O2 Chemoreceptors Research Articles

Developing zebrafish utilize taste-signaling pathways for oxygen chemoreception

A fundamental requirement for all animals is to sense and respond to changes in environmental O2 availability. Low O2 (hypoxia) typically stimulates breathing, a universal and critical response termed the hypoxic ventilatory response (HVR). In this study, we test the hypothesis that taste-signaling pathways are used for O2 sensing and activation of the HVR. We show that Merkel-like cells (MLCs), which are part of the taste-bud complex, function as O2 chemoreceptor cells in larval zebrafish and that transduction of the O2 signal uses taste-signaling pathways. Specifically, MLCs responded to hypoxia invivo with an increase in Ca2+ activity that can drive the HVR. In addition, MLCs transmit O2 signals to afferent cranial nerves IX and X (nIX/X), which project into the area postrema within the hindbrain and synapse with interneurons that are in contact with vagal motor neurons. Hypoxia or chemo-activation of nIX/X caused Ca2+ activity to increase within the area postrema and elicited hyperventilation. The results provide the first demonstration of an O2 signaling pathway that commences with the activation of taste receptors (MLCs) to yield a critical physiological reflex, the HVR.

Hypoxia sensing in the body: An update on the peripheral and central mechanisms.

An adequate supply of O2 is essential for the maintenance of cellular activity. Systemic or local hypoxia can be experienced during decreased O2 availability or associated with diseases, or a combination of both. Exposure to hypoxia triggers adjustments in multiple physiological systems in the body to generate appropriate homeostatic responses. However, with significant reductions in the arterial partial pressure of O2, hypoxia can be life-threatening and cause maladaptive changes or cell damage and death. To mitigate the impact of limited O2 availability on cellular activity, O2 chemoreceptors rapidly detect and respond to reductions in the arterial partial pressure of O2, triggering orchestrated responses of increased ventilation and cardiac output, blood flow redistribution and metabolic adjustments. In mammals, the peripheral chemoreceptors of the carotid body are considered to be the main hypoxic sensors and the primary source of excitatory feedback driving respiratory, cardiovascular and autonomic responses. However, current evidence indicates that the CNS contains specialized brainstem and spinal cord regions that can also sense hypoxia and stimulate brain networks independently of the carotid body inputs. In this manuscript, we review the discoveries about the functioning of the O2 chemoreceptors and their contribution to the monitoring of O2 levels in the blood and brain parenchyma and mounting cardiorespiratory responses to maintain O2 homeostasis. We also discuss the implications of the chemoreflex-related mechanisms in paediatric and adult pathologies.

The control of breathing in fishes – historical perspectives and the path ahead

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing.

The effects of dissolved organic carbon on the reflex ventilatory responses of the neotropical teleost (Colossoma macropomum) to hypoxia or hypercapnia

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O2 and/or CO2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l−1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l−1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro.

Does blood flow limit acute hypoxia performance in larval zebrafish (Danio rerio)?

Oxygen uptake (ṀO2) in larval zebrafish prior to maturation of the gill relies on cutaneous O2 transfer. Under normoxic conditions, rates of cutaneous O2 transfer are unaffected by haemoglobin availability but are diminished in fish lacking a functional circulatory system, suggesting that internal convection is critically involved in setting the resting ṀO2 in zebrafish larvae, even when relying on cutaneous O2 transfer. The reliance of ṀO2 on blood circulation led to the first objective of the current study, to determine whether loss of internal convection would reduce acute hypoxia performance (as determined by measuring critical PO2; Pcrit) in larval zebrafish under conditions of moderate hypoxia (PO2 = 55mmHg) at 28.5 and 34°C. Internal convection was eliminated by preventing development of blood vessels using morpholino knockdown of vascular endothelial growth factor (VEGF); these fish are termed VEGF morphants. Breathing frequency (fV) and heart rate (fH) also were measured (at 28.5°C) to determine whether any detriment in performance might be linked to cardiorespiratory dysfunction. Although ṀO2 was reduced in the VEGF morphants, there was no significant effect on Pcrit at 28.5°C. Raising temperature to 34°C resulted in the VEGF morphants exhibiting a higher Pcrit than the shams, suggesting an impairment of hypoxia tolerance in the morphants at the higher temperature. The usual robust increase in fV during hypoxia was absent or attenuated in VEGF morphants at 4 and 5days post fertilization (dpf), respectively. Resting fH was reduced in the VEGF morphants and unlike the sham fish, the morphants did not exhibit hypoxic tachycardia at 4 or 5 dpf. The number of cutaneous neuroepithelial cells (presumptive O2 chemoreceptors) was significantly higher in the VEGF morphants and thus the cardiorespiratory impairment in the morphants during hypoxia was unlikely related to inadequate peripheral O2 sensing.

