory responses. On the contrary, Hinrichsen et al. (13) demonstrated that a posterior hypothalamus lesion increased ventilatory responses to hypoxia. These seemingly opposite findings concerning hypoxic ventilatory responses might be due to the fact that the hypothalamus is a heterogeneous structure. In this context, the use of ORX-KO mice is strategically very helpful for us to investigate the hypothalamic area in a chemically specific manner. Although we have not clarified the site at which orexin regulates ventilation, we can safely say that orexin neurons are one part of the suprapontine architectures that serves to modulate breathing in a sleep-wake state-specific manner.

Limitation of the present study was lack of the metabolic parameters, one of the key factors that regulate ventilation. However, there was no significant difference in body temperature between the two genotypes. Body weight of ORX-KO mice tended to be larger but did not reach statistical significance as has been reported (12). Moreover, we observed no significant difference in arterial PCO2 and in respiratory minute volume. These suggested that CO2 production was normal in ORX-KO. However, because arterial blood was sampled during presumable QW state, we cannot rule out possible differences in metabolism during sleep.

To date, there is not enough data to determine the clinical features of disordered breathing in narcolepsy patients. Further clinical studies on narcolepsy would not only provide insights into its underlying pathophysiological mechanisms but also enrich knowledge about the physiological, endogenous role of orexin system in humans.

In summary, we found attenuated hypercapnic ventilatory responses during wake period and higher incidences of sleep apneas in ORX-KO mice. These results suggest critical roles of orexin for modulating breathing in physiological state-dependent manner. The diversity of synaptic control of the respiratory neurons seems necessary for animals to adapt themselves toward ever-changing life circumstances and behavioral states. Orexin system is likely to work as one of the essential modulators for coordinating circuits controlling respiration and behavior.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
Part of the study was supported by a Grant-in-Aid for Scientific Research (17590183, 1859023, 18790533) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by grants from the Smoking Research Foundation and the Mitsui Life Social Welfare Foundation.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Megumi Shimoyama for valuable discussion and help in editing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kuwaki, Dept. of Molecular and Integrative Physiology, Chiba Univ. Graduate School of Medicine, 1-8-1 Chuo-ku, Chiba 260-8670, Japan (e-mail: kuwaki{at}faculty.chiba-u.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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