Ventilatory Reflex Research Articles

Intrinsic molecular proton sensitivity underlies GPR4 effects on retrotrapezoid nucleus (RTN) neuronal activation and CO2-stimulated breathing.

An interoceptive homeostatic reflex monitors levels of CO2/H+ to maintain blood gas homeostasis and rapidly regulate tissue acid-base balance by driving lung ventilation and CO2 excretion - this CO2-evoked increase in respiration is the hypercapnic ventilatory reflex (HCVR). Retrotrapezoid nucleus (RTN) neurons provide crucial excitatory drive to downstream respiratory rhythm/pattern-generating circuits, and their activity is directly modulated by changes in CO2/H+ RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO2/H+ activation of RTN neurons and the HCVR. It has not been determined if the intrinsic pH sensitivity of these proton detectors is required for these effects. We used CRISPR/Cas9 genome editing to generate mice with mutations in either of two pH-sensing histidine residues in GPR4 to determine effects on RTN neuronal CO2/H+ sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO2-stimulated breathing and CO2-induced RTN neuronal activation were strongly blunted, with no effect on hypoxia-stimulated breathing. In brainstem slices from GPR4(H81F) mice, peak firing of RTN neurons during bath acidification was significantly reduced compared to GPR4 wild type mice, and a subpopulation of RTN neurons was rendered pH-insensitive, phenocopying previous results from GPR4-deleted mice. These effects were independent of changes in RTN number/distribution, neuronal excitability or transcript levels for GPR4 and TASK-2. CO2-stimulated breathing was reduced to a similar extent in GPR4(H81F) and TASK-2-deleted mice, with combined mutation yielding no additional deficit in the HCVR. Together, these data demonstrate that the intrinsic pH sensitivity of GPR4 is necessary for full elaboration of the HCVR.Significance Statement Among the critical mechanisms for whole-body homeostasis, the hypercapnic ventilatory reflex (HCVR) regulates lung ventilation in order to maintain physiological levels of arterial PCO2 and acid-base balance. GPR4 is a proton-activated receptor and putative molecular proton sensor in retrotrapezoid nucleus (RTN) neurons, which are a crucial neural component of this respiratory reflex. In this work, we developed multiple lines of mice in which the intrinsic pH sensitivity of GPR4 was globally disabled by mutations in key histidine residues; in those mice, CO2/H+-sensitivity of RTN neurons and the HCVR were strongly blunted. These data support a role for GPR4 as a direct molecular sensor for CO2/H+ in support of this important homeostatic reflex.

Loss-of-function of chemoreceptor neurons in the retrotrapezoid nucleus: What have we learned from it?

Central respiratory chemoreceptors are cells in the brain that regulate breathing in relation to arterial pH and PCO2. Neurons located at the retrotrapezoid nucleus (RTN) have been hypothesized to be central chemoreceptors and/or to be part of the neural network that drives the central respiratory chemoreflex. The inhibition or ablation of RTN chemoreceptor neurons has offered important insights into the role of these cells on central respiratory chemoreception and the neural control of breathing over almost 60 years since the original identification of acid-sensitive properties of this ventral medullary site. Here, we discuss the current definition of chemoreceptor neurons in the RTN and describe how this definition has evolved over time. We then summarize the results of studies that use loss-of-function approaches to evaluate the effects of disrupting the function of RTN neurons on respiration. These studies offer evidence that RTN neurons are indispensable for the central respiratory chemoreflex in mammals and exert a tonic drive to breathe at rest. Moreover, RTN has an interdependent relationship with oxygen sensing mechanisms for the maintenance of the neural drive to breathe and blood gas homeostasis. Collectively, RTN neurons are a genetically-defined group of putative central respiratory chemoreceptors that generate CO2-dependent drive that supports eupneic breathing and stimulates the hypercapnic ventilatory reflex.

Long‐term hypercapnia does not alter Galanin immunoreactivity in mouse brain respiratory and chemoreceptor regions

