Sexually dimorphic effects of prenatal diazepam exposure on respiratory control and the monoaminergic system of neonate and young rats.

Pregnancy is highly affected by anxiety disorders, which may be treated with benzodiazepines, especially diazepam (DZP), that can cross the placental barrier and interact with the fetal GABAergic system. We tested whether prenatal exposure to DZP promotes sex-specific postnatal changes in the respiratory control of rats. We evaluated ventilation ([Formula: see text]) and oxygen consumption ([Formula: see text] O2) in resting conditions and under hypercapnia (7% CO2) and hypoxia (10% O2) in newborn [postnatal day (P) 0-1 and P12-13)] and young (P21-22) rats from mothers treated with DZP during pregnancy. We also analyzed brainstem monoamines at the same ages. DZP exposure had minimal effects on room air-breathing variables in females, but caused hypoventilation (drop in [Formula: see text]/[Formula: see text] O2) in P12-13 males, lasting until P21-22. The hypercapnic ventilatory response was attenuated in P0-1 and P12-13 DZP-treated females mainly by a decrease in tidal volume (VT), whereas males had a reduction in respiratory frequency (fR) at P12-13. Minor changes were observed in hypoxia, but an attenuation in [Formula: see text] was seen in P12-13 males. In the female brainstem, DZP increased dopamine concentration and decreased 5-hydroxyindole-3-acetic acid (5-HIAA) and the 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine ratio at P0-1, and reduced DOPAC concentration at P12-13. In males, DZP decreased brainstem noradrenaline at P0-1. Our results demonstrate that prenatal DZP exposure reduces CO2 chemoreflex only in postnatal females and does not affect hypoxia-induced hyperventilation in both sexes. In addition, prenatal DZP alters brainstem monoamine concentrations throughout development differently in male and female rats.

Melanin-concentrating hormone regulates the hypercapnic chemoreflex by acting in the lateral hypothalamic area.

NEW FINDINGS: What is the central question of this study? Melanin-concentrating hormone (MCH) suppresses the hypercapnic chemoreflex: what is the mechanism by which this effect is produced? What is the main finding and its importance? MCH acting in the lateral hypothalamic area but not in the locus coeruleus in rats, in the light period, attenuates the hypercapnic chemoreflex. The data provide new insight into the role of MCH in the modulation of the hypercapnic ventilatory response. ABSTRACT: Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide involved in a broad range of homeostatic functions including regulation of the hypercapnic chemoreflex. We evaluated whether MCH modulates the hypercapnic ventilatory response by acting in the lateral hypothalamic area (LHA) and/or in the locus coeruleus (LC). Here, we measured pulmonary ventilation ( V ̇ E ${\dot V_{\rm{E}}}$ ), body temperature, electroencephalogram (EEG) and electromyogram (EMG) of unanaesthetized adult male Wistar rats before and after microinjection of MCH (0.4 mM) or MCH receptor 1 (MCH1-R) antagonist (SNAP-94847; 63 mM) into the LHA and LC, in room air and 7% CO2 conditions during wakefulness and sleep in the dark and light periods. MCH intra-LHA caused a decreased CO2 ventilatory response during wakefulness and sleep in the light period, while SNAP-94847 intra-LHA increased this response, during wakefulness in the light period. In the LC, MCH or the MCH1-R antagonist caused no change in the hypercapnic ventilatory response. Our results suggest that MCH, in the LHA, exerts an inhibitory modulation of the hypercapnic ventilatory response during the light-inactive period in rats.

Metabolic and respiratory chemosensitivity and brain monoaminergic responses to cold exposure in chicks subjected to thermal manipulation during incubation.

