Hypercapnic Ventilatory Response Research Articles

Exercise derived myokine irisin as mediator of cardiorespiratory, metabolic and thermal adjustments during central and peripheral chemoreflex activation

Exercise elicits physiological adaptations, including hyperpnea. However, the mechanisms underlying exercise-induced hyperpnea remain unresolved. Skeletal muscle acts as a secretory organ, releasing irisin (IR) during exercise. Irisin can cross the blood–brain barrier, influencing muscle and tissue metabolism, as well as signaling in the central nervous system (CNS). We evaluated the effect of intracerebroventricular or intraperitoneal injection of IR in adult male rats on the cardiorespiratory and metabolic function during sleep–wake cycle under room air, hypercapnia and hypoxia. Central IR injection caused an inhibition on ventilation (VE) during wakefulness under normoxia, while peripheral IR reduced VE during sleep. Additionally, central IR exacerbates hypercapnic hyperventilation by increasing VE and reducing oxygen consumption. As to cardiovascular regulation, central IR caused an increase in heart rate (HR) across all conditions, while no change was observed following peripheral administration. Finally, central IR attenuated the hypoxia-induced regulated hypothermia and increase sleep episodes, while peripheral IR augmented CO2-induced hypothermia, during wakefulness. Overall, our results suggest that IR act mostly on CNS exerting an inhibitory effect on breathing under resting conditions, while stimulating the hypercapnic ventilatory response and increasing HR. Therefore, IR seems not to be responsible for the exercise-induced hyperpnea, but contributes to the increase in HR.

Changes in hypoxic and hypercapnic ventilatory responses at high altitude measured using rebreathing methods.

Ventilatory responses to hypoxia and hypercapnia play a vital role in maintaining gas exchange homeostasis, and in adaptation to high-altitude environments. This study investigates the mechanisms underlying sensitization of hypoxic and hypercapnic ventilatory responses (HVR and HCVR, respectively) in individuals acclimatized to moderate high altitude (3800 m). Thirty-one participants underwent chemoreflex testing using the Duffin modified rebreathing technique. Measures were taken at sea level and after 2 days of acclimatization to high altitude. Ventilatory recruitment thresholds (VRT), HCVR, and HVR were quantified. Acclimatization to high altitude resulted in increased HVR (p<0.001) and HCVR (p<0.001), as expected. We also observed that the decrease in VRT under hypoxic test conditions significantly contributed to the elevated HVR at high altitude since the change in VRT across hyperoxic and hypoxic test conditions was greater at high altitude compared to baseline sea level tests (p=0.043). Pre-VRT ventilation also increased at high altitude (p<0.001), but the change did not differ between oxygen conditions. Taken together, this data suggests that the increase in HVR at high altitude is at least partially driven by a larger decrease in the VRT in hypoxia versus hyperoxia at high altitude compared to sea level. This study highlights the intricacies of respiratory adaptations during acclimatization to moderate high altitude, shedding light on the roles of the VRT, baseline respiratory drive, and two-slope HCVR in this process. These findings contribute to our understanding of how the human respiratory control responds to hypoxic and hypercapnic challenges at high altitude.

