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J Appl Physiol 103: 1772-1779, 2007. First published August 23, 2007; doi:10.1152/japplphysiol.00075.2007
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Contribution of orexin in hypercapnic chemoreflex: evidence from genetic and pharmacological disruption and supplementation studies in mice

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

Departments of 1Molecular and Integrative Physiology and 2Autonomic 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 January 2007 ; accepted in final form 18 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
We have previously shown that hypercapnic chemoreflex in prepro-orexin knockout mice (ORX-KO) is attenuated during wake but not sleep periods. In that study, however, hypercapnic stimulation had been chronically applied for 6 h because of technical difficulty in changing the composition of the inspired gas mixture without distorting the animal's vigilance states. In the present study we examined possible involvement of orexin in acute respiratory chemoreflex during wake periods. Ventilation was recorded together with electroencephalography and electromyography before and after intracerebroventricular administration of orexin or an orexin receptor antagonist, SB-334867. A hypercapnic (5 or 10% CO2) or hypoxic (15 or 10% O2) gas mixture was introduced into the recording chamber for 5 min. Respiratory parameters were analyzed only for quiet wakefulness. When mice breathed normal room air, orexin-A and orexin-B but not vehicle or SB-334867 increased minute ventilation in both ORX-KO and wild-type (WT) mice. As expected, hypercapnic chemoreflex in vehicle-treated ORX- KO mice (0.22 ± 0.03 ml·min–1·g–1·% CO2–1) was significantly blunted compared with that in WT mice (0.51 ± 0.05 ml·min–1·g–1·% CO2–1). Supplementation of orexin-A or -B (3 nmol) partially restored the hypercapnic chemoreflex in ORX-KO mice (0.28 ± 0.03 ml·min–1·g–1·% CO2–1 for orexin-A and 0.32 ± 0.04 ml·min–1·g–1·% CO2–1 for orexin-B). In addition, injection of SB-334867 (30 nmol) in WT mice decreased the hypercapnic chemoreflex (0.39 ± 0.04 ml·min–1·g–1·% CO2–1). On the other hand, hypoxic chemoreflex in vehicle-treated ORX-KO and SB-334867-treated WT mice was not different from that in corresponding controls. Our findings suggest that orexin plays a crucial role in CO2 sensitivity at least during wake periods in mice.

breathing control; chemostimulation; hypothalamus; orexin receptor antagonist; respiration

OREXINS (orexin-A and orexin-B), also named hypocretins (hypocretin-1 and hypocretin-2), were first discovered as hypothalamic neuropeptides facilitating appetite and consciousness (36). Further studies showed that orexins modulate circulation (8, 28, 44), respiration (40, 42), and analgesia (34, 37). Orexinergic neurons exist only in the dorsal part of the hypothalamus including the dorsomedial hypothalamus, perifornical area, and lateral hypothalamus, but their axons project widely to various regions of the central nervous system (17, 22, 24). These hypothalamic areas are reported to be responsible for integrating defense response against stressors (7, 14, 27, 39).

These physiological and anatomical features together suggest that orexinergic neurons play a role in integrating defense response. Respiration, an important component of defense response, is also likely to be controlled by orexins. Actually, physiological studies have indicated that orexins increase ventilation (40, 42) and that orexin deficiency causes attenuated ventilatory responses in fight-or-flight behavior (15, 43, 44). In addition, anatomical studies have demonstrated the connections between orexinergic neurons and respiratory motor neurons, such as the hypoglossal nucleus (11), pre-Bötzinger complex, and phrenic motor neurons in the spinal cord (40).

Our group (21) also found that prepro-orexin knockout (ORX-KO) mice have attenuated hypercapnic chemoreflex during wake but not sleep periods. This observation is consistent with the notion that the activity of orexinergic neurons is higher during wake than sleep periods (16, 19). In that study, however, hypercapnic stimulation had been applied chronically for 6 h because of the technical difficulty of changing the composition of the inspired gas mixture without distorting the vigilance states of animals.

