Hypoxic Respiratory Depression Research Articles

Postnatal Development of the Purinergic Signaling System in the preBötzinger Complex; Implications for the Hypoxic Ventilatory Response

The hypoxic ventilatory response (HVR) comprises a carotid body‐mediated increase in ventilation followed by a secondary, CNS‐mediated decrease, the hypoxic respiratory depression (HRD), that is larger and potentially life‐threatening in newborn mammals. The mechanisms responsible for the greater HRD in newborns are unknown, but purinergic signaling via P2 and P1 receptor (R) mechanisms helps shape this 2° phase. Shortly after the 1° increase in ventilation, astrocytes release ATP in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation), which stimulates breathing via P2Y1Rs and attenuates the HRD. Extracellular ATP (ATPe) is degraded to adenosine (ADOe), which inhibits breathing and is implicated in the HRD. Thus, the impact of ATP on the HVR is determined by the balance between the actions of ATP and ADOe, that could change developmentally and contribute to the greater HRD of newborns. Our goal was to characterize how the many factors that determine the balance between ATP and ADOe signaling change during development, including: P2Y1Rs; ADO A1Rs; ectonucleotidases (ECTOs) that degrade ATPe to ADOe; ADOe clearance by equilibrative nucleoside transporters (ENTs) via transmembrane transport of ADO down its concentration gradient; and ADO kinase (ADK), which is not mature in the cortex until postnatal day (P) 14, but facilitates ADOe clearance by converting ADOi into AMP so that ADOi<ADOe. We used rhythmic preBötC slice preparations to characterize developmental changes in: i) the effects of P2Y1R and ADOR signaling on preBötC rhythm; ii) ECTO activity; and iii) the impact of ENTs and ADK on baseline preBötC rhythm and preBötC responses to ATP and ADO that were locally injected to simulate hypoxia. iv) In vivo head‐out plethysmography was used to assess the role of ADOe clearance mechanisms in shaping the developing HVR.i) The frequency (freq) increase evoked by ATP in the preBötC doubled between P0‐6 and P9‐12, but the P2Y1R agonist MRS 2365‐evoked increase did not change. The A1R inhibition of freq was of similar magnitude but greater duration at P0‐6 compared to P9‐12. ii) ECTO activity, assessed by incubating preBötC tissue in ATP and measuring PO4, was greater at P9‐12. iii) The ADO freq inhibition was 2‐fold longer in ENT knockout mice compared to WT mice at all ages. ADK inhibition (ABT 702) inhibited baseline freq only at P9‐12. iv) Knockout of ENTs blunted the freq response to hypoxia (10% O2) in vivo, but increased the VT response at all ages. Introduction of constitutively active ADK into the brain produced P0‐2 ADKtg mice that responded to hypoxia with an adult‐like, sustained increase in VE/VO2; P0‐2 WT C57BL6 did not respond to hypoxia. This difference disappeared by P6‐8. Surprisingly, the mature HVR of P0‐2 ADKtg mice reflected a greater hypoxic depression of VO2, not a greater increase in VE.Data suggest preBötC sensitivity to P2Y1 and A1Rs does not change, while ECTO activity increases developmentally. ADO actions are prolonged in P0‐6 mice, suggesting that ADOe clearance mechanisms mature postnatally. ENTs appear to play a similar role from birth, which may involve clearing ADOe from the preBötC during hypoxia. ADK maturation also appears important in the development of an adaptive HVR. However, the role of ADK in modulating VE vs metabolic responses to hypoxia remains to be elucidated.