Chronic Hyperoxia Augments the Hypercapnic Ventilatory Response in Adult Rats

Rats exposed to 4 – 14 days of 60% O2 as adults exhibit a modest enhancement of their hypoxic ventilatory response (HVR) (Danielson &amp; Bavis, Physiology 38(S1): 5730119, 2023). To determine whether this plasticity is unique to the HVR or a general increase in excitability of the respiratory control system, we tested the hypothesis that chronic hyperoxia would also enhance the hypercapnic ventilatory response (HCVR). The ventilatory response to 7% CO2 was measured by whole-body plethysmography in adult, Sprague-Dawley rats immediately after 4 and 14 days of exposure to 60% O2 or an equivalent time period in room air. Baseline ventilation and the HCVR were unchanged after 4 days of hyperoxia. The HCVR was increased after 14 days (Treatment × FICO2, P=0.004), with hypercapnic ventilation being approximately 15% greater than in control rats. However, this effect was no longer significant after accounting for variation in metabolic rate, and the HCVR was fully recovered when rats were restudied four weeks after they were returned to room air. In a follow-up experiment, pulse oximetry was performed in isoflurane-anesthetized rats breathing room air or 12% O2. Exposure to 4 – 14 days of 60% O2 did not impair gas exchange, so augmented ventilatory responses likely reflect changes in the neural control of breathing and/or metabolism rather than differences in the levels of hypoxemia and hypercapnia experienced by rats during the tests. In conclusion, chronic hyperoxia elicits plasticity in both the HVR and HCVR of adult rats, but these responses recover in less than one month after rats are returned to room air. Supported by NIH grant P20 GM-103423 (Maine INBRE). This is the full abstract presented at the American Physiology Summit 2024 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.

Targeting Melanocortin 4 Receptor to Treat Sleep-Disordered Breathing

There is no effective pharmacotherapy for sleep-disordered breathing (SDB). A melanocortin receptor 4 (MCR4) agonist, setmelanotide (SET), is used to treat genetic obesity caused by abnormal melanocortin and leptin signaling. We hypothesized that SET can treat SDB in diet-induced obese mice. We performed a proof-of-concept randomized crossover trial of a single dose of SET vs vehicle and a two-week daily SET vs vehicle trial in obese mice. We also examined co-localization of Mcr4 mRNAs with a marker of CO2 sensing neurons PHOX2b in the brainstem and performed chemogenetic studies expressing Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) in Mcr4-Cre mice. SET increased minute ventilation across sleep/wake states, greatly enhanced the hypercapnic ventilatory response (HCVR), and abolished apneas during sleep. PHOX2b+ neurons in the nucleus of the solitary tract (NTS) and the parafacial region expressed Mc4r. Chemogenetic stimulation of the MC4R+ neurons in the parafacial region, but not in the NTS, augmented the HCVR without any changes in metabolism. Parafacial MC4R neurons projected to the respiratory pre-motor neurons expressing cholera toxin B after C3-C4 spinal cord injections. In conclusion, MC4R agonists enhance the HCVR and treat SDB by acting on the parafacial MC4R+ neurons. NIH (R01 HL128970, R01 HL133100, and R01 HL138932), AHA (#915167). This is the full abstract presented at the American Physiology Summit 2024 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.

Sex Differences In The Phenotype Of Sleep-Disordered Breathing In C57BL/6J Mice

Sex differences in sleep-disordered breathing (SDB) are widely recognized. SDB is more prevalent in men, but women appear to exhibit more symptoms at lower levels of SDB severity, including insomnia symptoms. Mechanisms underlying sex differences in SDB are unknown. In this study, we compared the sleep architecture and breathing patterns during sleep between male and female mice and examined the effects of ovariectomy on SDB. We hypothesized that female and male mice differed in sleep and SDB, which are related to differences in carbon dioxide (CO2) chemosensitivity between sexes. Adult male (n=9) and female (n=10) C57BL/6J mice underwent full-polysomnography inside a whole-body plethysmography chamber. Arousals from sleep and apneas were manually scored. Breathing variability was assessed with Poincaré plots of minute ventilation. Hypercapnic ventilatory response (HCVR) was assessed during wakefulness and NREM sleep by acute administration of 8% CO2. In female mice, we performed bilateral ovariectomy and repeated sleep studies after 14 days. We found that arousal index was 55% increased in females compared to males, but sleep fragmentation was mainly attributed to spontaneous arousals in both groups. Surprisingly, apnea severity was significantly increased in female mice compared to males (32.4±6.7 vs 11.8±4.5, P=0.001). Breathing instability was also increased in females. HCVR was positively correlated with apnea index (r=0.55, P=0.05) and was significantly augmented in females compared to males. Ovariectomy did not affect sleep fragmentation, but attenuated CO2 sensitivity and improved breathing stability during sleep. Our results have suggested different SDB phenotypes between male and female C57BL/6J mice. SDB in female mice is associated with a hyperarousal state and increased HCVR compared to males. An exacerbated CO2 responsiveness related to ovarian hormones in females is an important determinant of differences in SDB severity and respiratory patterns between male and female sex. American Heart Association Postdoctoral Fellowship Award 828142 (to L.J.K.), NHLBI grant 2R01 HL133100-05, NIH R01 HL128970, and R01 HL13892 (to V.Y.P.), 1K23HL155730 and American Academy of Sleep Medicine Foundation 238-BS-20 (to L.V.P.). This is the full abstract presented at the American Physiology Summit 2024 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.

