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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.

Ample evidence has been reported to show a probable contribution of orexin in the central cardiovascular regulation. Although cardiovascular and respiratory centers in the brain are located close to each other and are interconnected, the possible participation of orexin in respiratory regulation has not been fully documented. Here we examined the effects of intracerebroventricular administration of orexin-A on respiratory and cardiovascular parameters in urethane-anesthetized mice. Respiratory frequency and tidal volume were recorded simultaneously with blood pressure and heart rate. Orexin-A (0.003-3 nmol in 2 microL) or vehicle was administered into the lateral ventricle or cisterna magna. Lateral ventricular administration induced a rise in respiratory frequency (by 11% at the highest dose), tidal volume (76%), blood pressure (13%) and heart rate (6%) in a dose-dependent manner. With intracisternal administration, however, respiratory frequency did not change while a similar increase in tidal volume (75%) was observed. A relatively larger cardiovascular response was elicited with intracisternal administration (blood pressure 26%, heart rate 9%). On the other hand, with either administration route, orexin-A did not affect reflex increases in respiratory frequency and tidal volume in response to hypoxia and hypercapnia. These results show possible participation of orexin-A not only in the cardiovascular regulation but also in the respiratory control system. Moreover, orexin can affect the cardiorespiratory control system at multiple sites in different ways. Orexin-A seems not to be involved in respiratory reflex regulation in mice at least under anesthetized condition.

H-Dmt- d -Arg-Phe-Lys-NH 2 ([Dmt 1 ]DALDA, dDAL), a highly selective mu-opioid peptide, produces potent analgesia without respiratory depression after intrathecal administration. Despite carrying 3+ net charge, dDAL is also a potent analgesic after systemic administration. We compared the respiratory effects of dDAL and morphine after subcutaneous administration in mice using whole body plethysmography. Analgesic doses of 3 and 10 times ED 50 were examined. Both drugs dose-dependently decreased respiratory frequency and minute volume in room air. Tidal volume was increased by the lower dose of morphine, while it was decreased by the higher dose of dDAL. The decrease in minute volume by dDAL and morphine was completely reversed by naloxone. No difference in ventilatory response to CO 2 was observed between dDAL and morphine at three times ED 50 . Ventilatory response to hypoxia was significantly diminished by dDAL compared to morphine and saline, and this effect of dDAL was naloxone-irreversible. Thus dDAL likely reduces the sensitivity of the peripheral chemoreflex loop through a non-opioid action.

Sleep apnea occurs in humans and experimental animals. We examined whether it also arises in adult mice. Ventilation in male adult 129/Sv mice was recorded concomitantly by electroencephalograms and electromyograms for 6 h by use of body plethysmography. Apnea was defined as cessation of plethysmographic signals for longer than two respiratory cycles. While mice breathed room air, 32.3 ± 6.9 (mean ± SE, n = 5) apneas were observed during sleep but not in quiet awake periods. Sleep apneas were further classified into two types. Postsigh apneas occurred exclusively during slow-wave sleep (SWS), whereas spontaneous apneas arose during both SWS and rapid eye movement sleep. Compared with room air (9.1 ± 1.4/h of SWS), postsigh apneas were more frequent in hypoxia (13.7 ± 2.1) and less frequent in hyperoxia (3.6 ± 1.7) and hypercapnia (2.8 ± 2.1). Our data indicated that significant sleep apnea occurs in normal adult mice and suggested that the mouse could be a promising experimental model with which to study the genetic and molecular basis of respiratory regulation during sleep.

Blood pressure (BP) and heart rate (HR) in endothelin-3 (ET-3) null (-/-) knockout mice and ET(A) receptor (-/-) mice were measured using the servo null pressure measuring technique under halothane anaesthesia. In infant ET-3 (-/-) mice (2-3 weeks old), mean BP and HR were 55+/-2 mmHg and 436+/-30 beats/min respectively. These values were not different from those in age-matched wild-type mice (53+/-3 mmHg and 430+/-18 beats/min respectively). Baroreflex sensitivity, which was calculated as the slope of the relationship between systolic BP and RR interval on an ECG, was also similar in ET-3 (-/-) mice (0.84+/-0.20 ms/mmHg) and wild-type mice (1.07+/-0.38 ms/mmHg). ET(A) receptor (-/-) mice were obtained by caesarean section on the expected day of delivery and tracheotomized, so that they would live for more than 24 h. Mean BP and HR in ET(A) receptor (-/-) mice were 15+/-1 mmHg and 333+/-6 beats/min respectively. These values were not different from those in age-matched, similarly treated wild-type mice (16+/-3 mmHg and 308+/-10 beats/min respectively). Baroreflex sensitivity in the newborn ET(A) receptor (-/-) mice (0.45+/-0.15 ms/mmHg) and wild-type mice (0.31+/-0.06 ms/mmHg) were very low compared with the values in infant wild-type mice, but not different between the mutant mice and their littermates. Moreover, HR in awake ET(A) receptor (-/-) mice (396+/-13 beats/min) was not different from that in wild-type mice (409+/-13 beats/min). These results show that the ET(A) receptor and ET-3 are not involved in cardiovascular regulation, at least during the very early life of the mice. A possible involvement of the ET(A) receptor in BP regulation, if any, seems to occur at later times and/or in some pathological settings.