Long‐term hypercapnia has been implicated in respiratory disorders such as sleep apnea and chronic obstructive pulmonary disease (COPD). However, the mechanisms by which long‐term hypercapnia contributes to central respiratory dysfunction in these conditions are unclear. The brainstem respiratory circuit, including the ventral respiratory column (VRC), comprises distinct neuronal populations that govern expiratory and inspiratory drive. The neuropeptide galanin is extensively distributed in brainstem chemoreceptor regions and the VRC, and microinjection of galanin into the VRC inhibits breathing and attenuates ventilatory reflex responses to acute hypercapnia. Nevertheless, the normal pattern of regional expression of galanin in the mouse brain is not known and the impact of long‐term hypercapnia on galanin immunoreactivity (Gal‐ir) in the vicinity of brainstem respiratory and chemoreceptor regions has not yet been studied. Therefore, we aimed to determine Gal‐ir across the mouse brain and investigate the impact of prolonged hypercapnia exposure on Gal‐ir in brainstem respiratory and chemoreceptor regions (pons and medulla). Adult male Phox2b‐EGFP (MGI ID: 5776545) mice (31±2 g, n=5 mice/group) were exposed to hypercapnia (8% CO2; balance room air) or room air (control) for 10 or 14 days. To determine Gal‐ir, brain slices were collected and processed for immunohistochemistry. Quantitative measurement of Gal‐ir was performed using ImageJ 1.53a (a QuPath‐integrated version of ImageJ) where a positive area fraction, reflecting the proportion of positively labeled pixels, was calculated for each region of interest [locus coeruleus, the retrotrapezoid nucleus, caudal‐nucleus of the solitary tract (cNTS), rostral‐NTS (rNTS), VRC, lateral reticular nucleus, and caudoventrolateral reticular nucleus]. Under control conditions, we found that Gal‐ir was broadly present across the medulla oblongata, and abundant in the caudal aspects when compared with the rostral levels. The cNTS had a higher average percentage of Gal‐ir area (p = 0.043, p = 0.05, 2‐way ANOVA) compared to rNTS following 10‐ and 14‐days of room air exposure, respectively. A significant difference in Gal‐ir (p = 0.011, 2‐way ANOVA) was observed between the cNTS and rNTS following 14‐days of hypercapnia exposure. Overall, there was no significant difference of Gal‐ir in all regions assessed after long‐term hypercapnia compared to controls. Collectively, we found that long‐term hypercapnia does not induce changes in galanin neuropeptide levels within respiratory and chemoreceptor regions in the mouse brain. Under control and hypercapnia conditions, cNTS contains higher Gal‐ir compared to rNTS. Further investigation is required to elucidate the involvement of NTS‐galaninergic subsets in respiratory homeostasis.

The Role of Noradrenergic and Glutamatergic Signaling in Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are a critical component of the brainstem respiratory network that maintains blood CO2 and O2 levels through respiratory chemoreflexes. NA dysfunction is implicated in numerous developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS), Rett Syndrome, and Congenital Central Hypoventilation Syndrome (CCHS), in which abnormal chemosensory function is implicated. However, which NA neurons modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Previous studies in our lab utilizing intersectional chemogenetic (hM4D and hM3D DREADD mediated) inhibition and excitation identified multiple rhombomere (transient genetically-defined hindbrain segments arising during early development) specific NA populations that play a role in the adult hypercapnic chemoreflex. NA neurons are typically thought to communicate with synaptic partners through noradrenaline as their primary neurotransmitter. However, the excitatory neurotransmitter, glutamate, is also expressed in subpopulations of NA neurons including those derived from rhombomeres playing a role in the hypercapnic reflex. Prior studies have implicated NA derived glutamatergic signaling in respiratory control. Thus, we seek to determine the relative roles of noradrenergic and glutamatergic signaling in rhombomeric NA populations that are involved in adult respiratory chemoreflexes. To determine the requirement of noradrenergic signaling in the hypercapnic reflex, we aim to conditionally ablate noradrenergic signaling in rhombomeric NA sub-populations by crossing rhombomere specific Credrivers with the THflox/flox(tyrosine hydroxylase) allele to remove dopamine and downstream (nor) adrenaline production in targeted neurons. We also aim to determine if the glutamate producing NA neurons represented a larger functional cohort in breathing control. We therefore used our intersectional strategy to target and chemogenetically activate (hM3D) or inhibite (hM4De) glutamate co-expressing NA neurons (defined by partially overlapping Vglut2Cre; DBHFLPo activity) which are expected to comprise a super-set of rhombomere specific NA neurons that play a role in respiratory chemoreflexes. Whole-body barometric plethysmography was used to measure the respiratory function of the mice under room air (21%O2,79%N2) and hypercapnia (21%O2, 74%N2, 5% CO2). Our preliminary data suggests that removing noradrenergic signaling in a key rhombomeric NA subpopulation does not inhibit the hypercapnic reflex while DREADD mediated inhibition does reduce the hypercapnic reflex. Acute inhibition of glutamate-expressing NA neurons shows a reduction in the hypercapnic ventilatory reflex while acute activation of these same neurons shows a significantly enhanced reflex. Altogether, these preliminary data suggest that glutamate production may delineate a functionally specific NA subpopulation in the hypercapnic reflex and offers additional clues to the respective roles of noradrenaline and glutamatergic signaling in the noradrenergic system.

Lesion of Serotonergic Afferents to the Retrotrapezoid Nucleus Impairs the Tachypneic Response to Hypercapnia in Unanesthetized Animals

Hypercapnia promotes an increase in pulmonary ventilation due to the stimulation of brainstem chemosensory cells that are connected to the respiratory network. Among these cells are the raphe serotonergic neurons which widely send projections to distinct central respiratory compartments. Nevertheless, the physiological role of specific raphe serotonergic projections to other chemosensitive sites on the emergence of hypercapnia ventilatory response in vivo still remains to be elucidated. Here we investigated whether the ventilatory response to hypercapnia requires serotonergic inputs to the chemosensitive cells of the retrotrapezoid nucleus (RTN) in the ventrolateral medulla. To test this, pulmonary ventilation was evaluated under baseline conditions and during hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60–90 g) that received bilateral microinjections of either vehicle (control) or anti-SERT-SAP (0.1 mM, 10 pmol/100 nl) toxin in the RTN to retrogradely destroy serotonergic afferents to this region. Fifteen days after microinjections, baseline ventilation was not different between anti-SERT-SAP (n = 8) and control animals (n = 9). In contrast, the ablation of RTN-projecting serotonergic neurons markedly attenuated the hypercapnia-induced increase in respiratory frequency which was correlated with reduced numbers of serotonergic neurons in the raphe obscurus and magnus, but not in the raphe pallidus. The increase in tidal volume during hypercapnia was not significantly affected by anti-SERT-SAP microinjections in the RTN. Our data indicate that serotoninergic neurons that send projections to the RTN region are required for the processing of ventilatory reflex response during exposure to high CO2 in unanesthetized conditions.

