Acute Hypoxic Ventilatory Response Research Articles

Exercise-induced potentiation of the acute hypoxic ventilatory response: Neural mechanisms and implications for cerebral blood flow.

A given dose of hypoxia causes a greater increase in pulmonary ventilation during physical exercise than during rest, representing an exercise-induced potentiation of the acute hypoxic ventilatory response (HVR). This phenomenon occurs independently from hypoxic blood entering the contracting skeletal muscle circulation or metabolic byproducts leaving skeletal muscles, supporting the contention that neural mechanisms per se can mediate the HVR when humoral mechanisms are not at play. However, multiple neural mechanisms might be interacting intricately. First, we discuss the neural mechanisms involved in the ventilatory response to hypoxic exercise and their potential interactions. Current evidence does not support an interaction between the carotid chemoreflex and central command. In contrast, findings from some studies support synergistic interactions between the carotid chemoreflex and the muscle mechano- and metaboreflexes. Second, we propose hypotheses about potential mechanisms underlying neural interactions, including spatial and temporal summation of afferent signals into the medulla, short-term potentiation and sympathetically induced activation of the carotid chemoreceptors. Lastly, we ponder how exercise-induced potentiation of the HVR results in hyperventilation-induced hypocapnia, which influences cerebral blood flow regulation, with multifaceted potential consequences, including deleterious (increased central fatigue and impaired cognitive performance), inert (unchanged exercise) and beneficial effects (protection against excessive cerebral perfusion).

Influence of chronic hypoxia on the hypoxic ventilatory response of juvenile and adult rats

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition – from blunting to augmenting the HVR – occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 – 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one “typical” pattern for VAH in adult rats.

Chronic hyperoxia augments the hypoxic ventilatory response in adult rats

Chronic hyperoxia during postnatal development causes long-lasting attenuation of the hypoxic ventilatory response (HVR) in rats, but similar exposures during adulthood do not attenuate the HVR (Ling et al. J Physiol 405: 561-571, 1996). However, the HVR has not been assessed in adult-exposed rats immediately after chronic hyperoxia (i.e., without several months of recovery in room air). This is important since chronic hyperoxia depresses breathing and blunts the HVR in newborn rats, and only some of these effects persist after return to room air. Accordingly, adult rats were studied after 4 and 14 days of exposure to either 60% O 2 (Hyperoxia) or 21% O 2 (Control). Ventilation was measured by whole-body plethysmography; metabolic O 2 consumption and CO 2 production were also measured in a subset of rats. Normoxic ventilation was unchanged, but Hyperoxia rats exhibited a slight increase in their acute HVR after both durations of chronic hyperoxia. Overall, minute ventilation was approximately 10% greater in Hyperoxia rats while breathing 12% O 2 and 10% O 2 than in Control rats; this effect did not differ significantly between female and male rats. The HVR was also enhanced after 4 days of hyperoxia after controlling for metabolic rate, but the results were less clear after 14 days. After the 14-day exposure, the HVR tended to be enhanced when tested at 12% O 2 but not at 10% O 2 . These data demonstrate that chronic hyperoxia has different effects on the control of breathing in neonatal and adult rats when assessed immediately after the exposure. Supported by NIH grant P20 GM-103423 (Maine INBRE). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Orexin facilitates the hypoxic ventilatory response via the activation of corticotropin-releasing hormone neurons that project to the nucleus of the solitary tract

The paraventricular nucleus of the hypothalamus (PVN) contributes significantly to the peripheral chemoreflex-mediated respiratory and sympathetic responses to hypoxia, in part via reciprocal interactions with nucleus of the solitary tract (nTS). Previously we showed that orexin neurons in the perifornical hypothalamus are activated by hypoxia and facilitate the hypoxic ventilatory response (HVR) in the active phase of the circadian cycle . The neural circuitry through which orexin facilitates the HVR is unknown. Orexin receptors (OxR) are expressed in the PVN, and we recently showed that OxR blockade reduced the activation of both corticotropin-releasing hormone neurons in the PVN as well as nTS neurons. In addition, hypoxia activated orexin neurons projecting to the PVN, but not those projecting to nTS. Here we further explored the hypothesis that orexin facilitates the HVR and arterial blood pressure (ABP) response to hypoxia via the activation of nTS-projecting CRH neurons, and that specific OxR1 knockdown in the PVN would reduce the HVR. To test these hypotheses, we nanoinjected fluorescent retrobeads bilaterally into the nTS to label nTS-projecting neurons. Rats were then exposed to hypoxia (2hrs at 11% O2 during the active phase) following injection of either suvorexant (OxR antagonist; 20 mg/kg i.p.; n=4), or vehicle (DMSO; n=4). Using immunohistochemistry (IHC) we then quantified the number of activated (i.e., Fos-immunoreactive; IR) nTS-projecting CRH neurons. In separate groups of rats we nanoinjected shRNA to knockdown Ox1R expression specifically in the PVN (n=8). PVN of control rats was nanoinjected with scrambled RNA (scRNA; n=3) or aCSF (n=2). Three weeks later, rats were exposed to graded hypoxia (from 21% to 10% inspired O2) over 3-5 minutes to assess the acute HVR and ABP response to different levels of hypoxia. OxR blockade reduced the number of Fos-IR, nTS-projecting CRH neurons by ~32% (drug: p<0.0001). Images confirmed successful targeting of shRNA to PVN, and OxR1 knockdown (by ~50%) was confirmed in subsets of samples by Western Blot and IHC. Ox1R knockdown specifically in the PVN had no effect on resting ventilation, but significantly reduced the HVR (main effect: p=0.02; 22% decrease in VE/VCO2 at 13% O2), due to a reduction in the respiratory frequency response to hypoxia (main effect: p=0.004; 38 breath/min decrease at 13% O2). Ox1R knockdown also decreased ABP (~5 mmHg at all O2 levels; main effect: p=0.0015), but not its response to hypoxia. These data suggest that orexin neurons facilitate the HVR via OxR1 Grants: 5R01HL098602 (EH, DK), 5R01HL136710 (KC) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Catecholamines modulate the hypoxic ventilatory response of larval zebrafish (Danio rerio).

