Evolution of vertebrate respiratory central rhythm generators.

Tracing the evolution of the central rhythm generators associated with ventilation in vertebrates is hindered by a lack of information surrounding key transitions. To begin with, central rhythm generation has been studied in detail in only a few species from four vertebrate groups, lamprey, anuran amphibians, turtles, and mammals (primarily rodents). Secondly, there is a lack of information regarding the transition from water breathing fish to air breathing amniotes (reptiles, birds, and mammals). Specifically, the respiratory rhythm generators of fish appear to be single oscillators capable of generating both phases of the respiratory cycle (expansion and compression) and projecting to motoneurons in cranial nerves innervating bucco-pharyngeal muscles. In the amniotes we find oscillators capable of independently generating separate phases of the respiratory cycle (expiration and inspiration) and projecting to pre-motoneurons in the ventrolateral medulla that in turn project to spinal motoneurons innervating thoracic and abdominal muscles (reptiles, birds, and mammals). Studies of the one group of amphibians that lie at this transition (the anurans), raise intriguing possibilities but, for a variety of reasons that we explore, also raise unanswered questions. In this review we summarize what is known about the rhythm generating circuits associated with breathing that arise from the different rhombomeric segments in each of the different vertebrate classes. Assuming oscillating circuits form in every pair of rhombomeres in every vertebrate during development, we trace what appears to be the evolutionary fate of each and highlight the questions that remain to be answered to properly understand the evolutionary transitions in vertebrate central respiratory rhythm generation.

Different strategies for convective O2 transport in high altitude birds: A graphical analysis.

For illustrative purposes, in this article we use "Johansen Plots" as a graphical way of simultaneously visualizing the inter-connected variables that compose the convective steps of the gas transport cascade. These plots are used to reflect on some of the physiological characteristics seen in five species of birds, four of which sojourn to, or are native to, high altitudes (the barnacle goose, bar-headed goose, Andean goose, speckled teal and ruddy duck). These species were chosen to emphasize the diversity of responses to hypoxia that can exist within a single family. This diversity likely arose for many possible reasons, including local adaptation to hypoxia, differences in flight or diving abilities, or as a result of other phylogenetically-based differences across waterfowl in physiology, behaviour, and/or life style.

Heart mitochondria from naked mole-rats (Heterocephalus glaber) are more coupled, but similarly susceptible to anoxia-reoxygenation stress than in laboratory mice (Mus musculus).

Naked mole-rats (Heterocephalus glaber; NMRs) are among the most hypoxia-tolerant mammals described to date and exhibit plastic responses during hypoxia exposure. The goal of the present study was to determine if heart mitochondria from NMRs functionally differ from those of hypoxia-intolerant common laboratory mice (Mus musculus). We assessed heart mitochondrial respiratory flux, proton leak kinetics, responses to in vitro anoxia-recovery, and maximal complex enzyme activities. When investigated at their respective body temperatures (28 °C for NMR and 37 °C for mice), NMR heart mitochondria had lower respiratory fluxes relative to mice, particularly for state 2 and oligomycin-induced state 4 leak respiration rates. When leak respiration rates were standardized to the same membrane potential, NMR mitochondria had lower complex II-stimulated state 2 respiration rates than mice. Both mice and NMRs responded similarly to an in vitro anoxia-recovery challenge and decreased state 3 respiration rate post-anoxia. Finally, NMRs had overall lower maximal complex enzyme activities compared with mice, but the magnitude of the difference did not correspond with observed differences in respiratory fluxes. Overall, heart mitochondria from NMRs appear more coupled than those of mice, but in both species the heart appears equally susceptible to ischemic-reperfusion injury.

The roller coaster flight strategy of bar-headed geese conserves energy during Himalayan migrations.

The physiological and biomechanical requirements of flight at high altitude have been the subject of much interest. Here, we uncover a steep relation between heart rate and wingbeat frequency (raised to the exponent 3.5) and estimated metabolic power and wingbeat frequency (exponent 7) of migratory bar-headed geese. Flight costs increase more rapidly than anticipated as air density declines, which overturns prevailing expectations that this species should maintain high-altitude flight when traversing the Himalayas. Instead, a "roller coaster" strategy, of tracking the underlying terrain and discarding large altitude gains only to recoup them later in the flight with occasional benefits from orographic lift, is shown to be energetically advantageous for flights over the Himalayas.