P2Y 1 receptor modulation of Ih in the preBötzinger inspiratory rhythm generator may underlie the ATP‐mediated attenuation of the hypoxic respiratory depression

Premature infants often stop breathing briefly (apnea) because the brainstem respiratory network is still immature (apnea of prematurity, AOP). During these apneas, oxygen levels fall (hypoxia) triggering a biphasic hypoxic ventilatory response comprising an initial increase in ventilation followed by a centrally mediated secondary depression that can be life-threatening. Caffeine is used to stimulate breathing in AOP. However, ~20% of these infants do not respond to caffeine. Thus, alternate therapies are needed. Recent data suggest that during hypoxia, ATP is released in the preBötzinger Complex (preBötC, inspiratory rhythm generator) where it acts via P2Y1 receptors (Rs) to excite inspiratory neurons, increase ventilation and attenuate the secondary hypoxic depression. A mechanistic understanding of this P2Y1R excitation is lacking. Preliminary whole-cell analysis of inspiratory preBötC neurons in vitro revealed P2Y1R currents that reverse near -40 mV and increase in amplitude with neuron hyperpolarization. Based on these data, we tested the hypotheses that the increase in inspiratory frequency evoked by ATP in the preBötC in vitro and the ATP-mediated increase in ventilation during hypoxia in vivo reflects P2Y1R potentiation of the hyperpolarization-activated inward current, Ih (carried by cyclic nucleotide–gated, HCN, channels). Application of MRS2365 (P2Y1R agonist, 100 µM) to Ih-expressing inspiratory preBötC neurons in rhythmic brainstem slices from neonatal rat potentiated Ih by 32±6% (at -100 mV, n=18) and depolarized voltage-dependence of Ih activation by ~10 mV. The MRS2365 inward current and its potentiation of Ih were blocked by the Ih blocker, ZD7288 (100 μM). ZD7288 also attenuated the MRS2365-evoked frequency increase by 60-90%. Ih currents in preBötC neurons were sensitive to ZD7288 (100 µM) but not ivabradine (30 µM), suggesting HCN2 but not HCN1,4 subunit involvement. In paralyzed, anesthetized, vagotomized ventilated adult rats, bilateral microinfusion of ZD7288 (250 µM, but not 100 µM) into the preBötC increased the secondary hypoxic (10% O2) depression by 68±23% (n=6) compared to rats that received aCSF microinfusion (n=4). This effect was regionally specific. If the bilateral microinfusions of ZD7288 were not both in the preBötC (e.g., 1 in preBötC and the other in BötC or rVRG), ZD7288 had no effect on the hypoxic ventilatory response. Finally, in anesthetized, vagotomized, spontaneously-breathing adult rats, unilateral reverse microdialysis of ZD7288 (100 µM) in the preBötC area (n=13) increased the hypoxic frequency depression by 20±6% (compared to aCSF, n=6). These data suggest that the P2Y1R excitation of the preBötC network is produced, in part, via potentiation of Ih in a subgroup of inspiratory neurons, and that potentiation of Ih in the preBötC attenuates the hypoxic ventilatory depression in adult rats. Data also identify the HCN2 subunit as a potential target for new strategies to counteract respiratory depression in AOP.

Limited Capacity for Clearance of Adenosine from the preBötzinger Complex Inspiratory Network Contributes to the Profound Depression of Breathing by Hypoxia in the Very Young

Breathing in premature infants often stops briefly (apnea) because the brainstem network that controls breathing is immature (apnea of prematurity, AOP). Apneas cause hypoxia, triggering the hypoxic ventilatory response (HVR), which comprises an initial increase in ventilation followed by a centrally mediated 2° decrease. In adults, breathing remains above baseline during this 2° phase, but in the very young it falls below baseline leading to a life-threatening feedback loop where apnea causes hypoxia, respiratory depression, further hypoxia and so on. Understanding the cause of this powerful hypoxic inhibition of breathing in the very young, a major question in perinatal physiology, could inform new therapies for AOP. The transmitter ATP is released in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation) during hypoxia where it increases breathing and attenuates the 2° depression. However, extracellular ATP (ATPe) is degraded to adenosine (ADO), which inhibits breathing and is implicated in AOP; e.g., caffeine blocks ADOe actions and is the first line treatment for AOP. We hypothesize that the greater 2° inhibition of breathing in prematurity is due to an immature system for clearing ADOe. ADOe-mediated inhibition stops with its removal from the extracellular space, which depends on i) equilibrative nucleoside transporters (ENTs) that move ADOe across cell membranes down its concentration gradient, and ii) ADO kinase (ADK), an enzyme that keeps the level of ADO inside cells lower than outside so ENTs clear ADOe. How ENT activity develops in the preBötC is unknown. However, the form of ADK that influences ADOe in the brain is minimally functional until 2 weeks of age in rodents. To assess the importance of ADOe clearance in the development of the HVR, we used plethysmography to measure the HVR (10% O2) in 0-56 day old (P0-14) wild-type (WT), ENT knockout (KO), and ADKtg mice engineered to have functional ADK throughout life. In WT and ENT KO neonates (P0-2), ventilation fell below baseline after ~7 min in hypoxia, but the hypoxia-evoked frequency increase was markedly reduced in the ENT KO. Strikingly, P0-2 ADKtg mice showed the mature breathing response; ventilation remained above baseline levels throughout hypoxia. At P12 in WT and ENT KO, the HVR had still not matured; ventilation fell below baseline during the 2° phase, yet it remained above baseline in P12 ADKtg mice. To assess the importance of ENT and ADK specifically in the preBötC, we isolated the preBötC network in rhythmic brain slices from P0-12 mice. The ADO inhibition of preBötC frequency lasted twice as long in P0-12 ENT KO mice compared to WT. Conversely, the decrease in basal frequency caused by ADK inhibition (ABT-702) increased with age (P0-2, 15%; P9-11, 23%). These data suggest that i) ENTs are important for ADO clearance in the preBötC after birth and ii) low ADK activity at birth is a major contributor to the greater hypoxic respiratory depression in early development.