Some previous findings have suggested that the hypothalamus could modulate ventilation (12, 13, 32). For example, Horn and Waldrop (13) demonstrated that the posterior hypothalamus exerted an excitatory effect on both hypoxic and hypercapnic ventilatory responses. On the contrary, Hinrichsen et al. (12) 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 allows us to investigate the hypothalamic area in a chemically specific manner. In the present study, therefore, we examined whether our previous findings could be generalized to acute chemoreceptor reflex and whether orexin-related drugs could affect chemoreflex during wake periods in ORX-KO mice and their wild-type (WT) littermates.


   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 (5) and backcrossed to C57BL/6 more than nine times. They totally lacked orexin-A and orexin-B, because both peptides are the product of the one precursor, prepro-orexin. The animals were maintained in heterozygotes and crossed to obtain null mutants and WT littermates. The genotype of ORX-KO mice was identified by PCR on DNA extracted from the tail as previously reported (15). Mice used in this study were 28- to 44-wk-old male ORX-KO homozygote (n = 24, body weight 36 ± 1 g) and WT mice (n = 28, body weight 34 ± 1 g). Body weight of ORX-KO mice tended to be higher than that of WT mice, but there was no statistical difference. The animals were kept in an air-conditioned room (20–25°C) under a 12:12-h light-dark cycle with lights on from 7:00 AM. Mice were provided food and water 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 were 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 for this study were the same as those for previous studies by our group (20, 21) except that an additional cannula was implanted for intracerebroventricular (icv) administration of the drugs. In brief, mice were surgically implanted with electrodes for EEG and EMG and an icv guide cannula (C315GS-5/2.5; Plastics One, Roanoke, VA) to the lateral ventricle (1 mm lateral to bregma, 2.5 mm deep in the skull) (10) under isoflurane anesthesia at least 7 days before the experiments. An antibiotic, cefazolin (100 mg/kg), was injected subcutaneously before the surgery. 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. In the present study, a trembling sensor was attached to the chamber for detection of the animal's movement. EEG, EMG, movement, 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, digitally filtered EEG (0.25–4, 4–8, and 8–30 Hz), and movement signals. Vigilance states were classified into quiet wakefulness (QW), active wakefulness (AW), slow-wave sleep, and rapid eye movement sleep. QW and AW were discriminated by the amplitude of the EMG volleys and movement signals. Respiratory frequency (fR) and amplitude of plethysmography signals (representing tidal volume) were calculated using signal analysis software (Chart, ADInstrument). Ambient temperature and pressure and chamber temperature and humidity were recorded for each gas condition (see Protocols). Rectal temperature was taken at the beginning and end of each experiment. Based on these values, tidal volume (VT) was calculated according to the formula used by Epstein et al. (9). Minute volume (MV) was defined as the product of inspiratory VT and fR, normalized to the animal's body weight. Respiratory parameters were calculated only for QW, because administration of orexins almost completely deprived the mice of their sleep states (see RESULTS) and the animal's movement might distort the plethysmographic signals during AW. To quantify the animal's motor activity during the AW period, signals from the trembling sensor were full-wave rectified, integrated for 1 s, and averaged over the AW time. The absolute value may have varied among the experiments because a slight difference in the animal's body weight and in the contact between the trembling sensor and the chamber largely affected the amplitude of output signals. Therefore, the above-calculated movement values were normalized to the value observed during the control period.

Protocols

Experiment 1: orexin-A and orexin-B supplementation.   Nine ORX-KO and six WT mice were used. Room air at a flow rate of 0.6 l/min was supplied through the inflow of the chamber. Sufficient bedding material and food were provided in the chamber. Air-tightness was confirmed by checking all the connections to the chambers with soapy water. Each experiment was started around 10:00 AM when a mouse was put into the chamber and allowed to adapt to the environment for more than 1 h. The appearance of sleep, proven by the EEG and EMG recordings and behavior, was considered the sign of acclimatization.