Pulmonary responses to ozone exposure (1.0 ppm) were investigated in mice sensitized to ovalbumin compared with control mice receiving saline. Pulmonary function parameters were measured by pneumotachography. Arterial blood gases and the concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor(TNF-) in bronchoalveolar lavage fluid were analyzed. Ozone exposure, when compared with filtered air exposure, caused significantly larger decreases in dynamic compliance (P 0.05) and minute ventilation (P 0.05) in ovalbumin-sensitized mice but not in control mice. Moreover, the decrease in minute ventilation in response to ozone exposure was significantly greater (P0.01) in ovalbumin-sensitized mice than in control mice. Ozone exposure caused a significant decrease in PaO2 in ovalbumin-sensitized mice but not in control mice. PaO2 after ozone exposure tended to be smaller in ovalbumin-sensitized mice than in control mice. The concentration of sICAM-1 in bronchoalveolar lavage fluid increased in ovalbumin-sensitized mice, but effects of ozone exposure were not observed. These results indicated that sensitization of the immune system to ovalbumin might be a risk factor which aggravates the effects of ozone exposure on the respiratory system. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Congenital central hypoventilation syndrome (CCHS) and Hirschsprung's disease (HSCR) are often classified as neurocristopathies and are thought to share a common molecular pathogenesis related to the genes that control the development of neural crest cells. We examined whether endothelin-3 (ET-3), one of the developmental regulators of neural crest cells and of which null mutation results in aganglionic megacolon in mice, fulfills the requirements for such a common molecule. To investigate the possible involvement of ET-3 in central ventilatory control, we measured ventilation in mutant mice deficient in ET-3 by whole body plethysmography. Tidal volume and breathing frequency were measured during breathing of room air, hypoxic, hyperoxic, or hypercapnic gas mixtures in awake and anesthetized mice. There were no significant differences in resting ventilation as well as ventilatory responses to hypoxia and hypercapnia between ET-3-knockout mice and wild-type mice. Our results indicate that ET-3 can not be considered as a common pathogenic mechanism for CCHS and HSCR at least in mice.

A medullary-spinal cord preparation without the pons isolated from the neonatal rat was used to investigate the role of inhibitory neurotransmitters in the respiratory depression induced by hypoxia (hypoxic respiratory depression; HRD). The burst frequency (C(4)-f) and peak amplitudes of the integrated activity of the C(4) roots (integral C(4)) and of the hypoglossal nerve (integral XII) were recorded. A marked decrease in C(4)-f (to 36+/-6% of control, P

The activation of peripheral chemoreceptors by hypoxia or electrical stimulation of the carotid sinus nerve elicited a hypoxic respiratory response consisting of both stimulatory and subsequent or simultaneous inhibitory components (hypoxic respiratory stimulation and depression). Both components have different time domains of responses (time-dependent response), providing an integrated respiratory response to hypoxia. This review has focused on the neuroanatomical and neurophysiological correlations responsible for these responses and their neuropharmacological mechanisms. Hypoxic respiratory depression is characterized by the initial activation of respiration followed by a progressive and gradual decline in ventilation during prolonged and/or severe hypoxic exposure (biphasic response). The responsible mechanisms for the depression are located within the central nervous system and may be dependent upon activity from peripheral chemoreceptor. Two underlying mechanisms contributing to the depression have been advocated. (1) Change in synaptic transmission: Within the neuronal network controlling the hypoxic respiratory response, hypoxia might induce the enhancement of inhibitory neurotransmission (modulation), disfacilitation of excitatory neruotransmission or both. (2) Change in the membrane property of respiratory neurons: Hypoxia might suppress the membrane excitability of respiratory neurons composing the hypoxic respiratory response via modulating ion channels, leading to hyperpolarization or depolarization blocking of the neurons. However, the quantitative aspects of Pao(2) (degree and duration of hypoxic exposure) to induce these changes and the susceptibility of both mechanisms to the Pao(2) level have not yet been clearly elucidated.

This study was designed to evaluate the use of hepatic venous hemoglobin oxygen saturation (ShVO2) as an indicator of hepatic oxygen supply-demand relation and also regenerative status of the liver after partial hepatectomy in rats. We assessed the ShVO2 levels for 7 days, as well as hepatic hemodynamics, oxygen consumption, DNA synthesis and energy charge of the remnant liver for 3 days after 50% hepatectomy or sham operation. Total hepatic oxygen consumption (HVO2) per liver weight, hepatic oxygen extraction ratio (HO2ER), and DNA synthesis were significantly elevated at days 1 and 3 after hepatectomy, compared with the preoperative levels. Meanwhile, significantly decreased ShVO2 levels were observed at days 1 and 3, and the ShVO2 levels were significantly correlated with the HVO2. Furthermore, the decreased ShVO2 levels were synchronized with the increased DNA synthesis in the remnant liver. Energy charge levels were also significantly decreased at day 1 after hepatectomy. These results suggest that the regenerating liver demands an increased amount of oxygen for mitochondrial oxidative phosphorylation to restore hepatic energy charge. In conclusion, the ShVO2 after hepatectomy may reflect oxygen metabolic status in the remnant liver and could be useful for estimating liver regeneration.

An in vitro brainstem preparation of the neonatal rat with intact right vagal (X) innervation of the right atrium, and intact medullary roots of the left X and glossopharyngeal (IX) nerves for stimulation was developed. The preparation was continuously superfused with artificial CSF at 25 degrees C. The electrical activity of the right atrium was recorded to determine the heart rate. Applications of atropine or propranolol to the superfusate did not alter the heart rate. Electrical stimulation (0.5 ms pulse, 20 Hz) of the left IX and X afferents elicited a reduction in the heart rate from 70.3 +/- 13.2 to 50.6 +/- 13.2 beats/min (mean +/- SD, p < 0.05), which was abolished after division of the right X or application of atropine to the superfusing solution. A similar reflex bradycardia was seen in a preparation with intact left vagal-right atrium innervation during right IX and X afferent stimulation. Cervical spinal cord transection affected neither the baseline heart rate nor the magnitude of the reflex bradycardia. Longitudinal sectioning of the medulla oblongata in the mid-line down to the level of the posterior inferior cerebellar artery abolished the heart rate response. After bilateral cervical vagotomies, electrical stimulation (0.5 ms pulse, 20 Hz, up to 100 microA) of the ventrolateral medulla oblongata, lateral funiculus at C2 or intermediate nucleus of the spinal cord at Th1-4 did not affect the heart rate. These results indicate that the functions in the lower brainstem are preserved in this preparation, at least in regard to the generation of reflex bradycardia. The results also suggest that the laterality of cardiac vagal innervation and sympathetic innervation will develop during the postnatal period. This preparation may be useful for the study of the central neuronal network controlling the heart rate.