Validation of the i-STAT and HemoCue systems for the analysis of blood parameters in the bar-headed goose, Anser indicus.

Every year, bar-headed geese (Anser indicus) perform some of the most remarkable trans-Himalayan migrations, and researchers are increasingly interested in understanding the physiology underlying their high-altitude flight performance. A major challenge is generating reliable measurements of blood parameters on wild birds in the field, where established analytical techniques are often not available. Therefore, we validated two commonly used portable clinical analysers (PCAs), the i-STAT and the HemoCue systems, for the analysis of blood parameters in bar-headed geese. The pH, partial pressures of O2 and CO2 (PO2 and PCO2), haemoglobin O2 saturation (sO2), haematocrit (Hct) and haemoglobin concentration [Hb] were simultaneously measured with the two PCA systems (i-STAT for all parameters; HemoCue for [Hb]) and with conventional laboratory techniques over a physiological range of PO2, PCO2 and Hct. Our results indicate that the i-STAT system can generate reliable values on bar-headed goose whole blood pH, PO2, PCO2 and Hct, but we recommend correcting the obtained values using the linear equations determined here for higher accuracy. The i-STAT is probably not able to produce meaningful measurements of sO2 and [Hb] over a range of physiologically relevant environmental conditions. However, we can recommend the use of the HemoCue to measure [Hb] in the bar-headed goose, if results are corrected. We emphasize that the equations that we provide to correct PCA results are applicable only to bar-headed goose whole blood under the conditions that we tested. We encourage researchers to validate i-STAT or HemoCue results thoroughly for their specific study conditions and species in order to yield accurate results.

The effect of sustained hypoxia on the cardio-respiratory response of bowfin Amia calva: implications for changes in the oxygen transport system.

This study examined mechanisms underlying cardio-respiratory acclimation to moderate sustained hypoxia (6.0 kPa for 7 days at 22° C) in the bowfin Amia calva, a facultative air-breathing fish. This level of hypoxia is slightly below the critical oxygen tension (pcrit ) of A. calva denied access to air (mean ± s.e. = 9.3 ± 1.0 kPa). Before exposure to sustained hypoxia, A. calva with access to air increased air-breathing frequency on exposure to acute progressive hypoxia while A. calva without access to air increased gill-breathing frequency. Exposure to sustained hypoxia increased the gill ventilation response to acute progressive hypoxia in A. calva without access to air, regardless of whether they had access to air or not during the sustained hypoxia. Additionally, there was a decrease in Hb-O2 binding affinity in these fish. This suggests that, in A. calva, acclimation to hypoxia elicits changes that increase oxygen delivery to the gas exchange surface for oxygen uptake and reduce haemoglobin affinity to enhance oxygen delivery to the tissues.

The paradox of extreme high-altitude migration in bar-headed geese Anser indicus.

Bar-headed geese are renowned for migratory flights at extremely high altitudes over the world's tallest mountains, the Himalayas, where partial pressure of oxygen is dramatically reduced while flight costs, in terms of rate of oxygen consumption, are greatly increased. Such a mismatch is paradoxical, and it is not clear why geese might fly higher than is absolutely necessary. In addition, direct empirical measurements of high-altitude flight are lacking. We test whether migrating bar-headed geese actually minimize flight altitude and make use of favourable winds to reduce flight costs. By tracking 91 geese, we show that these birds typically travel through the valleys of the Himalayas and not over the summits. We report maximum flight altitudes of 7290 m and 6540 m for southbound and northbound geese, respectively, but with 95 per cent of locations received from less than 5489 m. Geese travelled along a route that was 112 km longer than the great circle (shortest distance) route, with transit ground speeds suggesting that they rarely profited from tailwinds. Bar-headed geese from these eastern populations generally travel only as high as the terrain beneath them dictates and rarely in profitable winds. Nevertheless, their migration represents an enormous challenge in conditions where humans and other mammals are only able to operate at levels well below their sea-level maxima.

The phylogeny of central chemoreception.

