Fish gill chemosensing: knowledge gaps and inconsistencies.

In this review, we explore the inconsistencies in the data and gaps in our knowledge that exist in what is currently known regarding gill chemosensors which drive the cardiorespiratory reflexes in fish. Although putative serotonergic neuroepithelial cells (NEC) dominate the literature, it is clear that other neurotransmitters are involved (adrenaline, noradrenaline, acetylcholine, purines, and dopamine). And although we assume that these agents act on neurons synapsing with the NECs or in the afferent or efferent limbs of the paths between chemosensors and central integration sites, this process remains elusive and may explain current discrepancies or species differences in the literature. To date it has been impossible to link the distribution of NECs to species sensitivity to different stimuli or fish lifestyles and while the gills have been shown to be the primary sensing site for respiratory gases, the location (gills, oro-branchial cavity or elsewhere) and orientation (external/water or internal/blood sensing) of the NECs are highly variable between species of water and air breathing fish. Much of what has been described so far comes from studies of hypoxic responses in fish, however, changes in CO2, ammonia and lactate have all been shown to elicit cardio-respiratory responses and all have been suggested to arise from stimulation of gill NECs. Our view of the role of NECs is broadening as we begin to understand the polymodal nature of these cells. We begin by presenting the fundamental picture of gill chemosensing that has developed, followed by some key unanswered questions about gill chemosensing in general.

The relationship between hypoxia exposure and circulating cortisol levels in social and solitary African mole-rats: An initial report.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole-rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground burrows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma glucocorticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited significantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface-dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

Control of Breathing in Ectothermic Vertebrates.

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

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

Ventilatory Sensitivity to Ambient CO2 at Different Hibernation Temperatures in Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus).

AbstractMammals entering hibernation undergo drastic reductions in metabolic rate and body temperature (Tb; to as low as ∼2% of euthermic metabolic rate and 1°C to -2°C). Although ventilation (V˙E) is also greatly reduced in hibernating ground squirrels, their relative ventilatory response (%ΔV˙E) to increases in inspired CO2 (∼400% increase to 7% CO2) dwarfs that of euthermic squirrels (∼60% increase). On the basis of data from earlier studies on hypothermic animals, we hypothesized that this switch in apparent ventilatory sensitivity was the result of the change in state (from euthermic to hibernating) and not due to the change in core Tb. Thus, we used whole-body plethysmography to assess the hypercapnic ventilatory response (HCVR) in thirteen-lined ground squirrels in steady-state hibernation at 20°C, 15°C, 10°C, 7°C, and 5°C. With the transition into hibernation as Tb fell, the breathing pattern became irregular and then episodic. Total V˙E and the oxygen consumption rate (V˙O2) decreased progressively as Tb fell. Hibernating squirrels with a core Tb of 20°C increased V˙E by 150% from normocapnic levels when given 7% CO2 to breathe, while squirrels with a Tb of 7°C increased V˙E by 650% when exposed to the same inspired CO2. When Tb was cooled from 7°C to 5°C, however, the increase in the HCVR fell to 450% and was associated with a rise in V˙O2 and total V˙E. These results reveal progressive changes in breathing pattern and the HCVR with decreasing Tb and suggest that the effects of hibernation state may be Tb dependent. V˙E did not fall in proportion to metabolic rate, and the HCVR increased progressively in both absolute terms and relative terms until a Tb of 7°C, both of which potentially constrain the extent of the metabolic suppression.

Changes in CO2 sensitivity during entrance into, and arousal from hibernation in Ictidomys tridecemlineatus.

