Biphasic Hypoxic Ventilatory Response Research Articles

Recovery of the biphasic hypoxic ventilatory response in neonatal rats after chronic hyperoxia

Newborn mammals exhibit biphasic hypoxic ventilatory responses (HVR) characterized by an initial increase in ventilation and a secondary ventilatory depression. The magnitude of the hypoxic ventilatory decline (HVD) in the late phase of the HVR normally decreases with age, but this occurs sooner in rats reared in 60% O2. We investigated whether a lower level of hyperoxia (30% O2) or a short period of recovery (1 or 3 d in 21% O2) would affect the expression of this plasticity. Similar to 60% O2, rat pups reared in 30% O2 until 3–4 days of age exhibited a less biphasic HVR to 12% O2. When pups reared in 60% O2 were returned to normoxia, the magnitude of HVD increased such that pups expressed a biphasic HVR appropriate for their chronological age. Blocking synaptic input from the carotid bodies revealed that CNS hypoxia depressed ventilation less in hyperoxia-reared rats immediately following hyperoxia and after 1 d in normoxia despite recovery of the biphasic HVR. This suggests that recovery of the biphasic HVR occurs in pathways regulating HVD that depend on carotid body activity. The early, carotid body-mediated phase of the HVR was also blunted immediately and 1 d after the hyperoxia exposure, but not after 3 d of recovery. These data confirm that short exposures to mild-to-moderate hyperoxia elicit developmental plasticity in the HVR. However, reemergence of the biphasic HVR after return to normoxia argues against a heterokairic process for the premature transition from biphasic HVR to sustained HVR in hyperoxia-reared rat pups.

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.

Influence of chronic hyperoxia on the developmental time course of the hypoxic ventilatory response relative to other traits in rats

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a decline back toward baseline levels. The magnitude of the secondary decline diminishes with postnatal age, but this transition occurs earlier in rat pups reared in moderate hyperoxia. This pattern is consistent with heterokairy, a form of developmental plasticity in which environmental factors alter the timing of developmental events. The present study investigated whether this plasticity is specific to the HVR or if hyperoxia instead accelerates overall development. Rat pups reared in 60 % O2 (Hyperoxia) exhibited a less biphasic ventilatory response to 12 % O2 than pups reared in 21 % O2 (Control) at 4 days of age (P4) and transitioned to a sustained HVR by P10-11; Control rats exhibited a biphasic HVR at both ages. However, the average ages at which pups attained other key developmental milestones (i.e., fur development at P5, incisor eruption at P9, and eye opening at P15) were similar between treatment groups. Moreover, growth rates and maturation of the metabolic response to cooling were not accelerated, and may have been delayed slightly, relative to Control rats. For example, the capacity for pups to increase their metabolic rate at low ambient temperatures increased with age, but this thermogenic capacity tended to be reduced in Hyperoxia pups at both P4 and P10-11 (i.e., lower CO2 production rates below the lower critical temperature). Collectively, these data support the conclusion that hyperoxia specifically advances the age at which rat pups exhibit a sustained HVR, altering the relative timing of developmental events rather than compressing the entire period of development.

Early Maturation of the Hypoxic Ventilatory Response in Hyperoxic Rat Pups: Evidence for Trait‐Specific Heterokairy

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a decline back toward baseline levels. The magnitude of the secondary decline diminishes with age, but rat pups reared in moderate hyperoxia make this transition earlier (Bavis et al., J. Appl. Physiol. 109(3): 796–803, 2010). This is consistent with heterokairy, a form of developmental plasticity in which environmental factors alter the rate and/or timing of developmental events. The present study investigated whether this effect is specific to the HVR or if hyperoxia instead accelerates overall development. Rat pups reared in 60% O2 (Hyperoxia) exhibited a less biphasic HVR at both P4 and P10‐11 compared to pups reared in 21% O2 (Control). However, growth curves and the average ages at which pups attained key developmental milestones (i.e., fur development at P5, incisor eruption at P9, and eye opening at P15) were similar between treatment groups. We also studied the relationship between metabolic rate and ambient temperature as another indicator of physiological maturity. The lower critical temperature (LCT) decreased with age, but the LCT was the same for Control and Hyperoxia rats at both P4 (~32°C) and P10‐11 (~30°C). The ability to increase metabolic rate at low ambient temperatures also increased with age, but this thermogenic capacity tended to be reduced in Hyperoxia rats at both P4 and P10‐11 (i.e., lower rates of metabolic CO2 production at temperatures below the LCT). We conclude that hyperoxia accelerates the rate at which the HVR matures without affecting the maturation of other morphological and physiological traits.