Control of breathing by orexinergic signaling in the nucleus tractus solitarii

Orexin signaling plays a facilitatory role in respiration. Abnormalities in orexin levels correlate with disordered breathing patterns and impaired central respiratory chemoreception. Nucleus tractus solitarii (NTS) neurons expressing the transcription factor Phox2b contribute to the chemoreceptive regulation of respiration. However, the extent to which orexinergic signaling modulates respiratory activity in these Phox2b-expressing NTS neurons remains unclear. In the present study, the injection of orexin A into the NTS significantly increased the firing rate of the phrenic nerve. Further analysis using fluorescence in situ hybridization and immunohistochemistry revealed that orexin 1 receptors (OX1Rs) were primarily located in the ventrolateral subdivision of the NTS and expressed in 25% of Phox2b-expressing neurons. Additionally, electrophysiological recordings showed that exposure to orexin A increased the spontaneous firing rate of Phox2b-expressing neurons. Immunostaining experiments with cFos revealed that the OX1R-residing Phox2b-expressing neurons were activated by an 8% CO2 stimulus. Crucially, OX1R knockdown in these NTS neurons notably blunted the ventilatory response to 8% CO2, alongside an increase in sigh-related apneas. In conclusion, orexinergic signaling in the NTS facilitates breathing through the activation of OX1Rs, which induces the depolarization of Phox2b-expressing neurons. OX1Rs are essential for the involvement of Phox2b-expressing NTS neurons in the hypercapnic ventilatory response.

Expression of endogenous epitope-tagged GPR4 in the mouse brain.

GPR4 is a proton-sensing G protein-coupled receptor implicated in many peripheral and central physiological processes. GPR4 expression has previously been assessed only via detection of the cognate transcript or indirectly, by use of fluorescent reporters. In this work, CRISPR/Cas9 knock-in technology was used to encode a hemagglutinin (HA) epitope tag within the endogenous locus of Gpr4 and visualize GPR4-HA in the mouse central nervous system using a specific, well characterized HA antibody; GPR4 expression was further verified by complementary Gpr4 mRNA detection. HA immunoreactivity was found in a limited set of brain regions, including in the retrotrapezoid nucleus (RTN), serotonergic raphe nuclei, medial habenula, lateral septum, and several thalamic nuclei. GPR4 expression was not restricted to cells of a specific neurochemical identity as it was observed in excitatory, inhibitory, and aminergic neuronal cell groups. HA immunoreactivity was not detected in brain vascular endothelium, despite clear expression of Gpr4 mRNA in endothelial cells. In the RTN, GPR4 expression was detected at the soma and in proximal dendrites along blood vessels and the ventral surface of the brainstem; HA immunoreactivity was not detected in RTN projections to two known target regions. This localization of GPR4 protein in mouse brain neurons corroborates putative sites of expression where its function has been previously implicated (e.g., CO2-regulated breathing by RTN), and provides a guide for where GPR4 could contribute to other CO2/H+ modulated brain functions. Finally, GPR4-HA animals provide a useful reagent for further study of GPR4 in other physiological processes outside of the brain.Significance Statement GPR4 is a proton-sensing G-protein coupled receptor whose expression is necessary for a number of diverse physiological processes including acid-base sensing in the kidney, immune function, and cancer progression. In the brain, GPR4 has been implicated in the hypercapnic ventilatory response mediated by brainstem neurons. While knockout studies in animals have clearly demonstrated its necessity for normal physiology, descriptions of GPR4 expression have been limited due to a lack of specific antibodies for use in mouse models. In this paper, we implemented a CRISPR/Cas9 knock-in approach to incorporate the coding sequence for a small epitope tag into the locus of GPR4. Using these mice, we were able to describe GPR4 protein expression directly for the first time.