Respiratory chemoreceptors responsive to changes in CO(2)/H(+) appear to be present in all vertebrates from fish to birds and mammals. They appear to have arisen first in the periphery sensitive to the external environment. Thus, in most fish CO(2)/H(+) chemoreceptors reside primarily in the gills and respond to changes in aquatic rather than arterial P(CO)2 . In the air-breathing tetrapods (amphibians, mammals, reptiles and birds), the branchial arches regress developmentally and the derivatives of the branchial arteries are now exclusively internal. The receptors associated with these arteries now sense only arterial (not environmental) P(CO)2/pH . Central CO(2)/H(+) chemoreception also appears to have arisen with the advent of air breathing, presumably as a second line of defense. These receptors may have arisen multiple times in association with several (but not all) of the independent origins of air breathing in fishes. There is strong evidence for multiple central sites of CO(2)/H(+) sensing, at least in amphibians and mammals, suggesting that it may not only have originated multiple times in different species but also multiple times within a single species.

Species differences in respiratory rhythm generation in rodents.

We examined the role of riluzole (RIL)- and flufenamic acid (FFA)-sensitive mechanisms in respiratory rhythmogenesis in rats and hamsters using the in situ arterially perfused preparation. Based on the hypothesis that respiratory networks in animals capable of autoresuscitation would have a greater prevalence of membrane mechanisms that promote endogenous bursting, we predicted that older (weaned) hamsters (a hibernating species) would be more sensitive to the blockade of RIL- and FFA-sensitive mechanisms than age-matched rats and that younger (preweaned) rats would behave more like hamsters. Consistent with this, we found that respiratory motor output in weaned hamsters [>21 days postnatal (P21)] was highly sensitive to RIL (0.2-20 muM), while in young rats (P12-14) it was less so (only affected at higher concentrations of RIL), and weaned rats were not affected at all. On the other hand, respiratory motor output was equally reduced by FFA (0.25-25 muM) in both young and weaned rats but was unaffected in weaned hamsters. Coapplication of RIL and FFA (RIL + FFA) produced greater inhibition of respiration in both young and weaned rats compared with either drug alone. In contrast, in weaned hamsters, FFA coapplication offset the inhibitory effect of RIL alone. Increasing respiratory drive with hypercapnia/acidosis ameliorated the respiratory inhibition produced by RIL + FFA in weaned rats but had no effect in young rats. Data from the present study indicate that respiratory rhythmogenesis in young rats is more dependent on excitatory RIL-sensitive and FFA-sensitive mechanisms than older rats and that fundamental differences exist in the respiratory rhythmogenic mechanisms between rats and hamsters.

NMDA receptor-mediated processes in the Parabrachial/Kölliker fuse complex influence respiratory responses directly and indirectly via changes in cortical activation state.

We tested the hypothesis that glutamate, acting via NMDA-type receptors (NMDAr) in the Parabrachial/Kölliker fuse (PBrKF) nucleus of the pons, is involved both directly and indirectly (via changes in cortical activation state) in modulating breathing and ventilatory responses to hypoxia. To this end we examined the effects of MK-801, injected either systemically or directly into the PBrKF, on the breathing patterns of urethane-anaesthetized rats breathing air or an hypoxic gas mixture as electroencephalographic (EEG) activity alternated between State I (awake-like) and State III (NREM sleep-like) EEG patterns. Regardless of EEG state, systemic MK-801 reduced ventilation primarily by reducing tidal volume while microinjection of MK-801 into the PBrKF reduced ventilation by reducing breathing frequency. With both injections, EEG pattern changed from State I to III mimicking the change from wakefulness to NREM sleep that occurs in unanaesthetized rats given MK-801 systemically. Systemic injection of MK-801 delayed and reduced the response to hypoxia while microinjection of MK-801 into the PBrKF did not reduce the HVR but sustained the hypoxic increase in tidal volume well into the post-hypoxic recovery period. Thus, while NMDAr in the PBrKF complex of the pons play a role in modulating sleep/wake-like states as well as changes in breathing pattern associated with changes in cortical activation state, they are neither involved in the hypoxic ventilatory response nor in the change in hypoxic sensitivity associated with the changes in cortical activation state.

