Respiratory activity during seizures induced by pentylenetetrazole.

This study investigated the respiratory activity in adult Wistar rats across different behavioral seizure severity induced by pentylenetetrazole (PTZ). Animals underwent surgery for electrodes implantation, allowing simultaneous EEG and diaphragm EMG (DIAEMG) recordings and the respiratory frequency and DIAEMG amplitude were measured. Seizures were acutely induced through PTZ injection and classified based on a pre-established score, with absence-like seizures (spike wave discharge (SWD) events on EEG) representing the lowest score. The respiratory activity was grouped into the different seizure severities. During absence-like and myoclonic jerk seizures, the breathing frequency decreased significantly (∼50% decrease) compared to pre- and post-ictal periods. Pronounced changes occurred with more severe seizures (clonic and tonic) with periods of apnea, especially during tonic seizures. Apnea duration was significantly higher in tonic compared to clonic seizures. Notably, during PTZ-induced tonic seizures the apnea events were marked by tonic DIAEMG contraction (tonic-phase apnea). In the majority of animals (5 out of 7) this was a fatal event in which the seizure-induced respiratory arrest preceded the asystole. In conclusion, we provide an assessment of the respiratory activity in the PTZ-induced acute seizures and showed that breathing dysfunction is more pronounced in seizures with higher severity.

Dietary Vitamin D Mitigates Coronavirus-Induced Lung Inflammation and Damage in Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.

A 5-Lipoxygenase Inhibitor, Zileuton, Modulates Host Immune Responses and Improves Lung Function in a Model of Severe Acute Respiratory Syndrome (SARS) Induced by Betacoronavirus.

Exacerbated inflammatory responses are a hallmark of severe coronavirus disease 2019 (COVID-19). Zileuton (Zi) is a selective inhibitor of 5-lipoxygenase, an enzyme involved in the production of several inflammatory/pro-resolving lipid mediators. Herein, we investigated the effect of Zi treatment in a severe acute respiratory syndrome (SARS) model. Mouse hepatitis virus (MHV)3-infected mice treated with Zi significantly improved the clinical score, weight loss, cardiopulmonary function, and survival rates compared with infected untreated animals. The protection observed in Zi-treated mice was associated with a lower inflammatory score, reduced dendritic cell-producing tumor necrosis factor (TNF), and increased neutrophil-producing interleukin (IL)-10 in the lungs three days after infection (dpi). At 5 dpi, the lungs of treated mice showed an increase in Th2-, Treg CD4+-, and Treg CD8+-producing IL-10 and reduced Th1 infiltrating cells. Furthermore, similar results were found upon Zi treatment after SARS-CoV-2 infection in transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2), significantly improving the clinical score, weight loss, and lung inflammatory score compared with untreated animals. Our data suggest that Zi protects against developing severe lung disease during SARS induced by betacoronavirus without affecting the host's capacity to deal with infection.

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.

Strategies adopted by undergraduate teaching assistants in physiology and biophysics education during the COVID-19 pandemic.

The COVID-19 pandemic affected almost all aspects of our lives, including the education sector and the way of teaching and learning. In March 2020, health authorities in Brazil imposed social isolation and the interruption of on-site activities in schools and universities. In this context, the Federal University of Minas Gerais (UFMG), one of the largest universities in Brazil and Latin America, developed an emergency remote learning (ERL) plan that allowed the return of classes in an online format and supported students to obtain access to equipment and internet network. Within this new perspective, the Undergraduate Teaching Assistant (UTA) program of the Department of Physiology and Biophysics (DFIB) explored strategies to minimize the impact of the absence of face-to-face classes. Using different available tools in online platforms and social media such as Microsoft Teams, YouTube animated video classes, and Instagram, the UTA program assisted >500 undergraduate students and strongly supported professors during ERL. In just over a year, our video classes on YouTube Channel reached ∼40,000 views. Most of the students reported that their questions were fully and quickly solved by the UTA program. Collectively, our results indicate that the strategies implemented by the UTA program helped the undergraduate students and professors to adapt to a remote learning format.

Inhibition of nNOS in the paraventricular nucleus of hypothalamus decreases exercise-induced hyperthermia.