Antagonistic Effects of Tetramethylpyrazine on Hypoxic Respiratory Depression in Rats.

Objective Tetramethylpyrazine (TMP) is an alkaloid extracted from the root and stem of the traditional Chinese herbal medicine called Chuanxiong. The present study aims to study the effects of TMP on hypoxic respiratory depression in rats. Materials and methods. The effects of TMP on respiratory responses of rats induced by hypoxia were observed by diaphragm electromyogram (EMG) recording. The effects of TMP on the protein expression of FOS and acid sensing ion channel1a (ASIC1a) in the brainstem induced by hypoxia were investigated by immunohistochemistry. Results The respiration of rats was first excited and then depressed during hypoxia treatment, while TMP pretreatment could significantly antagonize the respiratory depression induced by hypoxia (P < 0.01). Hypoxia obviously induced the protein expression of FOS (P < 0.01) and ASIC1a(P < 0.05) in the brainstem, which can be also significantly inhibited by TMP pretreatment. Conclusions TMP has protective effects on hypoxic respiratory depression, and the mechanisms might be concerned with its downregulation of FOS and ASIC1a in the brainstem induced by hypoxia.

Developmental Changes in the Role of Adenosine Clearance Mechanisms in Modulating the Activity of the preBötzinger Complex Inspiratory Network Under Basal and Hypoxic Conditions

Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) characterized by a carotid‐body‐mediated increase in ventilation followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is strongest and life‐threatening in premature infants (e.g., Apnea of Prematurity). Purinergic signaling plays a prominent role in shaping the HVR; hypoxia evokes release of ATP from astrocytes in the preBötzinger Complex (preBötC, key site of inspiratory rhythm generation), which increases breathing and attenuates the HRD. However, ATP is metabolized into adenosine (ADO), which inhibits the preBötC and is implicated in the HRD. Thus, the net effect of ATP in the preBötC during hypoxia is determined by the balance between the actions of ATP and ADO. The objective of this study is to determine during development how mechanisms important in clearing extracellular ADO (ADOe) influence preBötC activity and the HVR. First, we focused on equilibrative nucleoside transporters (ENTs) that passively move ADO across cell membranes down its concentration gradient. We compared the HVR (10 min 10% O2) of WT and ENT1/2 double KO mice (P0–3, P6–8, and P12–14) using whole‐body plethysmography. The HRD of ENT KO mice was significantly greater compared to WT mice at all ages, reflecting greater reductions in breathing frequency and a likely role for ENTs in removing ADOe during hypoxia. We then applied the ENT inhibitor NBMPR (100 μM) to the solution bathing rhythmic, preBötC‐containing medullary slices from neonatal mice to directly assess the influence of ENTs on basal preBötC rhythm. A significant, NBMPR‐mediated, 16% ± 7% (n=5) increase in frequency suggests that ENTs elevate basal ADOe tone, causing a modest reduction in frequency. In contrast, the reduction in inspiratory frequency evoked by injecting ADOe into the preBötC (to simulate a high ADOe load as may occur in hypoxia) was almost 2‐fold greater in slices from ENT1/2 KO compared to WT mice; i.e., under high load, ENTs appear to clear ADOe. We next examined ADO kinase (ADK), an enzyme that converts ADO into AMP, lowering intracellular ADO levels and facilitating clearance of ADOe by ENTs. Inhibition of ADK with ABT‐702 (10 μM) produced an adenosine‐mediated (i.e., the effect was reversed with the A1R antagonist, DPCPX, 100 nM) decrease in the baseline frequency of rhythmic medullary slices that was 16% ± 2% (n=7) at P0–2 and 24% ± 5% (n=8) by P9–11.In summary, these data suggest that under basal conditions ENTs and ADK are important in controlling ADOe tone, and that under conditions of high ADOe load, as produced here via local injection of ADO in vitro or hypoxia in vivo, ENTs and ADK become important in clearing ADOe, which increases ventilation. Finally, the observation in vitro that ADK activity in the preBötC appears to increase during development raises the intriguing possibility that the greater HRD of premature infants is due, at least in part, to an immature system for clearing ADOe.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFI, Alberta Lung AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular and ionic mechanisms underlying the P2Y1receptor‐mediated excitation of thepreBötzinger Complex inspiratory network in vitro

A host of adaptive responses have evolved to protect brain oxygen levels. Prominent among these is the biphasic hypoxic ventilatory response (HVR), in which a fall in arterial oxygen, detected at the peripheral chemoreceptors triggers a rapid increase in breathing that is followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is most pronounced in premature infants and can be life‐threatening. Our recent data challenge dogma that the only contribution of the CNS to the HVR is this secondary depression. Specifically, in vitro and in vivo data from rat suggest that astrocytes in the pre‐Bötzinger complex (pre‐BötC, critical site for inspiratory rhythm generation) release ATP in response to hypoxia, which acts via P2Y1 receptors (P2Y1Rs) on inspiratory neurons to increase ventilation and attenuate the HRD. In other parts of the brains, P2Y1Rs signal via the Gq second messenger pathway, but the signalling cascade(s) and ion channel(s) via which P2Y1Rs excite inspiratory neurons to increase inspiratory frequency remain unknown. Previous efforts from our lab to address these questions tested the effects of second messenger and ion channel blockers on ATP currents, but data were confounded by the finding that while P2Y1Rs account for the entire network frequency effect, they account for only a fraction (~30%) of the total ATP current. Thus, the first objective of this study was to revisit this question by examining the effects of Gq and ion channel blockers on currents evoked in inspiratory preBötC neurons by the P2Y1R agonist, MRS 2365. Our second goal was to determine whether the excitatory effect of P2Y1Rs on the preBötC network is mediated through activation of excitatory (glutamatergic) or inhibitory GABA/glycinergic) preBötC neurons.Analysis of whole‐cell recordings from inspiratory preBötC neurons in rhythmic medullary slices from neonatal (P0‐4) SD rats revealed that inward currents evoked by MRS 2365 (30 sec, 100 mM)immediately after obtaining whole‐cell configuration and again after 15 min of dialysis (via the pipette solution) with the protein kinase C (PKC) inhibitor (Chelerethryne, 10 mM, n = 5), the phospholipase C (PLC) inhibitor (U73122 4 mM, n = 6) or the BKCa channel blocker (paxilline 1 or 4 mM, n = 7), were reduced between 30–40%. To test whether P2Y1R‐sensitive, inspiratory neurons are excitatory or inhibitory, we prepared rhythmic slices from transgenic mice (P0‐4) in which glutamatergic (VGLUT2‐TdTomato) or GABAergic (GAD67‐YFP) neurons are fluorescently labeled. Local application of MRS 2365 evoked inward currents in zero of seven VGLUT2‐TdTomato inspiratory neurons. Analysis of GAD67‐YFP inspiratory neurons is ongoing. We are also applying RNAScope analysis to determine the transmitter phenotype of P2Y1R‐sensitive inspiratory neurons.These data suggest that P2Y1Rs acton non‐glutamatergic, inspiratory neurons, in part via the Gq signalling pathway and BKCa channels, to excite the preBötC inspiratory rhythm generating network.Support or Funding InformationSupported by CIHR, NSERC, CFI, WCHRI and NMHI.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of PHOX2B-derived astrocytes in chemosensory control of breathing and sleep homeostasis.