At the onset of entrance into hibernation in many mammals, there is a reduction in the respiratory exchange ratio (RER) thought to result in a retention of CO2 that contributes to the ensuing metabolic suppression. In steady-state hibernation, the relative hypercapnic ventilatory response (HCVR; the % change in ventilation to CO2 exposure) is elevated. These two observations, paradoxically, suggest a transient decrease in CO2 sensitivity at the onset of entrance into hibernation, allowing the retention of CO2, then a subsequent increase in CO2 sensitivity giving rise to the elevated HCVR in steady-state hibernation. We examined the time course of the changes in ventilation, O2 consumption rates ([Formula: see text]o2), CO2 excretion rates, body temperature, and hence the RER and ACR (air convection ratio, ventilation/[Formula: see text]o2) and the HCVR throughout entrance and arousal into and out of hibernation in 13-lined ground squirrels to confirm this. We observed a significant drop (entrance) and rise (arousal) in the RER produced by hypo- and hyperventilation, respectively. CO2 chemo-sensitivity while the RER was reduced on entrance was blunted and rose late in entrance. On arousal, CO2 chemo-sensitivity was elevated when the RER was elevated and fell immediately after RER returned to normal values. At any given Tb, the HCVR was lower during entrance compared to arousal producing a significant hysteresis. The HCVR, however, was the same at any given [Formula: see text]o2 during entrance and arousal. These data suggest that both the changes in [Formula: see text]o2 and in the HCVR are associated with changes in central regulation of the effector limbs establishing steady-state hibernation.

The high-altitude bird chronicles: lessons from field work with Frapps.

Over the past decade, Peter Frappell, aka Frapps, has been an integral part of an international group studying birds that migrate or reside at altitude. This research has taken the extended group from Terkhiin Tsagaan Lake on the Mongolian plateau to Chilika Lake in eastern India, Koonthankulum bird sanctuary in southern India, Lake Qinghai in Chinese Tibet, Summer Lake Wildlife and Malheur National Wildlife Refuge in Oregon, and San Pedro a Marca, Vichaycocha and Lake Titicaca National Reserve in Perú. It has been a productive project producing over 30 manuscripts, 15 of which were based on research in the field. What has not been published are the stories behind the research and the critical lessons learned along the way. Some of these are chronicled here.

Editorial: Untangling the oxygen transport cascade: a tribute to Peter Frappell (Frapps).

This collection of research articles was put together in honour of respiratory physiologist Professor Peter Frappell's (Frapps's) academic achievements. It encompasses various topics relating to the oxygen transport cascade, which was central to Frapps' career as a comparative physiologist. This issue highlights the diversity and outreach of his influence on the field and his pioneering spirit; promoting novel perspectives, methodologies and research techniques. This issue also demonstrates how Frapps' knowledge and scientific findings answered some of the fundamental questions within the field of respiratory physiology while creating and fostering a rather unique work atmosphere in the laboratories he led. We thank Frapps for the contributions he has made and the friendships he has nurtured over his career. Cheers, Frapps - we love you mate!

KCNQ Current Contributes to Inspiratory Burst Termination in the Pre-Bötzinger Complex of Neonatal Rats in vitro.

The pre-Bötzinger complex (preBötC) of the ventral medulla generates the mammalian inspiratory breathing rhythm. When isolated in explants and deprived of synaptic inhibition, the preBötC continues to generate inspiratory-related rhythm. Mechanisms underlying burst generation have been investigated for decades, but cellular and synaptic mechanisms responsible for burst termination have received less attention. KCNQ-mediated K+ currents contribute to burst termination in other systems, and their transcripts are expressed in preBötC neurons. Therefore, we tested the hypothesis that KCNQ channels also contribute to burst termination in the preBötC. We recorded KCNQ-like currents in preBötC inspiratory neurons in neonatal rat slices that retain respiratory rhythmicity. Blocking KCNQ channels with XE991 or linopirdine (applied via superfusion or locally) increased inspiratory burst duration by 2- to 3-fold. By contrast, activation of KCNQ with retigabine decreased inspiratory burst duration by ~35%. These data from reduced preparations suggest that the KCNQ current in preBötC neurons contributes to inspiratory burst termination.

Striped catfish (Pangasianodon hypophthalmus) use air-breathing and aquatic surface respiration when exposed to severe aquatic hypercarbia.