The ventilatory response to acute systemic hypoxia has been thought to be determined by the balance between hypoxic stimulation via peripheral chemoreceptors and hypoxic inhibition of the respiratory neurons. In moderate-severe hypoxia, the latter predominates the former resulting in ventilatory "depression" (HVD). However, ventilation relative to metabolic rate (V.O2) during HVD is "not depressed" but remains increased because of associated reduction in O2 uptake (V.O2). The experiment presented here was conducted to elucidate the changes in CO2 output (V.CO2) and acid-base status during hypoxia and their role in ventilatory regulation. Ventilation, metabolic rate (V.O2, V.CO2), acid-base status and blood lactate concentration were measured during and after inhalation of hypoxic gases in halothane-anesthetized and spontaneously breathing rats. The HVD occurred at FIO2 0.08 with increased blood lactate concentration, increased venous PCO2 and a large drop in venous pH without significant changes in arterial pH and PCO2. Furthermore, the amount of reduction in V.CO2 during HVD was much smaller than that of V.O2 and the V.CO2/V.O2 ratio increased. These findings suggest that CO2 output becomes relatively higher than O2 consumption in moderate-severe hypoxia. The possible origin of CO2 accumulation in the venous blood, such as the buffering of lactic acid by bicarbonate, and its role in ventilatory stimulation are discussed. Since there was no large increase in V.E and metabolic rate in the post-hypoxic period, "O2 debt" during HVD was small.

Study Design. This study investigated the effects of hypoxia and glucose on motor functions of spinal cord, monitoring ventral root motor-evoked potential in the in vitro cervical cord preparations. Objective. To study ischemia-induced changes in ventral root motor-evoked potential of spinal cord. Summary of Background Data. Previous studies demonstrated ischemic changes caused by local circulatory impairment might be a major pathophysiologic basis of neuron damage in cord compression. Methods. Ventral root motor-evoked potential elicited by stimulation of ventrolateral funiculus was recorded from the ventral root in the isolated spinal cord preparations obtained from a newborn rat. The preparations obtained form a newborn rat. The preparations were exposed to artificial cerebrospinal fluid equilibrated with severe or mild hypoxia for 90 minutes. Inhibitory and excitatory neurotransmitter antagonists were added to artificial cerebrospinal fluid to investigate synaptic transmission. The artificial cerebrospinal fluids containing various concentrations of glucose were used to study the glucose's effects. Results. Ventral root motor-evoked potential consisted of the early and late components, which were excitatory transsynaptic potentials. The amplitudes were increased in the early phase of severe hypoxia and declined in the prolonged exopsure. In mild hypoxia, there was a sustained increase of the amplitudes. The application of inhibitory neurotransmitter antagonists abolished the augmentation of the amplitudes in the early phase of severe hypoxia. Hypoxia without glucose accelerated hypoxic change. Conclusion. Inhibitory synaptic transmission was depressed preferentially in the early phase of severe hypoxia or in mild hypoxia. Excitatory and inhibitory transmissions were suppressed in prolonged severe hypoxia. Glucose deficiency aggravated hypoxic inhibition of synaptic transmissions.

The detailed morphology of rat phrenic motoneurons (PMs) was studied in 40 electrophysiologically identified cells with intracellular injection of Neurobiotin. In 15 cells, the dendritic trees were fully analyzed by using path-distance analysis, and total surface area and volume were estimated. Based on their relative onset times (ROT; i.e., the time of firing onset relative to the onset of whole phrenic activity), PMs were classified into three types; early recruited (type E; ROT10%), late recruited (type L; ROT12.5%), and quiescent (type Q; not recruited under normal conditions). Dendrites constituted 93.3% of the surface area of cells and 38.9% of the cell volumes. The number of primary dendrites (nPD) averaged 10.1, and the mean number of terminations was 38.8. The combined diameters of primary dendrites of PMs correlated well with the total dendritic surface area and the number of dendritic terminations. Comparisons among cell types revealed that type Q cells had greater dendritic surface areas and volumes than type E or type L cells. With path-distance analysis, this difference was found to be due to differences between the cell types in the numbers of their dendrites, their combined dendritic lengths, and the number of their branches. The differences between these data and those available for cat motoneurons are discussed. The input resistance of PMs correlated with their total surface area but did not correlate with their somal surface area, indicating that, in rat, PM input resistance is a function of the entire neuronal membrane rather than of the somal surface alone.

Endothelin-1 (ET-1) in the central nervous system has been suggested to produce suppressive effects on pain transmission. We investigated the manner by which ET-1 exerts this action. ET-1 administered intracerebroventricularly produced a dose-dependent antinociceptive effect in a thermal pain test that utilized a spinal reflex to determine nociceptive thresholds. This suggested that the antinociceptive effect of ET-1 involved a descending pain inhibitory system. The antinociceptive effect was blocked by an ET A receptor antagonist but not by an ET B receptor antagonist, indicating that the action was mediated through the ET A receptor. Antagonists of opioid receptors, serotonin receptors, alpha-2 adrenergic receptors, oxytocin receptors, and dopamine receptors did not block the antinociceptive effect of ET-1. Thus, major descending inhibitory systems were probably not involved. The antinociceptive effect was blocked by intracerebroventricular administration of an alpha-1 adrenergic receptor antagonist. This indicated that the antinociceptive effect involved the activation of a supraspinal noradrenergic pathway, which in turn may activate a still unknown descending pain inhibitory system.