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2weeks of severe hypoxia (PO2=35mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

Disruption of tph1 genes demonstrates the importance of serotonin in regulating ventilation in larval zebrafish (Danio rerio)

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O2 chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a−/− mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b−/− mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR.

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.

Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).

First demonstration of the neuroepithelial cells and their chemical code in the accessory respiratory organ and the gill of the sharptooth catfish, Clarias gariepinus: A preliminary study

AbstractAvailable studies that have examined O2 sensing in fish have indicated that oxygen‐sensitive neuroepithelial cells (NECs) are O2 sensors in the gills and initiate cardiorespiratory reflexes in aquatic vertebrates. This is the first study describing the occurrence of NECs in accessory respiratory organs in the air‐breathing catfish Clarias gariepinus. Immunocytochemical stainings with specific neuronal markers such as nNOS, VAchT, 5‐HT and TH have been shown to be very useful for location and distribution of these cells in the gill fans and suprabranchial chamber that take origin from the transformation of the gill tissue. But the response of these putative O2 chemoreceptors, their role in the respiratory reflexes and their innervation await investigation.

Translating carotid body function into clinical medicine.

The carotid body (CB) is considered the main O2 chemoreceptor, which contributes to cardiorespiratory homeostasis and ventilatory acclimatization. In clinical medicine, the most common pathologies associated with the CB are tumours. However, a growing body of evidence supports the novel idea that an enhanced CB chemosensory discharge contributes to the autonomic dysfunction and pathological consequences in obstructive sleep apnoea (OSA), hypertension, systolic heart failure (HF) and cardiometabolic diseases. Heightened CB chemosensory reactivity elicited by oxidative stress has been involved in sympathetic hyperactivity, cardiorespiratory instability, hypertension and insulin resistance. CB ablation, which reduces sympathetic hyperactivity, decreases hypertension in animal models of OSA and hypertension, eliminates breathing instability and improves animal survival in HF, and restores insulin tolerance in cardiometabolic models. Thus, data obtained from preclinical studies highlight the importance of the CB in the progression of sympathetic-related diseases, supporting the idea that appeasing the enhanced CB chemosensory drive may be useful in improving cardiovascular, respiratory and endocrine alterations. Accordingly, CB ablation has been proposed and used as a treatment for moderating resistant hypertension and HF-induced sympathetic hyperactivity in humans. First-in-human studies have shown that CB ablation reduces sympathetic overactivity, transiently reduces severe hypertension and improves quality of life in HF patients. Thus, CB ablation would be a useful therapy to reverse sympathetic overactivation in HF and severe hypertension, but caution is required before it is widely used due to the crucial physiological function played by the CB. Further studies in preclinical models are required to assess side-effects of CB ablation.

Identification of Vesicular Monoamine Transporter 2 (vmat2)‐Containing O2 Chemoreceptors in the Gills of Zebrafish

