Mitochondrial function in skeletal muscle contributes to reproductive endothermy in tegu lizards (Salvator merianae).

AIM: In cyclic climate variations, including seasonal changes, many animals regulate their energy demands to overcome critical transitory moments, restricting their high-demand activities to phases of resource abundance, enabling rapid growth and reproduction. Tegu lizards (Salvator merianae) are ectotherms with a robust annual cycle, being active during summer, hibernating during winter, and presenting a remarkable endothermy during reproduction in spring. Here, we evaluated whether changes in mitochondrial respiratory physiology in skeletal muscle could serve as a mechanism for the increased thermogenesis observed during the tegu's reproductive endothermy. METHODS: We performed high-resolution respirometry and calorimetry in permeabilized red and white muscle fibers, sampled during summer (activity) and spring (high activity and reproduction), in association with citrate synthase measurements. RESULTS: During spring, the muscle fibers exhibited increased oxidative phosphorylation. They also enhanced uncoupled respiration and heat production via adenine nucleotide translocase (ANT), but not via uncoupling proteins (UCP). Citrate synthase activity was higher during the spring, suggesting greater mitochondrial density compared to the summer. These findings were consistent across both sexes and muscle types (red and white). CONCLUSION: The current results highlight potential cellular thermogenic mechanisms in an ectothermic reptile that contribute to transient endothermy. Our study indicates that the unique feature of transitioning to endothermy through nonshivering thermogenesis during the reproductive phase may be facilitated by higher mitochondrial density, function, and uncoupling within the skeletal muscle. This knowledge contributes significant elements to the broader picture of models for the evolution of endothermy, particularly in relation to the enhancement of aerobic capacity.

Evaluation of sex-based differences in central control of breathing in American bullfrogs.

The neural control of breathing exhibits sex differences. There is now a large effort to account for biological sex in mammalian research, but the degree to which ectothermic vertebrates exhibit sex differences in the central control of breathing is not well-established. Therefore, we compared respiratory-related neural activity in brainstem-spinal cord preparations from female and male bullfrogs to determine if important aspects of the central control of breathing vary with sex. We found that the breathing pattern was similar across males and females, but baseline frequency of the respiratory network was faster in females. The magnitude of the central response to hypercapnia was similar across sexes, but the time to reach maximum burst rate occurred more slowly in females. These results suggest that sex differences may account for variation in traits associated with the control of breathing and that future work should carefully account for sex of the animal in analysis.

Inhibition of Pro-Inflammatory Microglia with Minocycline Improves Cognitive and Sleep-Wake Dysfunction Under Respiratory Stress in a Sporadic Model for Alzheimer's Disease.

BACKGROUND: Neuroinflammation in Alzheimer's disease (AD) can occur due to excessive activation of microglia in response to the accumulation of amyloid-ß peptide (Aß). Previously, we demonstrated an increased expression of this peptide in the locus coeruleus (LC) in a sporadic model for AD (streptozotocin, STZ; 2 mg/kg, ICV). We hypothesized that the STZ-AD model exhibits neuroinflammation, and treatment with an inhibitor of microglia (minocycline) can reverse the cognitive, respiratory, sleep, and molecular disorders of this model. OBJECTIVE: To evaluate the effect of minocycline treatment in STZ model disorders. METHODS: We treated control and STZ-treated rats for five days with minocycline (30 mg/kg, IP) and evaluated cognitive performance, chemoreflex response to hypercapnia and hypoxia, and total sleep time. Additionally, quantification of Aß, microglia analyses, and relative expression of cytokines in the LC were performed. RESULTS: Minocycline treatment improved learning and memory, which was concomitant with a decrease in microglial cell density and re-establishment of morphological changes induced by STZ in the LC region. Minocycline did not reverse the STZ-induced increase in CO2 sensitivity during wakefulness. However, it restored the daytime sleep-wake cycle in STZ-treated animals to the same levels as those observed in control animals. In the LC, levels of A and expression of Il10, Il1b, and Mcp1 mRNA remained unaffected by minocycline, but we found a strong trend of minocycline effect on Tnf- α. CONCLUSION: Our findings suggest that minocycline effectively reduces microglial recruitment and the inflammatory morphological profile in the LC, while it recovers cognitive performance and restores the sleep-wake pattern impaired by STZ.