After acclimatization, the baseline parameters were collected when the mouse breathed room air for 20 min (termed room air 1). Next, the mouse was taken out of the chamber, administered artificial cerebrospinal fluid (aCSF) through the icv cannula, and immediately returned to the chamber. Another 20-min period in room air (termed room air 2) was allowed to measure the effect the drug administration on baseline parameters. After this period, a hypercapnic gas mixture of 5% CO2-21% O2 and residual nitrogen was introduced into the chambers. When the CO2 concentration at the outlet port reached the target concentration (usually within 2 min after introduction), a 5-min recording was made. Subsequently, hypercapnic gas of 10% CO2-21% O2 and residual nitrogen was introduced and another 5-min recording was taken. Thereafter, the mouse breathed room air for 20 min (termed room air 3). After the experiments with hypercapnic gases, hypoxic gas of 15% O2 with residual nitrogen and hypoxic gas of 10% O2 with residual nitrogen were introduced into the chambers. A 5-min recording was taken for each hypoxic condition. There was a 20-min interval with room air breathing (termed room air 4) between the hypoxic stimulations. Finally, the mice breathed room air for 20 min for recovery (termed room air 5). In total, nine sequential data values (during room air 1 before icv, room air 2 after icv, 5% CO2, 10% CO2, room air 3, 15% O2, room air 4, 15% O2, and room air 5) were obtained in each experiment. Calibration gas of 60 µl was introduced with a pipette into the reference chamber before the mouse was taken out of the chamber. The same protocol was repeated with orexin-A and orexin-B in a random sequence for each animal, with at least a 2-day interval between experiments.

Experiment 2: orexin receptor blockade.   Only WT mice were used (n = 7). The experimental protocol was the same as that of experiment 1, except that the administered drugs were aCSF, vehicle, and the orexin receptor 1 antagonist SB-334867 (29, 37).

Blood Gas Analysis and CO2 Production

In a separate group of mice (n = 15 for each genotype), a catheter was implanted into the right carotid artery of each animal under isoflurane anesthesia. A hole was made in the skull (1 mm lateral to bregma) of the animal for icv administration of the drugs. After a recovery period of more than 3 h, the mouse was placed in the body plethysmography chamber and breathed room air for 20 min. Next, the mouse was taken out of the chamber and administered aCSF, orexin-A, or orexin-B through the hole in the skull with a microsyringe, whose needle tip lay 3.5 mm deep in the skull. Immediately after the administration, the animal was returned to the chamber and another 20-min period of room air was allowed. At the end of this period, up to 70 µl of arterial blood were drawn from the indwelling catheter, the distal end of which had been exteriorized from the chamber through a small port. After completion of the first blood sampling, the chamber was filled with the 10% CO2-21% O2 and residual nitrogen gas mixture for 10 min. A second blood sampling was performed while the animals were breathing the hypercapnic gas mixture. Only one drug was administered in each animal in this experiment. CO2 production was calculated from the CO2 concentration in the chamber and the flow rate (0.6 l/min) and expressed as STPD.

Agents Administered

aCSF, orexin-A (Peptide Institute, Osaka, Japan) (3 nmol) and orexin-B (3 nmol) in aCSF, vehicle (see below), and orexin receptor 1 antagonist SB-334867 (Tocris Bioscience, Bristol, UK) (30 nmol) dissolved in the vehicle were prepared and administered in a volume of 2 µl into the lateral ventricle through the icv internal cannula (C315IS-5/2.5; Plastic One), which was fitted into the guide cannula with 1-mm projection.

The orexin receptor 1 antagonist SB-334867 is hydrophobic and insoluble in aCSF. Although dimethyl sulfoxide (DMSO) is sometimes used as a solvent for hydrophobic substances and recommended by the manufacturer of SB-334867, it interferes with the sleep architecture (4). In addition, our preliminary study showed icv administration of 50% and higher concentrations of DMSO caused severe seizure, confirmed by EEG and muscle twitching. To avoid such problems, we dissolved SB-334867 first in DMSO (to a concentration of 50 mM) and then diluted the solution using 45% (2-hydroxypropyl)-β-cyclodextrin in aCSF to make 15 mM SB-334867. Without using β-cyclodextrin, SB-334867 was precipitated out of 30% DMSO in aCSF. Neither the vehicle (31.5% β-cyclodextrin-30% DMSO in aCSF) nor 30% DMSO in ACSF had affected sleep or caused any seizure or any other apparent abnormality in our preliminary study.