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent β-adrenoreceptor (AR)-mediated increase in fV that does not require expression of β1-ARs, as the hyperventilatory response to β-AR stimulation was unaltered in adrb1-/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min-1 without propranolol to 68±17 breaths min-1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1-/- mutants. Thus, β1-AR is not required for the HVR and other β-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.

Chronic Hypoxia Blunts the Hypoxic Ventilatory Response in Neonatal and Adult Rats

Chronic hypoxia (CH) may affect the control of breathing differently in neonatal versus adult mammals. It is widely accepted that CH blunts the hypoxic ventilatory response (HVR) in neonatal rats but augments hypoxic ventilation in adult rats. However, there is also evidence that longer durations of CH can progressively attenuate the HVR in adult rats too. Therefore, we predicted that short durations of CH (3 days) would blunt the HVR in neonatal rats and augment the HVR in adult rats, but that longer durations of CH (7 days) would blunt the HVR in adult rats. To test these predictions, rats were exposed to 12% O2 (CH) or room air (Control) for 3 days as neonates (from P0 to P3, or from P7 to P10) or as young adults. Baseline ventilation (21% O2) and the acute HVR (12% O2) were measured by plethysmography immediately following the exposure. The adult rats were then returned to the CH and Control chambers for 4 more days before repeating the breathing measurements. Although Sprague‐Dawley rats were used in all experiments, adults from two different commercial suppliers (Charles River Laboratories and Envigo) were used to determine whether genetic background might influence the results. Baseline ventilation was greater in all CH groups regardless of their age, consistent with ventilatory acclimatization. With the exception of CH rats at P10 in which peak hypoxic ventilation (i.e., minute ventilation during the first minute of 12% O2) was also greater than in Controls, hypoxic ventilation was generally similar between CH and Control groups or reduced in the CH group. Accordingly, the change in ventilation from baseline (i.e., HVR) was blunted by CH regardless of the age of the rats, the duration of the CH exposure (adults only), or the substrain of Sprague‐Dawley rat (adults only). The order in which test gases were presented during breathing measurements (i.e., 21% O2 – 12% O2 vs. 12% O2 – 21% O2) did not influence the calculated HVR in adult rats. We conclude that the respiratory plasticity elicited by normobaric CH is similar across ages in Sprague‐Dawley rats.

Lack of influence of dexmedetomidine on rat glomus cell response to hypoxia, and on mouse acute hypoxic ventilatory response.

Background and Aims:There is a lack of basic science data on the effect of dexmedetomidine on the hypoxic chemosensory reflex with both depression and stimulation suggested. The primary aim of this study was to assess if dexmedetomidine inhibited the cellular response to hypoxia in rat carotid body glomus cells, the cells of the organs mediating acute hypoxic ventilatory response (AHVR). Additionally, we used a small sample of mice to assess if there was any large influence of subsedative doses of dexmedetomidine on AHVR.Material and Methods:In the primary study, glomus cells isolated from neonatal rats were used to study the effect of 0.1 nM (n = 9) and 1 nM (n = 13) dexmedetomidine on hypoxia-elicited intracellular calcium [Ca2%]i influx using ratiometric fluorimetry. Secondarily, whole animal unrestrained plethysmography was used to study AHVR in a total of 8 age-matched C57BL6 mice, divided on successive days into two groups of four mice randomly assigned to receive sub-sedative doses of 5, 50, or 500 μg.kg-1 dexmedetomidine versus control in a crossover study design (total n = 12 exposures to drug with n = 12 controls).Results:There was no effect of dexmedetomidine on the hypoxia-elicited increase in [Ca2%]i in glomus cells (a mean ± SEM increase of 95 ± 32 nM from baseline with control hypoxia, 124 ± 41 nM with 0.1 nM dexmedetomidine; P = 0.514). In intact mice, dexmedetomidine had no effect on baseline ventilation during air-breathing (4.01 ± 0.3 ml.g-1.min-1 in control and 2.99 ± 0.5 ml.g-1.min-1 with 500 μg.kg-1 dexmedetomidine, the highest dose; P = 0.081) or on AHVR (136 ± 19% increase from baseline in control, 152 ± 46% with 500 μg.kg-1 dexmedetomidine, the highest dose; P = 0.536).Conclusion:Dexmedetomidine had no effect on the cellular responses to hypoxia. We conclude that it unlikely acts via inhibition of oxygen sensing at the glomus cell. The respiratory chemoreflex effects of this drug remain an open question. In our small sample of intact mice, hypoxic chemoreflex responses and basal breathing were preserved.