Development of ventilatory chemoreflexes in Coturnix quail chicks

Compared to mammals, little is known about the development of the respiratory control system in birds. In the present study, ventilation and metabolism were measured in Coturnix quail chicks exposed to room air, hypoxia (11 % O2), and hypercapnia (4% CO2) at 0–1, 3–4, and 6–7 days posthatching (dph). Mass-specific ventilation and metabolic rate tended to increase between 0–1 and 3–4 dph and then decrease again between 3–4 and 6–7 dph. The magnitude of the hypoxic ventilatory response (HVR) increased with age. The HVR also exhibited a biphasic shape in younger quail: after the initial increase in ventilation, ventilation declined back to (0–1 dph), or toward (4 dph), baseline. Older chicks (6–7 dph) had a “sustained HVR” in which ventilation remained high throughout the hypoxic challenge. The biphasic HVR did not appear to be caused by a decline in metabolic rate; although hypoxic hypometabolism was observed in quail chicks in all three age groups, the metabolic response appeared to occur more slowly than the biphasic HVR. The biphasic ventilatory response was also specific to hypoxia since the hypercapnic ventilatory response (HCVR) was characterized by a sustained increase in ventilation in all three age groups. The magnitude of the HCVR decreased with age. These results point to several similarities in the development of ventilatory chemorflexes between Coturnix quail and newborn mammals, including age-dependent (1) increases in the HVR, (2) transitions from a biphasic to a sustained HVR, and (3) decreases in the HCVR. Whether homologous mechanisms underlie these developmental changes remains to be determined.

Ventilatory and carotid body responses to acute hypoxia in rats exposed to chronic hypoxia during the first and second postnatal weeks

Chronic hypoxia (CH) during postnatal development causes a blunted hypoxic ventilatory response (HVR) in neonatal mammals. The magnitude of the HVR generally increases with age, so CH could blunt the HVR by delaying this process. Accordingly, we predicted that CH would have different effects on the respiratory control of neonatal rats if initiated at birth versus initiated later in postnatal development (i.e., after the HVR has had time to mature). Rats had blunted ventilatory and carotid body responses to hypoxia whether CH (12 % O2) occurred for the first postnatal week (P0 to P7) or second postnatal week (P7 to P14). However, if initiated at P0, CH also caused the HVR to retain the “biphasic” shape characteristic of newborn mammals; CH during the second postnatal week did not result in a biphasic HVR. CH from birth delayed the transition from a biphasic HVR to a sustained HVR until at least P9–11, but the HVR attained a sustained (albeit blunted) phenotype by P13–15. Since delayed maturation of the HVR did not completely explain the blunted HVR, we tested the alternative hypothesis that the blunted HVR was caused by an inflammatory response to CH. Daily administration of the anti-inflammatory drug ibuprofen (4 mg kg−1, i.p.) did not alter the effects of CH on the HVR. Collectively, these data suggest that CH blunts the HVR in neonatal rats by impairing carotid body responses to hypoxia and by delaying (but not preventing) postnatal maturation of the biphasic HVR. The mechanisms underlying this plasticity require further investigation.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

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.

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.

Postnatal Development of Hypoxic and Hypercapnic Ventilatory Responses in Coturnix Quail

Newborn mammals generally exhibit a biphasic hypoxic ventilatory response (HVR) in which the initial increase in ventilation is followed by a progressive decline toward (or below) baseline; the magnitude of the secondary decline diminishes with age. Considerably less is known about postnatal development of the HVR in birds, although a recent study did not detect a biphasic HVR in nestling red‐winged blackbirds (Dzialowski et al., Am J Physiol 311:R1105–R1112, 2016). We used head‐body plethysmography to characterize the ventilatory responses of Coturnix quail chicks to 15‐min challenges of 11% O2 or 4% CO2 at 0–1, 3–4, and 6–7 days of age. The magnitude of the HVR increased with postnatal age (21±4, 35±3, and 42±4 % increase from baseline, respectively; mean±SEM, P<0.01). The HVR was biphasic at P0–1 and (to a lesser extent) at P3–4, but not at P6–7. In contrast, the magnitude of the hypercapnic ventilatory response decreased with age (76±8, 54±4, 31±3 % increase from baseline, respectively; P<0.001). The hypercapnic ventilatory response was characterized by a relatively sustained increase in ventilation in all age groups, suggesting that the biphasic ventilatory response is specific to hypoxia. Additional experiments assessed hypoxic hypometabolism, and decreased metabolic rate does not appear to explain the biphasic HVR in the youngest quail chicks. We conclude that newborn quail exhibit a biphasic HVR similar to that observed in newborn mammals.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Equilibrative Nucleoside (Adenosine) Transporters on the preBötzinger Complex Inspiratory Network and the Hypoxic Ventilatory Response in Newborn Mice

Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) comprising of an initial increase in ventilation followed by a secondary depression. The secondary depression is much stronger and life‐threatening in premature infants and it is implicated in apneic events (Apnea of Prematurity). Despite the clinical significance, mechanisms underlying this secondary depression are poorly understood. Recent data suggest that during hypoxia, astrocytes in the preBötzinger complex (preBötC) release ATP, which increases breathing and attenuates the secondary depression. However, ATP is rapidly metabolized into extracellular adenosine (ADOe), which is implicated in the secondary depression. Thus, the overall effects of ATP are determined by a balance between its excitatory actions and the inhibitory actions of its by‐product, ADO. The importance of ADO is clear in that infants with apnea of prematurity are given caffeine (ADO receptor antagonist) to stimulate breathing. The ATP‐ADO balance is controlled by many factors, including equilibrative nucleoside transporters (ENTs) that move ADO across cell membranes along its concentration gradient. Our goal was to determine the role of ENTs in modulating preBötC network activity under baseline and hypoxic conditions. Bath application of the ENT‐1 inhibitor (NBMPR, 100 μM) to rhythmically‐active medullary slices from neonatal mice caused a significant 9.9±4% increase in frequency (n=5). However, when the preBötC was challenged with an ADO load (as might happen during hypoxia) via local injection of ADO (500 μM, 30 s) ENT‐1 inhibition enhanced the inhibitory effect. Similarly, when we compared the responses of wild type (WT) and ENT 1/2 knockout (ENT KO) mice to preBötC ADO injections, the duration of the frequency inhibition was almost doubled in the KO mice. We next compared the HVR of WT and ENT KO mice (1–3 days old) using whole‐body plethysmography (15 min control, 10 min 10% O2, 10 min recovery). Ventilation increased in both groups by 20% and gradually fell below baseline in WT mice after 4 min in hypoxia. In marked contrast, ventilation fell below baseline during the first 40 s of hypoxia in ENT KO mice by as much as 25% and never increased above baseline throughout the hypoxic challenge. These in vitro data suggest that ENTs contribute to setting ADOe tone in the preBötC under baseline conditions by transporting ADO into the extracellular space. However, under an extra load as might occur in hypoxia, ENTs become important in removing ADOe. In vivo data are consistent with this hypothesis, but it will be critical to determine whether the differences in the HVR of WT and ENT KO mice in vivo reflects loss of ENT activity in the carotid bodies or central nervous system.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of GABA Receptor Antagonists on the Biphasic Hypoxic Ventilatory Response of Newborn Rats

Newborn mammals exhibit a characteristically biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a progressive decline toward (or below) baseline values. Studies in anesthetized, newborn piglets suggest that the secondary hypoxic depression is partially attributable to γ‐aminobutyric acid (GABA) signaling through GABAA and GABAB receptors (Huang et al. J Appl Physiol 77:1006–1010, 1994). To determine whether GABAA and GABAB receptors similarly contribute to the biphasic HVR of newborn rats, we measured normoxic (21% O2) and hypoxic (12% O2) ventilation of newborn (P3–5) rats by head‐body plethysmography 30 minutes after intraperitoneal injection with vehicle or a GABA receptor antagonist. Two different GABAB receptor antagonists, CGP‐35348 (200 mg kg−1) and CGP‐46381 (50 or 100 mg kg−1), were found to have no effect on normoxic ventilation or the HVR (all P>0.05). Similar results were obtained for the GABAA receptor antagonist (+)‐bicuculline (2 or 3 mg kg−1). Collectively, these data do not support a role for GABAergic signaling in the biphasic HVR of newborn rats.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Caffeine does not alter the hypoxic ventilatory response of neonatal rats (1177.3)

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, control rats (Bavis et al., J Appl Physiol 109:796‐803, 2010). It has been suggested that adenosine receptors contribute to hypoxic ventilatory depression in neonatal rats, so we investigated the effect of caffeine (a nonselective adenosine receptor antagonist) on the HVR of rats reared in room air (Control) or 60% O2 (Hyperoxia) through 4‐5 days of age (P4‐5). Rats were injected with saline or caffeine (20 mg kg‐1 caffeine citrate, i.p.) approximately 30 min prior to measuring normoxic and hypoxic (12% O2) ventilation via head‐body plethysmography. As expected, Control rats exhibited a biphasic HVR and Hyperoxia rats exhibited a sustained HVR after saline injection. However, caffeine treatment had no effect on the HVR of either group. These data do not support a role for adenosine receptors in the late phase of the biphasic HVR of neonatal rats.