The icv administration of 3 nmol of orexin in 2 µl of aCSF would exceed the physiological concentration found in the cerebral spinal fluid (25, 41). Possible effects of volume and concentration on vigilance state and respiration cannot be excluded. However, other studies of icv orexin administration have adopted similar doses, and the changes in the concentration within the physiological range did not affect the parameters observed so far (6, 18, 42). Therefore, we decided to use a pharmacological dose of 3 nmol for convenience of comparison.

Statistical Analysis

All data are means ± SE. Possible effects of the genotypes and drugs on the vigilance state and the basal ventilation were assessed using two-way repeated-measures ANOVA. Where appropriate, a post hoc Student-Newman-Keuls procedure or contrast test was used. Because orexin-A and orexin-B significantly increased the baseline ventilation before the test of chemoreflex (see RESULTS), absolute values of MV during chemostimulation could not be directly compared among the different drug groups. Therefore, the magnitude of the chemoreflex was evaluated by calculating the slope of the CO2 (or O2)-MV relationships using linear regression analysis. Two-way repeated-measures ANOVA was used for comparison among slopes. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Vigilance States and Motor Activity

Administration of aCSF transiently increased the AW periods in WT mice for no more than 20 min (Fig. 1, A and E). Thereafter, AW periods progressively decreased toward the end of the experiment and were not apparently affected by hypercapnic or hypoxic stimuli. On the other hand, QW periods were hardly affected by animal handling and/or volume injection but were largely increased by severe chemostimuli (10% CO2 and 10% O2; Fig. 1). Comparing WT and ORX-KO mice, AW time in the ORX-KO mice was significantly (P < 0.05) shorter than in the WT mice. However, no difference was observed in QW time.


Figure 1
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  		Fig. 1. Time-related changes in vigilance states. Quiet wake time (QW; solid lines) and active wake time (AW; dashed lines) are plotted against the sequence of breathing gas conditions (see METHODS for details). Data are means ± SE of ratio of duration of each vigilance state relative to the recording time. Hypercapnic and hypoxic stimuli lasted for 5 min, and room air breathing periods were 20 min long. A–C: wild-type mice (WT; n = 6) and orexin knockout mice (KO; n = 9) received intracerebroventricular (icv) administration of artificial cerebrospinal fluid (aCSF; 2 µl), orexin-A (ORX-A; 3 nmol), and orexin-B (ORX-B; 3 nmol). Timing of injection is indicated by an arrow. D–F: another set of WT (n = 7) received aCSF, vehicle, and an orexin receptor antagonist, SB-334667 (30 nmol). *P < 0.05 compared with WT. {dagger}P < 0.05 compared with aCSF.

 
Administration of orexin-A dramatically increased AW periods and moderately increased QW periods (Fig. 1B). Total wake time (AW plus QW periods) exceeded 90% even at 2 h after injection, when the last measurement during room air breathing was performed. Compared with aCSF, orexin-A significantly (P < 0.05) increased both AW and QW periods. Administration of orexin-B also increased AW and QW periods (Fig. 1C). However, the effect of orexin-B lasted shorter than that of orexin-A. Therefore, the difference between AW (or QW) period after aCSF and that after orexin-B did not reach statistical significance. After administration of orexin-A or orexin-B, there was no statistical difference in AW or QW between ORX-KO and WT mice, indicating normally preserved orexin receptors in ORX-KO mice. The effects of SB-334867 and its vehicle on AW and QW periods were not different from those of aCSF (Fig. 1, D–F).

We also analyzed the animal's motor activity during the AW period (Fig. 2). Inhalation of CO2 increased motor activity in both WT and ORX-KO mice, irrespective of the injected drugs. Although orexin-A apparently increased motor activity in ORX-KO mice, the difference did not reach statistical significance. There was no difference between WT and ORX-KO mice in motor activity even after administration of aCSF. Together, these results show that orexin mainly affected the wake duration and had a lesser effect on the motor activity per AW time.