We evaluated ventilation (V˙E), body temperature (TB), oxygen consumption (V˙ O2), respiratory equivalent (V˙E/ V˙ O2), and monoamine concentrations of 14-day-old (14d) male and female chicks from eggs incubated at low (LT, 36 °C), control (CT, 37.5 °C) and high (HT, 39 °C) temperature during the early embryonic phase, to normoxia, hypercapnia and hypoxia under exposure to cold environment (20 °C). At normoxia, acute cold exposure did not affect the ventilatory variables, with the exception of HT males, in which cold prevented the reduced V˙E observed under thermoneutral conditions. Exposure to 20 °C caused a decrease in TB in both sexes, and LT and HT females presented a greater hypothermic response. Hypercapnia combined with cold did not alter the ventilatory variables, but LT females and CT males and females showed a blunted CO2-induced hyperventilation due to a higher V˙ O2, compared to the same groups in thermoneutral conditions. Unlike with thermoneutral conditions, the blunted hypercapnic hyperventilation observed in the HT groups was not observed during cold challenge. CO2 exposure promoted a similar decrease in TB in the thermoneutral and acutely cold exposed groups, while LT females under cold condition presented a blunted hypothermic response. During hypoxia, cold challenge attenuated the increase in V˙E in LT females and HT males, due to changes in VT. Hypoxic metabolic depression was greater in LT females and males and HT males during cold exposure, while no change in V˙E/ V˙ O2 was observed. The only alteration in monoaminergic concentration under cold challenge was an increase in brainstem 5-HIAA and 5-HIAA/5-HT ratio in HT females, and an enhanced 5-HT concentration in HT males. In summary, thermal manipulation during embryogenesis induces 14d old chicks to respond differently to cold stress with LT females and HT males being more sensitive.

Regulated hypothermia in response to endotoxin in birds.

KEY POINTS: The costs associated with immune and thermal responses may exceed the benefits to the host during severe inflammation. In this case, regulated hypothermia instead of fever can occur in rodents as a beneficial strategy to conserve energy for vital functions with consequent tissue protection and hypoxia prevention. We tested the hypothesis that this phenomenon is not exclusive to mammals, but extends to the other endothermic group, birds. A decrease in metabolic rate without any failure in mitochondrial respiration, nor oxygen delivery, is the main evidence supporting the regulated nature of endotoxin-induced hypothermia in chicks. Thermolytic mechanisms such as tachypnea and cutaneous vasodilatation can also be recruited to facilitate body temperature decrease under lipopolysaccharide treatment, especially in the cold. Our findings bring a new perspective for evolutionary medicine studies on energy trade-off in host defence because regulated hypothermia may be a phenomenon spread among vertebrates facing a severe immune challenge. ABSTRACT: A switch from fever to regulated hypothermia can occur in mammals under circumstances of reduced physiological fitness (e.g. sepsis) to direct energy to defend vital systems. Birds in which the cost to resist a pathogen is additive to the highest metabolic rate and body temperature (Tb ) among vertebrates may also benefit from regulated hypothermia during systemic inflammation. Here, we show that the decrease in Tb observed during an immune challenge in birds is a regulated hypothermia, and not a result of metabolic failure. We investigated O2 consumption (thermogenesis index), ventilation (respiratory heat loss), skin temperature (sensible heat loss) and muscle mitochondrial respiration (thermogenic tissue) during Tb fall in chicken chicks challenged with endotoxin [lipopolysaccharide (LPS)]. Chicks injected with LPS were also tested regarding the capacity to raise O2 consumption to meet an increased demand driven by 2,4-dinitrophenol. LPS decreased Tb and the metabolic rate of chicks without affecting muscle uncoupled, coupled and non-coupled mitochondrial respiration. LPS-challenged chicks were indeed capable of increasing metabolic rate in response to 2,4-dinitrophenol, indicating no O2 delivery limitation. Additionally, chicks did not attempt to prevent Tb from falling during hypothermia but, instead, activated cutaneous and respiratory thermolytic mechanisms, providing an additional cooling force. These data provide the first evidence of the regulated nature of the hypothermic response to endotoxin in birds. Therefore, it changes the current understanding of bird's thermoregulation during severe inflammation, indicating that regulated hypothermia is either a convergent trait for endotherms or a conserved response among vertebrates, which adds a new perspective for evolutionary medicine research.

A5 noradrenergic neurons and breathing control in neonate rats.

The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DßH-SAP) to specifically ablate noradrenergic neurons. Thus, DßH-SAP (420 ng/µL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).

A thermoregulatory role of the medullary raphe in birds.