Hyperoxia-induced developmental plasticity of the hypoxic ventilatory response in neonatal rats: Contributions of glutamate-dependent and PDGF-dependent mechanisms

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, Control rats. Given the similarity between the sustained HVR obtained after chronic exposure to developmental hyperoxia and the mature HVR, it was hypothesized that hyperoxia-induced plasticity and normal maturation share common mechanisms such as enhanced glutamate and nitric oxide signaling and diminished platelet-derived growth factor (PDGF) signaling. Rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth until 4–5 days of age were studied after intraperitoneal injection of drugs targeting these pathways. Hyperoxia rats receiving saline showed a sustained HVR to 12% O2, but blockade of NMDA glutamate receptors (MK-801) restored the biphasic HVR typical of newborn rats. Blockade of PDGF-β receptors (imatinib) had no effect on the pattern of the HVR in Hyperoxia rats, although it attenuated ventilatory depression during the late phase of the HVR in Control rats. Neither nitric oxide synthase inhibitor used in this study (nNOS inhibitor I and l-NAME) altered the pattern of the HVR in Control or Hyperoxia rats. Drug-induced changes in the biphasic HVR were not correlated with changes in metabolic rate. Collectively, these results suggest that developmental hyperoxia hastens the transition from a biphasic to sustained HVR by upregulating glutamate-dependent mechanisms and downregulating PDGF-dependent mechanisms, similar to the changes underlying normal postnatal maturation of the biphasic HVR.

Developmental hyperoxia alters CNS mechanisms underlying hypoxic ventilatory depression in neonatal rats

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR), but the relative contributions of carotid body-initiated CNS mechanisms versus central hypoxia on ventilatory depression during the late phase of the HVR are not well understood. Neonatal rats (P4-5 or P13-15) were treated with a nonselective P2 purinergic receptor antagonist (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, or PPADS; 125mgkg−1, i.p.) to pharmacologically denervate the peripheral chemoreceptors. At P4-5, rats reared in normoxia showed a progressive decline in ventilation during a 10-min exposure to 12% O2 (21–28% decrease from baseline). No hypoxic ventilatory depression was observed in the older group of neonatal rats (i.e., P13-15), suggesting that the contribution of central hypoxia to hypoxic ventilatory depression diminishes with age. In contrast, rats reared in moderate hyperoxia (60% O2) from birth exhibited no hypoxic ventilatory depression at either age studied. Systemic PPADS had no effect on the ventilatory response to 7% CO2, suggesting that the drug did not cross the blood–brain barrier. These findings indicate that (1) CNS hypoxia depresses ventilation in young, neonatal rats independent of carotid body activation and (2) hyperoxia alters the development of CNS pathways that modulate the late phase of the hypoxic ventilatory response.

Caffeine alters sleep and breathing without an increase in brain serotonin in developing rat pups

In rat pups a reduction in serotonin (5HT) input to respiratory networks impairs anoxic auto‐resuscitation and impairs arousal during hypoxia. In contrast, blockade of adenosine receptors enhances auto‐resuscitation and attenuates the VE decline during the biphasic hypoxic ventilatory response. Caffeine (Caf), an adenosine receptor antagonist, used to treat apnea of prematurity in human infants, has well known effects on sleep and vigilance in adults and in some reports increases the levels of brain 5HT. We therefore examined the pharmacodynamics and the physiological effects of single IP injections of Caf (base) at 5 to 40 mg/kg in P9–P11 pups. Plasma Caf levels reached 10.9 and 76.3 mg/l for 5 and 40 mg/kg, respectively, at 60 min, and were stable until at least 90 min. Caf increased the % time awake at 10 and 40 mg/kg and at 40 mg/kg decreased the % time in active sleep. Caf did not change baseline heart or respiratory rate but the effect of Caf on VT and VE was dependent on state and dose. At 40 mg/kg, VT was increased during wake and quiet sleep whereas VE was increased only during wake. No increases in brainstem 5HT were found at either 10 or 40 mg/kg. In rat pups, a low dose of Caf alters sleep and at higher doses stimulates respiration. The effects of Caf on auto‐resuscitation and ventilation during hypoxia do not appear to be related to an increase in brainstem 5HT levels. NICHD 5‐PO1 HD036379 15