Figure 2
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  		Fig. 2. Time-related changes in motor activity during active wake periods. A: WT (open symbols, n = 6) and KO mice (filled symbols, n = 9) received aCSF, ORX-A, and ORX-B. B: another set of WT mice (n = 7) received aCSF, vehicle, and an orexin receptor antagonist, SB-334667. Timing of injection is indicated by an arrow. Data are means ± SE. a.u., Arbitrary units. Note that there is no difference between KO and WT or among drug treatment groups.

 
Effects on Basal Breathing

Before icv drug administration, spontaneous breathing was not different between WT and ORX-KO mice or among treatment groups (Fig. 3). Administration of orexin-A significantly increased MV in both ORX-KO ({Delta}MV = 67 ± 18%, n = 9) and WT mice (43 ± 14%, n = 6). In a similar manner, administration of orexin-B increased MV in both ORX-KO (106 ± 13%) and WT mice (83 ± 28%). Orexin-B had significantly stronger effects than orexin-A in both genotypes. There was no significant difference between MV in ORX-KO and MV in WT after the treatment of any drug. Neither aCSF ({Delta}MV = 9 ± 10% for WT in experiment 1, 2 ± 5% for ORX-KO, and 15 ± 8% for WT in experiment 2, n = 7) nor SB-334867 (5 ± 4%) or its vehicle (10 ± 6%) influenced spontaneous ventilation.


Figure 3
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  		Fig. 3. Time-related changes in minute ventilation during quiet wake periods. A: WT (open symbols, n = 6) and KO mice (filled symbols, n = 9) received aCSF, ORX-A, and ORX-B. B: another set of WT mice (n = 7) received aCSF, vehicle, and an orexin receptor antagonist, SB-334667. Timing of injection is indicated by an arrow. Data are means ± SE. Insets: effects of icv administration of the drugs on the minute ventilation when the mice breathed normal room air. To show the effect of drug alone, the area encompassing room air 1 and room air 2 in A and B are shown enlarged. *P < 0.05 compared with before icv. {dagger}P < 0.05 compared with aCSF. {ddagger}P < 0.05 compared with ORX-A.

 
Hypercapnic Chemoreflex

In all of the treatment groups, inhalation of both 5 and 10% CO2 progressively increased MV (Figs. 3 and 4). Since orexin-A and orexin-B increased basal MV before chemoreflex stimulation, slopes of the dose-response curves were calculated and are shown at right in Fig. 4. Results can be summarized into three main points. First, aCSF-treated ORX-KO mice (0.22 ± 0.03 ml·min–1·g–1·% CO2–1) significantly attenuated the hypercapnic chemoreflex compared with the aCSF-treated WT mice (0.51 ± 0.05 ml·min–1·g–1·% CO2–1). Second, neither orexin-A nor orexin-B affected the slope in the WT mice. On the other hand, in the ORX-KO mice, orexin-A (0.28 ± 0.03 ml·min–1·g–1·% CO2–1) and orexin-B (0.32 ± 0.04 ml·min–1·g–1·% CO2–1) partially but significantly restored the attenuated hypercapnic chemoreflex (P < 0.05). In contrast to the effects on vigilance state and on basal ventilation, there was no clear difference between the effects of orexin-A and of orexin-B on the slope of the hypercapnic chemoreflex in the ORX-KO mice. Third, SB-334867 (0.39 ± 0.04 ml·min–1·g–1·% CO2–1) but not its vehicle attenuated the hypercapnic chemoreflex in WT mice (Fig. 4C).


Figure 4
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  		Fig. 4. Hypercapnic responses of minute ventilation. Absolute values of minute ventilation during quiet wake periods are plotted against inspired CO2 concentrations at left (reproduced from the same data shown in Fig. 3), and slopes calculated using linear regression analysis are shown at right. WT (n = 6; A) and KO mice (n = 9; B) received aCSF, ORX-A, and ORX-B. C: another set of WT mice (n = 7) received aCSF, vehicle, and an orexin receptor antagonist, SB-334667. Data are means ± SE. *P < 0.05 compared with aCSF-treated WT in A. {dagger}P < 0.05 compared with aCSF-treated KO. {ddagger}P < 0.05 compared with aCSF- and vehicle-treated WT in C.

