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.

Effects of aerial hypoxia and temperature on pulmonary breathing pattern and gas exchange in the South American lungfish, Lepidosiren paradoxa.

The South American lungfish Lepidosiren paradoxa is an obligatory air-breathing fish possessing well-developed bilateral lungs, and undergoing seasonal changes in its habitat, including temperature changes. In the present study we aimed to evaluate gas exchange and pulmonary breathing pattern in L. paradoxa at different temperatures (25 and 30°C) and different inspired O2 levels (21, 12, 10, and 7%). Normoxic breathing pattern consisted of isolated ventilatory cycles composed of an expiration followed by 2.4±0.2 buccal inspirations. Both expiratory and inspiratory tidal volumes reached a maximum of about 35mlkg-1, indicating that L. paradoxa is able to exchange nearly all of its lung air in a single ventilatory cycle. At both temperatures, hypoxia caused a significant increase in pulmonary ventilation (V̇E), mainly due to an increase in respiratory frequency. Durations of the ventilatory cycle and expiratory and inspiratory tidal volumes were not significantly affected by hypoxia. Expiratory time (but not inspiratory) was significantly shorter at 30°C and at all O2 levels. While a small change in oxygen consumption (V̇O2) could be noticed, the carbon dioxide release (V̇CO2, P=0.0003) and air convection requirement (V̇E/V̇O2, P=0.0001) were significantly affected by hypoxia (7% O2) at both temperatures, when compared to normoxia, and pulmonary diffusion capacity increased about four-fold due to hypoxic exposure. These data highlight important features of the respiratory system of L. paradoxa, capable of matching O2 demand and supply under different environmental change, as well as help to understand the evolution of air breathing in lungfish.

Acute effects of temperature and hypercarbia on cutaneous and branchial gas exchange in the South American lungfish, Lepidosiren paradoxa.

The South American lungfish, Lepidosiren paradoxa inhabits seasonal environments in the Central Amazon and Paraná-Paraguay basins that undergo significant oscillations in temperature throughout the year. They rely on different gas exchange organs, such as gills and skin for aquatic gas exchange while their truly bilateral lungs are responsible for aerial gas exchange; however, there are no data available on the individual contributions of the skin and the gills to total aquatic gas exchange in L. paradoxa. Thus, in the present study we quantify the relative contributions of skin and gills on total aquatic gas exchange during warm (35°C) and cold exposure (20°C) in addition to the effects of aerial and aquatic hypercarbia on aquatic gas exchange and gill ventilation rate (fG; 25°C), respectively. Elevated temperature (35°C) caused a significant increase in the contribution of cutaneous (from 0.61±0.13 to 1.34±0.26ml. STPD.h-1kg-1) and branchial (from 0.54±0.17 to 1.73±0.53ml. STPD.h-1kg-1) gas exchange for V̇CO2 relative to the lower temperature (20°C), while V̇O2 remained relatively unchanged. L. paradoxa exhibited a greater branchial contribution in relation to total aquatic gas exchange at lower temperatures (20 and 25°C) for oxygen uptake. Aerial hypercarbia decreased branchial V̇O2 whereas branchial V̇CO2 was significantly increased. Progressive increases in aquatic hypercarbia did not affect fG. This response is in contrast to increases in pulmonary ventilation that may offset any increase in arterial partial pressure of CO2 owing to CO2 loading through the animals' branchial surface. Thus, despite their reduced contribution to total gas exchange, cutaneous and branchial gas exchange in L. paradoxa can be significantly affected by temperature and aerial hypercarbia.

Effect of chronic ethanol exposure on rat ventilatory responses to hypoxia and hypercapnia.

OBJECTIVE: The effect of chronic ethanol exposure on chemoreflexes has not been extensively studied in experimental animals. Therefore, this study tested the hypothesis that known ethanol-induced autonomic, neuroendocrine and cardiovascular changes coincide with increased chemoreflex sensitivity, as indicated by increased ventilatory responses to hypoxia and hypercapnia. METHODS: Male Wistar rats were subjected to increasing ethanol concentrations in their drinking water (first week: 5% v/v, second week: 10% v/v, third and fourth weeks: 20% v/v). At the end of each week of ethanol exposure, ventilatory parameters were measured under basal conditions and in response to hypoxia (evaluation of peripheral chemoreflex sensitivity) and hypercapnia (evaluation of central chemoreflex sensitivity). RESULTS: Decreased respiratory frequency was observed in rats exposed to ethanol from the first until the fourth week, whereas minute ventilation remained unchanged. Moreover, we observed an increased tidal volume in the second through the fourth week of exposure. The minute ventilation responses to hypoxia were attenuated in the first through the third week but remained unchanged during the last week. The respiratory frequency responses to hypoxia in ethanol-exposed rats were attenuated in the second through the third week but remained unchanged in the first and fourth weeks. There was no significant change in tidal volume responses to hypoxia. With regard to hypercapnic responses, no significant changes in ventilatory parameters were observed. CONCLUSIONS: Our data are consistent with the notion that chronic ethanol exposure does not increase peripheral or central chemoreflex sensitivity.