Heat stress affects breathing and metabolism of chicks incubated at high temperature.

Global warming poses serious implications to animal physiology and a gradual increase in ambient temperature affects all living organisms, particularly fast-growing selected species. We recorded ventilation (V̇E), body temperature (TB), oxygen consumption (V̇O2) and respiratory equivalent (V̇E/V̇O2) of 14-day-old (14d) male and female chicks at room air conditions, hypercapnia and hypoxia at heat stress (HS, 32 °C). These chicks had previously been exposed to control (CI, 37.5 °C) and high (HI, 39 °C) temperatures during the first 5 days of incubation. Under resting conditions, acute HS increased V̇E in HI females but not in HI males. Hypercapnia combined with heat promoted a potentiation of CO2-hyperventilatory response in HI females when compared with thermoneutral condition, whereas in HI incubated males a hypoventilation under hypercapnia and heat stress was observed compared to the CI group. Hypoxia associated with heat stress increased V̇E only in HI females. Our data indicates that females are more sensitive to thermal manipulation during incubation and it seems that the thermal embryonic manipulation, at least during the first days of development, does not improve the adaptive response of chicks to heat stress.

Sex- and age-specific respiratory alterations induced by prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 in rats.

BACKGROUND AND PURPOSE: Cannabis legalization has risen in many countries, and its use during pregnancy has increased. The endocannabinoid system is present in the CNS at early stages of embryonic development, and regulates functional brain maturation including areas responsible for respiratory control, data on the influence of external cannabinoids on the development of the respiratory system and possible consequences during postnatal life are limited. EXPERIMENTAL APPROACH: We evaluated the effects of prenatal exposure to synthetic cannabinoid (WIN 55,212-2 [WIN], 0.5 mg·kg-1 ·day-1 ) on the respiratory control system in neonatal (P0, P6-7 and P12-13) and juvenile (P27-28) male and female rats. KEY RESULTS: WIN administration to pregnant rats interfered sex-specifically with breathing regulation of offspring, promoting a greater sensitivity to CO2 at all ages in males (except P6-7) and in juvenile females. An altered hypoxic chemoreflex was observed in P0 (hyperventilation) and P6-7 (hypoventilation) males, which was absent in females. Along with breathing alterations, brainstem analysis showed an increase in the number of catecholaminergic neurons and cannabinoid receptor type 1 (CB1 ) and changes in tissue respiration in the early males. A reduction in pulmonary compliance was observed in juvenile male rats. Preexposure to WIN enhanced spontaneous apnoea and reduced the number of serotoninergic (5-HT) neurons in the raphe magnus nucleus of P0 females. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that excess stimulation of the endocannabinoid system during gestation has prolonged and sex-specific consequences for the respiratory control system.

Intra-uterine diazepam exposure decreases the number of catecholaminergic and serotoninergic neurons of neonate rats.

Benzodiazepines, such as diazepam (DZP), are used to treat anxiety disorders, and are prescribed to pregnant woman for therapeutic purposes. Concerns regarding their consequences on postnatal development rise as they cross the placenta and interact with the embryo. Occurrence of malformation and behavioral syndromes have been reported for different ages, but little is known about their effects on the brain after exposure during intrauterine life. Thus, we sought to evaluate the effects of intrauterine exposure to DZP on the number of brainstem's catecholaminergic and serotonergic neurons, implicated in respiratory control, in male and female rats on postnatal (P) day 12-13, using immunofluorescence labeling for tyrosine-hydroxylase (TH) and serotonin (5-HT). We observed a reduction in the number of catecholaminergic neurons for males and females. Special attention is given to the reduction in the density of neurons in the A6 region, involved in ventilatory responses to CO2. Interestingly, only males showed a reduction in the number of serotonergic neurons, while females were not affected. These findings suggest that in utero exposure to DZP results in deleterious neuroanatomical effects on P12-13 rats and raises a note of concern for women clinicians to make more informed choices about the use of anxiolytic treatments during gestation.