Ethanol and opioids do not act synergistically to depress excitation in carotid body type I cells.

ObjectiveThe combination of opioids and ethanol can synergistically depress breathing and the acute ventilatory response to hypoxia. Multiple studies have shown that the underlying mechanisms for this may involve calcium channel inhibition in central neurons. But we have previously identified opioid receptors in the carotid bodies and shown that their activation inhibits calcium influx into the chemosensitive cells. Given that the carotid bodies contribute to the drive to breathe and underpin the acute hypoxic ventilatory response, we hypothesized that ethanol and opioids may act synergistically in these peripheral sensory organs to further inhibit calcium influx and therefore inhibit ventilation.MethodsCarotid bodies were removed from 56 Sprague–Dawley rats (1021 days old) and then enzymatically dissociated to allow calcium imaging of isolated chemosensitive type I cells. Cells were stimulated with high K+ in the presence and absence of the µ-opioid agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) (10 µM), a maximal sublethal concentration of ethanol (3 g L-1, 65.1 mM) or a combination of both.ResultsDAMGO alone significantly inhibited Ca2+ influx but this effect was not potentiated by the high concentration of ethanol.ConclusionThese results indicate for the first time that while opioids may suppress breathing via an action at the level of the carotid bodies, ethanol is unlikely to potentiate inhibition via this pathway. Thus, the synergistic effects of ethanol and opioids on ventilatory parameters are likely mediated by central rather than peripheral actions.

Orexin facilitates the hypoxic ventilatory response in rats

Orexin is a hypothalamic neuropeptide involved in a variety of functions including arousal, body temperature regulation, and feeding. Orexin neurons are active in wakefulness (or the dark phase in rats) and mostly silent in sleep (light phase), implying the effects of orexin are greater in wakefulness and/or dark phase. Recent evidence implicates orexin in the control of cardiorespiratory function, including the respiratory response to CO2 and blood pressure regulation. Whether orexin influences normal breathing in a state-dependent manner is unresolved. In addition, although the role of orexin in the hypercapnic ventilatory response is well established, its effects on the acute hypoxic ventilatory response (HVR) has not been investigated. We hypothesized that orexin receptor blockade would lead to hypoventilation in wakefulness but not sleep and would reduce the HVR. To test these hypotheses, we conducted two separate experiments, both of which were performed in the light phase. First, we used whole-body plethysmography to measure the ventilation of adult rats in room air. Rats were implanted with EEG and EMG electrodes to determine vigilance state, which was confirmed by applying Fast Fourier Transform on the EEG signals. After a 1hr baseline period, we injected i.p. either suvorexant (an OxR1 and OxR2 antagonist, 20 mg/kg i.p.) or vehicle (100% DMSO, i.p.) and allowed the rats to freely behave for 4 hours in the chamber while breathing room air. Separate groups of rats, also instrumented with EEG and EMG electrodes, were exposed to graded hypoxia (15%, 13%, 11%, and 9% O2) following administration of either 20 mg/kg i.p. suvorexant or vehicle. We found that, irrespective of state of vigilance, OxR1 and OxR2 blockade had no effect on baseline breathing, but significantly reduced the HVR (treatment × O2 level: p=0.03). The effect on the HVR was especially evident at 13% and 11% O2 when the response was reduced by ~30%. We show that orexin facilitates the HVR in the light phase, even though the orexin neurons are silent. Whether orexin has a larger effect on the HVR during the dark phase, when orexin neurons are active, has yet to be determined.