The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus. We identify in mice a population of ∼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

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

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

Thalamic mediation of hypoxic respiratory depression in lambs.

Immaturity of respiratory controllers in preterm infants dispose to recurrent apnea and oxygen deprivation. Accompanying reductions in brain oxygen tensions evoke respiratory depression, potentially exacerbating hypoxemia. Central respiratory depression during moderate hypoxia is revealed in the ventilatory decline following initial augmentation. This study determined whether the thalamic parafascicular nuclear (Pf) complex involved in adult nociception and sensorimotor regulation (Bentivoglio M, Balerecia G, Kruger L. Prog Brain Res 87: 53-80, 1991) also becomes a postnatal controller of hypoxic ventilatory decline. Respiratory responses to moderate isocapnic hypoxia were studied in conscious lambs. Hypoxic ventilatory decline was compared with peak augmentation. Pf and/or adjacent thalamic structures were destroyed by the neuron-specific toxin ibotenic acid (IB). IB lesions involving the thalamic Pf abolished hypoxic ventilatory decline. Lesions of adjacent thalamic nuclei that spared Pf and control injections of vehicle failed to blunt hypoxic respiratory depression. Our findings reveal that the thalamic Pf region is a critical controller of hypoxic ventilatory depression and thus a key target for exploring molecular concomitants of forebrain pathways regulating hypoxic ventilatory depression in early development.

ATP acts via P2Y1 receptors in the preBötzinger Complex in vivo to attenuate the secondary hypoxic respiratory depression

The ventilatory response to hypoxia comprises an initial increase in ventilation followed by a secondary depression that can be life‐threatening in premature infants. The mechanisms underlying the kinetics and magnitude of this secondary depression are not known, but ATP released within the ventrolateral medulla acts via purinergic (P2) receptors to attenuate this depression. We explored the hypothesis developed from analysis of the rhythmic medullary slice preparation that ATP acts via P2Y1 receptors in the preBötzinger Complex (preBötC, a critical site for inspiratory rhythm generation) to mediate this attenuation. Adult, urethane‐anesthetized, spontaneously‐breathing, vagotomized rats were exposed to 10% O2 (90% N2) for 5 min immediately following local unilateral injection of vehicle (0.2μl HEPES buffer) or 500 μM MRS2279 (P2Y1 receptor antagonist) into the preBötC. Respiratory frequency (fR) and minute ventilation (VE) increased rapidly by ~40% in both groups. fR fell to baseline levels in the control group but to 0.8 of baseline in the MRS2279‐injected animals. VE fell to levels that were 20% above baseline in the control group but to baseline in the MRS2279 group. Data suggest that during hypoxia endogenously‐released ATP acts via P2Y1 receptors in the preBötC to reduce the magnitude of the secondary hypoxic respiratory depression. Supported by CIHR, AIHS, CFI, WCHRI and ALA.

Adenosine A2areceptors and O2sensing in development

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O2sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5′-nucleotidase and the resulting activation of adenosine A2Areceptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A2Areceptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A2Areceptors mediate hypoxic inhibition of breathing and rapid eye movements. A2Areceptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A2Areceptors play virtually no role in O2sensing by the carotid bodies, but brain A2Areceptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A2Areceptors have been implicated in O2sensing by carotid glomus cells, while central A2Areceptors likely blunt hypoxic hyperventilation. In conclusion, A2Areceptors are crucially involved in the transduction mechanisms of O2sensing in fetal carotid bodies and brains. Postnatally, central A2Areceptors remain key mediators of hypoxic respiratory depression, but they are less critical for O2sensing in carotid chemoreceptors, particularly in developing lambs.