Paradoxical breathing during sleep is associated with increased sleep apnea and reduced ventilatory capacities in high-level spinal cord injury.

Sleep-disordered breathing is highly prevalent in individuals with high-level spinal cord injury. In addition, chest mechanics are known to be altered, leading to paradoxical breathing. Here we investigated the interaction between paradoxical breathing and sleep quality in these patients, and its association with measurements of respiratory function, hypercapnic ventilatory response and peak exercise ventilation. Home-based polysomnography was performed in 13 patients with spinal cord injury (C4 to T4) untreated for sleep-disordered breathing. We defined paradoxical breathing as counterphase between thoracic and abdominal movements during slow-wave and rapid eye movement sleep. Sleep quality, pulmonary function, hypercapnic ventilatory responses and peak exercise ventilation were compared between those with and without paradoxical breathing. Half of individuals presented with nocturnal paradoxical breathing. Despite similar age, body mass index, injury level, time since injury, and respiratory function, those with paradoxical breathing had higher apnea-hypopnea index (13 ± 8 versus 5 ± 3 events per hr) and average sleep heart rate (67 ± 12 versus 54 ± 4 bpm; p < 0.05). Moreover, paradoxical breathing was associated with lower hypercapnic ventilatory response (slope: 0.35 ± 0.17 versus 0.96 ± 0.38) and lower peak exercise ventilation (33 ± 4 versus 48 ± 12 L min-1 ; p < 0.05). Nocturnal respiratory muscle desynchronization could play a role in the pathophysiology of sleep apnea, and could relate to low ventilatory responses to both hypercapnia and exercise in high-level spinal cord injury. Polysomnography may be an important diagnostic tool for these patients for whom therapeutic approaches should be considered to treat this abnormality.

Revisiting the Role of Serotonin in Sleep-Disordered Breathing.

Serotonin or 5-hydroxytryptamine (5-HT) is a ubiquitous neuro-modulator-transmitter that acts in the central nervous system, playing a major role in the control of breathing and other physiological functions. The midbrain, pons, and medulla regions contain several serotonergic nuclei with distinct physiological roles, including regulating the hypercapnic ventilatory response, upper airway patency, and sleep-wake states. Obesity is a major risk factor in the development of sleep-disordered breathing (SDB), such as obstructive sleep apnea (OSA), recurrent closure of the upper airway during sleep, and obesity hypoventilation syndrome (OHS), a condition characterized by daytime hypercapnia and hypoventilation during sleep. Approximately 936 million adults have OSA, and 32 million have OHS worldwide. 5-HT acts on 5-HT receptor subtypes that modulate neural control of breathing and upper airway patency. This article reviews the role of 5-HT in SDB and the current advances in 5-HT-targeted treatments for SDB.

Leptin signaling in the dorsomedial hypothalamus couples breathing and metabolism in obesity

Mismatch between CO2 production (Vco2) and respiration underlies the pathogenesis of obesity hypoventilation. Leptin-mediated CNS pathways stimulate both metabolism and breathing, but interactions between these functions remain elusive. We hypothesized that LEPRb+ neurons of the dorsomedial hypothalamus (DMH) regulate metabolism and breathing in obesity. In diet-induced obese LeprbCre mice, chemogenetic activation of LEPRb+ DMH neurons increases minute ventilation (Ve) during sleep, the hypercapnic ventilatory response, Vco2, and Ve/Vco2, indicating that breathing is stimulated out of proportion to metabolism. The effects of chemogenetic activation are abolished by a serotonin blocker. Optogenetic stimulation of the LEPRb+ DMH neurons evokes excitatory postsynaptic currents in downstream serotonergic neurons of the dorsal raphe (DR). Administration of retrograde AAV harboring Cre-dependent caspase to the DR deletes LEPRb+ DMH neurons and abolishes metabolic and respiratory responses to leptin. These findings indicate that LEPRb+ DMH neurons match breathing to metabolism through serotonergic pathways to prevent obesity-induced hypoventilation.

