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.

Influence of light/dark cycle and orexins on breathing control in green iguanas (Iguana iguana).

Light/dark cycle affects the physiology of vertebrates and hypothalamic orexin neurons (ORX) are involved in this function. The breathing pattern of the green iguana changes from continuous to episodic across the light/dark phases. Since the stimulatory actions of ORX on breathing are most important during arousal, we hypothesized that ORX regulates changes of breathing pattern in iguanas. Thus, we: (1) Localized ORX neurons with immunohistochemistry; (2) Quantified cyclic changes in plasma orexin-A levels by ELISA; (3) Compared breathing pattern at rest and during hypoxia and hypercarbia; (4) Evaluated the participation of the ORX receptors in ventilation with intracerebroventricular microinjections of ORX antagonists during light and dark phases. We show that the ORX neurons of I. iguana are located in the periventricular hypothalamic nucleus. Orexin-A peaks during the light/active phase and breathing parallels these cyclic changes: ventilation is higher during the light phase than during the dark phase. However, inactivation of ORX-receptors does not affect the breathing pattern. Iguanas increase ventilation during hypoxia only during the light phase. Conversely, CO2 promotes post-hypercarbic hyperpnea during both phases. We conclude that ORXs potentiate the post-hypercarbic (but not the hypoxic)-drive to breathe and are not involved in light/dark changes in the breathing pattern.

Effect of temperature on chemosensitive locus coeruleus neurons of savannah monitor lizards, Varanus exanthematicus.

Savannah monitor lizards (Varanus exanthematicus) are unusual among ectothermic vertebrates in maintaining arterial pH nearly constant during changes in body temperature in contrast to the typical α-stat regulating strategy of most other ectotherms. Given the importance of pH in the control of ventilation, we examined the CO2/H+ sensitivity of neurons from the locus coeruleus (LC) region of monitor lizard brainstems. Whole-cell patch-clamp electrophysiology was used to record membrane voltage in LC neurons in brainstem slices. Artificial cerebral spinal fluid equilibrated with 80% O2, 0.0-10.0% CO2, balance N2, was superfused across brainstem slices. Changes in firing rate of LC neurons were calculated from action potential recordings to quantify the chemosensitive response to hypercapnic acidosis. Our results demonstrate that the LC brainstem region contains neurons that can be excited or inhibited by, and/or are not sensitive to CO2 in V. exanthematicus While few LC neurons were activated by hypercapnic acidosis (15%), a higher proportion of the LC neurons responded by decreasing their firing rate during exposure to high CO2 at 20°C (37%); this chemosensitive response was no longer exhibited when the temperature was increased to 30°C. Further, the proportion of chemosensitive LC neurons changed at 35°C with a reduction in CO2-inhibited (11%) neurons and an increase in CO2-activated (35%) neurons. Expressing a high proportion of inhibited neurons at low temperature may provide insights into mechanisms underlying the temperature-dependent pH-stat regulatory strategy of savannah monitor lizards.

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.

Orexin in the toad Rhinella schneideri: The location of orexinergic neurons and the role of orexin in ventilatory responses to hypercarbia and hypoxia.

Recent reports have suggested that orexins, also known as hypocretins, play an important role in the modulation of respiratory control in mammals, but there are no data available describing the role of the orexinergic system in the peripheral and central chemoreception of non-mammalian vertebrates. Therefore, the present study was designed to examine the localization of orexin-immunoreactive neurons in the brain of toads (Rhinella schneideri) and to investigate the contribution of orexin receptor-1 (OX1R) to the hypoxic and hypercarbic ventilatory responses of these animals during light and dark phases. Our results demonstrated that the orexinergic neurons of R. schneideri are located in the suprachiasmatic nucleus of the diencephalon. Additionally, the intracerebroventricular injection of SB-334867 (OX1R selective antagonist) attenuated the ventilatory response to hypercarbia during the dark phase by acting on tidal volume and breathing frequency, while during the light phase, SB-334867 attenuated the ventilatory response to hypoxia by acting on tidal volume only. We conclude that in the toad R. schneideri, orexinergic neurons are located in the suprachiasmatic nucleus and that OX1R contributes to hypercarbic and hypoxic chemoreflexes.

The breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in the frog Pipa carvalhoi.

Anuran amphibians are known to exhibit an intermittent pattern of pulmonary ventilation and to exhibit an increased ventilatory response to hypoxia and hypercarbia. However, only a few species have been studied to date. The aquatic frog Pipa carvalhoi inhabits lakes, ponds and marshes that are rich in nutrients but low in O(2). There are no studies of the respiratory pattern of this species and its ventilation during hypoxia or hypercarbia. Accordingly, the aim of the present study was to characterize the breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in this species. With this purpose, pulmonary ventilation (V(I)) was directly measured by the pneumotachograph method during normocapnic normoxia to determine the basal respiratory pattern and during aerial and aquatic hypercarbia (5% CO(2)) and hypoxia (5% O(2)). Our data demonstrate that P. carvalhoi exhibits a periodic breathing pattern composed of single events (single breaths) of pulmonary ventilation separated by periods of apnea. The animals had an enhanced V(I) during aerial hypoxia, but not during aquatic hypoxia. This increase was strictly the result of an increase in the breathing frequency. A pronounced increase in V(I) was observed if the animals were simultaneously exposed to aerial and aquatic hypercarbia, whereas small or no ventilatory responses were observed during separately administered aerial or aquatic hypercarbia. P. carvalhoi primarily inhabits an aquatic environment. Nevertheless, it does not respond to low O(2) levels in water, although it does so in air. The observed ventilatory responses to hypercarbia may indicate that this species is similar to other anurans in possessing central chemoreceptors.