The brainstem region medullary raphe modulates non-shivering and shivering thermogenesis and cutaneous vasomotion in rodents. Whether the same scenario occurs in the other endothermic group, i.e. birds, is still unknown. Therefore, we hypothesised that the medullary raphe modulates heat gain and loss thermoeffectors in birds. We investigated the effect of glutamatergic and GABAergic inhibitions in this specific region on body temperature (Tb), oxygen consumption (thermogenesis), ventilation (O2 supply in cold, thermal tachypnea in heat) and heat loss index (cutaneous vasomotion) in one-week-old chicken exposed to neutral (31°C), cold (26°C) and heat (36°C) conditions. Intra-medullary raphe antagonism of NMDA glutamate (AP5; 0.5, 5 mM) and GABAA (bicuculline; 0.05, 0.5 mM) receptors reduced Tb of chicks at 31°C and 26oC, due mainly to an O2 consumption decrease. AP5 transiently increased breathing frequency during cold exposure. At 31°C, heat loss index was higher in the bicuculline and AP5 groups (higher doses) than vehicle at the beginning of the Tb reduction. No treatment affected any variable tested at 36oC. The results suggest that glutamatergic and GABAergic excitatory influences on the medullary raphe of chicks modulate thermogenesis and glutamatergic stimulation prevents tachypnea, without having any role in warmth-defence responses. A double excitation influence on the medullary raphe may provide a protective neural mechanism for supporting thermogenesis during early life, when energy expenditure to support growth and homeothermy is high. This novel demonstration of a thermoregulatory role for the raphe in birds suggests a convergent brainstem neurochemical regulation of body temperature in endotherms.

Orexin-A inhibits fictive air breathing responses to respiratory stimuli in the bullfrog tadpole (Lithobates catesbeianus).

In pre-metamorphic tadpoles, the neural network generating lung ventilation is present but actively inhibited; the mechanisms leading to the onset of air breathing are not well understood. Orexin (ORX) is a hypothalamic neuropeptide that regulates several homeostatic functions, including breathing. While ORX has limited effects on breathing at rest, it potentiates reflexive responses to respiratory stimuli mainly via ORX receptor 1 (OX1R). Here, we tested the hypothesis that OX1Rs facilitate the expression of the motor command associated with air breathing in pre-metamorphic bullfrog tadpoles (Lithobates catesbeianus). To do so, we used an isolated diencephalic brainstem preparation to determine the contributions of OX1Rs to respiratory motor output during baseline breathing, hypercapnia and hypoxia. A selective OX1R antagonist (SB-334867; 5-25 µmol l-1) or agonist (ORX-A; 200 nmol l-1 to 1 µmol l-1) was added to the superfusion media. Experiments were performed under basal conditions (media equilibrated with 98.2% O2 and 1.8% CO2), hypercapnia (5% CO2) or hypoxia (5-7% O2). Under resting conditions gill, but not lung, motor output was enhanced by the OX1R antagonist and ORX-A. Hypercapnia alone did not stimulate respiratory motor output, but its combination with SB-334867 increased lung burst frequency and amplitude, lung burst episodes, and the number of bursts per episode. Hypoxia alone increased lung burst frequency and its combination with SB-334867 enhanced this effect. Inactivation of OX1Rs during hypoxia also increased gill burst amplitude, but not frequency. In contrast with our initial hypothesis, we conclude that ORX neurons provide inhibitory modulation of the CO2 and O2 chemoreflexes in pre-metamorphic tadpoles.

Differential modulation of active expiration during hypercapnia by the medullary raphe in unanesthetized rats.