Inhibition of SOCS3 signaling in the nucleus tractus solitarii and retrotrapezoid nucleus alleviates hypoventilation in diet-induced obese male mice

The central leptin signaling system has been found to facilitate breathing and is linked to obesity-related hypoventilation. Activation of leptin signaling in the nucleus tractus solitarii (NTS) and retrotrapezoid nucleus (RTN) enhances respiratory drive. In this study, we investigated how medullary leptin signaling contributes to hypoventilation and whether respective deletion of SOCS3 in the NTS and RTN could mitigate hypoventilation in diet-induced obesity (DIO) male mice. Our findings revealed a decrease in the number of CO2-activated NTS neurons and downregulation of acid-sensing ion channels in DIO mice compared to lean control mice. Moreover, NTS leptin signaling was disrupted, as evidenced by the downregulation of phosphorylated STAT3 and the upregulation of SOCS3 in DIO mice. Importantly, deleting SOCS3 in the NTS and RTN significantly improved the diminished hypercapnic ventilatory response in DIO mice. In conclusion, our study suggests that disrupted medullary leptin signaling contributes to obesity-related hypoventilation, and inhibiting the upregulated SOCS3 in the NTS and RTN can alleviate this condition.

The effects of P2Y12 adenosine receptors' inhibitors on central and peripheral chemoreflexes.

Introduction: The most common side effect of ticagrelor is dyspnea, which leads to premature withdrawal of this life-saving medication in 6.5% of patients. Increased chemoreceptors' sensitivity was suggested as a possible pathophysiological explanation of this phenomenon; however, the link between oversensitization of peripheral and/or central chemosensory areas and ticagrelor intake has not been conclusively proved. Methods: We measured peripheral chemoreceptors' sensitivity using hypoxic ventilatory response (HVR), central chemoreceptors' sensitivity using hypercapnic hyperoxic ventilatory response (HCVR), and dyspnea severity before and 4 ± 1 weeks following ticagrelor initiation in 11 subjects with chronic coronary syndrome undergoing percutaneous coronary intervention (PCI). The same tests were performed in 11 age-, sex-, and BMI-matched patients treated with clopidogrel. The study is registered at ClinicalTrials.com at NCT05080478. Results: Ticagrelor significantly increased both HVR (0.52 ± 0.46 vs. 0.84 ± 0.69 L min-1%-1; p < 0.01) and HCVR (1.05 ± 0.64 vs. 1.75 ± 1.04Lmin-1mmHg-1; p < 0.01). The absolute change in HVR correlated with the change in HCVR. Clopidogrel administration did not significantly influence HVR (0.63 ± 0.32 vs. 0.58 ± 0.33 L min-1%-1; p = 0.53) and HCVR (1.22 ± 0.67 vs. 1.2 ± 0.64Lmin-1mmHg-1; p = 0.79). Drug-related dyspnea was reported by three subjects in the ticagrelor group and by none in the clopidogrel group. These patients were characterized by either high baseline HVR and HCVR or excessive increase in HVR following ticagrelor initiation. Discussion: Ticagrelor, contrary to clopidogrel, sensitizes both peripheral and central facets of chemodetection. Two potential mechanisms of ticagrelor-induced dyspnea have been identified: 1) high baseline HVR and HCVR or 2) excessive increase in HVR or HVR and HCVR. Whether other patterns of changes in chemosensitivities play a role in the pathogenesis of this phenomenon needs to be further investigated.

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.