The kreisler mutation leads to the loss of intrinsically hypoxia-activated spots in the region of the retrotrapezoid nucleus/parafacial respiratory group

Acute hypoxia elicits a biphasic respiratory response characterized in the newborn by a transient hyperventilation followed by a severe decrease in respiratory drive known as hypoxic respiratory depression. Medullary O(2) chemosensitivity is known to contribute to respiratory depression induced by hypoxia, although precise involvement of cell populations remains to be determined. Having a thorough knowledge of these populations is of relevance because perturbations in the respiratory response to hypoxia may participate in respiratory diseases in newborns. We aimed to analyze the hypoxic response of ponto-medullary cell populations of kreisler mutant mice. These mice have defects in a gene expressed in two rhombomeres encompassing a part of the medulla oblongata implicated in hypoxic respiratory depression. Central responses to hypoxia were analyzed in newborn mice by measuring respiratory rhythm in ex vivo caudal pons-medullary-spinal cord preparations and c-fos expression in wild-type and kreisler mutants. The homozygous kreisler mutation, which eliminates most of rhombomere 5 and mis-specifies rhombomere 6, abolished (1) an early decrease in respiratory frequency within 10 min of hypoxia and (2) an intrinsic hypoxic activation, which is characterized by an increase in c-fos expression in the region of the ventral medullary surface encompassing the retrotrapezoid nucleus/parafacial respiratory group expressing Phox2b. This increase in c-fos expression persisted in wild-type Phox2b-negative and Phox2b-positive cells after blockade of synaptic transmission and rhythmogenesis by a low [Ca(2+)](0). Another central response was retained in homozygous kreisler mutant mice; it was distinguished by (1) a delayed (10-30 min) depression of respiratory frequency and (2) a downregulation of c-fos expression in the ventrolateral reticular nucleus of the medulla, the nucleus of the solitary tract, and the area of the A5 region. Thus, two types of ponto-medullary cell groups, with distinct anatomical locations, participate in central hypoxic respiratory depression in newborns.

ATP-Depletion durch Hyperventilation, Gewebehypoxie und Hypermetabolismus als Ursache für Plötzlichen Kindstod – eine Hypothese

Despite a decreasing incidence, sudden infant death syndrome (SIDS) is still the most frequent cause of death in industrial nations during the first year of life. Hypoxia plays a major role in the pathogenesis, but the exact mechanism is not fully understood. This study was based on personal considerations and a selective online literature search. SIDS is the result of a frequently protracted ATP-depletion. Especially in combination, all risk factors for SIDS favour an ATP-deficiency by increasing ATP-catabolism and/or by diminishing ATP-synthesis. Prenatal chronic hypoxaemia and an insufficient supply with nutrients lead to low birth-weight, reduced adipose tissue, elevated haemoglobin F, increased sympathetic activity, hypermetabolism, and diminished hypoxia tolerance in the neonates. Because of reduced adipose tissue, more energy for thermogenesis is needed after birth. In reaction to hypoxaemia, infants with risk factors show hyperventilation instead of hypoxic hypometabolism and respiratory depression. Enhanced breathing, however, requires additional ATP and causes increasing oxygen affinity, which is elevated physiologically during the first months of life. Thereby, tissue-hypoxia and diminished ATP-synthesis may arise. Besides, enhanced sympathetic activity leads to hypermetabolism and increased ATP-catabolism. While innate risk factors may reduce ATP-production in burdening situations, like food deprivation, postnatal hyperthermia and stress augment ATP-catabolism by hyperventilation and hypermetabolism and empty energy stores. For term newborns, the peak incidence of SIDS might be explained by the haemoglobin nadir of physiological anaemia and by the therefore reduced capacity for oxygen transport. Thereby, the risk of tissue-hypoxia, which follows increased oxygen affinity and vanishing ability to hypoxic hypometabolism, is further enhanced. The almost identical symptoms of SIDS and ATP-deficiency diseases like hypophosphataemia, heat stroke, and carbon monoxide poisoning support the presented hypothesis.