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.

CO2 exposure enhances Fos expression in hypothalamic neurons in rats during the light and dark phases of the diurnal cycle.

Orexinergic (OX) neurons in the lateral hypothalamus (LH), perifornical area (PFA) and dorsomedial hypothalamus (DMH) play a role in the hypercapnic ventilatory response, presumably through direct inputs to central pattern generator sites and/or through interactions with other chemosensitive regions. OX neurons can produce and release orexins, excitatory neuropeptides involved in many functions, including physiological responses to changes in CO2/pH. Thus, in the present study, we tested the hypothesis that different nuclei (LH, PFA and DMH) where the orexinergic neurons are located, show distinct activation by CO2 during the light-dark cycle phases. For this purpose, we evaluated the Fos and OXA expression by immunohistochemistry to identify neurons that co-localize Fos + OXA in the LH, LPeF, MPeF and DMH in the light-inactive and dark-active phase in Wistar rats subjected to 3 h of normocapnia or hypercapnia (7% CO2). Quantitative analyses of immunoreactive neurons show that hypercapnia caused an increase in the number of neurons expressing Fos in the LH, LPeF, MPeF and DMH in the light and dark phases. In addition, the number of Fos + OXA neurons increased in the LPeF and DMH independently of the phases of the diurnal cycle; whereas in the MPeF, this increase was observed exclusively in the light phase. Thus, we suggest that OX neurons are selectively activated by hypercapnia throughout the diurnal cycle, reinforcing the differential role of nuclei in the hypothalamus during central chemosensitivity.

Cutaneous TRPV4 Channels Activate Warmth-Defense Responses in Young and Adult Birds.

Transient receptor potential vanilloid 4 (TRPV4) channels are sensitive to warm ambient temperatures (Tas), triggering heat loss responses in adult rats in a Tas range of ∼26-30°C. In birds, however, the thermoregulatory role of TRPV4 has never been shown. Here, we hypothesized that stimulation of TRPV4 induces thermolytic responses for body temperature (Tb) maintenance in birds, and that this function is already present in early life, when the Ta range for TRPV4 activation does not represent a warm condition for these animals. We first demonstrated the presence of TRPV4 in the dorsal and ventral skin of chickens (Gallus gallus domesticus) by immunohistochemistry. Then, we evaluated the effects of the TRPV4 agonist, RN1747, and the TRPV4 antagonists, HC067047 and GSK2193874, on Tb and thermoeffectors at different Tas in 5-day-old chicks and 60-day-old adult chickens. For the chicks, RN1747 transiently reduced Tb both in thermoneutrality (31°C) and in a cold Ta for this phase (26°C), which relied on huddling behavior inhibition. The TRPV4 antagonists alone did not affect Tb or thermoeffectors but blocked the Tb decrease and huddling inhibition promoted by RN1747. For the adults, TRPV4 antagonism increased Tb when animals were exposed to 28°C (suprathermoneutral condition for adults), but not to 19°C. In contrast, RN1747 decreased Tb by reducing metabolic rate and activating thermal tachypnea at 19°C, a Ta below the activation range of TRPV4. Our results indicate that peripheral TRPV4 receptors are functional in early life, but may be inhibited at that time when the range of activation (∼26-30°C) represents cold Ta for chicks, and become physiologically relevant for Tb maintenance when the activation Ta range for TRPV4 becomes suprathermoneutral for adult chickens.

Control of Breathing in Ectothermic Vertebrates.