The Role of Noradrenergic and Glutamatergic Signaling in Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are a critical component of the brainstem respiratory network that maintains blood CO2 and O2 levels through respiratory chemoreflexes. NA dysfunction is implicated in numerous developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS), Rett Syndrome, and Congenital Central Hypoventilation Syndrome (CCHS), in which abnormal chemosensory function is implicated. However, which NA neurons modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Previous studies in our lab utilizing intersectional chemogenetic (hM4D and hM3D DREADD mediated) inhibition and excitation identified multiple rhombomere (transient genetically‐defined hindbrain segments arising during early development) specific NA populations that play a role in the adult hypercapnic chemoreflex. NA neurons are typically thought to communicate with synaptic partners through noradrenaline and adrenaline as their primary neurotransmitter. However, the excitatory neurotransmitter, glutamate, is also expressed in subpopulations of NA neurons including rhombomeric NA neurons playing a role in the hypercapnic reflex. Prior studies have implicated NA derived glutamatergic signaling in respiratory control. Thus, we seek to determine the relative roles of noradrenergic and glutamatergic signaling in rhombomeric NA populations that are involved in adult respiratory chemoreflexes. To determine the requirement of noradrenergic signaling in the hypercapnic reflex, we aim to conditionally ablate noradrenergic signaling in rhombomeric NA sub‐populations by crossing rhombomere specific Credrivers with the THflox/flox(tyrosine hydroxylase) allele to remove dopamine and downstream (nor) adrenaline production in targeted neurons. We also aim to determine if the glutamate producing NA neurons represent a larger functional cohort in breathing control. We therefore used our intersectional strategy to target and chemogenetically activate (hM3D) or inhibite (hM4De) glutamate co‐expressing NA neurons (defined by partially overlapping Vglut2Cre; DBHFLPo activity) which are expected to comprise a super‐set of rhombomere specific NA neurons that play a role in respiratory chemoreflexes. Whole‐body barometric plethysmography was used to measure the respiratory function of the mice under room air (21%O2, 79%N2) and hypercapnia (21%O2, 74%N2, 5% CO2). Our preliminary data suggests that removing noradrenergic signaling in a key rhombomeric NA subpopulation does not inhibit the hypercapnic reflex while DREADD mediated inhibition does reduce the hypercapnic reflex. Acute inhibition of glutamate‐expressing NA neurons shows a reduction in the hypercapnic ventilatory reflex while acute activation of these same neurons shows a significantly enhanced reflex. Altogether, these preliminary data suggest that glutamate production may delineate a functionally specific NA subpopulation in the hypercapnic reflex and offers additional clues to the respective roles of noradrenaline and glutamatergic signaling in the noradrenergic system.Support or Funding InformationR01HL153505

Off-Target Effects of Clozapine-N-Oxide on the Chemosensory Reflex Are Masked by High Stress Levels.

Respiratory chemosensory circuits are implicated in several physiological and behavioral disorders ranging from sudden infant death syndrome to panic disorder. Thus, a comprehensive map of the chemosensory network would be of significant value. To delineate chemosensory neuronal populations, we have utilized pharmacogenetic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) perturbations for acute neuronal perturbations in respiratory circuit mapping. Recent studies show that the biologically inert DREADD ligand clozapine-N-oxide (CNO) is back-metabolized into the bioactive compound clozapine in rodents, emphasizing the need for CNO-only DREADD-free controls, which have been carried out in several studies. However, we show that high CNO doses used in several chemosensory circuit mapping studies nonetheless affect the chemosensory ventilatory reflexes in control mice, which is unmasked by extensive habituation. Here, unhabituated control animals showed no differences in respiratory parameters after CNO administration, whereas habituated animals receiving the commonly used dose of 10 mg/kg of CNO show a deficit in the hypercapnic (high CO2) chemosensory reflex, which is not present in 1 mg/kg CNO treated or saline control groups. Our findings indicate that even in appropriately controlled studies, additional masked CNO off-target effects may exist and underscore the importance of using minimal doses of activating ligand in combination with high levels of habituation.