Gill chemoreceptors and cardio-respiratory reflexes in the neotropical teleost pacu, Piaractus mesopotamicus.

This study examined the location and distribution of O(2) chemoreceptors involved in cardio-respiratory responses to hypoxia in the neotropical teleost, the pacu (Piaractus mesopotamicus). Intact fish and fish experiencing progressive gill denervation by selective transection of cranial nerves IX and X were exposed to gradual hypoxia and submitted to intrabuccal and intravenous injections of NaCN while their heart rate, ventilation rate and ventilation amplitude were measured. The chemoreceptors producing reflex bradycardia were confined to, but distributed along all gill arches, and were sensitive to O(2) levels in the water and the blood. Ventilatory responses to all stimuli, though modified, continued following gill denervation, however, indicating the presence of internally and externally oriented receptors along all gill arches and either in the pseudobranch or at extra-branchial sites. Chemoreceptors located on the first pair of gill arches and innervated by the glossopharyngeal nerve appeared to attenuate the cardiac and respiratory responses to hypoxia. The data indicate that the location and distribution of cardio-respiratory O(2) receptors are not identical to those in tambaqui (Colossoma macropomum) despite their similar habitats and close phylogenetic lineage, although the differences between the two species could reduce to nothing more than the presence or absence of the pseudobranch.

Cardiorespiratory responses to hypercarbia in tambaqui Colossoma macropomum: chemoreceptor orientation and specificity.

Experiments were carried out to test the hypothesis that ventilatory and cardiovascular responses to hypercarbia (elevated water P(CO2)) in the tambaqui Colossoma macropomum are stimulated by externally oriented receptors that are sensitive to water CO(2) tension as opposed to water pH. Cardiorespiratory responses to acute hypercarbia were evaluated in both the absence and presence of internal hypercarbia (elevated blood P(CO2)), achieved by treating fish with the carbonic anhydrase inhibitor acetazolamide. Exposure to acute hypercarbia (15 min at each level, final water CO(2) tensions of 7.2, 15.5 and 26.3 mmHg) elicited significant increases in ventilation frequency (at 26.3 mmHg, a 42% increase over the normocarbic value) and amplitude (128%), together with a fall in heart rate (35%) and an increase in cardiac stroke volume (62%). Rapid washout of CO(2) from the water reversed these effects, and the timing of the changes in cardiorespiratory variables corresponded more closely to the fall in water P(CO2) (Pw(CO2)) than to that in blood P(CO2) (Pa(CO2)). Similar responses to acute hypercarbia (15 min, final Pw(CO2) of 13.6 mmHg) were observed in acetazolamide-treated (30 mg kg(-1)) tambaqui. Acetazolamide treatment itself, however, increased Pa(CO2) (from 4.81+/-0.58 to 13.83+/-0.91 mmHg, mean +/-S.E.M.; N=8) in the absence of significant change in ventilation, heart rate or cardiac stroke volume. The lack of response to changes in blood P(CO2) and/or pH were confirmed by comparing responses to the bolus injection of hypercarbic saline (5% or 10% CO(2); 2 ml kg(-1)) into the caudal vein with those to the injection of CO(2)-enriched water (1%, 3%, 5% or 10% CO(2); 50 ml kg(-1)) into the buccal cavity. Whereas injections of hypercarbic saline were ineffective in eliciting cardiorespiratory responses, changes in ventilation and cardiovascular parameters accompanied injection of CO(2)-laden water into the mouth. Similar injections of CO(2)-free water acidified to the corresponding pH of the hypercarbic water (pH 6.3, 5.6, 5.3 or 4.9, respectively) generally did not stimulate cardiorespiratory responses. These results are in agreement with the hypothesis that in tambaqui, externally oriented chemoreceptors that are predominantly activated by increases in water P(CO2), rather than by accompanying decreases in water pH, are linked to the initiation of cardiorespiratory responses to hypercarbia.

Effect of hypothermia on respiratory rhythm generation in hamster brainstem-spinal cord preparations.