Maturation Influences effects of long‐term facilitation induced changes in inspiratory network complexity in urethane‐anesthetized neonatal rats

Long‐term facilitation (LTF) is a form of respiratory plasticity, which is expressed as a persistent increase in inspiratory motor output following intermittent, but not continuous, hypoxia. Although numerous studies have examined timing and magnitude changes in respiratory LTF, changes in inspiratory network complexity (quantified by approximate entropy ApEn analysis) in neonatal Sprague‐Dawley rats have only begun to be explored. Here we extend earlier analyses by examining ApEn of diaphragm EMG bursts recorded from spontaneously breathing urethane‐anesthetized neonatal rats (P12–21 n=18) before and at 5‐min intervals for at least 60‐min following three 5‐min episodes of hypoxia (8% O2); all rats were continuously supplied with 4% CO2 to minimize hypoxic respiratory depression. Under these conditions, respiratory LTF was seen mainly as an increase in burst frequency although in some cases an increase in amplitude was observed. During LTF, ApEn values increased from 0.37±0.04 to 0.42±0.02 (P=0.004) P12–P13 rats(n=7) and from 0.40±0.03 to 0.43±0.03 (P=0.04) in P21 rats(n=3), with the peak response seen at ≥50 min. In contrast, ApEn values decreased from 0.40±0.03 to 0.36±0.03 (P=0.007) in P14–P18 rats(n=8), with the peak response seen at ≤45 min. These data show that inspiratory network complexity is modified during LTF, and that the stage of development affects LTF‐related network reconfiguration.

Caffeine in the milk prevents respiratory disorders caused by in utero caffeine exposure in rats

Consequences of postnatal caffeine exposure by the milk on ponto-medullary respiratory disturbances observed following an in utero caffeine exposure were analysed. Ponto-medullary-spinal cord preparations from newborn rats exposed to caffeine during gestation but not after the birth display an increase in respiratory frequency and an exaggeration of the hypoxic respiratory depression compared to not treated preparations. These data suggest that tachypneic and apneic episodes encountered in human newborns whose mother consumed caffeine during pregnancy are due in large part to central effect of caffeine at the ponto-medullary level. Both baseline respiratory frequency increase and emphasis of hypoxic respiratory depression are not encountered if rat dams consumed caffeine during nursing. Our hypothesis is that newborn rats exposed to caffeine during gestation but not after the birth would be in withdrawal situation whereas, when caffeine is present in drinking fluid of lactating dams, it goes down the milk and is able to prevent ponto-medullary respiratory disturbances.

Consequences of in utero caffeine exposure on respiratory output in normoxic and hypoxic conditions and related changes of Fos expression: a study on brainstem-spinal cord preparations isolated from newborn rats.

Several aspects of the central regulation of respiratory control have been investigated on brainstem-spinal cord preparations isolated from newborn rats whose dam was given 0.02% caffeine in water as drinking fluid during the whole period of pregnancy. Analysis of the central respiratory drive estimated by the recording of C4 ventral root activity was correlated to Fos ponto-medullary expression. Under normoxic conditions, preparations obtained from the caffeine-treated group of animals displayed a higher respiratory frequency than observed in the control group (9.2 +/- 0.5 versus 7.2 +/- 0.6 burst/min). A parallel Fos detection tends to indicate that the changes of the respiratory rhythm may be due to a decrease in neuronal activity of medullary structures such as the ventrolateral subdivision of the solitary tract, the area postrema, and the nucleus raphe obscurus. Under hypoxic conditions, the preparations displayed a typical hypoxic respiratory depression associated with changes in the medullary Fos expression pattern. In addition, the hypoxic respiratory depression is clearly emphasized after in utero exposure to caffeine and coincides with an increased Fos expression in the area postrema and nucleus raphe obscurus, two structures in which it is not increased in the absence of caffeine. Taken together, these results support the idea that in utero caffeine exposure could affect central respiratory control.