Active expiration represents an important mechanism to improve ventilation in conditions of augmented ventilatory demand, such as hypercapnia. While a rostral ventromedullary region, the parafacial respiratory group (pFRG), has been identified as a conditional expiratory oscillator, little is known about how central chemosensitive sites contribute to modulate active expiration under hypercapnia. In this study, we investigated the influence of the medullary raphe in the emergence of phasic expiratory abdominal activity during hypercapnia in unanesthetized adult male rats, in a state-dependent manner. To do so, reverse microdialysis of muscimol (GABAA receptor agonist, 1 mM) or 8-OH-DPAT (5-HT1A agonist, 1 mM) was applied in the MR during sleep and wakefulness periods, both in normocapnic (room air) and hypercapnic conditions (7% CO2). Electromyography (EMG) of diaphragm and abdominal muscles was performed to measure inspiratory and expiratory motor outputs. We found that active expiration did not occur in room air exposure during wakefulness or sleep. However, hypercapnia did recruit active expiration, and differential effects were observed with the drug dialyses in the medullary raphe. Muscimol increased the diaphragm inspiratory motor output and also increased the amplitude and frequency of abdominal expiratory rhythmic activity during hypercapnia in wakefulness periods. On the other hand, the microdialysis of 8-OH-DPAT attenuated hypercapnia-induced active expiration in a state-dependent manner. Our data suggest that the medullary raphe can either inhibit or potentiate respiratory motor activity during hypercapnia, and the balance of these inhibitory or excitatory outputs may determine the expression of active expiration.

Hypothalamic TRPV4 channels participate in the medial preoptic activation of warmth-defence responses in Wistar male rats.

Recently, we have described, in non-genetically modified rats, that peripheral transient receptor potential vanilloid-4 (TRPV4) channels are activated and trigger warmth-defence responses at ambient temperatures of 26-30 °C. Evidence points to the presence of TRPV4 in the medial preoptic area, a region described to be involved in the activation of thermoeffector pathways, including those involved in heat loss. Thus, we tested the hypothesis that TRPV4 in the medial preoptic area modulates thermoregulation under warm conditions. To this end, under two ambient temperatures (21 and 28 °C), body temperature was measured in rats following blockade of preoptic TRPV4 with two antagonists, HC-067047 and GSK 2193874. Oxygen consumption, heat loss index and preferred ambient temperature were also determined in order to assess thermoeffector activity. Antagonism of central TRPV4 caused an increase in body temperature in rats exposed to 28 °C, but not in those exposed to 21 °C. The body temperature increase at 28 °C was accompanied by an increase in oxygen consumption and an earlier reduction of the heat loss index. In behavioural experiments, control animals previously exposed to warm ambient temperatures (28-30 °C) for 2 h selected colder temperatures in a thermogradient compared to those injected with HC-067047. Our results support the idea that preoptic TRPV4 modulates thermoregulation in a warm environment by activating both autonomic and behavioural heat loss responses. Thus, according to the present study and to that published recently by our group, the activation of warmth-defence responses by TRPV4 seems to be dependent on the activity of both peripheral and central channels.

Sex differences in breathing.

Breathing is a vital behavior that ensures both the adequate supply of oxygen and the elimination of CO2, and it is influenced by many factors. Despite that most of the studies in respiratory physiology rely heavily on male subjects, there is much evidence to suggest that sex is an important factor in the respiratory control system, including the susceptibility for some diseases. These different respiratory responses in males and females may be related to the actions of sex hormones, especially in adulthood. These hormones affect neuromodulatory systems that influence the central medullary rhythm/pontine pattern generator and integrator, sensory inputs to the integrator and motor output to the respiratory muscles. In this article, we will first review the sex dependence on the prevalence of some respiratory-related diseases. Then, we will discuss the role of sex and gonadal hormones in respiratory control under resting conditions and during respiratory challenges, such as hypoxia and hypercapnia, and whether hormonal fluctuations during the estrous/menstrual cycle affect breathing control. We will then discuss the role of the locus coeruleus, a sexually dimorphic CO2/pH-chemosensitive nucleus, on breathing regulation in males and females. Next, we will highlight the studies that exist regarding sex differences in respiratory control during development. Finally, the few existing studies regarding the influence of sex on breathing control in non-mammalian vertebrates will be discussed.

Seasonal variation of hypoxic and hypercarbic ventilatory responses in the lizard Tropidurus torquatus.