This study examined the effect of hypothermia on respiratory neural output from brainstem-spinal cord preparations of a cold tolerant rodent, the Syrian hamster. Brainstem-spinal cords from neonatal hamsters (0-6 days) were placed in a recording dish and respiratory-like neural activity was recorded from roots of the first cervical nerve. The preparations were cooled and warmed in a continuous or stepwise fashion. Inputs from the pons completely inhibited neural activity under steady state conditions. With the pons removed, fictive breathing was robust. Cooling caused respiratory arrest, followed by spontaneous resumption of activity on re-warming. Preparations from older hamsters (4-6 days old) were more cold tolerant than younger preparations (0-3 days old). Motor discharge was episodic during continuous cooling, and seizure-like discharge was observed during continuous warming. These phenomena were not observed with stepwise temperature changes suggesting that transient temperature effects on membrane properties may be involved. These preparations were not as cold tolerant as hamster pups in vivo but they retained the ability to autoresuscitate at all ages studied.

Respiratory chemoreflexes and effects of cortical activation state in urethane anesthetized rats.

Urethane anesthetized (< 1 .3 g/kg), Sprague-Dawley (SD) rats spontaneously cycled between a cortically desynchronized state (State I) and a cortically synchronized state (State III), which were very similar to awake and slow wave sleep (SWS) states in unanesthetized animals, based on EEG criteria. These low levels of urethane anaesthesia did not cause significant respiratory depression or reductions in sensitivity to hypoxia (10% O2 in nitrogen) or hypercapnia (5% CO2 in air) in rats in either State I or State III. Thus, breathing frequency (fR), tidal volume (VT) and total ventilation (VTOT) all increased on cortical activation in urethane-anaesthetized rats whether breathing air, the hypoxic or the hypercapnic gas mixture, in a manner that was very similar to that observed in unanaesthetized animals. The relative sensitivity to hypoxia was greater in State III than State I, the relative sensitivity to CO2, overall, was equal in both states, State III occurred less often during hypoxia and hypercapnia, and hypoxic, urethane-anaesthetized rats sighed frequently, particularly in State I. This is also similar to the situation seen in unanesthetized rats. Given the similarities seen between urethane anesthetized rats in the present study and literature values for unanesthetized rats, the data suggest that urethane-anaesthetized rats provide a good model system for studying respiratory patterns and chemoreflexes as a function of cortical activation state.

Interactive effects of mechano- and chemo-receptor inputs on cardiorespiratory outputs in the toad.

Arterial blood pressure (P(b)), pulmocutaneous blood flow (Q(pc)), heart rate (f(H)), and fictive ventilation (motor activity in the Vth cranial nerve, V(int)), were recorded from decerebrated, paralysed toads receiving unidirectional ventilation with experimental gas mixtures over a range of lung inflation. At the onset of spontaneous bouts of fictive ventilation, (Q(pc)) and P(b) increased immediately, often with changes in heart rate, implying central cardiorespiratory interactions. Inflation of the lungs with different gas mixtures revealed that the effect of hypercarbia on V(int) was reduced by lung inflation and that feedback from pulmonary stretch receptors may summate with central feedforward control of f(H) and (Q(pc)) in an interactive fashion. The results of bolus injections of cyanide into the carotid or the pulmonary circulations suggest there are oxygen sensitive receptors in both circuits that affect the cardiovascular system directly and respiratory activity by complex central interactions with inputs from central chemoreceptors and pulmonary stretch receptors.

A comparison of adrenergic stress responses in three tropical teleosts exposed to acute hypoxia.