All vertebrates “sense” changes in oxygen (O2) via specialized cells, called chemoreceptors, associated with the peripheral nervous system. Aquatic vertebrates are especially prone to fluctuations in environmental O2 levels. Thus it is essential for them to readily detect low O2 and make appropriate ventilatory adjustments. Teleost species, such as zebrafish, have O2 chemoreceptive neuroepithelial cells (NECs) embedded in the gill epithelium. These cells exhibit membrane depolarization and vesicular recycling upon exposure to hypoxia, and express the monoamine neurotransmitter, serotonin (5‐HT). Our research objectives are to identify gill O2 chemoreceptors in live tissue preparations and determine the role of intracellular Ca2+ in O2 sensing. We are currently employing a transgenic zebrafish line, expressing green fluorescent protein (GFP) under the control of vesicular monoamine transporter 2 (vmat2) regulatory elements, in order to identify NECs and characterize their hypoxic responses in vitro and in situ. Immunohistochemistry and confocal microscopy were used to determine the distribution and identity of GFP‐positive cells in gills. Our results show that the population of GFP‐positive cells in our transgenic line significantly overlaps with serotonergic NECs in the gill filaments. Preliminary studies using fura 2‐based imaging indicate that isolated GFP‐positive cells stimulated by hypoxia undergo an increase in intracellular Ca2+ concentration. Identification of NECs both in vitro and in situ using a vital marker will have a great impact on future work addressing the physiological properties of these cells.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).

Mitogenic action of hypoxia upon cutaneous neuroepithelial cells in developing zebrafish.

In zebrafish, cutaneous neuroepithelial cells (NECs) contain serotonin (5-HT) and are believed to initiate physiological and behavioral responses to hypoxia during embryonic and early larval development, when mature gills and O2 chemoreceptors are not yet present. The number of skin NECs rapidly declines as embryos develop into larvae, but acclimation to hypoxia leads to retention of a greater number of these cells. We hypothesized that reduction of the partial pressure of oxygen (PO2 ) in water would stimulate mitosis in cutaneous NECs in zebrafish. Zebrafish were exposed to 5-bromo-2'-deoxyuridine (BrdU) and immunolabeled with antibodies against serotonin and BrdU to identify mitotic skin cells, including NECs. Cells were imaged and quantified using confocal microscopy. From embryonic to larval stages, we observed an overall increase in the number of BrdU-positive cells in the skin, but a decrease in BrdU-positive serotonergic NECs. Exposure of larvae to hypoxia (PO2 = 30 mmHg) in vivo for 24 h produced a 1.7-fold increase in the number of NECs labeled with BrdU. We conclude that under normal environmental PO2 the population of cutaneous NECs declines due to a decrease in mitotic activity. During environmental hypoxia, the number of NECs undergoing cell division in the skin is increased, and this promotes retention of NECs under these conditions. These data demonstrate the direct action of hypoxia upon the cell cycle of cutaneous NECs in developing zebrafish, and support the notion that cutaneous NECs are embryonic O2 chemoreceptors. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 789-801, 2017.

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen.

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 μM (EC50 = 24.5 μM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced fV at 50 μM (EC50 = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Control of cardiorespiratory function in response to hypoxia in an air-breathing fish, the African sharptooth catfish, Clarias gariepinus

We evaluated the role of the first pair of gill arches in the control of cardiorespiratory responses to normoxia and hypoxia in the air-breathing catfish, Clarias gariepinus. An intact group (IG) and an experimental group (EG, bilateral excision of first gill arch) were submitted to graded hypoxia, with and without access to air. The first pair of gill arches ablations reduced respiratory surface area and removed innervation by cranial nerve IX. In graded hypoxia without access to air, both groups displayed bradycardia and increased ventilatory stroke volume (VT), and the IG showed a significant increase in breathing frequency (fR). The EG exhibited very high fR in normoxia that did not increase further in hypoxia, this was linked to reduced O2 extraction from the ventilatory current (EO2) and a significantly higher critical O2 tension (PcO2) than the IG. In hypoxia with access to air, only the IG showed increased air-breathing, indicating that the first pair of gill arches excision severely attenuated air-breathing responses. Both groups exhibited bradycardia before and tachycardia after air-breaths. The fH and gill ventilation amplitude (VAMP) in the EG were overall higher than the IG. External and internal NaCN injections revealed that O2 chemoreceptors mediating ventilatory hypoxic responses (fR and VT) are internally oriented. The NaCN injections indicated that fR responses were mediated by receptors predominantly in the first pair of gill arches but VT responses by receptors on all gill arches. Receptors eliciting cardiac responses were both internally and externally oriented and distributed on all gill arches or extra-branchially. Air-breathing responses were predominantly mediated by receptors in the first pair of gill arches. In conclusion, the role of the first pair of gill arches is related to: (a) an elevated EO2 providing an adequate O2 uptake to maintain the aerobic metabolism during normoxia; (b) a significant bradycardia and increased fAB elicited by externally oriented O2 chemoreceptors; (c) increase in the ventilatory variables (fR and VAMP) stimulated by internally oriented O2 chemoreceptors.