Contribution of retrotrapezoid nucleus and carotid bodies to asphyxia‐induced arousal in rats

Arousal in response to asphyxia is a life‐saving reflex that helps restore normal breathing and blood gas homeostasis. The carotid bodies (CB) are essential to hypoxia‐induced arousal whereas CNS serotonergic neurons and the lateral parabrachial nucleus contribute to CO2‐induced arousal. Here we wished to test whether the retrotrapezoid nucleus (RTN) is also implicated in CO2‐induced arousal. Our hypothesis was that RTN contributes to arousal elicited by hypercapnia but not hypoxia. The rationale is as follows. RTN is an important pluricellular CO2 detector that mediates the bulk of the hypercapnic ventilatory reflex (HCVR). RTN is likely absent since birth in central congenital hypoventilation syndrome, CCHS, a developmental disease in which the HCVR and asphyxia‐induced arousal are both greatly reduced. To test our hypothesis, we studied four groups of adult Sprague‐Dawley rats. In one group, we nearly completely destroyed RTN (“RTN lesion” group; n=8) with microinjections of saporin conjugated with substance‐P (2.4 ng/injection). The cognate control group (“RTN control”; n=7) received saline. The CBs were ablated in a third rat cohort (“CBx rats”; n=7). The fourth group (“CB control rats”; n=7) underwent sham surgery. Four weeks later, rats were placed in a plethysmograph chamber where breathing frequency and amplitude, EEG and EMG were recorded. Using mass flow controllers, the gas mixture perfusing the chamber was intermittently changed for exactly 1 min from normoxia (21% O2/balance N2) back to normoxia (control for flow interruption), from normoxia to hypercapnia (15% CO2/21% O2/balance N2), or from normoxia to hypoxia (10% O2, balance N2). Sleep survival curves representing the cumulative probability for SWS to persist after onset of the gas change were obtained. Two‐way ANOVA for repeated measures was used for statistical analysis of these curves. Sleep architecture was assessed by measuring the proportion of time the rats spent in SWS, REM sleep or quiet waking and the number of sleep stage transitions. Based on these measurements, sleep was unaffected by CBx or RTN lesion. However, the probability of SWS to persist 20 sec after the beginning of stimulus was significantly higher in RTN lesion rats compared to control (73.6 ± 14% vs. 54.8 ± 18 % at 20 sec from the beginning of stimulus). The corresponding figures at the 30 s time‐point were 57.8 ± 12 vs. 9.6 ± 13% and, at 40 sec, 40.1 ± 11 vs. 0 %. By contrast, RTN lesion had no detectable effect on hypoxia‐induced arousal. The arousal deficits elicited by CBx were different. CBx‐rats exposed to hypoxia had a significantly probability of SWS to persist compared to CB Control rats (89.9 ± 9 vs. 67.1 ± 9 % at 20 sec; 81.5 ± 15 vs. 56.4 ± 14 % at 30 sec, and 70.8 ± 16 vs. 39.9 ± 11 at 40 sec after the beginning of stimulus). In summary, we confirm the importance of the CBs to hypoxia‐induced arousal and demonstrate that arousal to hypercapnia is selectively reduced after RTN lesion. Neither CBx nor RTN lesions eliminated the arousal elicited by hypoxia or hypercapnia, however. Additional O2 and CO2 sensors (peripheral or central) therefore likely contribute to asphyxia‐induced arousal.Support or Funding InformationNational Institutes of Health (HL074011 and HL28785 to P.G.G.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Respiratory Compensation Point and the Deoxygenation Break Point Are Valid Surrogates for Critical Power and Maximum Lactate Steady State.

The Respiratory Compensation Point and the Deoxygenation Break Point Are Valid Surrogates for Critical Power and Maximum Lactate Steady State.

Breathing regulation and blood gas homeostasis after near complete lesions of the retrotrapezoid nucleus in adult rats.

The retrotrapezoid nucleus (RTN) drives breathing proportionally to brain PCO2 but its role during various states of vigilance needs clarification. Under normoxia, RTN lesions increased the arterial PCO2 set-point, lowered the PO2 set-point and reduced alveolar ventilation relative to CO2 production. Tidal volume was reduced and breathing frequency increased to a comparable degree during wake, slow-wave sleep and REM sleep. RTN lesions did not produce apnoeas or disordered breathing during sleep. RTN lesions in rats virtually eliminated the central respiratory chemoreflex (CRC) while preserving the cardiorespiratory responses to hypoxia; the relationship between CRC and number of surviving RTN Nmb neurons was an inverse exponential. The CRC does not function without the RTN. In the quasi-complete absence of the RTN and CRC, alveolar ventilation is reduced despite an increased drive to breathe from the carotid bodies. The retrotrapezoid nucleus (RTN) is one of several CNS nuclei that contribute, in various capacities (e.g. CO2 detection, neuronal modulation) to the central respiratory chemoreflex (CRC). Here we test how important the RTN is to PCO2 homeostasis and breathing during sleep or wake. RTN Nmb-positive neurons were killed with targeted microinjections of substance P-saporin conjugate in adult rats. Under normoxia, rats with large RTN lesions (92±4% cell loss) had normal blood pressure and arterial pH but were hypoxic (-8mmHg PaO2 ) and hypercapnic (+10mmHg ). In resting conditions, minute volume (VE ) was normal but breathing frequency (fR ) was elevated and tidal volume (VT ) reduced. Resting O2 consumption and CO2 production were normal. The hypercapnic ventilatory reflex in 65% FiO2 had an inverse exponential relationship with the number of surviving RTN neurons and was decreased by up to 92%. The hypoxic ventilatory reflex (HVR; FiO2 21-10%) persisted after RTN lesions, hypoxia-induced sighing was normal and hypoxia-induced hypotension was reduced. In rats with RTN lesions, breathing was lowest during slow-wave sleep, especially under hyperoxia, but apnoeas and sleep-disordered breathing were not observed. In conclusion, near complete RTN destruction in rats virtually eliminates the CRC but the HVR persists and sighing and the state dependence of breathing are unchanged. Under normoxia, RTN lesions cause no change in VE but alveolar ventilation is reduced by at least 21%, probably because of increased physiological dead volume. RTN lesions do not cause sleep apnoea during slow-wave sleep, even under hyperoxia.