Inspiratory (I) discharges of the phrenic (Phr) and glossopharyngeal (stylopharyngeal muscle branch, IX) nerves were compared in the urethane anesthetized, vagi-intact and artificially ventilated rat in which respiratory rhythm was generated in synchrony with cyclic changes in airway pressure (P(aw)) produced by a ventilator. Observations were made during respiratory suppression due to excess lung inflation and bilateral cooling of the ventral medullary surface (VMS). In the control condition, regular rhythmic I bursts appeared at low baseline P(aw) phase (lung deflation) and ceased when P(aw) increased (lung inflation) in each ventilator cycle. Increase in baseline P(aw) (P(aw) > 8 cmH2O, excess lung inflation) suppressed the initiation of rhythmic I discharge. However, threshold baseline P(aw) for suppressing I bursts was higher in the IX than in the Phr nerve, and regular rhythmic I activity remained in the IX even after cessation of Phr bursts. During VMS cooling, I bursts disappeared first in the Phr and subsequently in the IX nerve and emerged always first in the IX during recovery. The results suggest that I activity of the IX (stylopharyngeal) motoneurons, which are located in the rostral part of the nucleus ambiguus, are less suppressed than that of Phr motoneurons by vagal afferents arising probably from slowly adapting pulmonary stretch receptors or by a reduction in respiratory drive from VMS. These differential responses of Phr and IX motoneurons may be ascribed to differences in activation or inhibition processes between two motoneuron groups despite both being driven by a common rhythm generator.

Electrophysiological properties of phrenic motoneurons (PMs) were studied by intracellular recordings in anesthetized, paralyzed, and artificially ventilated adult rats. Our results revealed that rat PMs, as compared to cat PMs, had a shorter afterhyperpolarization duration (55 +/- 13 ms; mean +/- SD), a narrower half-width of action potential, a larger membrane input resistance (Rm; 2.0 +/- 0.6 M omega), a smaller rheobase (Irh) and a shorter minimum paired pulse interval to provoke the second spike (1.9 +/- 0.6 ms). These features indicate that the rat PMs possess higher frequency responsiveness than cat PMs. Based on the time of firing onset relative to the onset of whole phrenic nerve activity (relative onset time; ROT) or on the absence of discharge during inspiration, the PMs could be classified into four types: early recruited with ROT10.0%; late recruited with 12.5%ROT37.5%; very late recruited with ROT45%, and quiescent which was not recruited during normal experimental conditions. A number of differences in the membrane properties such as end-expiratory membrane potential, Rm, and Irh among the four cell types are discussed in relation to the recruitment order. Inhibitory postsynaptic potentials (IPSPs) were present during the late expiratory phase (stage II expiration) in all PMs tested.

Bilateral cooling of the parapyramidal region in the rostral ventral medullary surface (VMS) elicited a reduction in respiratory frequency and phrenic inspiratory activity in halothane anesthetized rats. A distinct cluster of neurons (nucleus parapyramidalis superficialis) was found in a superficial layer (10–150 μm from the surface) just beneath the area where cooling produced suppression of respiration. The rat VMS layer contains neural substrates which regulate the respiratory rhythm generation and inspiratory neural output.

The long-term adjustment of ventilation and blood gases throughout life was studied in halothane anaesthetized male Wistar rats of various ages (1.5-20 months). Basal metabolic rate (O2 consumption, CO2 production), ventilation and ventilatory response to CO2 changed significantly during growth and aging, whereas arterial partial pressure of CO2 (PaCO2) and pH remained unchanged. Changes in the rate of CO2 production were associated with proportional changes in alveolar ventilation and in the sensitivity of the ventilatory control system to a CO2 stimulus at various ages. Ventilatory equivalent (ratio of alveolar ventilation to CO2 production) and the slope of the CO2/ventilation response line normalized for CO2 production were maintained constant throughout life, despite significant changes in the breathing pattern. These findings suggest that PaCO2 homeostasis is maintained by ventilatory regulation coupled tightly with CO2 production throughout life.

To determine the neural mechanism of hypoxic respiratory inhibition, discharge patterns of efferent phrenic (Phr), vagal superior laryngeal (Xsl), and vagal pharyngeal (Xphar) nerves were analyzed during systemic hypoxia in the urethane-anesthetized, vagotomized and artificially ventilated rat. In the carotid sinus nerve (CSN) intact rat, moderate hypoxia (end-tidal Po2, 40-50 mmHg) caused an initial increase in respiratory activity which was followed by inhibition due to reduction in respiratory frequency (f). The decrease in f was associated with prolongation of decremental Xphar expiratory (E) activity and retardation of the onset of inspiratory (I) activity. Integrated peak Phr or Xs1 I and Xphar E activities remained augmented during respiratory inhibition. After bilateral CSN section, moderate hypoxia produced an extreme reduction in f due to delayed onset of I activity and a strong reduction in the Xphar E activity. Phr and Xs1 I activities were little affected, and changes in inspiratory time were small. These results suggest that hypoxia centrally inhibits the process of initiating the onset of rhythmic I activity and the activity of decremental Xphar E motoneurons. Carotid chemoreceptor stimulation was inadequate to offset the central inhibitory effect of hypoxia on the onset of I activity.

