Ventilatory long-term facilitation in mice can be observed during both sleep and wake periods and depends on orexin.

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 x 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.

Contribution of orexin in hypercapnic chemoreflex: evidence from genetic and pharmacological disruption and supplementation studies in mice.

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% CO(2)) or hypoxic (15 or 10% O(2)) 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 mlxmin(-1)xg(-1)x% CO(2)(-1)) was significantly blunted compared with that in WT mice (0.51 +/- 0.05 mlxmin(-1)xg(-1)x% CO(2)(-1)). Supplementation of orexin-A or -B (3 nmol) partially restored the hypercapnic chemoreflex in ORX-KO mice (0.28 +/- 0.03 mlxmin(-1).g(-1)x% CO(2)(-1) for orexin-A and 0.32 +/- 0.04 mlxmin(-1)xg(-1)x% CO(2)(-1) for orexin-B). In addition, injection of SB-334867 (30 nmol) in WT mice decreased the hypercapnic chemoreflex (0.39 +/- 0.04 mlxmin(-1)xg(-1)x% CO(2)(-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 CO(2) sensitivity at least during wake periods in mice.

Vigilance state-dependent attenuation of hypercapnic chemoreflex and exaggerated sleep apnea in orexin knockout mice.

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).%CO(2)(-1)) was significantly smaller than that in WT mice (0.38 +/- 0.04 ml.min(-1).g(-1).%CO(2)(-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 CO(2) sensitivity during wakefulness and in preserving ventilation stability during sleep.

Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors.

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.

Multiple components of the defense response depend on orexin: evidence from orexin knockout mice and orexin neuron-ablated mice.

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.

Persistent pain and stress activate pain-inhibitory orexin pathways.

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.

Differential respiratory effects of [Dmt1]DALDA and morphine in mice.

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.

Respiratory and cardiovascular actions of orexin-A 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.

Sleep apnea and effect of chemostimulation on breathing instability in mice.

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.

Inhibitory actions of endothelin-1 on pain processing.

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 ETA receptor antagonist but not by an ETB receptor antagonist, indicating that the action was mediated through the ETA 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.

Acute effects of ozone exposure on lung function in mice sensitized to ovalbumin.

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-alpha (TNF-alpha) 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 (P<0.01) 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.

Orexin neuron-mediated skeletal muscle vasodilation and shift of baroreflex during defense response in mice.

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.

Blood pressure of endothelin-3 null (-/-) knockout mice and endothelin A receptor null (-/-) knockout mice under anaesthesia.

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.

Attenuated defense response and low basal blood pressure in orexin knockout mice.

The perifornical area of the hypothalamus has been known as the center for the defense response, or "fight or flight" response, which is characterized by a concomitant rise in arterial blood pressure (AP), heart rate (HR), and respiratory frequency (Rf). We examined whether orexin, a recently identified hypothalamic neuropeptide, contributes to the defense response and basal cardiovascular regulation using orexin knockout mice. Microinjection of a GABA-A receptor antagonist, bicuculline methiodide (0.1-1 mM in 20 nl), to the perifornical area in urethane-anesthetized wild-type mice elicited dose-dependent increases in AP, HR, and Rf. Although similar changes were observed in orexin knockout mice, intensities were smaller and duration was shorter than those in wild-type mice. Moreover, in an awake and freely moving condition, telemeter-indwelling orexin knockout mice showed diminished cardiovascular and behavioral responses to emotional stress in the resident-intruder test. We also found that basal AP in orexin knockout mice was significantly lower in both anesthetized (117 +/- 8 mmHg in wild type and 92 +/- 3 in knockout) and conscious (125 +/- 6 mmHg in wild type and 109 +/- 2 in knockout) conditions. alpha-Adrenergic blockade with prazosin or ganglion blockade with hexamethonium canceled the difference in basal AP. HR and cardiac contractile parameters by echocardiography did not differ between the two strains of mice. These results indicate lower sympathetic vasoconstrictor tone in knockout mice. The present study suggests that orexin-containing neurons in the perifornical area play a role as one of the efferent pathways of defense response and also operate as a regulator of AP at basal condition by activating sympathetic outflow.

Interleukin-13 induces thymus and activation-regulated chemokine (CCL17) in human peripheral blood mononuclear cells.

BACKGROUND: In allergic inflammation involving allergic rhinitis, the predominance of Th(2) lymphocytes is one of the primary causal agents in promotion of the allergic condition. Thymus and activation-regulated chemokine (TARC/CCL17) is a recently identified chemokine that induces the development of Th(2) lymphocytes. One of the sources of TARC has been reported to be peripheral blood mononuclear cells (PBMCs). OBJECTIVE: We investigated TARC production from PBMCs by the stimulation of specific antigens and Th(2) type cytokines. METHOD: PBMCs were isolated from both allergic rhinitis patients and healthy volunteers. PBMCs were incubated with cytokine. TARC mRNA expression was examined by real time PCR methods and the amount of TARC production was examined by ELISA. RESULTS: IL-13 was found to be the most potent inducer for TARC mRNA expression and protein production in PBMCs. Furthermore, tumour necrosis factor alpha and IL-13 synergistically induce TARC. The amount of TARC from allergic rhinitis patients was significantly larger than that from healthy volunteers. Moreover, TARC was induced by a specific antigen, and was 35% inhibited by an anti-IL-13 neutralizing antibody. CONCLUSION: These results indicate that IL-13 is important in TARC mediated Th(2) lymphocytes infiltration in the nasal mucosa.