Developmental hyperoxia attenuates hypoxic ventilatory depression in neonatal rats

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, control rats (Bavis et al., J Appl Physiol 109:796–803, 2010). Since ventilatory decline in the late phase of the biphasic HVR is linked to inhibitory modulation of respiratory neurons in the CNS, we hypothesized that hyperoxia‐treated rats would exhibit less CNS‐mediated hypoxic ventilatory depression. We used the P2X receptor antagonist PPADS (125 mg kg−1, i.p.) to “chemically denervate” the carotid bodies (CB) of 4–5‐day old rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia). PPADS had no effect on the hypercapnic ventilatory response (7% CO2) but abolished the early, carotid body‐mediated phase of the HVR (12% O2) in Control rats. Control rats showed a progressive decline in ventilation during hypoxia (−28±4% by 10 min, n=18), but this effect was absent in Hyperoxia rats (−2±4%, n=18; P<0.001 vs. Control). We conclude that CNS hypoxia depresses ventilation in young, neonatal rats independent of CB activation. Moreover, our data suggest that hyperoxia alters the development of CNS pathways that modulate the hypoxic ventilatory response.

Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats

Chronic hyperoxia during the first 1-4 postnatal weeks attenuates the hypoxic ventilatory response (HVR) subsequently measured in adult rats. Rather than focusing on this long-lasting plasticity, the present study considered the influence of hyperoxia on respiratory control during the neonatal period. Sprague-Dawley rats were born and raised in 60% O2 until studied at postnatal ages (P) of 4, 6-7, or 13-14 days. Ventilation and metabolism were measured in normoxia (21% O2) and acute hypoxia (12% O2) using head-body plethysmography and respirometry, respectively. Compared with age-matched rats raised in room air, the major findings were 1) diminished pulmonary ventilation and metabolic O2 consumption in normoxia at P4 and P6-7; 2) decreased breathing stability during normoxia; 3) attenuation of the early phase of the HVR at P6-7 and P13-14; and 4) a sustained increase in ventilation during hypoxia (vs. the normal biphasic HVR) at all ages studied. Attenuation of the early HVR likely reflects progressive impairment of peripheral arterial chemoreceptors while expression of a sustained HVR in neonates before P7 suggests that hyperoxia also induces plasticity within the central nervous system. Together, these results suggest a complex interaction between inhibitory and excitatory effects of hyperoxia on the developing respiratory control system.

Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats

Perinatal hyperoxia causes long‐lasting attenuation of the hypoxic ventilatory response (HVR) in rats, but the timing of these ventilatory changes has not been investigated during the hyperoxic exposure. Rats were raised in room air (control) or 60% O2 from birth until studied at 4, 6‐7, and 13‐14 days of age. Ventilation was measured by head‐body plethysmography in 21% O2 and in 12% O2. Normoxic ventilation was significantly reduced at P4 and P6‐7 in hyperoxia‐treated rats, which may be partially explained by lower metabolic rates. Control rats exhibited a biphasic HVR at P4 and P6‐7; the secondary decline diminished with advancing age and was no longer observed by P13‐14. Hyperoxia‐treated rats exhibited a sustained increase in ventilation at all ages, however, suggesting that hyperoxia hastens postnatal maturation of the central components of the HVR. The early (carotid body‐mediated) phase of the HVR tended to be greater in hyperoxia‐treated rats relative to controls at P4 (P=0.09 at minute 1) but decreased as the hyperoxic exposure progressed (i.e., significantly reduced at P6‐7 and P13‐14 vs. controls). Thus, it appears that short exposures to chronic hyperoxia initially enhance the HVR but that this is gradually overcome by impaired carotid body function. Supported by NIH grant R15 HL‐083972.

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia by D'Agostino D, Mazza E, and Neubauer JA

the effect of hypoxia to stimulate breathing and the cardiovascular system has been well known for more than a century (for a review, see Ref. [11][1]). In 1938, Heymans was awarded the Nobel Prize for Physiology or Medicine for demonstrating the importance of carotid body chemoreceptors. However,