We investigated the extent to which the facultative air-breathing fish, the striped catfish (Pangasianodon hypophthalmus), uses air-breathing to cope with aquatic hypercarbia, and how air-breathing is influenced by the experimental exposure protocol and level of hypercarbia. We exposed individuals to severe aquatic hypercarbia (up to Pw CO2 = 81 mmHg) using step-wise and progressive exposure protocols while measuring gill ventilation rate, heart rate, mean arterial blood pressure, and air-breathing frequency, as well as arterial blood pH and PCO2 . We confirm that P. hypophthalmus is tolerant of hypercarbia. Under both protocols gill ventilation rate, heart rate, and mean arterial blood pressure were maintained near control levels even at very high CO2 levels. We observed a marked amount of individual variation in the PwCO2 at which air-breathing was elicited, with some individuals not responding at all. The experimental protocol also influenced the onset of air-breathing. Air-breathing began at lower Pw CO2 in the step-wise protocol (23 ± 4.1 mmHg) compared with the progressive protocol (46 ± 7.8 mmHg). Air-breathing was often followed by aquatic surface respiration, at higher PCO2 (71 ± 5.2 mmHg) levels. On average, the blood PCO2 was approximately 43% lower (46 ± 2.5 mmHg) than water Pw CO2 (~81 mmHg) at our highest tested CO2 level. While this suggests that aerial CO2 elimination is an effective, and perhaps critical, respiratory strategy used by P. hypophthalmus to cope with severe hypercarbia, this observation may also be explained by a long lag time required for equilibration.

Effects of hypoxia on the respiratory and metabolic responses to progressive cooling in newborn rodents that range in heterothermic expression.

NEW FINDINGS: What is the central question of this study? Adult homeotherms and heterotherms differ in cold and hypoxia tolerance and in how they match O2 supply and demand in response to these stressors. It has never been ascertained whether these differences reflect different developmental trajectories or whether they are already present at birth. What is the main finding and its importance? When exposed to cold and hypoxia, newborn rodents differed in how they matched O2 supply and demand, with responses reflecting the degree of heterothermic expression and tolerance. Our findings indicate that elements of the adult phenotype are already present at birth. ABSTRACT: There are physiological differences in how adult rodents regulate O2 supply and O2 demand when exposed to hypoxia in the cold. We examined whether these differences reflect divergent developmental trajectories of homeotherms and heterotherms or whether the differences are already present at birth. We exposed newborn rodents (0-4 days old) that ranged in heterothermic expression [a homeotherm, the rat (Rattus norvegicus); two facultative heterotherms, the mouse (Mus musculus) and the hamster (Mesocricetus auratus); and an obligate heterotherm, the ground squirrel (Ictidomys tridecemlineatus)] to either normoxia (21% O2 ) or hypoxia (7% O2 ) and measured their metabolic, thermoregulatory and ventilatory responses while progressively reducing the ambient temperature from 33 to 15°C. All newborns reduced their body temperature, O2 consumption rate and ventilation during progressive cooling, both in normoxia and in hypoxia. When progressively cooled in hypoxia, however, the homeothermic rats exhibited the greatest thermogenic response, depressed their O2 consumption rate the least and increased ventilation the most. In contrast, the obligate heterotherm, the ground squirrel, did not mount a thermogenic response, exhibited the greatest reduction in O2 consumption rate and increased O2 uptake not by increasing ventilation like the rat, but by extracting ≤80% of the O2 from each breath. Facultative heterotherms (mice and hamsters) exhibited responses in between these two extreme phenotypes. We conclude that even as newborns, homeotherms and heterotherms diverge in how they match O2 supply and O2 demand when progressively cooled in hypoxia, with responses reflecting the degree of heterothermic expression, in addition to reported hypoxia and cold tolerance.

Respiratory development in burrowing rodents: Effect of perinatal hypercapnia.

Burrowing rodents have a blunted hypercapnic ventilatory response compared to non-burrowing rodents, but semi-fossorial ground squirrels and hamsters are not born with this blunted response when raised in room conditions. This study examined the hypercapnic ventilatory response of rats, hamsters, and ground squirrels raised in burrow-like hypercapnia (∼3 % CO2) through development (embryonic day 16-18 to postnatal day 30) to determine if chronic hypercapnia exerts any effect on the developing and adult semi-fossorial response. Chronic hypercapnia attenuated the ventilatory response to 5 % CO2 by 60 % (rats), 150 % (hamsters), and 70 % (squirrels) in newborns when compared to newborns raised in normal conditions. When raised in burrow conditions, squirrels and hamsters reached the blunted adult response ∼8-12 days sooner in development than their room air counterparts, while burrow-reared rats maintained a consistently blunted response until removal from chronic hypercapnia. Our study revealed no lasting effect of chronic hypercarbia on the ventilatory responses to CO2 in burrowing rodents, but rather a change in the developmental profile such that the blunted adult response was reached earlier in development.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

A morphometric analysis of the lungs of high-altitude ducks and geese.