Obstructive sleep apnea in children with Down syndrome: contribution of upper airway and chemosensitivity.

The high prevalence of obstructive sleep apnea syndrome (OSAS) in children with Down syndrome (DS) has been attributed to a reduced upper airway size, while the role of ventilatory control is unclear. The objectives of our case-control study were to evaluate the upper airway reduction in children with DS and moderate to severe OSAS as compared to typically developing (TD) children with similar OSAS severity and to evaluate the degree of chemical loop gain modifications including its components: controller and plant gains (CG, PG). Thirteen children with DS were matched for age, sex, OSAS severity and ethnicity with 26 TD children. They had undergone acoustic rhinometry and pharyngometry, chemical LG obtained during awake tidal breathing measurement and hypercapnic-hyperoxic ventilatory response testing. As compared to TD, children with DS depicted reduced oropharyngeal dimensions, significantly lower CG and LG and no different PG. Their hypercapnic ventilatory response slopes were not different. We concluded that the decreased CG in DS was related to decreased peripheral chemoreceptor sensitivity, and while central chemosensitivity was normal, the former explained the increased end-tidal PCO2 observed in children with DS as compared to TD. Pharyngeal dimensions are reduced in children with DS and OSAS. Reduced upper airway size and nocturnal alveolar hypoventilation in children with Down syndrome (DS) have been previously reported. We confirmed that children with DS and moderate-to-severe OSA have reduced oropharyngeal size as compared to typically developing children with similar OSAS severity and demonstrated decreased peripheral chemosensitivity explaining the alveolar hypoventilation observed in children with DS. Central chemosensitivity appears to be intact in children with DS and moderate to severe OSAS Our results support growing evidence that Down syndrome is associated with autonomic nervous system dysfunction.

Central CO2 chemosensitivity and CO2 controller gain independently contribute to daytime PCO2 in young subjects with congenital central hypoventilation syndrome.

Whether peripheral chemoreceptor response is altered in congenital central hypoventilation syndrome (CCHS) remains debated. Our aim was to prospectively evaluate both peripheral and central CO2 chemosensitivity and to evaluate their correlations with daytime PCO2 and arterial desaturation during exercise in CCHS. To this end, tidal breathing was recorded in CCHS patients allowing the calculation of loop gain and its components (steady-state controller [assumed to mainly be peripheral chemosensitivity] and plant gains using a bivariate [PETCO2 and ventilation] constrained model), a hyperoxic, hypercapnic ventilatory response test (central chemosensitivity), and a six-minute walk test (arterial desaturation). The results of loop gain were compared to those previously obtained in a healthy group of similar age. The study prospectively included 23 subjects with CCHS, without daytime ventilatory support; the subjects had a median age of 10 (5.6 to 27.4) years (15 females) with moderate polyalanine repeat mutation (PARM: 20/25, 20/26, n=11), severe PARM (20/27, 20/33, n=8), or non-PARM (n=4). As compared to 23 healthy subjects (4.9 to 27.0 years), the subjects with CCHS had a decreased controller gain and an increased plant gain. Mean daytime PETCO2 level of subjects with CCHS correlated negatively to both Log(controller gain) and the slope of CO2 response. Genotype was not related to chemosensitivity. Arterial desaturation on exercise correlated negatively with Log(controller) gain but not with the slope of the CO2 response. In conclusion, we demonstrate that peripheral CO2 chemosensitivity is altered in some patients with CCHS and that the daytime PETCO2 depends on central and peripheral chemoreceptor responses.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

The effect of diet-induced obesity on sleep and breathing in female mice.