Respiratory long-term facilitation (LTF) is a long-lasting (>1 h) augmentation of respiratory motor output that occurs even after cessation of hypoxic stimuli, is serotonin-dependent, and is thought to prevent sleep-disordered breathing such as sleep apnea. Raphe nuclei, which modulate several physiological functions through serotonin, receive dense projections from orexin-containing neurons in the hypothalamus. We examined possible contributions of orexin to ventilatory LTF by measuring respiration in freely moving prepro-orexin knockout mice (ORX-KO) and wild-type (WT) littermates before, during, and after exposure to intermittent hypoxia (IH; 5 × 5 min at 10% O2), sustained hypoxia (SH; 25 min at 10% O2), or sham stimulation. Respiratory data during quiet wakefulness (QW), slow wave sleep (SWS), and rapid-eye-movement sleep were separately calculated. Baseline ventilation before hypoxic stimulation and acute responses during stimulation did not differ between the ORX-KO and WT mice, although ventilation depended on vigilance state. Whereas the WT showed augmented minute ventilation (by 20.0 ± 4.5% during QW and 26.5 ± 5.3% during SWS; n = 8) for 2 h following IH, ORX-KO showed no significant increase (by −3.1 ± 4.6% during QW and 0.3 ± 5.2% during SWS; n = 8). Both genotypes showed no LTF after SH or sham stimulation. Sleep apnea indexes did not change following IH, even when LTF appeared in the WT mice. We conclude that LTF occurs during both sleep and wake periods, that orexin is necessary for eliciting LTF, and that LTF cannot prevent sleep apnea, at least in mice.

We examined whether intrinsic orexin participates in the control of breathing in a vigilance state-dependent manner. Ventilation was recorded together with electroencephalography and electromyograp...

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.

We have previously shown that some features of the defense response, such as increases in arterial blood pressure (AP), heart rate (HR), and ventilation were attenuated in prepro-orexin knockout (ORX-KO) mice. Here, we examined whether the same was true in orexin neuron-ablated [orexin/ataxin-3 transgenic mice (ORX/ATX-Tg)] mice. In addition, we examined other features of the defense response: skeletal muscular vasodilation and shift of baroreceptor reflex. In both anesthetized and conscious conditions, basal AP in ORX/ATX-Tg mice was significantly lower by approximately 20 mmHg than in wild-type (WT) controls, as was the case in ORX-KO mice. The difference in AP disappeared after treatment with an alpha-blocker but not with a beta-blocker, indicating lower sympathetic vasoconstrictor outflow. Stimulation of the perifornical area (PFA) in urethane-anesthetized ORX/ATX-Tg mice elicited smaller and shorter-lasting increases in AP, HR, and ventilation, and skeletal muscle vasodilation than in WT controls. In addition, air jet stress-induced elevations of AP and HR were attenuated in conscious ORX/ATX-Tg mice. After pretreatment with a beta-blocker, atenolol, stimulation of PFA suppressed phenylephrine (50 microg/kg iv)-induced bradycardia (DeltaHR=-360+/-29 beats/min without PFA stimulation vs. -166+/-26 during stimulation) in WT. This demonstrated the resetting of the baroreflex. In ORX/ATX-Tg mice, however, no significant suppression was observed (-355+/-16 without stimulation vs. -300+/-30 during stimulation). The present study provided further support for our hypothesis that orexin-containing neurons in PFA play a role as a master switch to activate multiple efferent pathways of the defense response and also operate as a regulator of basal AP.

Previous studies have demonstrated the virtual lack of analgesia in mu opioid receptor knockout mice after systemic administration of morphine. Thus, it has been suggested that analgesic actions of morphine are produced via the mu opioid receptor, despite its ability to bind to kappa and delta receptors in vitro. However, it is not clear whether the results of these studies reflect the effect of morphine in the spinal cord. In the present study, we report study of the analgesic actions of spinally-administered morphine and other opioid receptor agonists in mu opioid receptor knockout and wild type mice. Morphine produced a dose-dependent antinociceptive effect in the tail flick test in the knockout mice, although higher doses were needed to produce antinociception than in wild type mice. The antinociceptive effect of morphine was completely blocked by naloxone (a non-selective opioid antagonist) and nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist), but not by naltrindole (a selective delta-opioid receptor antagonist). U-50,488H (a selective kappa-opioid receptor agonist) also produced a dose-dependent antinociceptive effect in knockout mice but presented lower analgesic potency in knockout mice than in wild type mice. Analgesic effects of [d-Pen2,d-Pen5]enkephalin (DPDPE, a selective delta-opioid receptor agonist) were observed in wild type mice but abolished in knockout mice. SNC80 (a selective delta-opioid receptor agonist) was not antinociceptive even in wild type mice. The present study demonstrated that morphine can produce thermal antinociception via the kappa opioid receptor in the spinal cord in the absence of the mu opioid receptor. Lower potency of U50,488H in mu opioid receptor knockout mice suggests interaction between kappa and mu opioid receptors at the spinal level.

Stressor induces not only cognitive, emotional and behavioral changes but also autonomic changes. Although research on the neural circuits underlying such autonomic changes has implicated the hypothalamus in the defense response against stressors, neurotransmitters in this multifaceted and coordinated response have not been revealed. In this brief review, here we summarize our recent discovery using orexin knockout mice and orexin neuron-ablated mice of possible contribution of orexin in the defense response and discuss future directions.

Orexins are synthesized by neurons in the hypothalamus and contribute to multiple physiological functions. Orexin fibers innervate many regions of the CNS, which include areas involved in descending control of pain. We examined the role orexins may play in endogenous modulation of pain transmission using prepro-orexin (precursor of orexin A and B) knockout mice. Baseline pain thresholds of knockout and wild type mice were not different. Knockout mice presented greater degree of hyperalgesia induced by peripheral inflammation and less stress-induced analgesia than wild type mice. Double staining of orexin and c-Fos in wild type mice revealed activation of orexin neurons under both conditions. These results suggest that persistent pain and stress activate orexin neurons, which act to inhibit pain transmission.