Effect of chronic ethanol exposure on rat ventilatory responses to hypoxia and hypercapnia

OBJECTIVE: The effect of chronic ethanol exposure on chemoreflexes has not been extensively studied in experimental animals. Therefore, this study tested the hypothesis that known ethanol-induced autonomic, neuroendocrine and cardiovascular changes coincide with increased chemoreflex sensitivity, as indicated by increased ventilatory responses to hypoxia and hypercapnia. METHODS: Male Wistar rats were subjected to increasing ethanol concentrations in their drinking water (first week: 5% v/v, second week: 10% v/v, third and fourth weeks: 20% v/v). At the end of each week of ethanol exposure, ventilatory parameters were measured under basal conditions and in response to hypoxia (evaluation of peripheral chemoreflex sensitivity) and hypercapnia (evaluation of central chemoreflex sensitivity). RESULTS: Decreased respiratory frequency was observed in rats exposed to ethanol from the first until the fourth week, whereas minute ventilation remained unchanged. Moreover, we observed an increased tidal volume in the second through the fourth week of exposure. The minute ventilation responses to hypoxia were attenuated in the first through the third week but remained unchanged during the last week. The respiratory frequency responses to hypoxia in ethanol-exposed rats were attenuated in the second through the third week but remained unchanged in the first and fourth weeks. There was no significant change in tidal volume responses to hypoxia. With regard to hypercapnic responses, no significant changes in ventilatory parameters were observed. CONCLUSIONS: Our data are consistent with the notion that chronic ethanol exposure does not increase peripheral or central chemoreflex sensitivity. .

Serotonergic neurons in the nucleus raphé obscurus are not involved in the ventilatory and thermoregulatory responses to hypoxia in adult rats.

The medullary raphé is an important component of the central respiratory network, playing a key role in CO2 central chemoreception. However, its participation in hypoxic ventilatory responses is less understood. In the present study, we assessed the role of nucleus raphé obscurus (ROb), and specifically 5-HT neurons confined in the ROb, on ventilatory and thermoregulatory responses to hypoxia. Chemical lesions of the ROb were performed using either ibotenic acid (non-specific lesion; control animals received PBS) or anti-SERT-SAP (5-HT specific lesion; control animals received IgG-SAP). Ventilation (V˙E; whole body plethysmograph) and body temperature (Tb; data loggers) were measured during normoxia (21% O2, N2 balance) and hypoxia exposure (7% O2, N2 balance, 1h) in conscious adult rats. Ibotenic acid or anti-SERT-SAP-induced lesions did not affect baseline values of V˙E and Tb. Similarly, both lesion procedures did not alter the ventilatory or thermoregulatory responses to hypoxia. Although evidence in the literature suggests a role of the rostral medullary raphé in hypoxic ventilatory responses, under the present experimental conditions our data indicate that caudal medullary raphé (ROb) and its 5-HT neurons neither participate in the tonic maintenance of breathing nor in the ventilatory and thermal responses to hypoxia.

Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats.

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats.

Central leptin replacement enhances chemorespiratory responses in leptin-deficient mice independent of changes in body weight.