Obesity and male sex are main risk factors for sleep-disordered breathing (SDB). We have shown that male diet-induced obesity (DIO) mice develop hypoventilation, sleep apnea, and sleep fragmentation. The effects of DIO on breathing and sleep architecture in females have not been investigated. We hypothesized that female mice are less susceptible to the detrimental effects of DIO on sleep and SDB compared to males. Female DIO-C57BL/6J and lean C57BL/6J mice underwent 24-hour metabolic studies and were exposed to 8% CO2 to measure the hypercapnic ventilatory response (HCVR), and sleep studies. Ventilatory response to arousals was calculated as ratio of the average and peak minute ventilation (VE) during each arousal relative to the baseline VE. Breathing stability was measured with Poincaré plots of VE. Female obesity was associated with decreased metabolism, indicated by reduced oxygen consumption (VO2) and CO2 production (VCO2). VE in 8% CO2 and HCVR were significantly attenuated during wakefulness. NREM sleep duration was reduced in DIO mice, but REM sleep was preserved. Ventilation during NREM and REM sleep was augmented compared to lean mice. Arousal frequency was similar between groups. Obesity increased the frequency of spontaneous arousals, whereas the apnea index was 4-fold reduced in DIO compared to lean mice. Obesity decreased pre- and post-apnea arousals. Obese mice had more stable breathing with reduced ventilatory response to arousals, compared to lean females. We conclude that obese female mice are protected against SDB, which appears to be related to an attenuated CO2 responsiveness, compared to the lean state.

0255 Female Mice Are Less Susceptible to the Effects of Diet-Induced Obesity on Sleep and Sleep-Disordered Breathing

Abstract Introduction Diet-induced obesity (DIO) in male mice is a well-established model of sleep-disordered breathing (SDB). Male DIO mice manifest hypoventilation, increased apnea severity, and sleep fragmentation. The effects of DIO on breathing during sleep and sleep architecture in females have been insufficiently investigated. We hypothesized that female mice are less susceptible to the detrimental effects of DIO on sleep and SDB compared to males. Methods Full-polysomnographies were performed in 15 lean female C57BL/6J and 14 female DIO-C57BL/6J mice using a whole-body plethysmography chamber. Ventilatory response to an arousal was calculated as ratio of the average minute ventilation (VE) during each arousal (2.5s) relative to the baseline VE. Breathing stability was measured using Poincaré plots of VE. Mice were exposed to 8% CO2 to measure the hypercapnic ventilatory response (HCVR). Mice underwent 24-h metabolic studies. Results Obesity was associated with hypometabolism, indicated by reduced oxygen consumption (VO2) and CO2 production in DIO mice. VE in 8% CO2 and HCVR were significantly suppressed suggesting a decreased CO2 sensitivity in obese animals. NREM sleep duration was reduced, but REM sleep was preserved. Arousal frequency and VE during NREM and REM sleep were similar between groups. However, apnea severity was 4-fold augmented in lean females compared to DIO mice (14.7±3.9 vs 74.2±11.9 events/h; P&amp;lt; 0.001). In lean mice, the increased frequency of apneas was mainly attributed to sleep fragmentation since the events were predominantly post-arousals. Breathing stability during sleep was significantly increased in lean mice. Ventilatory response to an arousal was 30% increased in lean females compared to DIO group. Conclusion Our results suggest potential sex differences in the effects of DIO on breathing during sleep and sleep architecture. DIO in females appears to be less detrimental regarding SDB severity. The mechanisms underlying the augmented SDB severity in lean females are unknown, but an exacerbated CO2 responsiveness during sleep related to sleep fragmentation could play an important role. Support (if any) American Heart Association Postdoctoral Fellowship Award 828142, NHLBI grant NIH R01s HL128970, HL133100, and HL13892, American Academy of Sleep Medicine Foundation 238-BS-20, and American Thoracic Society Unrestricted Award.