The ectothermic vertebrates are a diverse group that includes the Fishes (Agnatha, Chondrichthyes, and Osteichthyes), and the stem Tetrapods (Amphibians and Reptiles). From an evolutionary perspective, it is within this group that we see the origin of air-breathing and the transition from the use of water to air as a respiratory medium. This is accompanied by a switch from gills to lungs as the major respiratory organ and from oxygen to carbon dioxide as the primary respiratory stimulant. This transition first required the evolution of bimodal breathing (gas exchange with both water and air), the differential regulation of O2 and CO2 at multiple sites, periodic or intermittent ventilation, and unsteady states with wide oscillations in arterial blood gases. It also required changes in respiratory pump muscles (from buccopharyngeal muscles innervated by cranial nerves to axial muscles innervated by spinal nerves). The question of the extent to which common mechanisms of respiratory control accompany this progression is an intriguing one. While the ventilatory control systems seen in all extant vertebrates have been derived from common ancestors, the trends seen in respiratory control in the living members of each vertebrate class reflect both shared-derived features (ancestral traits) as well as unique specializations. In this overview article, we provide a comprehensive survey of the diversity that is seen in the afferent inputs (chemo and mechanoreceptor), the central respiratory rhythm generators, and the efferent outputs (drive to the respiratory pumps and valves) in this group. © 2022 American Physiological Society. Compr Physiol 12: 1-120, 2022.

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.

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.

Prenatal fluoxetine has long-lasting, differential effects on respiratory control in male and female rats.

Serotonin (5-HT) is an important modulator of brain networks that control breathing. The selective serotonin reuptake inhibitor fluoxetine (FLX) is the first-line antidepressant drug prescribed during pregnancy. We investigated the effects of prenatal FLX exposure on baseline breathing, ventilatory and metabolic responses to hypercapnia and hypoxia as well as number of brainstem 5-HT and tyrosine hydroxylase (TH) neurons of rats during postnatal development (P0-82). Prenatal FLX exposure of males showed a lower baseline V̇e that appeared in juveniles and remained in adulthood, with no sleep-wake state dependency. Prenatal FLX exposure of females did not affect baseline breathing. Juvenile male FLX showed increased CO2 and hypoxic ventilatory responses, normalizing by adulthood. Alterations in juvenile FLX-treated males were associated with a greater number of 5-HT neurons in the raphe obscurus (ROB) and raphe magnus (RMAG). Adult FLX-exposed males showed greater number of 5-HT neurons in the raphe pallidus (RPA) and TH neurons in the A5, whereas reduced number of TH neurons in A7. Prenatal FLX exposure of female rats was associated with greater hyperventilation induced by hypercapnia at P0-2 and juveniles, whereas P12-14 and adult FLX (non-rapid eye movement, NREM sleep) rats showed an attenuation of the hyperventilation induced by CO2. FLX-exposed females had fewer 5-HT neurons in the RPA and reduced TH A6 density at P0-2; and greater number of TH neurons in the A7 at P12-14. These data indicate that prenatal FLX exposure affects the number of some monoaminergic regions in the brain and results in long-lasting, sex-specific changes in baseline breathing pattern and ventilatory responses to respiratory challenges.NEW & NOTEWORTHY Selective serotonin reuptake inhibitors (SSRIs) readily cross the placental and the fetal blood-brain barrier where it will affect 5-HT levels in the developing brain. Although SSRI is used during pregnancy, there are no studies showing SSRI exposure during late pregnancy and postnatal effects on breathing control in males and females. We demonstrated that fluoxetine exposure during late pregnancy in rats was associated with long-lasting, sex-specific effects on breathing and brainstem monoaminergic groups.

Thermal Acclimation to the Highest Natural Ambient Temperature Compromises Physiological Performance in Tadpoles of a Stream-Breeding Savanna Tree Frog.

Amphibians may be more vulnerable to climate-driven habitat modification because of their complex life cycle dependence on land and water. Considering the current rate of global warming, it is critical to identify the vulnerability of a species by assessing its potential to acclimate to warming temperatures. In many species, thermal acclimation provides a reversible physiological adjustment in response to temperature changes, conferring resilience in a changing climate. Here, we investigate the effects of temperature acclimation on the physiological performance of tadpoles of a stream-breeding savanna tree frog (Bokermannohyla ibitiguara) in relation to the thermal conditions naturally experienced in their microhabitat (range: 18.8-24.6°C). We quantified performance measures such as routine and maximum metabolic rate at different test (15, 20, 25, 30, and 34°C) and acclimation temperatures (18 and 25°C). We also measured heart rate before and after autonomic blockade with atropine and sotalol at the respective acclimation temperatures. Further, we determined the critical thermal maximum and warming tolerance (critical thermal maximum minus maximum microhabitat temperature), which were not affected by acclimation. Mass-specific routine and mass-specific maximum metabolic rate, as well as heart rate, increased with increasing test temperatures; however, acclimation elevated mass-specific routine metabolic rate while not affecting mass-specific maximum metabolic rate. Heart rate before and after the pharmacological blockade was also unaffected by acclimation. Aerobic scope in animals acclimated to 25°C was substantially reduced, suggesting that physiological performance at the highest temperatures experienced in their natural habitat is compromised. In conclusion, the data suggest that the tadpoles of B. ibitiguara, living in a thermally stable environment, have a limited capacity to physiologically adjust to the highest temperatures found in their micro-habitat, making the species more vulnerable to future climate change.