Ablation of neuromedin B (NMB)‐expressing neurons located within retrotrapezoid nucleus (RTN) reduces the central respiratory chemoreflex (CRC) selectively in conscious rats

RTN senses PCO2 and regulates breathing in a CO2‐dependent manner but the full extent of RTN's contribution to the CRC is not known. Lesions produced by microinjecting a substance‐P analog conjugated with saporin (SSP‐SAP) elicit small and/or transient CRC reductions in rats. Interpretative limitations are many: development of compensatory mechanisms, especially of carotid body origin, difficulty to identify the critical neurons and to lesion them sufficiently. As shown recently, neuromedin B (Nmb) seems to be a selective marker of the Phox2b+/Vglut2+ RTN neurons that contribute to the CRC. In the present study, we microinjected saline or SSPSAP (3 injections per side; ng/injection: 0.6, 1.2 or 2.4 ng) to destroy RTN in ~250 g Sprague‐Dawley male rats. Physiological experiments were conducted three weeks later in unanesthetized animals, following which we counted the surviving Nmb+ neurons using fluorescent ISH (FISH) in a 1/6 series of sections. SSP‐lesioned rats were regrouped post hoc into two clusters according to the number of surviving RTN Nmb+ neurons: mild lesion group (ML rats; N= 13; > 55 Nmb+ cells) and large lesion group (LL rats; N=11; 4–55 Nmb+ cells remaining). Control rats (C rats; N= 11) had 324 ± 48 Nmb+ neurons (these and all subsequent values represent mean ± SD). Under normoxia, arterial pH was virtually the same in all groups (LL rats: 7.46 ± 0.03; ML: 7.48 ± 0.01; C: 7.49 ± 0.01). PaO2 was lower in LL rats than in C and ML animals (71 ± 7 vs. 80 ± 6 vs. 78 ± 6 mmHg) but PaCO2 was higher (49 ± 7 vs. 38 ± 3 vs. 39 ± 5 mmHg). BP measured via arterial catheter was the same in all groups. Under normoxia, LL rats had a reduced VT relative to C and ML rats during quiet waking (0.39 ± 0.1 vs. 0.49 ± 0.07 vs. 0.54 ± 0.1 mL/100g), SW sleep (0.32 ± 0.08 vs.0.45 ± 0.06 vs. 0.50 ± 0.1 mL/100g) and REM sleep (0.25 ± 0.06 vs. 0.35±0.05 vs. 0.36 ± 0.1 mL/100g). Short‐term hyperoxia (1 min of 65% FiO2, balance N2) caused a larger decrease in VE in LL and in ML rats compared to C (ΔVE: −11 ± 3 vs. −13 ± 6 vs. −8 ± 3 mL/100g/min). Of note, the hypoventilation still present after 20 min of continuous hyperoxia and was larger in LL than in ML and C rats (ΔVE: −9 ± 6 vs. −5 ± 10 vs. 0.9 ± 4 mL/100g/min). When exposed to 6 % FiCO2 in 65% FiO2 LL rats had a greatly reduced breathing stimulation (hypercapnic ventilatory reflex, HCVR) compared to C and ML rats (ΔVE: 17.8 ± 9 vs. 63.6 ± 12 vs. 49.5 ± 21 mL/100g/min). The HCVR to 9 % FiCO2 in 65% FiO2 was reduced in similar proportion (ΔVE: 25.6 ± 9 vs. 85.9 ± 14 vs. 76.3 ± 27 mL/100g/min). Hypoxia (10% FiO2, balance N2) activated VE similarly in all three groups (ΔVE: 34.1 ± 6 vs. 27.2 ± 8 vs. 28.2 ± 9 in mL/100g/min for LL, C and ML rats respectively) although ΔVT was enhanced and ΔFR reduced in LL rats. In conclusion, >80% RTN Nmb+ neurons need to be destroyed to produce a notable reduction of the HCVR. Large RTN lesions reduce the CRC by ≥ 71% without decreasing the hypoxic ventilatory reflex. Large RTN lesions raise the arterial PCO2 homeostatic set‐point by ≥10 mmHg and lower PaO2 by ~9 mmHg but have little influence on long‐term arterial pH. Finally, the loss of breathing stimulation contributed by RTN is largely compensated by a permanent state of hypoxia and increased carotid body activity.Support or Funding InformationNational Institutes of Health (Grants RO1 HL074011 and RO1 HL 028785 to P.G.G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of defensive airway reflexes during continuous positive airway pressure in the rabbit

Lung hyperinflation may alter the pattern of ventilatory reflexes in chronic respiratory disorders. The aim of the study was to test the effect of continuous positive airway pressure (CPAP) on ventilatory responses to mechanical stimulation of the trachea.

Impaired chemosensory control of breathing after depletion of bulbospinal catecholaminergic neurons in rats.