Carbon dioxide (CO2) and oxygen (O2) influence the breathing pattern of reptiles, especially when CO2 is in excess or O2 at low concentrations and the effects of these gases on the respiratory response varies according to the species. In addition to respiratory gases, seasonal changes can also modulate breathing pattern and ventilatory responses to hypoxia and hypercarbia. Therefore, the present study investigated the breathing pattern and ventilatory responses to hypercarbia (5% CO2) and hypoxia (5% O2) of the Neotropical lizard Tropidurus torquatus over a period of one year, covering all seasons (summer, autumn, winter and spring). Our data suggest that like other ectothermic sauropsids, Tropidurus torquatus possesses distinct ventilatory responses to hypoxia and hypercarbia, being more sensitive to changes in CO2 than in O2. Additionally, the ventilatory responses to hypoxia were more pronounced during summer and hypercanic and pos-hypercapnic ventilatory response was reduced during spring, suggesting that seasonality modulates the control of ventilation in this species.

Hypercapnia-induced active expiration increases in sleep and enhances ventilation in unanaesthetized rats.

KEY POINTS: Expiratory muscles (abdominal and thoracic) can be recruited when respiratory drive increases under conditions of increased respiratory demand such as hypercapnia. Studying hypercapnia-induced active expiration in unanaesthetized rats importantly contributes to the understanding of how the control system is integrated in vivo in freely moving animals. In unanaesthetized rats, hypercapnia-induced active expiration was not always recruited either in wakefulness or in sleep, suggesting that additional factors influence the recruitment of active expiration. The pattern of abdominal muscle recruitment varied in a state-dependent manner with active expiration being more predominant in the sleep state than in quiet wakefulness. Pulmonary ventilation was enhanced in periods with active expiration compared to periods without it. ABSTRACT: Expiration is passive at rest but becomes active through recruitment of abdominal muscles under increased respiratory drive. Hypercapnia-induced active expiration has not been well explored in unanaesthetized rats. We hypothesized that (i) CO2 -evoked active expiration is recruited in a state-dependent manner, i.e. differently in sleep or wakefulness, and (ii) recruitment of active expiration enhances ventilation, hence having an important functional role in meeting metabolic demand. To test these hypotheses, Wistar rats (280-330 g) were implanted with electrodes for EEG and electromyography EMG of the neck, diaphragm (DIA) and abdominal (ABD) muscles. Active expiratory events were considered as rhythmic ABDEMG activity interposed to DIAEMG . Animals were exposed to room air followed by hypercapnia (7% CO2 ) with EEG, EMG and ventilation ( V̇E ) recorded throughout the experimental protocol. No active expiration was observed during room air exposure. During hypercapnia, CO2 -evoked active expiration was predominantly recruited during non-rapid eye movement sleep. Its increased occurrence during sleep was evidenced by the decreased DIA-to-ADB ratio (1:1 ratio means that each DIA event is followed by an ABD event, indicating a high occurrence of ABD activity). Moreover, V̇E was also enhanced (P < 0.05) in periods with active expiration. V̇E had a positive correlation (P < 0.05) with the peak amplitude of ABDEMG activity. The data demonstrate strongly that hypercapnia-induced active expiration increases during sleep and provides an important functional role to support V̇E in conditions of increased respiratory demand.

Brainstem catecholaminergic neurones and breathing control during postnatal development in male and female rats.

KEY POINTS: The brainstem catecholaminergic (CA) modulation on ventilation changes with development. We determined the role of the brainstem CA system in ventilatory control under normocapnic and hypercapnic conditions during different phases of development [postnatal day (P)7-8, P14-15 and P20-21] in male and female Wistar rats. Brainstem CA neurones produce a tonic inhibitory drive that affects breathing frequency in P7-8 rats and provide an inhibitory drive during hypercapnic conditions in both males and females at P7-8 and P14-15. In pre-pubertal rats, brainstem CA neurones become excitatory for the CO2 ventilatory response in males but remain inhibitory in females. Diseases such as sudden infant death syndrome, congenital central hypoventilation syndrome and Rett syndrome have been associated with abnormalities in the functioning of CA neurones; therefore, the results of the present study contribute to a better understanding of this system. ABSTRACT: The respiratory network undergoes significant development during the postnatal phase, including the maturation of the catecholaminergic (CA) system. However, postnatal development of this network and its effect on the control of pulmonary ventilation ( V̇E ) is not fully understood. We investigated the involvement of brainstem CA neurones in respiratory control during postnatal development [postnatal day (P)7-8, P14-15 and P20-21], in male and female rats, through chemical injury with conjugated saporin anti-dopamine ß-hydroxylase (DßH-SAP). Thus, DßH-SAP (420 ng µL-1 ), saporin (SAP) or phosphate buffered solution (PBS) was injected into the fourth ventricle of neonatal Wistar rats of both sexes. V̇E and oxygen consumption were recorded 1 week after the injections in unanaesthetized neonatal and juvenile rats during room air and hypercapnia. The resting ventilation was higher in both male and female P7-8 lesioned rats by 33%, with a decrease in respiratory variability being observed in males. The hypercapnic ventilatory response (HCVR) was altered in male and female lesioned rats at all postnatal ages. At P7-8, the HCVR for males and females was increased by 37% and 30%, respectively. For both sexes at P14-15 rats, the increase in V̇E during hypercapnia was 37% higher for lesioned rats. A sex-specific difference in HCRV was observed at P20-21, with lesioned males showing a 33% decrease, and lesioned females showing an increase of 33%. We conclude that brainstem CA neurones exert a tonic inhibitory effect on V̇E in the early postnatal days of the life of a rat, increase variability in P7-8 males and modulate HCRV during the postnatal phase.