We examined the morphology of the lungs of five species of high-altitude resident ducks from Lake Titicaca in the Peruvian Andes (yellow-billed pintail [Anas georgica], cinnamon teal [Anas cyanoptera orinomus], puna teal [Anas puna], speckled teal [Anas flavirostris oxyptera], and ruddy duck [Oxyura jamaicensis ferruginea]) and compared them with those of the high-altitude migratory bar-headed goose (Anser indicus) and the low-altitude migratory barnacle goose (Branta leucopsis). We then determined the relationship between mass-specific lung volume, the volume densities of the component parts of the lung, and previously reported hypoxia-induced increases in pulmonary O2 extraction. We found that the mass-specific lung volumes and the mass-specific volume of the exchange tissue were larger in the lungs of high-altitude resident birds. The bar-headed goose had a mass-specific lung volume that fell between those of the low-altitude species and the high-altitude residents, but a mass-specific volume of exchange tissue that was not significantly different than that of the high-altitude residents. The data suggest that the mass-specific volume of the lung may increase with evolutionary time spent at altitude. We found an inverse relationship between the percentage increase in pulmonary O2 extraction and the percentage increase in ventilation across species that was independent of the volume density of the exchange tissue, at least for the resident Andean birds.

Cardiovascular responses to progressive hypoxia in ducks native to high altitude in the Andes.

The cardiovascular system is critical for delivering O2 to tissues. Here, we examined the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared with four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct) and blood haemoglobin (Hb) concentration. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal and speckled teal) had higher Hct and Hb concentration, increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and Hb concentration between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or Hb concentration compared with their low-altitude relatives, revealing yet another avian strategy for coping with high altitude.

Cardiovascular responses to progressive hypoxia in ducks native to high altitude in the Andes.

The cardiovascular system is critical for delivering O2 to tissues. Here we examine the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared to four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct), and blood haemoglobin concentration [Hb]. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration, and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal, and speckled teal) had higher Hct and [Hb], increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and [Hb] between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or [Hb] compared to their low-altitude relatives, revealing yet another avian strategy for coping with high altitude.

Postnatal changes in O2 and CO2 sensitivity in rodents.

In rodents, the ventilatory responses to hypoxia (low O2) and hypercarbia (high CO2) change significantly over postnatal development. In hypoxia, most adult rodents increase ventilation and decrease metabolism to some degree. Hypercarbia, however, leads to an increase in ventilation with little, to no change in metabolism. Neonates, on the other hand, respond to hypoxia with a profound metabolic depression, and a severely attenuated ventilatory response. In hypercarbia, they exhibit a strong ventilatory response early in development that blunts, reaches a nadir, and then rises back to the adult-like response, thus, stabilizing postnatally. In this review we discuss how the O2 and CO2 ventilatory responses develop in rodents, the possible mechanisms that drive these postnatal changes, and how being raised in a burrow, an environment putatively low in O2 and high in CO2, may affect the development of O2 and CO2 sensitivity in rodents.

Effect of temperature, age and the pons on respiratory rhythm in the rat brainstem-spinal cord.

Neonatal animals are extremely tolerant of hypothermia. However, cooling will ultimately lead to ventilatory arrest, or cessation of respiratory movements. Upon rewarming, ventilation can recover spontaneously (autoresuscitation). This study examined the effect of age (P0-P5) and the pons on respiratory-related output during hypothermic ventilatory arrest and recovery using a brainstem-spinal cord preparation of neonatal rats. As temperature fell, burst frequency slowed, burst duration increased, burst shape became fragmented and eventually respiratory arrest occurred in all preparations. Removing the pons had little effect on younger preparations (P0-P2). Older preparations (P4-P5) with the pons removed continued to burst at cooler temperatures compared to pons-intact preparations and burst durations were significantly longer. Episodic breathing patterns were observed in all preparations (all ages, pons on or off) at lower temperatures. At 27 °C, however, episodic breathing was only observed in younger preparations with the pons on. These data suggest that developmental changes occurring at the level of the pons underlie the loss of hypothermic tolerance and episodic breathing.