The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic control, which integrates thermoregulation centers and sympathetic outflow to thermoeffector organs. PVN neurons express the neuronal isoform of nitric oxide synthase (nNOS) whose expression is locally upregulated by physical exercise. Thus, the aim of the present study was to evaluate the role of nNOS in the PVN in the exercise-induced hyperthermia. Seven days after surgery, male Wistar rats received bilateral intra-PVN microinjections of the selective nNOS inhibitor Nw-Propyl-L-Arginine (NPLA) or vehicle (saline) and were submitted to an acute progressive exercise session on a treadmill until fatigue. Abdominal and tail skin temperature (Tabd and Ttail, respectively) were measured, and the threshold (Hthr; °C) and sensitivity (Hsen) for heat dissipation calculated. Performance variables were also collected. During the progressive exercise protocol, all animals displayed an increase in the Tabd. However, compared to vehicle group, the microinjection of NPLA in the PVN attenuated the exercise-induced hyperthermia. There was no difference in Ttail or Hthr between NPLA and control rats. In contrast, Hsen was increased in the NPLA group compared to vehicle. In addition, heat storage was lower in NPLA-treated animals. Despite the temperature differences, inhibition of nNOS in the PVN did not affect running performance on the treadmill. These results suggest that nitrergic signaling within the PVN, under nNOS activation, drives the increase of body temperature, being necessary for the proper thermal regulatory mechanisms during progressive exercise-induced hyperthermia.

Role of hydrogen sulfide in ventilatory responses to hypercapnia in the medullary raphe of adult rats.

NEW FINDINGS: What is the central question of this study? There is evidence that H2 S plays a role in the control of breathing: what are its actions on the ventilatory and thermoregulatory responses to hypercapnia via effects in the medullary raphe, a brainstem region that participates in the ventilatory adjustments to hypercapnia? What is the main finding and its importance? Hypercapnia increased the endogenous production of H2 S in the medullary raphe. Inhibition of endogenous H2 S attenuated the ventilatory response to hypercapnia in unanaesthetized rats, suggesting its excitatory action via the cystathionine ß-synthase-H2 S pathway in the medullary raphe. ABSTRACT: Hydrogen sulfide (H2 S) has been recently recognized as a gasotransmitter alongside carbon monoxide (CO) and nitric oxide (NO). H2 S seems to modulate the ventilatory and thermoregulatory responses to hypoxia and hypercapnia. However, the action of the H2 S in the medullary raphe (MR) on the ventilatory responses to hypercapnia remains to be elucidated. The present study aimed to assess the role of H2 S in the MR (a brainstem region that contains CO2 -sensitive cells and participates in the ventilatory adjustments to hypercapnia) in the ventilatory responses to hypercapnia in adult unanaesthetized Wistar rats. To do so, aminooxyacetic acid (AOA; a cystathionine ß-synthase (CBS) enzyme inhibitor), propargylglycine (PAG; a cystathionine γ-lyase enzyme inhibitor) and sodium sulfide (Na2 S; an H2 S donor) were microinjected into the MR. Respiratory frequency (fR ), tidal volume (VT ), ventilation ( V̇E ), oxygen consumption ( V̇O2 ) and body temperature (Tb ) were measured under normocapnic (room air) and hypercapnic (7% CO2 ) conditions. H2 S concentration within the MR was determined. Microinjection of the drugs did not affect fR , VT and V̇E during normocapnia when compared to the control group. However, the microinjection of AOA, but not PAG, attenuated fR and V̇E during hypercapnia in comparison to the vehicle group, but had no effects on Tb . In addition, we observed an increase in the endogenous production of H2 S in the MR during hypercapnia. Our findings indicate that endogenously produced H2 S in the MR plays an excitatory role in the ventilatory response to hypercapnia, acting through the CBS-H2 S pathway.

Lesion of Serotonergic Afferents to the Retrotrapezoid Nucleus Impairs the Tachypneic Response to Hypercapnia in Unanesthetized Animals.