Bulbospinal catecholaminergic neurons located in the rostral aspect of the ventrolateral medulla (C1 neurons) or within the ventrolateral pons (A5 neurons) are involved in the regulation of blood pressure and sympathetic outflow. A stimulus that commonly activates the C1 or A5 neurons is hypoxia, which is also involved in breathing activation. Although pharmacological and optogenetic evidence suggests that catecholaminergic neurons also regulate breathing, a specific contribution of the bulbospinal neurons to respiratory control has not been demonstrated. Therefore, in the present study, we evaluated whether the loss of bulbospinal catecholaminergic C1 and A5 cells affects cardiorespiratory control during resting, hypoxic (8% O2), and hypercapnic (7% CO2) conditions in unanesthetized rats. Thoracic spinal cord (T4-T8) injections of the immunotoxin anti-dopamine β-hydroxylase-saporin (anti-DβH-SAP-2.4ng/100nl) and the retrograde tracer Fluor-Gold or ventrolateral pontine injections of 6-OHDA were performed in adult male Wistar rats (250-280g, N=7-9/group). Anti-DβH-SAP or 6-OHDA eliminated most bulbospinal C1 and A5 neurons or A5 neurons, respectively. Serotonergic neurons and astrocytes were spared. Depletion of the bulbospinal catecholaminergic cells did not change cardiorespiratory variables under resting condition, but it did affect the response to hypoxia and hypercapnia. Specifically, the increase in the ventilation, the number of sighs, and the tachycardia were reduced, but the MAP increased during hypoxia in anti-DβH-SAP-treated rats. Our data reveal that the bulbospinal catecholaminergic neurons (A5 and C1) facilitate the ventilatory reflex to hypoxia and hypercapnia.

Decreased purinergic signaling in the RTN is associated with respiratory disorders in HFpEF rats

Heart failure with preserved ejection fraction (HFpEF) is characterized by the presence of respiratory disorders. Interestingly, it has been described that ATP‐mediated purinergic signaling (PSATP), in chemosensitive areas from the brainstem, contribute to central respiratory chemoreception. We recently described that HFpEF rats displays both resting breathing pattern irregularities and enhanced central chemoreflex drive. Therefore, we aimed to determine if HFpEF rats display alterations in the PSATP in the Retrotrapezoid nucleus (RTN), a major central chemosensitive area, and its possible role in the development of irregular breathing patterns in HFpEF. Adult male Sprague‐Dawley rats were subjected to volume overload to induce HFpEF. Whole body plethysmography was used to assess the ventilatory reflex response to hypercapnia to determine central chemoreceptors (CC) sensitivity. Resting breathing pattern was analyzed to score apneas/hypopneas incidence (AHI), as well as breath‐to‐breath (BB) interval variability. RTN micropunches were obtained for the study of PSATP protein expression and molecular markers of neuronal/astrocyte activation using immunoblot. ATP bioavailability in the RTN was determined using a high sensitive bioluminescence assay. Compared to control rats, HFpEF rats (Sham vs. CHF) display: increased AHI (9,7±1,1 vs. 20,2±1,6 events/hour, p<.05), increased both the short‐term (SD1) and long‐term (SD2) BB interval variability (SD1: 42.8±8.0 vs.115,6±28,2; SD2: 54.5±9.0 vs. 138.4±33.1, p<.05) and enhanced CC sensitivity (127.3±10.3 vs. 165.3±9.1 ml/min/100g, p<.05). No changes in normoxic minute ventilation was found between sham and CHF condition. Also, we found that HFpEF rats compared to control rats (Sham vs. CHF) display similar FosB protein expression (p>.05), decreased expression of GFAP (100±2.0% vs. 65±7.0%, p<.05) without changes in S100β expression (85±11.8% vs. 100±9.5%, p>.05) and decreased ATP bioavailable (78±2 vs. 21±10 pmoles/100μg of protein, p<.05) in the RTN. In addition, we found that the P2X7 receptor was constitutively expressed both at the mRNA and protein levels in the RTN from sham rats and that CHF rats showed a significant decrease in the P2X7 receptor expression (45±8% vs. 100±15%, Sham vs. CHF, respectively, p<.05). Remarkably, stereotaxic injection (2.5mm caudal to lambda, 1.8mm lateral to the midline and 7.5mm below the dura mater) of BzATP (200 μM/100 nl) into the RTN, a selective agonist of P2X7 receptor, markedly decrease the BB interval variability in sham rats (SD1: 100±8% vs. 45.7±5%; SD2: 100±8% vs. 40.7±5% pre vs. post BzATP, respectively, p<.05) without changing resting tidal volume and/or respiratory frequency (p>.05). In summary, our results show that HFpEF rats displayed astrocyte inhibition, reduced ATP bioavailability and decreased P2X7 receptor expression in the RTN along with a higher incidence of breathing irregularities compared to healthy animals. Together, our results suggest that altered breathing patterns in HFpEF may be associated with a decrease purinergic signalling in the RTN acting preferentially on P2X7 receptor within the RTN.Support or Funding InformationFONDECYT 1140275

Ventilatory and chemoreceptor responses to hypercapnia in neonatal rats chronically exposed to moderate hyperoxia