Neuronal Loss in the Nucleus Tractus Solitarius and Associated Disturbances of the Hypoxic Ventilatory Response in Rat Offspring Following in‐utero Morphine Exposure

The incidence of opioid use during pregnancy, including prescriptions, has become a growing public health concern and has been linked to neonatal abstinence syndrome (NAS), poor neurodevelopmental outcome and increased risk of sudden infant death syndrome (SIDS). Abnormal cardiorespiratory regulation in infants born following in-utero opioid exposure includes reduced vagal tone, delayed arousal, and altered ventilatory responses to hypoxia and hypercapnia. Impairments in ventilatory defense responses and abnormal development of brainstem cardiorespiratory control regions may contribute to infant vulnerability and dampen responses to exogenous stressors leading to SIDS. Although infants with NAS may be treated with morphine, not all infants require pharmacologic treatment. To mimic the latter group of infants, we used a rat model of in utero morphine exposure to investigate the long-term effects it has on respiratory control and brainstem neuronal development of the offspring. Using a rat model of in utero morphine exposure to characterize long-term (10 days) effects on the offspring including: 1) the acute hypoxic (HVR) and hypercapnic ventilatory response (HCVR) as indices of respiratory control (dys)function; and 2) brainstem neuron counts as an indicator of neural development. Pregnant dams received twice daily (morning and afternoon) subcutaneous injections of various doses of morphine (0.5, 1, and 5mg/kg) or saline (control group) during the final week of pregnancy to mimic 3rd trimester maternal opioid use. Following birth, nursing dams and rat pups were left untreated. On postnatal day 10, whole body plethysmography was used to assess the HVR and HCVR in the pups. Brains were removed and prepared for immunohistochemical assessment of neuron cell counts in key brainstem cardiorespiratory control regions. In-utero morphine exposure at any dose (0.5, 1, 5mg/kg) caused a long-term attenuation of the HVR (Fig. 1A), but not the HCVR when compared to pups from in-utero saline treated rats. At the highest dose (5mg/kg), neuron cell numbers were reduced by approximately 50% compared to saline rats (Fig. 1B-C). These data demonstrate a long-term effect of in-utero morphine exposure on respiratory control, which may in part be explained by disrupted brainstem neural development. These cardiorespiratory anomalies could offer insight into the pathophysiology of the increased risk of SIDS seen in infants born from mothers with opioid use.

Caffeine prevents prostaglandin E1-induced disturbances in respiratory control in neonatal rats: implications for infants with critical congenital heart disease.

Continuous infusion of prostaglandin E1 (PGE1) is used to maintain ductus arteriosus patency in infants with critical congenital heart disease, but it can also cause central apnea suggesting an effect on respiratory neural control. In this study, we investigated whether 1) PGE1 inhibits the various phases of the acute hypoxic ventilatory response (HVR; an index of respiratory control dysfunction) and increases apnea incidence in neonatal rats; and 2) whether these changes would be reversible with caffeine pretreatment. Whole body plethysmography was used to assess the HVR and apnea incidence in neonatal rats 2 h following a single bolus intraperitoneal injection of PGE1 with and without prior caffeine treatment. Untreated rats exhibited a biphasic HVR characterized by an initial increase in minute ventilation followed by a ventilatory decline of the late phase (~5th minute) of the HVR. PGE1 had a dose-dependent effect on the HVR. Contrary to our hypothesis, the lowest dose (1 µg/kg) of PGE1 prevented the ventilatory decline of the late phase of the HVR. However, PGE1 tended to increase postsigh apnea incidence and the coefficient of variability (CV) of breathing frequency, suggesting increased respiratory instability. PGE1 also decreased brainstem microglia mRNA and increased neuronal nitric oxide synthase (nNOS) and platelet-derived growth factor-β (PDGF-β) gene expression. Caffeine pretreatment prevented these effects of PGE1, and the adenosine A2A receptor inhibitor MSX-3 had similar preventative effects. Prostaglandin appears to have deleterious effects on brainstem respiratory control regions, possibly involving a microglial-dependent mechanism. The compensatory effects of caffeine or MSX-3 treatment raises the question of whether prostaglandin may also operate on an adenosine-dependent pathway.

Influence of estrus cycle and timing of lipopolysaccharide (LPS)‐induced inflammation on inspiratory motor activity and the acute hypoxic ventilatory response (HVR) in urethane‐anesthetized spontaneously breathing adult female Sprague‐Dawley rat