 
Hypoxic Chemoreflex

As was the case in hypercapnic chemoreflex, inhalation of 15 and 10% O2 progressively increased MV in all of the treatment groups (Figs. 3 and 5), although the increases were not as prominent as those in the hypercapnia group. Analysis similar to that employed for the hypercapnic response revealed that the slope of the hypoxic chemoreflex was not affected by orexin deficiency or any drug treatment.


Figure 5
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  		Fig. 5. Hypoxic responses of minute ventilation. Absolute values of minute ventilation during quiet wake periods are plotted against inspired O2 concentrations at left (reproduced from the same data shown in Fig. 3 except that room air data was the average value obtained during room air 3 and room air 4 periods), and slopes calculated using linear regression analysis are shown at right. WT (n = 6; A) and KO mice (n = 9; B) received aCSF, ORX-A, and ORX-B. C: another set of WT mice (n = 7) received aCSF, vehicle, and an orexin receptor antagonist, SB-334667. Data are means ± SE. Note that slopes of the hypoxic chemoreflex are not different between WT and KO or among drug treatment groups.

 
Blood Gas and CO2 Production

Possible effects of orexin on arterial partial pressure of CO2 (PaCO2) and CO2 production were evaluated using another set of mice. In the WT mice, icv administration of orexin-A and orexin-B slightly increased arterial partial pressure of O2 (PaO2) and decreased PaCO2 (Table 1). However, these changes did not reach statistical significance (P = 0.18 for PaO2 and P = 0.15 for PaCO2). On the other hand, inhalation of 10% CO2 significantly increased PaO2 and PaCO2. There was no interaction between the drugs (aCSF, orexin-A, or orexin-B) and the gas conditions (room air or 10% CO2). The same was true in the ORX-KO mice. Moreover, there was no difference between the blood gases in WT mice and those in the ORX-KO mice for any particular condition.


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  		Table 1. PaO2, PaCO2, and CO2 production during inhalation of 10% CO2 or room air after intracerebroventicular injection of orexin-A, orexin-B, or vehicle

 
Neither orexin-A nor orexin-B affected CO2 production in either the WT or ORX-KO mice (Table 1). CO2 production in the ORX-KO mice tended to be smaller than that in the WT mice, indicating lower basal metabolism in the ORX-KO mice. However, the difference did not reach statistical significance (P = 0.18). These observations suggest that alterations in PaCO2 and CO2 production do not contribute to the attenuated CO2 reflex in ORX-KO mice or its restoration by supplementation of orexins.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In this study, we investigated the role of orexins in respiratory chemoreflex. The results showed that 1) ORX-KO mice attenuated respiratory chemoreflex to hypercapnic but not hypoxic gases; 2) supplementation of orexin-A or orexin-B partially restored the attenuated chemoreflex in ORX-KO mice; and 3) an orexin receptor 1 antagonist, SB-334867, weakened the hypercapnic chemoreflex in the WT mice. These three lines of evidence clearly support our hypothesis that orexin participates in hypercapnic chemoreflex during wake periods in mice.

In our group's previous study (21), we showed that hypercapnic but not hypoxic chemoreflex in ORX-KO mice was blunted only when the mice were awake, even though the baseline ventilation was not different between the ORX-KO and WT littermates; it is worth noting that identical results were obtained in this study despite some differences in the experimental setup. First, chronic chemoreceptor stimulations (for 6 h) were used in the previous study because priority was set on observing vigilance states with minimal perturbations rather than studying chemoreflex. Second, animals were backcrossed five times to C57BL/6 in the previous study, whereas animals were backcrossed more than nine times in the current study. Third, ventilation was compared using naive mice in the previous study and after pretreatment with aCSF in the current study. Since these differences did not affect the conclusion, orexin deficiency in mice conceivably distorts the hypercapnic chemoreflex during wake periods.