Postnatal development of blood pressure, heart rate and their regulation by arterial baroreceptor reflex in mice was examined. We first confirmed that simultaneous recordings of pulsatile blood pressure by the “servo null” method and the conventional catheter method gave almost identical tracings in halothane-anesthetized adult mice. We then measured blood pressure by servo null method together with electrocardiograph in mice of various ages from newborn to adult. Mean blood pressure increased progressively with age from 19±2 mm Hg in P0 newborn to 74±1 in adult mice, while heart rate initially increased from 365±12 bpm in newborn to 441±15 in infant (7 days old), and then decreased to 337±15 in adult mice. Between 1 and 2 weeks of age, gain of arterial baroreceptor reflex abruptly increased from a newborn value of 0.3 to a near adult value of 1.1 ms/mm Hg. On the other hand, sensitivity to anesthesia did not differ except for P1 and P2 newborns. We conclude that pulsatile blood pressure can be accurately measured by the servo null method even in the newborn mice and that baroreflex heart rate control mature at around 2 weeks after birth in the mice.

To clarify a possible origin of 'respiratory like' rhythmic activities observed in in vitro brainstem preparation, the phrenic (Phr) and cranial nerve (XII or IX) inspiratory activities were analyzed in halothane-anesthetized, vagotomized and artificially ventilated newborn (2--6 days after birth) and young adult rats (30--50 days) during altered chemical stimuli and prolonged asphyxia at 25 degrees C. The newborn rat showed regular rhythmic inspiratory discharges of short duration, and their responses to CO(2) and hypoxia did not differ from those seen in adult rats. In the newborn rat the Phr and cranial nerve inspiratory discharges increased first, then respiratory frequency decreased and finally ceased completely for approximately 1--2 min during asphyxia. Thereafter, 'secondary' rhythmic inspiratory activity emerged at a slower rate with decremental inspiratory discharge profile, which persisted for a period more than 40 min of asphyxia. A normal respiratory activity recovered after resumption of artificial ventilation. Though young adult rats exhibited similar sequential changes in respiratory activity during asphyxia, the 'secondary' rhythmic activity persisted for a period of several min only. The pattern of 'secondary' respiratory activity corresponded well with that of rhythmic activities seen in the isolated medullary block preparation of newborn rat. 'Respiratory like' activity found in isolated medullary preparations of newborn animals may arise from a mechanism that generates 'secondary' (or so called 'gasping' type) rhythmic inspiratory activity during prolonged asphyxia in in vivo preparations.

It has been hypothesized that respiratory "gain" to hypoxic stimulus is not depressed in hypothermic animals though ventilation and that metabolic O(2) demand (Vo(2)) decreases with reduction in body temperature. The present study addressed this hypothesis by quantitative analysis of ventilatory and carotid chemoreceptor responsiveness to hypoxia during induced hypothermia in halothane anesthetized and spontaneously breathing rats. Rectal temperature was lowered from 37 degrees C (normothermia) to 30 and 25 degrees C by cooling body surface at comparable anesthetic depth without inducing shivering. Ventilation (V(E)), V(O2), PaO(2) and carotid chemoreceptor afferent discharges were measured during hyperoxic and hypoxic gas breathing. PaO(2) values at the same Fi(O2) (range 0. 35-0.08) decreased progressively as rectal temperature decreased. Both the V(E)/V(O2)- and chemoreceptor discharge-response curves shifted toward a lower PaO(2) range with a slight increase in the response slopes during hypothermia. The results indicated that the sensitivity of carotid chemoreceptor and ventilatory responses to hypoxia did not decrease at reduced body temperature. It is concluded that carotid chemoreceptor mediated regulation of ventilation is tightly coupled to changes in PaO(2 )range in halothane anesthetized rats during induced hypothermia.

Background: Changes in hepatic oxygen metabolism in relation to the extent of liver regeneration are expected after partial hepatectomy. There are few reports, however, about hepatic oxygen metabolism during liver regeneration. In this study, we evaluated changes in hepatic oxygen metabolism related to the regeneration rate, and the relationship between hepatic venous oxygen saturation (Shvo 2 ) and liver regeneration after partial hepatectomy. Methods: The work was done using 50% hepatectomized rats with continuous infusion of octreotide for inhibition of liver regeneration or with saline as control. The hepatic hemodynamics, oxygen metabolism, and Shvo 2 levels as well as the regenerating liver status were evaluated for 3 days after hepatectomy. Results: Administration of octreotide resulted in a significant reduction of the regenerating liver weight on days 1 and 3 after hepatectomy compared with the control group. Significantly decreased DNA synthesis and proliferating cell nuclear antigen labeling index were also found on day 1. Meanwhile, hepatic oxygen consumption (HVO 2 ) and oxygen extraction ratio were significantly decreased in the octreotide-treated group on day 1. In contrast, the Shvo 2 levels in the octreotide-treated group were significantly higher than those in the control group, and were inversely correlated with the HVO 2 . Conclusion: The remnant liver demands an increased amount of oxygen in relation to the extent of regeneration, and changes in the Shvo 2 are inversely correlated with the HVo 2 . Therefore, monitoring the Shvo 2 could be useful for estimating liver regeneration after partial hepatectomy.

Cyanate derivatives such as NaOCN have been known to increase the hypoxia tolerance of animals by increasing the affinity of hemoglobin (Hb) to O(2). To clarify the mechanism of this increase in hypoxia tolerance, we examined changes in metabolic rate and cardiovascular parameters during a hypoxia test in halothane-anesthetized, NaOCN-treated and spontaneously breathing rats (50 mg/kg/d S.C., 10 d). Control animals received saline. The capillary density in the skeletal muscle (sternocleidomastoid muscle), cardiac papillary muscle and medulla oblongata was also examined histologically. The Hb-O(2) affinity index, P(50), decreased from 38 (control rat) to 24 mmHg in NaOCN-treated rats. During hyperoxic gas breathing, the rat treated with NaOCN showed a significantly lower metabolic rate (V(.)O(2), V(.)CO(2)), higher cardiac stroke volume, slower heart rate, lower PvO(2), and lower O(2) extraction ratio than those in control rats. The NaOCN-treated rats exhibited well-maintained arterial blood pressure and a larger cardiac output response to reduction in FIO(2) to 0.10-0.08. The increase in O(2) extraction ratio with reduction in FIO(2) was larger in NaOCN-treated than in control rats. The circulatory and metabolic depressions at FIO(2) 0.05 were effectively attenuated in NaOCN-treated rats. The capillary density of the cardiac muscle and medulla oblongata but not the skeletal muscle was significantly higher in NaOCN-treated rats than in control rats. The greater hypoxia tolerance in NaOCN-treated rats is ascribed to the combined effects of left shift of Hb-O(2) dissociation curve, lower basal V(. )O(2), higher capillary density in the heart, and brain, and other adaptive mechanisms induced probably by prolonged tissue hypoxia.