Accumulating evidence suggests that estrogens can exert anti‐inflammatory and neuroprotective effects, which have the capacity to influence the onset, magnitude, and time course of inflammatory processes. The most commonly used experimental approach for inducing inflammation involves systemic administration of the bacterial endotoxin lipopolysaccharide (LPS), and the progression of the LPS‐regulated pro‐inflammatory gene/protein expression response has been well characterized, with an induction phase of inflammation occurring at ~2–4 hours following LPS exposure, a peak inflammatory phase occurring at ~4–6 hours following LPS exposure, and a resolution phase at ~24 hours following LPS exposure. It is likely that time‐dependent changes in expression of the upregulated pro‐inflammatory cytokines underlie the progression of alterations in various physiological processes, including ventilatory control mechanisms. We have recently begun to examine the impact of inflammation on various aspects of ventilatory control, and the current study was undertaken to begin to evaluate the potential influence of the hormonal changes associated with the estrous cycle on inflammation‐induced alterations in ventilatory control during the induction and resolution phases of the early inflammatory response. For the current study, we examined basal inspiratory (diaphragm) motor (EMG) activity and the acute hypoxic ventilatory response (HVR; 12% O2 for 90s) in spontaneously breathing urethane‐anesthetized female Sprague‐Dawley rats at either ~2–4 hr or 24‐hr after systemic administration of LPS (3 mg/kg, ip) or vehicle (0.9% saline, ip) delivered during proestrus (high estrogen) or diestrus (low estrogen). We found that basal EMG burst timing and patterning features were slightly modified by LPS exposure at both time points albeit only trends were observed at the 24 hr time point. In contrast, at the 2–4 hr time point, significantly increased burst frequency, decreased TI, reduced TI/Ttot, and lower ApEn values were noted in diestrus LPS‐treated rats. We also found that in proestrus LPS‐, proestrus saline‐, and diestrus saline‐treated rats at 2–4 hr post administration, HVR increases in burst amplitude and frequency were similar, while in diestrus LPS‐treated rats, there was a markedly blunted increase in burst amplitude. In contrast at 24 hr post administration, in proestrus LPS‐, proestrus saline‐, diestrus LPS‐, and diestrus saline‐treated rats, we found that HVR increases in burst amplitude and frequency were comparable across all groups. These observations suggest that both LPS administration during different phases of the estrus cycle as well as time‐dependent phases of inflammation influence the impact of LPS‐induced inflammation on inspiratory motor activity and HVR ventilatory control.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

Neuronal HIF-1α in the nucleus tractus solitarius contributes to ventilatory acclimatization to hypoxia.

We hypothesized that hypoxia inducible factor 1α (HIF-1α) in CNS respiratory centres is necessary for ventilatory acclimatization to hypoxia (VAH); VAH is a time-dependent increase in baseline ventilation and the hypoxic ventilatory response (HVR) occurring over days to weeks of chronic sustained hypoxia (CH). Constitutive deletion of HIF-1α in CNS neurons in transgenic mice tended to blunt the increase in HVR that occurs in wild-type mice with CH. Conditional deletion of HIF-1α in glutamatergic neurons of the nucleus tractus solitarius during CH significantly decreased ventilation in acute hypoxia but not normoxia in CH mice. These effects are not explained by changes in metabolic rate, nor CO2 , and there were no changes in the HVR in normoxic mice. HIF-1α mediated changes in gene expression in CNS respiratory centres are necessary in addition to plasticity of arterial chemoreceptors for normal VAH. Chronic hypoxia (CH) produces a time-dependent increase of resting ventilation and the hypoxic ventilatory response (HVR) that is called ventilatory acclimatization to hypoxia (VAH). VAH involves plasticity in arterial chemoreceptors and the CNS [e.g. nucleus tractus solitarius (NTS)], although the signals for this plasticity are not known. We hypothesized that hypoxia inducible factor 1α (HIF-1α), an O2 -sensitive transcription factor, is necessary in the NTS for normal VAH. We tested this in two mouse models using loxP-Cre gene deletion. First, HIF-1α was constitutively deleted in CNS neurons (CNS-HIF-1α-/- ) by breeding HIF-1α floxed mice with mice expressing Cre-recombinase driven by the calcium/calmodulin-dependent protein kinase IIα promoter. Second, HIF-1α was deleted in NTS neurons in adult mice (NTS-HIF-1α-/- ) by microinjecting adeno-associated virus that expressed Cre-recombinase in HIF-1α floxed mice. In normoxic control mice, HIF-1α deletion in the CNS or NTS did not affect ventilation, nor the acute HVR (10-15min hypoxic exposure). In mice acclimatized to CH for 1week, ventilation in hypoxia was blunted in CNS-HIF-1α-/- and significantly decreased in NTS-HIF-1α-/- compared to control mice (P<0.0001). These changes were not explained by differences in metabolic rate or CO2 . Immunofluorescence showed that HIF-1α deletion in NTS-HIF-1α-/- was restricted to glutamatergic neurons. The results indicate that HIF-1α is a necessary signal for VAH and the previously described plasticity in glutamatergic neurotransmission in the NTS with CH. HIF-1α deletion had no effect on the increase in normoxic ventilation with acclimatization to CH, indicating this is a distinct mechanism from the increased HVR with VAH.

Long‐term Cardiorespiratory Anomalies in Rat Offspring Following Prenatal Morphine Exposure