Awake-promoting effect of orexins have been repeatedly reported in normal animals (1, 30, 36) and in orexin-deficient mice (18). Such an effect is likely to result in stimulating respiration. If so, orexin-A would exert a stronger or at least a similar ventilation-promoting effect compared with that of orexin-B, because the awake-promoting effect of orexin-A is stronger and longer lasting than that of orexin-B (Fig. 1). On the contrary, orexin-B had a stronger ventilation-promoting effect than orexin-A (Fig. 3). Moreover, the ventilation-promoting effect of orexin-A can be observed under anesthetized conditions (42). Thus the ventilation-promoting effect of orexins cannot be fully explained by their awake-promoting effect. The awake-promoting effect of orexins seemed to result mainly from their action on alertness rather than on motor activity (Fig. 2) or metabolism (Table 1). Detailed analysis of the animals' behavior (such as orientation/exploring vs. motor hyperactivity) might support such a characterization. Unfortunately, however, we did not record the animals' behavior in this study.

The current experiment showed that orexin-B had a stronger effect than orexin-A on increasing spontaneous ventilation. Orexin-B mainly acts on orexin receptor 2 (26); thus orexin receptor 2 appears to have played a larger part in the current results. On the other hand, blockage of orexin receptor 1 by SB-334867 weakened hypercapnic chemoreflex of the WT mice, indicating that orexin receptor 1 is necessary for chemoreflex control. On the basis of the results of this study, we speculate that blockade of orexin receptor 2 is likely to weaken hypercapnic chemoreflex to a larger degree. This is a question worth investigating further when a specific antagonist to orexin receptor 2 becomes available.

ORX-KO mice did not respond differently from WT mice when they breathed hypoxic gas mixtures. Therefore, orexins are possibly not involved in respiratory hypoxic chemoreflex. On the other hand, the mouse strain we used (C57BL/6) is not sensitive to hypoxic stimulation (31). The slope of hypoxic response after aCSF pretreatment was not as prominent as that of hypercapnia (compare the scale difference in the vertical axis between Figs. 4 and 5). Therefore, the difference between ORX-KO and WT mice may be too small to be detected. Orexin supplementation also did not cause any significant difference for the same reason. Backcrossing to a more hypoxia-sensitive strain such as DBA/2J (31) may resolve this issue.

The chemoreflex-promoting effect of orexins may be relevant to respiratory augmentation during many (patho)physiological conditions such as exercise, fight-or-flight response, and hyperventilation syndrome. Actually, our group (15,43) previously showed that hypothalamic stimulation resulted in attenuated respiratory responses in orexin-deficient mice. In the same animal model, stress-induced increases in blood pressure and heart rate and stress-induced analgesia were also blunted. During fight-or-flight response, chemoreflex is augmented (33), whereas baroreflex is attenuated (23). Our group (43) confirmed the latter phenomenon was blunted in orexin neuron-ablated mice, another animal model of orexin deficiency. Therefore, it seems reasonable to speculate possible contribution of orexin in respiratory augmentation during the fight-or-flight response, although this issue awaits the findings of further experiments.

Orexin neurons are activated by hypoglycemia (3) and inhibited by glucose (2, 38). Therefore, hyperventilation during hypoglycemia may be related not only to metabolic acidosis but also to the respiratory-promoting effect of orexin. During revision of this article, a report appeared showing activation of the orexinergic neurons in hypothalamic slice preparations by CO2 and H+ (35). This is in direct support of our hypothesis and may explain hyperventilation during hypoglycemia, although the precise physiological relevance should be studied further in vivo.

In conclusion, our results together with those of previous studies indicate that orexins are involved in integrity of overall ventilation control. To our knowledge, this is the first study to clearly demonstrate the involvement of orexin in respiratory chemoreflex using both loss-of-function (genetic and antagonistic) and gain-of-function (agonistic) strategies.


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 METHODS
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 DISCUSSION
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ACKNOWLEDGMENTS
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Part of this study was supported by Grants-in-Aid for Scientific Research (17590183, 18590203, 18790533) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by grants from the Smoking Research Foundation and Mitsui Life Social Welfare Foundation.


   ACKNOWLEDGMENTS
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 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Megumi Shimoyama for valuable discussion.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kuwaki, Dept. of Molecular & 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.


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 REFERENCES
 

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