Hypercapnia promotes an increase in pulmonary ventilation due to the stimulation of brainstem chemosensory cells that are connected to the respiratory network. Among these cells are the raphe serotonergic neurons which widely send projections to distinct central respiratory compartments. Nevertheless, the physiological role of specific raphe serotonergic projections to other chemosensitive sites on the emergence of hypercapnia ventilatory response in vivo still remains to be elucidated. Here we investigated whether the ventilatory response to hypercapnia requires serotonergic inputs to the chemosensitive cells of the retrotrapezoid nucleus (RTN) in the ventrolateral medulla. To test this, pulmonary ventilation was evaluated under baseline conditions and during hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60-90 g) that received bilateral microinjections of either vehicle (control) or anti-SERT-SAP (0.1 mM, 10 pmol/100 nl) toxin in the RTN to retrogradely destroy serotonergic afferents to this region. Fifteen days after microinjections, baseline ventilation was not different between anti-SERT-SAP (n = 8) and control animals (n = 9). In contrast, the ablation of RTN-projecting serotonergic neurons markedly attenuated the hypercapnia-induced increase in respiratory frequency which was correlated with reduced numbers of serotonergic neurons in the raphe obscurus and magnus, but not in the raphe pallidus. The increase in tidal volume during hypercapnia was not significantly affected by anti-SERT-SAP microinjections in the RTN. Our data indicate that serotoninergic neurons that send projections to the RTN region are required for the processing of ventilatory reflex response during exposure to high CO2 in unanesthetized conditions.

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.

Cardiorespiratory and thermal responses to hypercapnia in chickens exposed to CO2 during embryonic development.

The concentration of CO2 in the environment surrounding the embryo impacts development and may also influence the cardiorespiratory responses after hatching. Therefore, we aimed to evaluate the cardiorespiratory and thermal responses to hypercapnia in chicks that were exposed to CO2 during embryonic development, i.e., incubation. Embryos were incubated without and with a gradual increase in CO2 concentration up to 1 % during the first ten days of incubation. Ten-day-old chicks (males and females) were again acutely exposed to hypercapnia (7 % CO2), or to room air (normocapnia) and pulmonary ventilation, arterial pH and blood gases, arterial blood pressure and heart rate, body temperature (Tb) and oxygen consumption (V⋅O2) were measured. Compared to control animals, male chicks incubated with 1 % CO2 presented an attenuated ventilatory response to hypercapnia (P < 0.05), whereas no difference was found in the hypercapnic ventilatory response in both female chick groups (0 % vs 1 % CO2 incubation). Hypercapnia induced bradycardia in all groups (P < 0.001). The CO2 exposure during incubation did not alter the cardiovascular responses to hypercapnia in post-hatch animals. There were no significant effects of incubation treatment (0 % vs 1 % CO2) or sex in the mean arterial pressure, Tb, and V⋅O2 of animals in normocapnia and hypercapnia. As for the V⋅E/V⋅O2, hypercapnia caused an increase in both groups (P < 0.05), regardless of incubation treatment. In conclusion, among cardiorespiratory and metabolic variables, the ventilatory response to hypercapnia can be attenuated by pre-exposure to 1 % CO2 during embryonic development, especially in male chicks up to 10 days.

Respiratory control of acid-base status in lungfish.

The acid-base status is a tightly regulated physiological process, resulting from a balance of ions in the organism relevant to acid-base. The efficiency of the regulatory systems importantly determines the compensatory pH changes for a given disturb. Vertebrates minimize (or compensate) an acid-base disturb by general processes, which include ion transfer and/or PCO2 changes. Acid-base adjustment in fish is predominantly achieved by branchial exchange of acid-base relevant ions with correlated change in plasma HCO3- levels. Conversely, land vertebrates change blood PCO2 through ventilatory process and hence respiratory control of acid-base regulation plays an important role as a compensatory mechanism. Lungfishes (Dipnoi) have central position on vertebrate's evolution being considered as the sister group to the tetrapods. With an aquatic life mode, lungfish share similarities of respiratory function with tetrapods. This article reviews evidence showing that lungfish's respiratory system regulates acid-base status, like terrestrial ectothermic vertebrates. In the South American lungfish, Lepidosiren paradoxa, the presence of central CO2/pH chemoreceptors was unequivocally described. Also, the blood PCO2 and acid-base status are typical of a terrestrial vertebrate. These aspects are discussed under different environmental conditions that require respiratory acid-base adjustments, such as, exposure to hypercarbia, hypoxia, high temperature and aestivation. Interesting questions regarding the location and cell phenotype of CO2/pH central and peripheral chemoreceptors remain an open field to be explored in lungfish.