Rats reared in hyperoxia hypoventilate in normoxia and exhibit progressive blunting of the hypoxic ventilatory response, changes which are at least partially attributed to abnormal carotid body development. Since the carotid body also responds to changes in arterial CO2/pH, we tested the hypothesis that developmental hyperoxia would attenuate the hypercapnic ventilatory response (HCVR) of neonatal rats by blunting peripheral and/or central chemoreceptor responses to hypercapnic challenges. Rats were reared in 21% O2 (Control) or 60% O2 (Hyperoxia) until studied at 4, 6–7, or 13–14days of age. Hyperoxia rats had significantly reduced single-unit carotid chemoafferent responses to 15% CO2 at all ages; CO2 sensitivity recovered within 7days after return to room air. Hypercapnic responses of CO2-sensitive neurons of the caudal nucleus tractus solitarius (cNTS) were unaffected by chronic hyperoxia, but there was evidence for a small decrease in neuronal excitability. There was also evidence for augmented excitatory synaptic input to cNTS neurons within brainstem slices. Steady-state ventilatory responses to 4% and 8% CO2 were unaffected by developmental hyperoxia in all three age groups, but ventilation increased more slowly during the normocapnia-to-hypercapnia transition in 4-day-old Hyperoxia rats. We conclude that developmental hyperoxia impairs carotid body chemosensitivity to hypercapnia, and this may compromise protective ventilatory reflexes during dynamic respiratory challenges in newborn rats. Impaired carotid body function has less of an impact on the HCVR in older rats, potentially reflecting compensatory plasticity within the CNS.

Is plasticity within the retrotrapezoid nucleus responsible for the recovery of the PCO2 set-point after carotid body denervation in rats?

Arterial PCO2 is kept constant via breathing adjustments elicited, at least partly, by central chemoreceptors (CCRs) and the carotid bodies (CBs). The CBs may be active in a normal oxygen environment because their removal reduces breathing. Thereafter, breathing slowly returns to normal. In the present study, we investigated whether an increase in the activity of CCRs accounts for this return. One week after CB excision, the hypoxic ventilatory reflex was greatly reduced as expected, whereas ventilation and blood gases at rest under normoxia were normal. Optogenetic inhibition of Phox2b-expressing neurons including the retrotrapezoid nucleus, a cluster of CCRs, reduced breathing proportionally to arterial pH. The hypopnoea was greater after CB excision but only in a normal or hypoxic environment. The difference could be simply explained by the loss of fast feedback from the CBs. We conclude that, in rats, CB denervation may not produce CCR plasticity. We also question whether the transient hypoventilation elicited by CB denervation means that these afferents are active under normoxia. Carotid body denervation (CBD) causes hypoventilation and increases the arterial PCO2 set-point; these effects eventually subside. The hypoventilation is attributed to reduced CB afferent activity and the PCO2 set-point recovery to CNS plasticity. In the present study, we investigated whether the retrotrapezoid nucleus (RTN), a group of non-catecholaminergic Phox2b-expressing central respiratory chemoreceptors (CCRs), is the site of such plasticity. We evaluated the contribution of the RTN to breathing frequency (FR ), tidal volume (VT ) and minute volume (VE ) by inhibiting this nucleus optogenetically for 10s (archaerhodopsinT3.0) in unanaesthetized rats breathing various levels of O2 and/or CO2 . The measurements were made in seven rats before and 6-7 days after CBD and were repeated in seven sham-operated rats. Seven days post-CBD, blood gases and ventilation in 21% O2 were normal, whereas the hypoxic ventilatory reflex was still depressed (95.3%) and hypoxia no longer evoked sighs. Sham surgery had no effect. In normoxia or hypoxia, RTN inhibition produced a more sustained hypopnoea post-CBD than before; in hyperoxia, the responses were identical. Post-CBD, RTN inhibition reduced FR and VE in proportion to arterial pH or PCO2 (ΔVE : 3.3±1.5% restingVE /0.01 pHa). In these rats, 20.7±8.9% of RTN neurons expressed archaerhodopsinT3.0. Hypercapnia (3-6% FiCO2 ) increased FR and VT in CBD rats (n=4). In conclusion, RTN regulates FR and VE in a pH-dependent manner after CBD, consistent with its postulated CCR function. RTN inhibition produces a more sustained hypopnoea after CBD than before, although this change may simply result from the loss of the fast feedback action of the CBs.

State-dependent control of breathing by the retrotrapezoid nucleus.