To clarify the ion/water secretion mechanism in the nasal epithelial cells, the Ussing chamber method was applied to the nasal mucosa isolated from guinea pigs. The preparation, which contained surface epithelial cells, showed a small but consistent potential difference between mucosal and submucosal sides (mucosal surface negative to submucosa). The short-circuit current (Isc) across the epithelial layer was measured, and the effects of Na+ and/or Cl- transport inhibitors and methacholine (MCh) on Isc were analyzed. The basal Isc was almost totally suppressed by the combined application of amiloride (Na+ transport inhibitor) and low-Cl- Krebs Ringer (KR) solution or solutions containing Cl- transport inhibitors (furosemide or DPC). The application of MCh elicited triphasic Isc responses, i.e., initial transient increase (phase 1) followed by a small decrease (phase 2) and further sustained increase (phase 3) in Isc. A possible ionic mechanism underlying phase 1 and 3 responses was analyzed. The Phase 1 response was greatly reduced by low-Cl- KR solution or furosemide but not influenced by amiloride. The Phase 3 response was augmented by amiloride and suppressed by low-Cl- KR solution, furosemide or DPC. These findings indicated that the basal Isc was associated with Cl- secretion and/or Na+ absorption across epithelial cells under short-circuit condition and that MCh increased Isc probably via enhancing Cl- secretion in the nasal surface epithelial cell.

Recently generated knockout mice with disrupted genes encoding endothelin (ET)-1 showed an elevation of arterial blood pressure (AP) and supplied an evidence for intrinsic ET-1 as one of the physiological regulators of systemic AP. Little is yet known, however, why deficiency of ET-1, which was originally found as a potent vasoconstrictor, led to higher AP in these mice. To address this apparent paradox, we first developed a method to measure renal sympathetic nerve activity (RSNA) in mice using rats as reference and successively compared it between normal and ET-1 deficient mice. RSNA was successfully recorded in urethane-anesthetized and artificially ventilated mice by a slight modification of the method used for rats. At basal condition, mean AP (MAP) and RSNA in ET-1 deficient mice (105+/-2 mmHg and 9.71+/-1.49 muVs, n=20) were significantly higher than those in wild-type mice (96+/-2 mmHg and 5. 07+/-0.70 muVs, n=25). Basal heart rate (HR) and baroreflex-control of HR was not significantly different between the two. On the other hand, resting RSNA, RSNA range, and maximum RSNA were significantly greater in ET-1 deficient mice, and thus MAP-RSNA relationship was upwards reset. Hypoxia-induced increase in RSNA was not different between ET-1 deficient (73.4+/-9.4%) and wild-type mice (91.2+/-12.0%), while hypercapnia-induced one was significantly attenuated in ET-1 deficient mice (18.8+/-3.6 vs. 39.1+/-5.2% at 10% CO2). These results indicate that endogenous ET-1 participates in the central chemoreception of CO2 and reflex control of the RSNA. Baroreceptor resetting and normally preserved hypoxia-induced chemoreflex may explain a part of the elevation of AP in ET-1 deficient mice.

To clarify the control mechanism of ventilation during posture change, ventilatory parameters, PETCO2, and ventilatory response to CO2 were examined in 11 healthy male subjects at supine (0 degrees) and 75 degrees head-up tilt positions. Minute expiratory ventilation (V.E), tidal volume (VT), respiratory frequency (f), end-tidal and transcutaneous PCO2 and CO2 output (V.CO2), and ventilatory response to CO2 were measured during a steady state condition. V.E (V.A) and VT increased significantly at 75 degrees tilt with significant decrease in PETCO2 from 40.1 mmHg (0 degrees) to about 36.1 mmHg (75 degrees). Transcutaneous PCO2 also decreased during tilt, by 3.3 mmHg. Physiological dead space (VD/VT) and V.CO2, however, remained unchanged, and ventilatory equivalent (V.E/V.CO2, V.A/V.CO2) increased significantly. The CO2-ventilatory response curve shifted upward (or leftward) without significant change in the response slope. At 75 degrees tilt, EMG activity of gastro-cnemius muscle increased. These findings suggested that PETCO2 decreased because of increased V.E (V.A) with a leftward shift of CO2-ventilatory response curve. Various signals such as afferents from lower extremities might have net stimulatory effects on a CO2-ventilation control system to reset the controlled level of PETCO2 to a lower range, but without significant change in CO2-ventilatory response during upright position.