ATP in the lateral hypothalamus/perifornical area enhances the CO2 chemoreflex control of breathing.

NEW FINDINGS: What is the central question of this study? ATP is known to modulate the chemosensitivity of some brain areas. However, whether the ATP contributes specifically to the mechanism of chemoreception in the lateral hypothalamus/perifornical area (LH/PFA) remains to be determined. What is the main finding and its importance? ATP, acting on the LH/PFA, enhances the hypercapnic ventilatory response in rats during wakefulness, in the dark period. Our results highlight the importance of ATP as a modulator of central chemoreception and provide new insight regarding the mechanisms involved in LH/PFA chemosensitivity and the sleep-wake differences in the CO2 /H+ -dependent drive to breathe. ABSTRACT: The lateral hypothalamus/perifornical area (LH/PFA) is a central chemoreceptor site, which acts in an arousal state-dependent manner. It has been shown that purinergic signalling through ATP influences the CO2 /H+ responsiveness of other chemosensitive regions, but it is unknown whether ATP is also involved in the mechanisms that underlie LH/PFA chemoreception. Here, we studied the effects of microdialysis of a P2X-receptor agonist [α,ß-methylene ATP (α,ß-meATP), 10 mm] and a non-selective P2-receptor antagonist [pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 1 mm] into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2 . In the dark (active) phase, but not in the light, microdialysis of α,ß-meATP caused an augmented hypercapnic ventilatory response during wakefulness, but not during non-REM sleep (P < 0.001). PPADS caused no change in CO2 ventilatory responses in either the dark period or the light period. Our data suggest that ATP in LH/PFA contributes to the hypercapnic ventilatory response in conscious rats during wakefulness in the dark phase of the diurnal cycle.

Mu and kappa opioid receptors of the periaqueductal gray stimulate and inhibit thermogenesis, respectively, during psychological stress in rats.

The periaqueductal gray matter (PAG) is rich in mu and kappa opioid receptors, and this system is involved in thermoregulation, analgesia, and defensive behaviors. No study approached the involvement of the PAG opioids in body temperature (Tb) regulation during psychological stress such as restraint. Because activation of mu and kappa receptors increases and reduces Tb, respectively, we tested the hypothesis that they exert excitatory and inhibitory modulation, respectively, of the restraint-induced fever in rats. To this end, Tb, heat loss index (HLI, inference for peripheral vasoconstriction/vasodilation), and oxygen consumption (inference for thermogenesis) were monitored in unanesthetized rats, restrained or unrestrained, before and after intra-PAG microinjection of the selective mu opioid receptor antagonist (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 cyclic, CTAP; 1 and 10 µg/100 nL) or the selective kappa opioid receptor antagonist (nor-binaltorphimine dihydrochloride, nor-BNI; 1 and 4 µg/100 nL) or saline (100 nL). CTAP and nor-BNI did not change the Tb or HLI of euthermic animals. During restraint, Tb increased (1.0 ± 0.1 °C) in all groups; however, this effect was lower in those animals treated with CTAP and higher in animals treated with nor-BNI. The HLI decreased during restraint and increased after animals were released, but this response was not affected by any treatment. Restraint stress increased oxygen consumption (35.9 ± 3.9% elevation), but this response was diminished by CTAP and overstimulated by nor-BNI. Confirming our hypothesis, the results indicate that the mu and kappa opioid receptors in the PAG of rats play a pyrogenic and antipyretic role, respectively, during fever induced by restraint by affecting the thermogenic but not the heat conservation effector.