This study explores the state dependence of the hypercapnic ventilatory reflex (HCVR). We simulated an instantaneous increase or decrease of central chemoreceptor activity by activating or inhibiting the retrotrapezoid nucleus (RTN) by optogenetics in conscious rats. During quiet wake or non-REM sleep, hypercapnia increased both breathing frequency (fR ) and tidal volume (VT ) whereas, in REM sleep, hypercapnia increased VT exclusively. Optogenetic inhibition of RTN reduced VT in all sleep-wake states, but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Phasic RTN stimulation produced active expiration and reduced early expiratory airflow (i.e. increased upper airway resistance) only during wake. We conclude that the HCVR is highly state-dependent. The HCVR is reduced during REM sleep because fR is no longer under chemoreceptor control and thus could explain why central sleep apnoea is less frequent in REM sleep. Breathing has different characteristics during quiet wake, non-REM or REM sleep, including variable dependence on PCO2. We investigated whether the retrotrapezoid nucleus (RTN), a proton-sensitive structure that mediates a large portion of the hypercapnic ventilatory reflex, regulates breathing differently during sleep vs. wake. Electroencephalogram, neck electromyogram, blood pressure, respiratory frequency (fR ) and tidal volume (VT ) were recorded in 28 conscious adult male Sprague-Dawley rats. Optogenetic stimulation of RTN with channelrhodopsin-2, or inhibition with archaerhodopsin, simulated an instantaneous increase or decrease of central chemoreceptor activity. Both opsins were delivered with PRSX8-promoter-containing lentiviral vectors. RTN and catecholaminergic neurons were transduced. During quiet wake or non-REM sleep, hypercapnia (3 or 6% FI,CO2 ) increased both fR and VT whereas, in REM sleep, hypercapnia increased VT exclusively. RTN inhibition always reduced VT but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Blood pressure was unaffected by either stimulation or inhibition. Except in REM sleep, phasic RTN stimulation entrained and shortened the breathing cycle by selectively shortening the post-inspiratory phase. Phasic stimulation also produced active expiration and reduced early expiratory airflow but only during wake. VT is always regulated by RTN and CO2 but fR is regulated by CO2 and RTN only when the brainstem pattern generator is in autorhythmic mode (anaesthesia, non-REM sleep, quiet wake). The reduced contribution of RTN to breathing during REM sleep could explain why certain central apnoeas are less frequent during this sleep stage.

Breathing Frequency (fR) Is No Longer Under Chemoreflex Control During REM Sleep

ObjectiveTo define the contribution of central chemoreception in general and the retrotrapezoid nucleus (RTN) in particular to the regulation of breathing in REM sleep. MethodsEEG, neck EMG, blood pressure, fR and tidal volume (VT) were recorded in 23 conscious adult male Sprague‐Dawley rats. RTN was optogenetically stimulated with channelrhodopsin‐2 (ChR2, 20s) or inhibited with archaerhodopsin (Arch; 10 s) during natural sleep and quiet wake periods. ChR2 and Arch were delivered using previously described PRSX8‐promoter containing lentiviral vectors. Main ResultsIn quiet wake or non‐REM sleep, hypercapnia (3 or 6% FiCO2) increased both fR and VT; in REM sleep, hypercapnia increased VT but fR was unaffected. Optogenetic inhibition of RTN neurons reduced VT in all three states but reduced fR only during quiet wake and non‐REM sleep. Optogenetic stimulation of RTN neurons always increased VT but raised fR only in quiet wake and non‐REM sleep. Phasic stimulation of RTN at frequencies above resting fR entrained and shortened the breathing cycle except in REM sleep. Phasic RTN stimulation produced active expiration only during wake and increased fR exclusively by reducing the duration of the post‐inspiratory phase.ConclusionsDuring REM sleep, VT regulation by both hypercarbia and RTN is retained but the overall hypercapnic ventilatory reflex is reduced because fR is no longer under chemoreceptor control. Furthermore, respiratory drive contributed by RTN is remarkably state‐dependent, active expiration being elicited only during wake and tachypnea only during non‐REM sleep and quiet wake. Support: HL074011.

Nasal involvement in obstructive sleep apnea syndrome.

Numerous studies have reported an association between nasal obstruction and obstructive sleep apnea syndrome (OSAS), but the precise nature of this relationship remains to be clarified. This paper aimed to summarize data and theories on the role of the nose in the pathophysiology of sleep apnea as well as to discuss the benefits of surgical and medical nasal treatments. A number of pathophysiological mechanisms can potentially explain the role of nasal pathology in OSAS. These include the Starling resistor model, the unstable oral airway, the nasal ventilatory reflex, and the role of nitric oxide (NO). Pharmacological treatment presents some beneficial effects on the frequency of respiratory events and sleep architecture. Nonetheless, objective data assessing snoring and daytime sleepiness are still necessary. Nasal surgery can improve the quality of life and snoring in a select group of patients with mild OSAS and septal deviation but is not an effective treatment for OSA as such. Despite the conflicting results in the literature, it is important that patients who are not perfectly adapted to CPAP are evaluated in detail, in order to identify whether there are obstructive factors that could be surgically corrected.

Analgesic effects of dyspnoea: “Air hunger” does not inhibit the spinal nociception reflex in humans

Dyspnoea has distinct sensory modalities, including air hunger and the sensation of excessive breathing “work/effort”. Both have analgesic properties. In the case of work/effort, spinal mechanisms have been documented (inhibitory effect on the spinal nociceptive flexor reflex, RIII). This mechanism involves C-fibres. As C-fibres are unlikely to play a major role in air hunger, we hypothesised that inducing this type of dyspnoea would not result in RIII inhibition. Eight healthy volunteers were exposed to a hypercapnic hyperoxic gas mixture (5% CO2 and 95% O2) and asked to voluntarily fight the corresponding ventilatory reflex response by reducing tidal volume below its spontaneous level. Ventilatory variables and dyspnoea intensity (ordinal scale) were measured. Electromyography of the biceps femoris was used to record the amplitude of RIII in response to painful electrical sural nerve stimulation. Air hunger failed to inhibit the RIII reflex. We conclude that the mechanisms of air hunger induced analgesia do not include a spinal contribution and are therefore mostly central.