Experiments were performed to assess the afferent and efferent limbs of the hypoxia-mediated humoral adrenergic stress response in selected hypoxia-tolerant tropical fishes that routinely experience environmental O(2) depletion. Plasma catecholamine (Cat) levels and blood respiratory status were measured during acute aquatic hypoxia [water Po(2) (Pw(O(2))) = 10-60 mmHg] in three teleost species, the obligate water breathers Hoplias malabaricus (traira) and Piaractus mesopotamicus (pacu) and the facultative air breather Hoplerythrinus unitaeniatus (jeju). Traira displayed a significant increase in plasma Cat levels (from 1.3 +/- 0.4 to 23.3 +/- 15.1 nmol/l) at Pw(O(2)) levels below 20 mmHg, whereas circulating Cat levels were unaltered in pacu at all levels of hypoxia. In jeju denied access to air, plasma Cat levels were increased markedly to a maximum mean value of 53.6 +/- 19.1 nmol/l as Pw(O(2)) was lowered below 40 mmHg. In traira and jeju, Cat release into the circulation occurred at abrupt thresholds corresponding to arterial Po(2) (Pa(O(2))) values of approximately 8.5-12.5 mmHg. A comparison of in vivo blood O(2) equilibration curves revealed low and similar P(50) values (i.e., Pa(O(2)) at 50% Hb-O(2) saturation) among the three species (7.7-11.3 mmHg). Thus Cat release in traira and jeju occurred as blood O(2) concentration was reduced to approximately 50-60% of the normoxic value. Intravascular injections of nicotine (600 nmol/kg) elicited pronounced increases in plasma Cat levels in traira and jeju but not in pacu. Thus the lack of Cat release during hypoxia in pacu may reflect an inoperative or absent humoral adrenergic stress response in this species. When allowed access to air, jeju did not release Cats into the circulation at any level of aquatic hypoxia. The likeliest explanation for the absence of Cat release in these fish was that air breathing, initiated by aquatic hypoxia, prevented Pa(O(2)) values from falling to the critical threshold required for Cat secretion. The ventilatory responses to hypoxia in each species were similar, consisting generally of increases in both frequency and amplitude. These responses were not synchronized with or influenced by plasma Cat levels. Thus the acute humoral adrenergic stress response does not appear to stimulate ventilation during acute hypoxia in these tropical species.

The ventilatory response to environmental hypercarbia in the South American rattlesnake, Crotalus durissus.

To study the effects of environmental hypercarbia on ventilation in snakes, particularly the anomalous hyperpnea that is seen when CO(2) is removed from inspired gas mixtures (post-hypercapnic hyperpnea), gas mixtures of varying concentrations of CO(2) were administered to South American rattlesnakes, Crotalus durissus, breathing through an intact respiratory system or via a tracheal cannula by-passing the upper airways. Exposure to environmental hypercarbia at increasing levels, up to 7% CO(2), produced a progressive decrease in breathing frequency and increase in tidal volume. The net result was that total ventilation increased modestly, up to 5% CO(2) and then declined slightly on 7% CO(2). On return to breathing air there was an immediate but transient increase in breathing frequency and a further increase in tidal volume that produced a marked overshoot in ventilation. The magnitude of this post-hypercapnic hyperpnea was proportional to the level of previously inspired CO(2). Administration of CO(2) to the lungs alone produced effects that were identical to administration to both lungs and upper airways and this effect was removed by vagotomy. Administration of CO(2) to the upper airways alone was without effect. Systemic injection of boluses of CO(2)-rich blood produced an immediate increase in both breathing frequency and tidal volume. These data indicate that the post-hypercapnic hyperpnea resulted from the removal of inhibitory inputs from pulmonary receptors and suggest that while the ventilatory response to environmental hypercarbia in this species is a result of conflicting inputs from different receptor groups, this does not include input from upper airway receptors.

Phylogeny of CO2/H+ chemoreception in vertebrates.

The traditional view has been that respiratory chemoreceptors responsive to changes in P(CO(2))/pH first evolved in air breathing vertebrates at both peripheral and central sites. Recent evidence, however, suggests that fish also possess chemoreceptors responsive to changes in P(CO(2)) per se. In many species these receptors reside in the gills and respond primarily to changes in aquatic rather than arterial P(CO(2)). There is also scattered evidence to suggest that central CO(2)/H(+)-sensitive chemoreceptors may be present in representatives of all fish groups but only the data for air breathing fish are strong and convincing. The phylogenetic trends that are emerging indicate that the use of CO(2) chemoreception for cardiorespiratory processes arose much earlier than previously believed, (arguably) that CO(2) chemoreception may first have arisen in the periphery sensitive to the external environment and that central CO(2)/H(+) chemoreception subsequently arose multiple times in association with several of the independent origins of air breathing, and that the mechanisms of CO(2)/H(+) chemotransduction may be as varied as the different receptor groups involved.