Aquatic surface respiration and swimming behaviour in adult and developing zebrafish exposed to hypoxia.

Severe hypoxia elicits aquatic surface respiration (ASR) behaviour in many species of fish, where ventilation of the gills at the air-water interface improves O2 uptake and survival. ASR is an important adaptation that may have given rise to air breathing in vertebrates. The neural substrate of this behaviour, however, is not defined. We characterized ASR in developing and adult zebrafish (Danio rerio) to ascertain a potential role for peripheral chemoreceptors in initiation or modulation of this response. Adult zebrafish exposed to acute, progressive hypoxia (PO2 from 158 to 15 mmHg) performed ASR with a threshold of 30 mmHg, and spent more time at the surface as PO2 decreased. Acclimation to hypoxia attenuated ASR responses. In larvae, ASR behaviour was observed between 5 and 21 days postfertilization with a threshold of 16 mmHg. Zebrafish decreased swimming behaviour (i.e. distance, velocity and acceleration) as PO2 was decreased, with a secondary increase in behaviour near or below threshold PO2 . In adults that underwent a 10-day intraperitoneal injection regime of 10 μg g(-1) serotonin (5-HT) or 20 μg g(-1) acetylcholine (ACh), an acute bout of hypoxia (15 mmHg) increased the time engaged in ASR by 5.5 and 4.9 times, respectively, compared with controls. Larvae previously immersed in 10 μmol l(-1) 5-HT or ACh also displayed an increased ASR response. Our results support the notion that ASR is a behavioural response that is reliant upon input from peripheral O2 chemoreceptors. We discuss implications for the role of chemoreceptors in the evolution of air breathing.

Heme oxygenase-1 (HO-1) mediated respiratory responses to hypoxia in the goldfish, Carassius auratus

In this study we investigated the role of heme oxygenase-1 (HO-1) in modulating the hypoxic and hyperoxic ventilatory responses of goldfish (Carassius auratus) acclimated to 7 and 25°C. HO-1 was present in the neuroepithelial cells (NECs; putative branchial O2 chemoreceptors) of fish acclimated to 7°C only. Hypoxia exposure increased gill HO-1 activity in 7°C fish (14.0±1.4 to 42.5±3.2pmolbilirubinmin−1mgprotein−1). Inhibition of HO-1 activity with zinc protophorphyrin IX (ZnPPIX) increased the ventilation frequency response to acute hypoxia (30mmHg); frequency increased from 48.3±5.1 to 137.4±16.0 breaths per min (BPM) in hypoxic 7°C fish treated with ZnPPIX compared to 46.2±4.2 to 77.9±5.3 BPM in control fish. Unlike in the control (untreated) 7°C fish exposed to hyperoxia, fish injected with ZnPPIX did not significantly decrease breathing frequency. Inhibiting HO-1 activity was without effect on the hypoxic or hyperoxic ventilatory responses of fish acclimated to 25°C. Based on these observations, we suggest that HO-1 plays an inhibitory role in regulating breathing frequency but only in goldfish acclimated to 7°C.

Control of Breathing in In Vitro Brain Stem Preparation from Goldfish (Carassius auratus; Linnaeus)