Pathophysiologic Mechanisms Underlying Apnea of Prematurity

After completing this article, readers should be able to: 1. Characterize the unique features of neonatal respiratory control mechanisms. 2. Clarify how immature respiratory control contributes to neonatal apnea. 3. Identify the contribution of upper airway obstruction to episodes of mixed apnea. Idiopathic apnea of prematurity is a common disorder that requires therapeutic intervention to avoid potential morbidity in preterm infants who require neonatal intensive care. Severe, recurrent apneic episodes may lead to multiple investigations to rule out secondary disorders leading to apnea (see accompanying article in this issue). Although the incidence of neonatal apnea is inversely related to gestational age and is probably as high as 100% in the most immature preterm infants, the onset may be delayed by the presence of lung disease in the first days of life. Apnea of prematurity, especially if severe or persistent, has been associated with poor developmental outcome in school-age children, although a cause-and-effect relationship is difficult to establish. The definition of apnea varies among studies. Apnea of prematurity has been defined most widely as cessation of breathing in excess of 15 seconds’ duration, typically accompanied by desaturation and bradycardia. However, shorter episodes of apnea, and even periodic breathing, may be accompanied by bradycardia or hypoxemia. Prolonged desaturation episodes also have been reported in the absence of apnea or bradycardia, both in healthy preterm infants and in infants who have chronic lung disease. These episodes might represent obstructive apnea, hypoventilation, or possibly intrapulmonary right-to-left shunting, although episodes of desaturation and bradycardia almost always are preceded by apnea or hypoventilation. Apnea is classified traditionally into three categories based on the presence or absence of upper airway obstruction: central, obstructive, and mixed apneas. Central apnea is characterized by total cessation of inspiratory efforts with no evidence of obstruction. In obstructed apnea, the infant tries to breathe against …

The effect of intracerebroventricular dopamine administration on the respiratory response to hypoxia.

Acute hypoxia produces an increase in ventilation. When the hypoxia is sustained, the initial increase in ventilation is followed a decrease in ventilation. The precise mechanism of this decline in ventilation during sustained hypoxia is unknown. Recent studies hypothesized that the accumulation of dopamine in the central nervous system might have a major role in production of hypoxic respiratory depression. The purpose of this study was to examine whether dopamine has an effect on occurance of central ventilatory depression which is seen in acute hypoxia in peripheral chemoreceptors denervated animals. The experiment were conducted in rabbits anesthetized with Na-pentobarbital (25 mg x kg(-1) i.v.). For intracerebroventricular (i.c.v.) injections of dopamine (1 microg) in each animal, canula was placed in left lateral cerebral ventricle by stereotaxic method. Respiratory frequency (f x min(-1)), tidal volume (V(T)) ventilation minute volume (V(E)) and systemic arterial blood pressure (BP) were recorded during air and 3 minutes hypoxic gas mixture (8%O2-92%N2) breathing. I.c.v. administration of dopamine during normoxia decreased V(T), f, V(E) and BP, significantly. When rabbits were injected with an i.c.v. dopamine on hypoxic gas mixture breathing in control animals, there was depression of hypoxic ventilatory responses. After i.c.v. administration of dopamine antagonists haloperidol (0.1 mg) and domperidone (0.07 mg) in chemodenervated rabbits, the significant increases in V(T), V(E) and BP were observed. On the breathing of hypoxic gas mixture of chemodenervated and i.c.v. dopamine antagonists administrated rabbits, hypoxic depression was completely abolished. These results of this study show that accumulation of dopamine in the brain seems to reduce the response of the central control mechanisms to chemoreceptor impulses during normoxia and hypoxia. In conclusion present study suggests important role played by central dopaminergic pathways in the occurence of acute hypoxic ventilatory depression.

Fos study of ponto-medullary areas involved in the in vitro hypoxic respiratory depression.

In this study, the brainstem-spinal cord preparation isolated from newborn rats, an established model for the study of the hypoxic respiratory depression (HRD), has been used. The comparison of Fos expression in ponto-medullary areas in these preparations placed either in normoxic or hypoxic conditions suggests that only the retrotrapezoid nucleus (RTN) and the ventrolateral medulla (VLM) are involved in the in vitro HRD. Hypoxic preparations exhibit a Fos expression enhanced in the RTN, suggesting that the RTN might play a crucial role in the HRD. As well as this, VLM neurons presented a decrease in Fos expression that could be related to the decline of the respiratory output induced by hypoxia.