Previous studies showed that leptin-deficient (ob/ob) mice develop obesity and impaired ventilatory responses to CO(2) (V(E) - CO(2)). In this study, we examined if leptin replacement improves chemorespiratory responses to hypercapnia (7 % CO(2)) in ob/ob mice and if these effects were due to changes in body weight or to the direct effects of leptin in the central nervous system (CNS). V(E) - CO(2) was measured via plethysmography in obese leptin-deficient- (ob/ob) and wild-type- (WT) mice before and after leptin (10 µg/2 µl day) or vehicle (phosphate buffer solution) were microinjected into the fourth ventricle for four consecutive days. Although baseline V(E) was similar between groups, obese ob/ob mice exhibited attenuated V(E) - CO(2) compared to WT mice (134 ± 9 versus 196 ± 10 ml min(-1)). Fourth ventricle leptin treatment in obese ob/ob mice significantly improved V(E) - CO(2) (from 131 ± 15 to 197 ± 10 ml min(-1)) by increasing tidal volume (from 0.38 ± 0.03 to 0.55 ± 0.02 ml, vehicle and leptin, respectively). Subcutaneous leptin administration at the same dose administered centrally did not change V(E) - CO(2) in ob/ob mice. Central leptin treatment in WT had no effect on V(E) - CO(2). Since the fourth ventricle leptin treatment decreased body weight in ob/ob mice, we also examined V(E) - CO(2) in lean pair-weighted ob/ob mice and found it to be impaired compared to WT mice. Thus, leptin deficiency, rather than obesity, is the main cause of impaired V(E) - CO(2) in ob/ob mice and leptin appears to play an important role in regulating chemorespiratory response by its direct actions on the CNS.

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.

Serotonergic neurons in the nucleus raphe obscurus contribute to interaction between central and peripheral ventilatory responses to hypercapnia.

Serotonergic (5-HT) neurons in the nucleus raphe obscurus (ROb) are involved in the respiratory control network. However, it is not known whether ROb 5-HT neurons play a role in the functional interdependence between central and peripheral chemoreceptors. Therefore, we investigated the role of ROb 5-HT neurons in the ventilatory responses to CO2 and their putative involvement in the central-peripheral CO2 chemoreceptor interaction in unanaesthetised rats. We used a chemical lesion specific for 5-HT neurons (anti-SERT-SAP) of the ROb in animals with the carotid body (CB) intact or removed (CBR). Pulmonary ventilation (V (E)), body temperature and the arterial blood gases were measured before, during and after a hypercapnic challenge (7% CO2). The lesion of ROb 5-HT neurons alone (CB intact) or the lesion of 5-HT neurons of ROb+CBR did not affect baseline V (E) during normocapnic condition. Killing ROb 5-HT neurons (CB intact) significantly decreased the ventilatory response to hypercapnia (p < 0.05). The reduction in CO2 sensitivity was approximately 15%. When ROb 5-HT neurons lesion was combined with CBR (anti-SERT-SAP+CBR), the V (E) response to hypercapnia was further decreased (-31.2%) compared to the control group. The attenuation of CO2 sensitivity was approximately 30%, and it was more pronounced than the sum of the individual effects of central (ROb lesion; -12.3%) or peripheral (CBR; -5.5%) treatments. Our data indicate that ROb 5-HT neurons play an important role in the CO2 drive to breathing and may act as an important element in the central-peripheral chemoreception interaction to CO2 responsiveness.

Blood gases and cardiovascular shunt in the South American lungfish (Lepidosiren paradoxa) during normoxia and hyperoxia.

The South American lungfish (Lepidosiren paradoxa) has an arterial P(O(2)) (Pa(O(2))) as high as 70-100 mmHg, corresponding to Hb-O(2) saturations from 90% to 95%, which indicates a moderate cardiovascular right to left (R-L) shunt. In hyperoxia (50% O(2)), we studied animals in: (1) aerated water combined with aerial hyperoxia, which increased Pa(O(2)) from 78+/-2 to 114+/-3 mmHg and (2) and aquatic hyperoxia (50% O(2)) combined room air, which gradually increased Pa(O(2)) from 75+/-4 mmHg to as much as 146+/-10 mmHg. Further, the hyperoxia (50%) depressed pulmonary ventilation from 58+/-13 to 5.5+/-3.0 mLBTPSkgh(-1), and Pa(CO(2)) increased from 20+/-2 to 31+/-4 mmHg, while pHa became reduced from 7.56+/-0.03 to 7.31+/-0.09. At the same time, venous P(O(2)) (Pv(O(2))) rose from 40.0+/-2.3 to 46.4+/-1.2 mmHg and, concomitantly, Pv(CO(2)) increased from 23.2+/-1.1 to 32.2+/-0.5 mmHg. R-L shunts were estimated to about 19%, which is moderate when compared to most amphibians.

Respiratory chemoreceptor function in vertebrates comparative and evolutionary aspects.