In vitro brain stem preparations from goldfish (Carassius auratus) were used to first determine whether this species possesses central chemoreceptors able to modulate respiratory activity. Preparations were superfused with an artificial cerebrospinal fluid (aCSF); fictive breathing was recorded extracellularly by placing a suction electrode on cranial nerve VII. Reducing the level of O2 in the gas mixture used to bubble the aCSF from a hyperoxic level (80% or 98.7% O2) to a relative hypoxic level (20% or 40% O2) increased the frequency of the fictive respiratory burst (P = 0.0002). Reducing the pH of the aCSF from 7.9 to 7.4 by increasing CO2 in the superfusate did not affect fictive breathing. Chloride-mediated neurotransmission (GABA/glycine) is a major modulator of respiratory activity; however, its effect on the neural circuits that regulate breathing in teleosts remains unknown. Bath application of GABA (0.5, 5.0 mM) decreased burst frequency but not amplitude; this effect was dose dependent (drug × concentration: P = 0.01). Superfusion of the preparations with aCSF containing 1.25 μM of bicuculline methochloride and 1.50 μM of strychnine hydrochloride (GABAA and glycine receptor antagonists, respectively) increased burst frequency (P = 0.002) and amplitude (P < 0.001). We conclude that respiratory activity produced by the goldfish brain stem is not responsive to the moderate CO2 levels used in this study; it may contain O2 chemoreceptors, but the relatively small response could also reflect nonspecific effects of hypoxia on the central nervous system. Cl(-)-mediated neurotransmission inhibits fictive breathing; this aspect of respiratory regulation is similar to other groups of vertebrates.

Fetal breathing movements and changes at birth.

The fetus, which develops within a fluid-filled amniotic sac, relies on the placenta for respiratory gas exchange rather than the lungs. While not involved in fetal oxygenation, fetal breathing movements (FBM) nevertheless have an important role in lung growth and in development of respiratory muscles and neural regulation. FBM are regulated differently in many respects than postnatal respiration, which results from the unique intrauterine environment. Prominent distinctions of FBM include its episodic nature and apnea-sensitivity to hypoxia. The latter characteristic is the basis for using FBM in the assessment of fetuses at risk for hypoxic injury. At birth, the transition to continuous postnatal respiration involves a fall in temperature, gaseous distention of the lungs, activation of the Hering-Breuer reflexes, and functional connectivity of afferent O2 chemoreceptor activity with respiratory motoneurons and arousal centers. Importantly, exposure to drugs or adverse conditions in utero not only can change patterns of FBM but also can lead to epigenetic dysregulation in postnatal respiration. Such changes, can blunt respiratory and arousal defenses against hypoxic challenges in sleep. Thus, fetal hypoxia and/or drug exposure may in later life dispose sleeping infants, children, and adults to hypertension, diabetes mellitus, brain injury, and sudden death.

The Effect of Adding CO2 to Hypoxic Inspired Gas on Cerebral Blood Flow Velocity and Breathing during Incremental Exercise

Hypoxia increases the ventilatory response to exercise, which leads to hyperventilation-induced hypocapnia and subsequent reduction in cerebral blood flow (CBF). We studied the effects of adding CO2 to a hypoxic inspired gas on CBF during heavy exercise in an altitude naïve population. We hypothesized that augmented inspired CO2 and hypoxia would exert synergistic effects on increasing CBF during exercise, which would improve exercise capacity compared to hypocapnic hypoxia. We also examined the responsiveness of CO2 and O2 chemoreception on the regulation ventilation (E) during incremental exercise. We measured middle cerebral artery velocity (MCAv; index of CBF), E, end-tidal PCO2, respiratory compensation threshold (RC) and ventilatory response to exercise (E slope) in ten healthy men during incremental cycling to exhaustion in normoxia and hypoxia (FIO2 = 0.10) with and without augmenting the fraction of inspired CO2 (FICO2). During exercise in normoxia, augmenting FICO2 elevated MCAv throughout exercise and lowered both RC onset andE slope below RC (P<0.05). In hypoxia, MCAv and E slope below RC during exercise were elevated, while the onset of RC occurred at lower exercise intensity (P<0.05). Augmenting FICO2 in hypoxia increased E at RC (P<0.05) but no difference was observed in RC onset, MCAv, or E slope below RC (P>0.05). The E slope above RC was unchanged with either hypoxia or augmented FICO2 (P>0.05). We found augmenting FICO2 increased CBF during sub-maximal exercise in normoxia, but not in hypoxia, indicating that the ‘normal’ cerebrovascular response to hypercapnia is blunted during exercise in hypoxia, possibly due to an exhaustion of cerebral vasodilatory reserve. This finding may explain the lack of improvement of exercise capacity in hypoxia with augmented CO2. Our data further indicate that, during exercise below RC, chemoreception is responsive, while above RC the ventilatory response to CO2 is blunted.