The incidence of opioid‐related pregnancies is a growing public health concern. Although not all infants born from opioid using mothers require hospitalization, many exhibit a variety of withdrawal symptoms (Neonatal Opioid Withdrawal Syndrome, NOWS) often requiring weeks of neonatal intensive care. There are longer‐term effects of prenatal opioid use including altered CNS function comprising cognitive impairments, neurobehavioral abnormalities and increased risk of Sudden Infant Death Syndrome (SIDS). In fact, abnormal cardiorespiratory regulation in infants born following prenatal opioid exposure includes reduced vagal tone, tachypnea, delayed arousal, and altered ventilatory responses to hypoxia and hypercapnia. In the present study, we used a rat model to characterize long‐term (postnatal (P) 10 days of age) cardiorespiratory responses of the offspring following in utero morphine exposure. Pregnant dams received twice daily (morning and evening) subcutaneous injections of various doses of morphine (1, 5 or 10mg/kg) during the 3rd week of pregnancy to mimic 3rd trimester maternal opioid use. Following birth, rat pups (and the nursing dam) were left untreated and whole‐body plethysmography was used to assess the acute hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses at P10 days of age. Heart rate was also assessed in rats fitted with a custom‐made jacket with built‐in ECG electrodes. Generally, the postnatal effects of morphine were dose‐dependent: 1) increasing doses of in utero morphine exposure caused a progressive attenuation of the HCVR in 10 day old rats; 2) there was a high rate of neonatal (within 24hrs of birth) mortality in the highest (10mg/kg) prenatal dose; 3) however, the rats who survived exhibited both an attenuated HVR and HCVR; and 4) rats also exhibited resting tachycardia and tachypnea (at 5–10 mg/kg) compared to in utero saline treated rats. These data demonstrate a long‐term (10 days) effect of prenatal morphine exposure on resting cardiorespiratory function, including impairments in ventilatory defense responses to acute hypoxia and hypercapnia. These cardiorespiratory anomalies could offer a window of insight into the pathophysiology of the increased risk of SIDS seen in infants born from mothers of substance use.

Influence of Estrus Cycle on Lipopolysaccharide (LPS)‐induced Modulation of Basal Inspiratory Motor Activity and Acute Hypoxic Ventilatory Response (HVR) in Urethane‐anesthetized Spontaneously Breathing Adult Female Sprague‐Dawley Rat

Accumulating evidence suggests that estrogens can exert anti‐inflammatory and neuroprotective effects, which have the capacity to influence the onset, magnitude, and time course of inflammatory processes. The most commonly used experimental approach for inducing inflammation involves systemic administration of the bacterial endotoxin lipopolysaccharide (LPS), and this approach is often used in ongoing studies addressing the impact of inflammation on various aspects of ventilatory control. The potential influence of the hormonal changes associated with the estrous cycle on inflammation‐induced alterations in ventilatory control, however, have not been identified. To begin to address this issue, we examined basal inspiratory (diaphragm) motor (EMG) activity and the acute hypoxic ventilatory response (HVR; 12% O2 for 90s) during proestrus (high estrogen) and diestrus (low estrogen) in urethane‐anesthetized spontaneously breathing female Sprague‐Dawley rats after acute (2–4 hrs post) administration of LPS (3 mg/kg, ip); saline administration served as a control in aditional groups of rats. For basal diaphragm EMG burst characteristics, we quantified burst frequency, burst duration (TI), time between bursts (TE), inspiratory duty cycle (TI/Ttot), time‐to‐peak (Tpeak/TI), and inspiratory burst complexity (ApEn) from 20 consecutive breathing cycles. We found that in proestrus, LPS‐ and saline‐treated rats exhibited similar timing and patterning features with the exception of TI/Ttot (P=0.0385) and TI (P=0.0694) which both tended to be shorter in LPS‐treated rats while in diestrus, LPS‐treated rats (compared to saline‐treated rats) exhibited increased burst frequency (P=0.0399) and decreased TI (P=0.0087). In addition, ApEn was significantly lower in diestrus than in proestrus (P=0.0421). For the HVR, we quantified burst amplitude and frequency for 5 consecutive breathing cycles at 30s intervals for 90s before, during, immediately following, and 5 min after the hypoxic gas exposure. We found that in proestrus LPS‐, proestrus saline‐, and diestrus saline‐treated rats, HVR burst amplitude and frequency responses were similar, with burst amplitude increasing by 30–35% above BL levels and burst frequency increasing by 35–41% above BL levels. While diestrus LPS‐treated rats exhibited an ~48% increase in burst frequency above BL levels, they had a markedly blunted increase in burst amplitude (~14% above BL levels), yielding a significantly different response than that noted for the other groups (P=0.009). It should also be noted that in a subset of diestrus LPS‐treated rats, the HVR challenge was terminated early due to development of severe respiratory depression. These observations demonstrate that LPS administration during diestrus slightly alters basal inspiratory motor activity and impairs the HVR in a manner similar to what we have seen in LPS‐treated male rats. Moreover, these observations may be taken to support a potential protective role for estrogen in ventilatory control.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Improved Acute Hypoxic Ventilatory Response (HVR) After Pharmacological Activation of 5‐HT1A Receptors in Two‐Hit Lipopolysaccharide (LPS)‐Treated Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat with Prior Single‐Bout Acute Intermittent Hypoxia Exposure