0040 Leptin Signaling in the Dorsomedial Hypothalamus Couples Metabolism and Breathing in Obesity

Abstract Introduction Individuals with obesity hypoventilation syndrome (OHS) are unable to increase ventilation to match increased CO2 production (VCO2). Obstructive sleep apnea (OSA), defined by the closure of the upper airway during sleep, is present in 90% of OHS patients. Leptin, an adipocyte-produced hormone, acts in leptin receptor (LEPRb) positive neurons and was implicated in the pathophysiology of OSA and OHS. Diet-induced obese (DIO) C57BL/6J mice shows features of OHS and OSA. Leptin-mediated neural pathways may link metabolism and breathing in obesity. We hypothesized that LEPRb-positive neurons in the dorsomedial hypothalamus (DMH) are necessary and sufficient to match control of breathing to VCO2 and that the stimulation of these neurons relieves OHS in DIO mice. Methods We expressed designer receptors exclusively activated by designer drugs (DREADDs) in DMH of DIO Leprb-Cre mice and measured breathing during sleep, the hypercapnic ventilatory response (HCVR), VCO2, and O2 consumption (VO2). Results Activation of LEPRb-positive DMH neurons increased inspiratory flow and minute ventilation during REM sleep, without affecting sleep architecture. The effect was pronounced during inspiratory flow limitation, suggeting that upper airway obstruction during REM sleep was relieved. Activation of these neurons increased the HCVR and this response was blunted by a serotonin receptor antagonist, methysergide. Activation of these neurons augmented VCO2, VO2, and VE/VCO2, suggesting that breathing was stimulated out of proportion to increases in metabolism. In vitro experiments revealed that LEPRb-positive DMH neurons project to 5-HT neurons in the dorsal raphe (DR) and that optogenetic stimulation of these neurons evoked excitatory postsynaptic currents in 5-HT neurons of the DR. Administration of retrograde Cre-dependent AAV harboring caspase to the DR in Leprb-Cre mice deleted LEPRb-positive DMH neurons and abolished respiratory and metabolic effects of chemogenetic stimulation. Apoptosis of LEPRb-positive DMH neurons projecting to the DR also prevented the increase in ventilation during REM sleep induced by intranasal leptin. Conclusion Leptin increases the HCVR, improves upper airway patency during sleep, and increases VCO2 acting on LEPRb-positive DMH neurons projecting to the DR. We conclude that LEPRb-positive DMH neurons projecting to the DR couple metabolism and breathing in obesity. Support (if any) NIH (R01 HL128970, R01 HL133100, and R01 HL138932), AHA (#915167).

The Molecular Circadian Clock of Phox2b-expressing Cells Drives Daily Variation of the Hypoxic but Not Hypercapnic Ventilatory Response in Mice.

While the suprachiasmatic nucleus (SCN) controls 24-h rhythms in breathing, including minute ventilation (VE), the mechanisms by which the SCN drives these daily changes are not well understood. Moreover, the extent to which the circadian clock regulates hypercapnic and hypoxic ventilatory chemoreflexes is unknown. We hypothesized that the SCN regulates daily breathing and chemoreflex rhythms by synchronizing the molecular circadian clock of cells. We used whole-body plethysmography to assess ventilatory function in transgenic BMAL1 knockout (KO) mice to determine the role of the molecular clock in regulating daily rhythms in ventilation and chemoreflex. Unlike their wild-type littermates, BMAL1 KO mice exhibited a blunted daily rhythm in VE and failed to demonstrate daily variation in the hypoxic ventilatory response (HVR) or hypercapnic ventilatory response (HCVR). To determine if the observed phenotype was mediated by the molecular clock of key respiratory cells, we then assessed ventilatory rhythms in BMAL1fl/fl; Phox2bCre/+ mice, which lack BMAL1 in all Phox2b-expressing chemoreceptor cells (hereafter called BKOP). BKOP mice lacked daily variation in HVR, similar to BMAL1 KO mice. However, unlike BMAL1 KO mice, BKOP mice exhibited circadian variations in VE and HCVR comparable to controls. These data indicate that the SCN regulates daily rhythms in VE, HVR, and HCVR, in part, through the synchronization of the molecular clock. Moreover, the molecular clock of Phox2b-expressing cells is specifically necessary for daily variation in the hypoxic chemoreflex. These findings suggest that disruption of circadian biology may undermine respiratory homeostasis, which, in turn, may have clinical implications for respiratory disease.