The sensing of blood gas tensions and/or pH is an evolutionarily conserved, homeostatic mechanism, observable in almost all species studied from invertebrates to man. In vertebrates, a shift from the peripheral O(2)-oriented sensing in fish, to the central CO(2)/pH sensing in most tetrapods reflects the specific behavioral requirements of these two groups whereby, in teleost fish, a highly O(2)-oriented control of breathing matches the ever-changing and low oxygen levels in water, whilst the transition to air-breathing increased the importance of acid-base regulation and O(2)-related drive, although retained, became relatively less important. The South American lungfish and tetrapods are probably sister groups, a conclusion backed up by many similar features of respiratory control. For example, the relative roles of peripheral and central chemoreceptors are present both in the lungfish and in land vertebrates. In both groups, the central CO(2)/pH receptors dominate the ventilatory response to hypercarbia (60-80%), while the peripheral CO(2)/pH receptors account for 20-30%. Some basic components of respiratory control have changed little during evolution. This review presents studies that reflect the current trends in the field of chemoreceptor function, and several laboratories are involved. An exhaustive review on the previous literature, however, is beyond the intended scope of the article. Rather, we present examples of current trends in respiratory function in vertebrates, ranging from fish to humans, and focus on both O(2) sensing and CO(2) sensing. As well, we consider the impact of chronic levels of hypoxia-a physiological condition in fish and in land vertebrates resident at high elevations or suffering from one of the many cardiorespiratory disease states that predispose an animal to impaired ventilation or cardiac output. This provides a basis for a comparative physiology that is informative about the evolution of respiratory functions in vertebrates and about human disease. Currently, most detail is known for mammals, for which molecular biology and respiratory physiology have combined in the discovery of the mechanisms underlying the responses of respiratory chemoreceptors. Our review includes new data on nonmammalian vertebrates, which stresses that some chemoreceptor sites are of ancient origin.

Locus coeruleus is a central chemoreceptive site in toads.

The locus coeruleus (LC) has been suggested as a CO2 chemoreceptor site in mammals. This nucleus is a mesencephalic structure of the amphibian brain and is probably homologous to the LC in mammals. There are no data available for the role of LC in the central chemoreception of amphibians. Thus the present study was designed to investigate whether LC of toads (Bufo schneideri) is a CO2/H+ chemoreceptor site. Fos immunoreactivity was used to verify whether the nucleus is activated by hypercarbia (5% CO2 in air). In addition, we assessed the role of noradrenergic LC neurons on respiratory and cardiovascular responses to hypercarbia by using 6-hydroxydopamine lesion. To further explore the role of LC in central chemosensitivity, we examined the effects of microinjection of solutions with different pH values (7.2, 7.4, 7.6, 7.8, and 8.0) into the nucleus. Our main findings were that 1) a marked increase in c-fos-positive cells in the LC was induced after 3 h of breathing a hypercarbic gas mixture; 2) chemical lesions in the LC attenuated the increase of the ventilatory response to hypercarbia but did not affect ventilation under resting conditions; and 3) microinjection with acid solutions (pH = 7.2, 7.4, and 7.6) into the LC elicited an increased ventilation, indicating that the LC of toads participates in the central chemoreception.

Morphometric comparison of the respiratory organs in the South American lungfish Lepidosiren paradoxa (Dipnoi).

In light of the relationship of lungfish to the origin of tetrapods, information on the respiratory biology of lungfish can give insight into the functional morphological and physiological prerequisites for the conquest of land by the first tetrapods. Stereological methods were employed in order to determine the respiratory surface area and thickness of the water-blood barrier or air-blood of the gills, lungs, and skin, respectively, of the South American lungfish Lepidosiren paradoxa. The morphometric diffusing capacity was then determined by multiplying by the appropriate Krogh diffusion constants (K). Our results indicate a total diffusing capacity of all respiratory organs of 0.11 mL min(-1) mmHg(-1) kg(-1), which is more than twice the value of the physiological diffusion capacity (approximately 0.04 mL min(-1) mmHg(-1) kg(-1)). Of this, 99.15% lies in the lungs, 0.85% in the skin, and only 0.0013% in the gills. Since K for CO(2) is 20-25 times greater than for O(2), diffusing capacity of CO(2) through the skin is potentially important. That of the gills, however, is negligible, raising the question as to their function. Our results indicate that the morphological prerequisites for terrestrial survival with regard to supporting aerobic metabolism already existed in the lungfish.