Accumulating evidence indicates that inflammation and serotonergic (5‐HT) neurotransmission interact thereby affecting a variety of physiological processes. Ongoing studies in our laboratory are examining the effects of peripheral inflammation, and its accompanying neuroinflammation, on the neural control of breathing, including the impact of LPS‐induced neuroinflammation on 5‐HT1A receptor‐mediated inspiratory motor behaviors. Here, we examined the effects of systemic administration of the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐npropylamino) tetralin (8‐OH DPAT) on the (diaphragm) EMG burst amplitude and frequency response to a single bout of acute hypoxia (HVR; 11% O2 for 90s) in spontaneously breathing urethane‐anesthetized adult male Sprague‐Dawley rats following a ‘two‐hit’ lipopolysaccharide (LPS) administration protocol in which systemic LPS (3 mg/kg, ip) was administered ~24 hr prior to an intratracheal LPS (0.5 mg/kg, IT) injection. Similar HVR experiments were performed in control rats that were either untreated or received two‐hit saline injections. All rats used for this study had also undergone an acute intermittent hypoxia (AIH) exposure protocol that was completed at least 90 min prior to administration of 8‐OH DPAT, and they were continuously supplied with 2% CO2 throughout the recording protocol. Following baseline (BL; 40% O2) recording of diaphragm EMG activity, 8‐OH DPAT (0.3 mg/kg, iv) was administered and allowed to exert its effects for 30 min, after which a 30s HVR challenge was performed (11% O2) followed by recovery under BL conditions. Data from these experiments were also compared to our previous observations in two‐hit LPS‐ and vehicle‐treated male rats that had not undergone an AIH exposure protocol or received 8‐OH DPAT (to serve as a control for two‐hit LPS HVR behaviors). In brief, we had reported that two‐hit LPS administration blunts the HVR amplitude response such that it increases by ≤10% above BL levels (versus ≥30% increase in vehicle‐treated rats) and produces an exaggerated frequency HVR response such that it increases by ≥30% above BL levels (versus~15–20% increase in vehicle‐treated rats). In contrast to these observations, in the current study, we found that following 8‐OH DPAT administration, the HVR exhibited similar increases in both burst frequency and amplitude in both control and LPS‐treated rats. In this case, HVR amplitude and frequency increased on average by ~23% and ~25%, respectively, although LPS‐treated rats exhibited variable magnitude HVR responses. Regardless, following 8‐OH DPAT administration, HVR inspiratory motor activity appeared to be indistinguishable between control and LPS‐treated rats. These observations suggest that following pharmacological activation of 5‐HT1A receptors, amplitude and frequency HVR behaviors are improved in two‐hit LPS‐treated rats. The specific mechanisms underlying these differences remain to be identified.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glutamatergic Receptors Modulate Normoxic but Not Hypoxic Ventilation and Metabolism in Naked Mole Rats.

Naked mole rats (Heterocephalus glaber) are among the most hypoxia-tolerant mammals, but their physiological responses to acute and chronic sustained hypoxia (CSH), and the molecular underpinnings of these responses, are poorly understood. In the present study we evaluated the acute hypoxic ventilatory response and the occurrence of ventilatory acclimatization to hypoxia following CSH exposure (8–10 days in 8% O2) of naked mole rats. We also investigated the role of excitatory glutamatergic signaling in the control of ventilation and metabolism in these conditions. Animals acclimated to normoxia (control) or CSH and then exposed to acute hypoxia (7% O2 for 1 h) exhibited elevated tidal volume (VT), but decreased breathing frequency (fR). As a result, total ventilation (E) remained unchanged. Conversely, VT was lower in CSH animals relative to controls, suggesting that there is ventilatory plasticity following acclimatization to chronic hypoxia. Both control and CSH-acclimated naked mole rats exhibited similar 60–65% decreases in O2 consumption rate during acute hypoxia, and as a result their air convection requirement (ACR) increased ∼2.4 to 3-fold. Glutamatergic receptor inhibition decreased fR, E, and the rate of O2 consumption in normoxia but did not alter these ventilatory or metabolic responses to acute hypoxia in either the control or CSH groups. Taken together, these findings indicate that ventilatory acclimatization to hypoxia is atypical in naked mole rats, and glutamatergic signaling is not involved in their hypoxic ventilatory or metabolic responses to acute or chronic hypoxia.

Ventilatory responses to acute hypoxia and hypercapnia in humans with a patent foramen ovale.