Influence of estrous cycle hormonal fluctuations and gonadal hormones on the ventilatory response to hypoxia in female rats.

Sex hormones may influence many physiological processes. Recently, we demonstrated that hormonal fluctuations of cycling female rats do not affect respiratory parameters during hypercapnia. However, it is still unclear whether sex hormones and hormonal fluctuations that occur during the estrous cycle can affect breathing during a hypoxic challenge. Our study aimed to evaluate respiratory, metabolic, and thermal responses to hypoxia in female rats on different days of the estrous cycle (proestrus, estrus, metestrus, and diestrus) and in ovariectomized rats that received replacement with oil (OVX), estradiol (OVX + E2), or a combination of estradiol and progesterone (OVX + E2P). Ventilation (V E), tidal volume (V T), respiratory frequency (fR), oxygen consumption (VO2), and V E/VO2 were not different during the estrous cycle in normoxia or hypoxia. Body temperature (Tb) was higher during estrus, but decreased similarly in all groups during hypoxia. Compared with intact females in estrus, gonadectomized rats also had lower Tb in normoxia, but not in hypoxia. OVX rats experienced a significant drop in the ventilatory response to hypoxia, but hormonal replacement did not restore values to the levels of an intact animal. Our data demonstrate that the different phases of the estrous cycle do not alter ventilation during normoxia and hypoxia, but OVX animals display lower ventilatory responses to hypoxia compared with ovary-intact rats. Because estradiol and progesterone replacement did not cause significant differences in ventilation, our findings suggest that a yet-to-be-defined non-steroidal ovarian hormone is likely to stimulate the ventilatory responses to hypoxia in females.

Temperature effects on the cardiorespiratory control of American bullfrog tadpoles based on a non-invasive methodology.

Temperature effects on cardiac autonomic tonus in amphibian larval stages have never been investigated. Therefore, we evaluated the effect of different temperatures (15, 25 and 30°C) on the cardiorespiratory rates and cardiac autonomic tonus of premetamorphic tadpoles of the bullfrog, Lithobates catesbeianus To this end, a non-invasive method was developed to permit measurements of electrocardiogram (ECG) and buccal movements (fB; surface electromyography of the buccal floor). For evaluation of autonomic regulation, intraperitoneal injections of Ringer solution (control), atropine (cholinergic muscarinic antagonist) and sotalol (ß-adrenergic antagonist) were performed. Ringer solution injections did not affect heart rate (fH) or fB across temperatures. Cardiorespiratory parameters were significantly augmented by temperature (fH: 24.5±1.0, 54.5±2.0 and 75.8±2.8 beats min-1 at 15, 25 and 30°C, respectively; fB: 30.3±1.1, 73.1±4.0 and 100.6±3.7 movements min-1 at 15, 25 and 30°C, respectively). A predominant vagal tone was observed at 15°C (32.0±3.2%) and 25°C (27.2±6.7%) relative to the adrenergic tone. At 30°C, the adrenergic tone increased relative to the lower temperature. In conclusion, the cholinergic and adrenergic tones seem to be independent of temperature for colder thermal intervals (15-25°C), while exposure to a hotter ambient temperature (30°C) seems to be followed by a significant increase in adrenergic tone and may reflect cardiovascular adjustments made to match oxygen delivery to demand. Furthermore, while excluding the use of implantable electrodes or cannulae, this study provides a suitable non-invasive method for investigating cardiorespiratory function (cardiac and respiratory rates) in water-breathing animals such as the tadpole.

Hypoxia during embryonic development increases energy metabolism in normoxic juvenile chicks.