Buccal jet streaming and dead space determination in the South American lungfish, Lepidosiren paradoxa.

The "jet stream" model predicts an expired flow within the dorsal part of the buccal cavity with small air mixing during buccal pump ventilation, and has been suggested for some anuran amphibians but no other species of air breathing animal using a buccal force pump has been investigated. The presence of a two-stroke buccal pump in lungfish, i.e. expiration followed by inspiration, was described previously, but no quantitative data are available for the dead-space of their respiratory system and neither a detailed description of airflow throughout a breathing cycle. The present study aimed to assess the degree of mixing of fresh air and expired gas during the breathing cycle of Lepidosiren paradoxa and to verify the possible presence of a jet stream during expiration in this species. To do so, simultaneous measurements of buccal pressure and ventilatory airflows were carried out. Buccal and lung gases (PCO2 and PO2) were also measured. The effective ventilation was calculated and the dead space estimated using Bohr equations. The results confirmed that the two-stroke buccal pump is present in lungfish, as it is in anuran amphibians. The present approaches were coherent with a small dead space, with a very small buccal-lung PCO2 difference. In the South American lungfish the dead space (VD) as a percentage of tidal volume (VT) (VD / VT) ranged from 4.1 to 12.5%. Our data support the presence of a jet stream and indicate a small degree of air mixing in the buccal cavity. Comparisons with the literature indicate that these data are similar to previous data reported for the toad Rhinella schneideri.

Central administration of aminooxyacetate, an inhibitor of H2S production, affects thermoregulatory but not cardiovascular and ventilatory responses to hypercapnia in spontaneously hypertensive rats.

Hydrogen sulfide (H2S) is classically known for its toxic effects. More recently H2S has been documented as a neuromodulator. Here we investigated the central effects of aminooxyacetate (AOA; inhibitor of the H2S-synthesizing enzyme cystathionine ß-synthase, CBS) on cardiovascular, respiratory and thermoregulatory responses to hypercapnia in spontaneously hypertensive rats (SHR). To attain this goal we measured mean arterial pressure (MAP), heart rate (HR), ventilation (VE), and deep body temperature (Tb) of SHR and (normotensive) Wistar Kyoto (WKY) rats before and after microinjection of AOA (9 nmol/µL) or saline into the fourth ventricle immediately followed by 30-min hypercapnia exposure (7% inspired CO2). In saline-treated WKY rats, hypercapnia caused an increase in MAP accompanied by bradycardia, an increase in VE, and a drop in Tb. In AOA-treated WKY rats exposed to hypercapnia, the drug did not affect the increased MAP, potentiated the bradycardic response, attenuated the increased VE, and potentiated the drop in Tb. In saline-treated SHR, in comparison to the saline-treated WKY rats, hypercapnia elicited a minor, shorter-lasting increase in MAP with no changes in HR, evoked a greater increase in VE, and did not induce a drop in Tb. In AOA-treated SHR exposed to hypercapnia, the drug did not change the hypercapnia-induced cardiovascular and ventilatory responses while permitted a drop in Tb. Our findings indicate that AOA, an inhibitor of H2S production, modulates cardiorespiratory and thermoregulatory responses to hypercapnia in normotensive rats, whereas hypertension development in SHR is accompanied by suppression of the AOA effect on the cardiovascular and respiratory responses.

Carotid body removal normalizes arterial blood pressure and respiratory frequency in offspring of protein-restricted mothers.