Subjects with a patent foramen ovale (PFO) have blunted ventilatory acclimatization to high altitude compared with subjects without PFO. The blunted response observed could be because of differences in central and/or peripheral respiratory chemoreflexes. We hypothesized that compared with subjects without a PFO (PFO-), subjects with a PFO (PFO+) would have blunted ventilatory responses to acute hypoxia and hypercapnia. Sixteen PFO+ subjects (9 female) and 15 PFO- subjects (8 female) completed four 20-min trials on the same day: 1) normoxic hypercapnia (NH), 2) hyperoxic hypercapnia (HH), 3) isocapnic hypoxia (IH), and 4) poikilocapnic hypoxia (PH). Hypercapnic trials were completed before the hypoxic trials, the order of the hypercapnic (NH & HH) and hypoxic (IH & PH) trials were randomized, and trials were separated by ≥40 min. During the NH trials but not the HH trials subjects who were PFO+ had a blunted hypercapnic ventilatory response compared with subjects who were PFO- (1.41 ± 0.46 l·min-1·mmHg-1 vs. 1.98 ± 0.71 l·min-1·mmHg-1, P = 0.02). There were no differences between the PFO+ and PFO- subjects with respect to the acute hypoxic ventilatory response during IH and PH trials. Hypoxic ventilatory depression was similar between subjects who were PFO+ and PFO- during IH. These data suggest that compared with subjects who were PFO-, subjects who were PFO+ have normal ventilatory chemosensitivity to acute hypoxia but blunted ventilatory chemosensitivity to carbon dioxide, possibly because of reduced carbon dioxide sensitivity of either the central and/or the peripheral chemoreceptors. NEW & NOTEWORTHY Patent foramen ovale (PFO) is found in ~25%-40% of the population. The presence of a PFO appears to be associated with blunted ventilatory responses during acute exposure to normoxic hypercapnia. The reason for this blunted ventilatory response during acute exposure to normoxic hypercapnia is unknown but may suggest differences in either central and/or peripheral chemoreflex contribution to hypercapnia.

Serotonin and Adenosine G-protein Coupled Receptor Signaling for Ventilatory Acclimatization to Sustained Hypoxia.

Different patterns of hypoxia evoke different forms of plasticity in the neural control of ventilation. For example, acute intermittent hypoxia produces long term facilitation (LTF) of ventilation, while chronic sustained hypoxia (CH) causes ventilatory acclimatization to hypoxia (VAH). In both LTF and VAH, ventilation in normoxia is greater than normal after the hypoxic stimulus is removed and the acute hypoxic ventilatory response can increase. However, the mechanisms of LTF and VAH are thought to be different based on previous results showing serotonin 5HT2 receptors, which are G protein coupled receptors (GPCR) that activate GQ signaling, contribute to LTF but not VAH. Newer results show that a different GPCR, namely adenosine A2A receptors and the GS signaling pathway, cause LTF with more severe intermittent hypoxia, i.e., PaO2 = 25–30 Torr for GS versus 35–45 Torr for LTF with the GQ signaling pathway. We hypothesized adenosine A2A receptors and GS signaling are involved in establishing VAH with longer term moderate CH and tested this in adult male rats by measuring ventilatory responses to O2 and CO2 with barometric pressure plethysmography after administering MSX-3 or ketanserin (A2A and 5HT2 antagonists, respectively, both 1 mg/Kg i.p.) during CH for 7 days. Blocking GS or GQ signals throughout CH exposure, significantly decreased VAH. After VAH was established, GQ blockade did not affect ventilation while GS blockade increased VAH. Similar to LTF, data support roles for both GQ and GS pathways in the development of VAH but after VAH has been established, the GS pathway inhibits VAH.

Consequences of postnatal myo‐inositol supplementation on respiratory control and nucleus tractus solitarius (nTS) neurons

Myo‐inositol is a naturally occurring sugar derivative formed endogenously from the breakdown of glucose; it is also found in a variety of foods including breast milk and is commercially available as a nutritional supplement. Serum levels of myo‐inositol in infants decrease naturally over the first few postnatal weeks; prior studies in preterm infants have demonstrated improved pulmonary function, reduced incidence of respiratory distress, and increased survival rates following myo‐inositol supplementation. In support of a potential beneficial effect of myo‐inositol supplementation on respiratory control, we have shown previously that myo‐inositol treatment over the first 2 postnatal weeks enhances the acute hypoxic ventilatory response (HVR). In the present study, we tested the hypothesis that the enhanced HVR following myo‐inositol treatment would be associated with increased excitability of neurons within the area of brainstem chemoafferent integration, the nucleus tractus solitarius (nTS). Mouse pups received subcutaneous injections of myo‐inositol (80 mg/kg/day) for the first two postnatal weeks. Whole cell patch clamp recordings were then used to examine the excitatory properties of nTS neurons monosynaptically connected to the solitary tract. Compared to saline treated mice, myo‐inositol exposure increased the rheobase and the failure rate of nTS neurons, and decreased the paired pulse ratio following 20 Hz stimulation. Spontaneous activity (EPSC frequency, area, rise time, decay time), and basic neuronal properties (membrane resistance, capacitance, resting membrane potential) did not differ between groups. In conclusion, and contrary to our hypothesis, the enhanced HVR following postnatal myo‐inositol treatment was associated with decreased excitability of nTS neurons. We speculate the reduced nTS neuron excitability may be a compensatory response to increased chemoafferent (presumably the carotid body) inputs during hypoxia. These data may be important to our understanding of how myo‐inositol supplementation could influence development and function of brainstem respiratory neural control regions and may have implications for the pathophysiology of various respiratory morbidities in preterm infants.Support or Funding InformationThe Gerber FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.