Environmental changes during perinatal development can affect the postnatal life. In this sense, chicken embryos that experience low levels of O2 over a specific phase of incubation can have their tissue growth reduced and the ventilatory response to hypoxia blunted, at least until hatching. Additionally, exposure to low level of O2 after birth reduces the thermogenesis as well. In the present study, we tested the hypothesis that hypoxia over the third week of incubation affects the thermoregulation of juvenile chicks at an age when thermogenesis is already expected to be well-developed. To this end, we measured body temperature (Tb) and oxygen consumption (V̇02) under acute hypoxia or different ambient temperatures (Ta) of 1 and 10day-old chicks that have been exposed to 21% O2 for entire incubation (Nx) or to 15% O2 in the last week of incubation (Hx). We also assessed the thermal preference under normoxia or acute hypoxia of the older chicks from both incubation groups in a thermocline. Hypoxia over incubation reduced growth but did not affect the cold-induced thermogenesis in hatchlings. Regarding the juvenile Hx, present data indicate a catch up growth with higher resting V̇02, a thermal preference for warmer Tas and a possible higher thermal conductance. In conclusion, our results show that hypoxia over the third week of incubation can affect the thermoregulation at least until 10days after hatch in chickens.

Orexinergic system in the locus coeruleus modulates the CO2 ventilatory response.

The orexins are hypothalamic neuropeptides involved in an array of functions such as regulation of sleep/wake states and chemoreception to CO2/pH. The locus coeruleus (LC) is a chemosensitive site and expresses an extensive population of orexin receptor 1 (OX1R). We tested the hypothesis that OX1Rs located in the LC participate in the ventilatory response to hypercapnia in a vigilance state and diurnal cycle-dependent manner. For this, we performed unilateral injections of SB-334867 (OX1R antagonist, 5 mM) into the LC of male Wistar rats and evaluated the ventilatory response to 7 % CO2 during wakefulness and sleep in the dark and light phases of the diurnal cycle. Hypercapnia induced an increase in ventilation (V E) in all groups compared to normocapnic values. However, during the dark phase, but not in the light phase, SB-334867 injection promoted an attenuation of the hypercapnic chemoreflex during wakefulness (V E: vehicle, 1502.6 ± 100 mL kg(-1) min(-1) vs SB-334867, 1200.3 ± 70.0 mL kg(-1) min(-1)) but not during sleep (V E: vehicle, 1383.0 ± 113.9 vs SB-334687, 1287.6 ± 92.1 mL kg(-1) min(-1)), due to changes in tidal volume (V T). We suggest that projections of orexin-containing neurons to the LC contribute, via OX1Rs, to the hypercapnic chemoreflex during wakefulness in the dark phase.

Nitric oxide and fever: immune-to-brain signaling vs. thermogenesis in chicks.

Nitric oxide (NO) plays a role in thermogenesis but does not mediate immune-to-brain febrigenic signaling in rats. There are suggestions of a different situation in birds, but the underlying evidence is not compelling. The present study was designed to clarify this matter in 5-day-old chicks challenged with a low or high dose of bacterial LPS. The lower LPS dose (2 µg/kg im) induced fever at 3-5 h postinjection, whereas 100 µg/kg im decreased core body temperature (Tc) (at 1 h) followed by fever (at 4 or 5 h). Plasma nitrate levels increased 4 h after LPS injection, but they were not correlated with the magnitude of fever. The NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester, l-NAME; 50 mg/kg im) attenuated the fever induced by either dose of LPS and enhanced the magnitude of the Tc reduction induced by the high dose in chicks at 31-32°C. These effects were associated with suppression of metabolic rate, at least in the case of the high LPS dose. Conversely, the effects of l-NAME on Tc disappeared in chicks maintained at 35-36°C, suggesting that febrigenic signaling was essentially unaffected. Accordingly, the LPS-induced rise in the brain level of PGE2 was not affected by l-NAME. Moreover, l-NAME augmented LPS-induced huddling, which is indicative of compensatory mechanisms to run fever in the face of attenuated thermogenesis. Therefore, as in rats, systemic inhibition of NO synthesis attenuates LPS-induced fever in chicks by affecting thermoeffector activity and not by interfering with immune-to-brain signaling. This may constitute a conserved effect of NO in endotherms.