The aim of this study is to evaluate the short-term and long-term effects elicited by carotid body removal (CBR) on ventilatory function and the development of hypertension in the offspring of malnourished rats. Wistar rats were fed a normo-protein (NP, 17% casein) or low-protein (LP, 8% casein) diet during pregnancy and lactation. At 29 days of age, the animals were submitted to CBR or a sham surgery, according to the following groups: NP-cbr, LP-cbr, NP-sham, or LP-sham. In the short-term, at 30 days of age, the respiratory frequency (RF) and immunoreactivity for Fos on the retrotrapezoid nucleus (RTN; brainstem site containing CO2 sensitive neurons) after exposure to CO2 were evaluated. In the long term, at 90 days of age, arterial pressure (AP), heart rate (HR), and cardiovascular variability were evaluated. In the short term, an increase in the baseline RF (~6%), response to CO2 (~8%), and Fos in the RTN (~27%) occurred in the LP-sham group compared with the NP-sham group. Interestingly, the CBR in the LP group normalized the RF in response to CO2 as well as RTN cell activation. In the long term, CBR reduced the mean AP by ~20 mmHg in malnourished rats. The normalization of the arterial pressure was associated with a decrease in the low-frequency (LF) oscillatory component of AP (~58%) and in the sympathetic tonus to the cardiovascular system (~29%). In conclusion, carotid body inputs in malnourished offspring may be responsible for the following: (i) enhanced respiratory frequency and CO2 chemosensitivity in early life and (ii) the production of autonomic imbalance and the development of hypertension.

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.

Analysis of the respiratory component of heart rate variability in the Cururu toad Rhinella schneideri.

Beat-to-beat variation in heart rate (f H ) has been used as a tool for elucidating the balance between sympathetic and parasympathetic modulation of the heart. A portion of the temporal changes in f H is evidenced by a respiratory influence (cardiorespiratory interaction) on heart rate variability (HRV) with heartbeats increasing and decreasing within a respiratory cycle. Nevertheless, little is known about respiratory effects on HRV in lower vertebrates. By using frequency domain analysis, we provide the first evidence of a ventilatory component in HRV similar to mammalian respiratory sinus arrhythmia in an amphibian, the toad Rhinella schneideri. Increases in the heartbeats arose synchronously with each lung inflation cycle, an intermittent breathing pattern comprised of a series of successive lung inflations. A well-marked peak in the HRV signal matching lung inflation cycle was verified in toads whenever lung inflation cycles exhibit a regular rhythm. The cardiac beat-to-beat variation evoked at the moment of lung inflation accounts for both vagal and sympathetic influences. This cardiorespiratory interaction may arise from interactions between central and peripheral feedback mechanisms governing cardiorespiratory control and may underlie important cardiorespiratory adjustments for gas exchange improvement especially under extreme conditions like low oxygen availability.

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.

Excitatory Modulation of the preBötzinger Complex Inspiratory Rhythm Generating Network by Endogenous Hydrogen Sulfide.

Hydrogen Sulfide (H2S) is one of three gasotransmitters that modulate excitability in the CNS. Global application of H2S donors or inhibitors of H2S synthesis to the respiratory network has suggested that inspiratory rhythm is modulated by exogenous and endogenous H2S. However, effects have been variable, which may reflect that the RTN/pFRG (retrotrapezoid nucleus, parafacial respiratory group) and the preBötzinger Complex (preBötC, critical for inspiratory rhythm generation) are differentially modulated by exogenous H2S. Importantly, site-specific modulation of respiratory nuclei by H2S means that targeted, rather than global, manipulation of respiratory nuclei is required to understand the role of H2S signaling in respiratory control. Thus, our aim was to test whether endogenous H2S, which is produced by cystathionine-ß-synthase (CBS) in the CNS, acts specifically within the preBötC to modulate inspiratory activity under basal (in vitro/in vivo) and hypoxic conditions (in vivo). Inhibition of endogenous H2S production by bath application of the CBS inhibitor, aminooxyacetic acid (AOAA, 0.1-1.0 mM) to rhythmic brainstem spinal cord (BSSC) and medullary slice preparations from newborn rats, or local application of AOAA into the preBötC (slices only) caused a dose-dependent decrease in burst frequency. Unilateral injection of AOAA into the preBötC of anesthetized, paralyzed adult rats decreased basal inspiratory burst frequency, amplitude and ventilatory output. AOAA in vivo did not affect the initial hypoxia-induced (10% O2, 5 min) increase in ventilatory output, but enhanced the secondary hypoxic respiratory depression. These data suggest that the preBötC inspiratory network receives tonic excitatory modulation from the CBS-H2S system, and that endogenous H2S attenuates the secondary hypoxic respiratory depression.

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.