Physiological roles of ventilatory responses to acute hypoxia in the regulation of O2 transport and consumption were evaluated quantitatively in halothane anesthetized and spontaneously breathing rats with or without peripheral chemoreceptor afferents. Ventilation (VE), blood gas values, cardiac output (Q), and whole-body O2 consumption (VO2) were measured at various levels of inspired O2 concentration (FIO2) before and after denervation of carotid chemoreceptor afferents. In the carotid sinus nerve (CSN)-intact rat, the reduction in FIO2 from0.27 (hyperoxia) to about 0.10 (mild-moderate hypoxia) elicited an augmentation of VE which was accompanied by a well maintained total amount of O2 delivered to tissue (Q x O2 content of arterial blood, DO2) and VO2. The increase in VE was, however, turned off and the VE value did not differ from that in hyperoxia at FIO2 lower than 0.10 (moderate-severe hypoxia) (hypoxic ventilatory depression, HVD). Significant decreases in Q, DO2, and VO2 were also seen at FIO20.10. After CSN section, VE, Q, DO2, and VO2 values decreased progressively with the reduction in FIO2 from hyperoxia to mild hypoxia (FIO2 = 0.20-0.15). These findings indicate that the normal DO2 and VO2 are preserved in the presence of carotid chemoreceptor afferents in the FIO2 range0.10 and that HVD is accompanied by the decrease in VO2. Summarizing all results, VO2 decreased linearly with the reduction in PaO2 lower than about 60 mmHg or DO2 lower than 4 ml/min/100 g bw (critical PaO2 or DO2 for metabolic suppression), above which VO2 remained unchanged. The decrease in VO2 in lower PaO2 or DO2 levels (hypoxic hypometabolism) ensues perhaps from "adaptive" mechanisms reducing O2 demand when adequate O2 supply to tissue is limited.

The effect of low-power He-Ne laser on synaptic transmission was studied in the superior cervical sympathetic ganglion of Wistar rats. Extracellular and intracellular recordings were made from isolated ganglia before and after laser irradiation. In addition, effects of ganglion irradiation on skin blood flow were observed in in vivo preparations. Irradiation on the isolated ganglion decreased the amplitude of evoked compound action potentials in the extracellular recordings. Intracellular recordings from ganglion cells revealed that the neuronal membrane was hyperpolarized, while threshold potential and latency of response were unchanged. The reduction of skin blood flow during preganglionic nerve stimulation lessened by irradiation on the ganglion. Our results indicate that low-power He-Ne laser depresses sympathetic ganglionic transmission, and hyperpolarization of ganglionic cells takes part in the mechanism of action.

Inspiratory (I) activities of glossopharyngeal (IX) and phrenic (Phr) nerves were compared during hypocapnia or hypoxia in the urethane anesthetized and carotid deafferented rat. Hypoxic or hypocapnic suppression of I activity was smaller in the IX than in the Phr nerve. A small ramp like rhythmic IX activity was seen even in the absence of Phr discharge during hypocapnia or hypoxia. Respiratory rhythmicity may be better recognized by IX motoneuron activity during suppression by altered chemical stimuli.

Central respiratory rhythmicity and/or timing has been determined by the trajectory of phrenic (Phr) nerve discharges. Cranial nerves innervating the upper airway muscles also display respiratory modulated activity synchronized with Phr activity. These cranial nerve motoneurons as well as spinal Phr motoneurons (or Phr driving medullary pre-motor neurons) are driven by a common rhythm generating mechanism. There are, however, significant differences in the discharge pattern and responses to chemical stimuli between Phr and cranial nerve respiratory activity1. The onset of inspiratory activity of the cranial nerves is much earlier than that of Phr nerve2, 3, 4. Changes in chemical stimuli (Pao2, Paco2) initiate differential effects on I activity among various respiratory nerves, including Phr and cranial nerves3, 5, 6, 7. On the other hand, cranial nerve respiratory activities are sensitive to anesthesia and/or sleep stage, and hence have not been considered as major output signals for observation of central respiratory rhythmicity8, 9, 10. In the present experiment we found that the glossopharyngeal (IX) nerve I activity showed much smaller suppression during hypocapnia or hypoxia than the Phr I discharge did. Furthermore a small ramp-like rhythmic IX activity even without Phr burst was seen during hypocapnic or hypoxic respiratory suppression.

The respiratory effect of progestin differs among various animal species and humans. The rat does not hyperventilate in response to exogenous progestin. The present study was conducted to determine whether administration of combined progestin and estrogen prompts ventilatory stimulation in the male rat. Ventilation, blood gases, and metabolic rates (O2 consumption and CO2 production) were measured in the awake and unrestrained male Wistar rat. The combined administration of a synthetic potent progestin (TZP4238) and estradiol for 5 days significantly increased tidal volume and minute expiratory ventilation (VE), reduced arterial PCO2, and enhanced the ventilatory response to CO2 inhalation (delta VE/delta PCO2). On the other hand, respiratory frequency, O2 consumption, CO2 production, and body temperature were not affected. The arterial pH increased slightly, with a concomitant decrease in plasma [HCO3-]. Administration of either TZP4238 or estradiol alone or vehicle (Tween 80) had no effect on respiration, blood gases, and ventilatory response to CO2. The results indicated that respiratory stimulation following combined progestin plus estradiol treatment in the male rat involves activation of process(es) that regulate tidal volume and its augmentation during CO2 stimulus.

To estimate whether H+ is the unique stimulus of the medullary chemosensor, ventilatory effects of HCO3- and/or CO2 applied on the ventral medullary surface using an improved superfusion technique and of CO2 inhalation were compared in halothane-anesthetized spontaneously breathing rats. Superfusion with low [HCO3-]-acid mock cerebrospinal fluid (CSF) (normal Pco2) induced a significant increase in ventilation, with an accompanying reduction in endtidal Pco2 (PETco2). High [HCO3-]-alkaline CSF depressed ventilation. Changes in Pco2 of superfusing CSF, on the other hand, had no significant effect despite the similar changes in pH. Simultaneous decrease in [HCO3-] and Pco2 of mock CSF with normal pH also maintained stimulated respiration. CO2 inhalation during superfusion with various [HCO3-] solutions caused further increase in ventilation as PETco2 increased. The results suggest that the surface area of the rat ventral medulla contains HCO3- (or H+)-sensitive respiratory neural substrates which are, however, little affected by CO2 in the subarachnoid fluid. A CO2 (or CO2-induced H+)-sensitive chemosensor responsible for the increase in ventilation during CO2 inhalation may exist elsewhere functionally apart from the HCO3- (or H+)-sensitive sensor in the examined surface area.