Embryonic Thermal Manipulation Affects Ventilation, Metabolism, Thermal Control and Central Dopamine in Newly Hatched and Juvenile Chicks.

The first third of incubation is critical for embryonic development, and environmental changes during this phase can affect the physiology and survival of the embryos. We evaluated the effects of low (LT), control (CT), and high (HT) temperatures during the first 5 days of incubation on ventilation ( V . E ), body temperature (Tb), oxygen consumption ( V . O2), respiratory equivalent ( V . E / V . O2), and brain monoamines on 3-days-old (3d) and 14-days-old (14d) male and female chickens. The body mass of LT animals of both ages and sexes was higher compared to HT and CT animals (except for 3d males). The heart mass of 14d HT animals was higher than that of CT animals. Thermal manipulation did not affect V . E , V . O2 or V . E / V . O2 of 3d animals in normoxia, except for 3d LT males V . E , which was lower than CT. Regarding 14d animals, the HT females showed a decrease in V . E and V . O2 compared to CT and LT groups, while the HT males displayed a lower V . O2 compared to CT males, but no changes in V . E / V . O2. Both sexes of 14d HT chickens presented a greater Tb compared to CT animals. Thermal manipulations increased the dopamine turnover in the brainstem of 3d females. No differences were observed in ventilatory and metabolic parameters in the 3d animals of either sexes, and 14d males under 7% CO2. The hypercapnic hyperventilation was attenuated in the 14d HT females due to changes in V . O2, without alterations in V . E . The 14d LT males showed a lower V . E , during hypercapnia, compared to CT, without changes in V . O2, resulting in an attenuation in V . E / V . O2. During hypoxia, 3d LT females showed an attenuated hyperventilation, modulated by a higher V . O2. In 14d LT and HT females, the increase in V . E was greater and the hypometabolic response was attenuated, compared to CT females, which resulted in no change in the V . E / V . O2. In conclusion, thermal manipulations affect hypercapnia-induced hyperventilation more so than hypoxic challenge, and at both ages, females are more affected by thermal manipulation than males.

5-HT neurons of the medullary raphe contribute to respiratory control in toads.

Air-breathing vertebrates undergo respiratory adjustments when faced with disturbances in the gas composition of the environment. In mammals, the medullary raphe nuclei are involved in the neuronal pathway that mediates the ventilatory responses to hypoxia and hypercarbia. We investigate whether the serotoninergic neurons of the medullary raphe nuclei of toads (Rhinella diptycha) play a functional role in respiratory control during resting conditions (room air), hypercarbia (5% CO2), and hypoxia (5% O2). The raphe nuclei were located and identified based on the location of the serotoninergic neurons in the brainstem. We then lesioned the medullary raphe (raphe pallidus, obscurus and magnus) with anti-SERT-SAP and measured ventilation in both control and lesioned groups and we observed that serotonin (5-HT) specific chemical lesions of the medullary raphe caused reduced respiratory responses to both hypercarbia and hypoxia. In summary, we report that the serotoninergic neurons of the medullary raphe of the cururu toad Rhinella diptycha participate in the chemoreflex responses during hypercarbia and hypoxia, but not during resting conditions. This current evidence in anurans, together with the available data in mammals, brings insights to the evolution of brain sites, such as the medullary raphe, involved in the ventilatory chemoreflex in vertebrates.

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.

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).

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.

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.