Central Respiratory Chemoreflex Research Articles

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.

Strategies to improve respiratory chemoreflex characterization by Duffin's rebreathing.

What is the central question of this study? We assessed the test-retest variability of respiratory chemoreflex characterization by Duffin's modified rebreathing method and explored whether signal averaging of repeated trials improves confidence in parameter estimation. What is the main finding and its importance? Modified rebreathing is a reproducible method to characterize responses of central and peripheral respiratory chemoreflexes. Signal averaging of multiple repeated tests minimizes within- and between-test variability, improves the confidence of chemoreflex characterization and reduces the minimal change in parameters required to establish an effect. Future experiments that apply this method might benefit from signal averaging to improve its discriminatory effect. We assessed the test-retest variability of central and peripheral respiratory chemoreflex characterization by Duffin's modified rebreathing method and explored whether signal averaging of repeated trials improves confidence in parameter estimation. Over four laboratory visits, 13 participants (mean±SD age, 25±5years) performed six repetitions of modified rebreathing in isoxic-hypoxic conditions [end-tidal ( ) = 50mmHg] and isoxic-hyperoxic conditions ( =150mmHg). End-tidal ( ), and minute ventilation ( E ) were measured breath-by-breath, by gas analyser and pneumotachograph. The E versus relationships were fitted with a piecewise model to estimate the ventilatory recruitment threshold (VRT) and the slope above the VRT ( E S). Breath-by-breath data from the three within- and between-day trials were averaged using two approaches [simple average (fit then average) and ensemble average (average then fit)] and compared with a single-trial fit. Variability was assessed by intraclass correlation (ICC) and coefficient of variance (CV), and the minimal detectable change was computed for each approach using two independent sets of three trials. Within days, the VRT and E S exhibited excellent test-retest variability in both hyperoxic conditions (VRT: ICC=0.965, CV=2.3%; E S: ICC=0.932, CV=15.5%) and hypoxic conditions (VRT: ICC=0.970, CV=2.9%; E S: ICC=0.891, CV=17.2%). Between-day reproducibility was also excellent (hyperoxia, VRT: ICC=0.930, CV=2.2%; E S: ICC=0.918, CV=14.2%; and hypoxia, VRT: ICC=0.940, CV=3.0%; E S: ICC=0.880, CV=18.1%). Compared with a single-trial fit, there were no differences in VRT or E S using the simple average or ensemble average approaches; however, ensemble averaging reduced the minimal detectable change for E S from 2.95 to 1.39Lmin-1 mmHg-1 (hyperoxia) and from 3.64 to 1.82 Lmin-1 mmHg-1 (hypoxia). Single trials of modified rebreathing are reproducible; however, signal averaging of repeated trials improves confidence in parameter estimation.

Acute Gravitational Stress Selectively Impairs Dynamic Cerebrovascular Reactivity in the Anterior Circulation Independent of Changes to the Central Respiratory Chemoreflex.

Cerebrovascular reactivity (CVR) to changes in the partial pressure of arterial carbon dioxide (PaCO2) is an important mechanism that maintains CO2 or pH homeostasis in the brain. To what extent this is influenced by gravitational stress and corresponding implications for the regulation of cerebral blood flow (CBF) remain unclear. The present study examined the onset responses of pulmonary ventilation (V̇E) and anterior middle (MCA) and posterior (PCA) cerebral artery mean blood velocity (Vmean) responses to acute hypercapnia (5% CO2) to infer dynamic changes in the central respiratory chemoreflex and cerebrovascular reactivity (CVR), in supine and 50° head-up tilt (HUT) positions. Each onset response was evaluated using a single-exponential regression model consisting of the response time latency [CO2-response delay (t0)] and time constant (τ). Onset response of V̇E and PCA Vmean to changes in CO2 was unchanged during 50° HUT compared with supine (τ: V̇E, p = 0.707; PCA Vmean, p = 0.071 vs. supine) but the MCA Vmean onset response was faster during supine than during 50° HUT (τ: p = 0.003 vs. supine). These data indicate that gravitational stress selectively impaired dynamic CVR in the anterior cerebral circulation, whereas the posterior circulation was preserved, independent of any changes to the central respiratory chemoreflex. Collectively, our findings highlight the regional heterogeneity underlying CBF regulation that may have translational implications for the microgravity (and hypercapnia) associated with deep-space flight notwithstanding terrestrial orthostatic diseases that have been linked to accelerated cognitive decline and neurodegeneration.

Chapter 2 - Central respiratory chemoreception

Brain PCO2 is sensed primarily via changes in [H+]. Small pH changes are detected in the medulla oblongata and trigger breathing adjustments that help maintain arterial PCO2 constant. Larger perturbations of brain CO2/H+, possibly also sensed elsewhere in the CNS, elicit arousal, dyspnea, and stress, and cause additional breathing modifications. The retrotrapezoid nucleus (RTN), a rostral medullary cluster of glutamatergic neurons identified by coexpression of Phoxb and Nmb transcripts, is the lynchpin of the central respiratory chemoreflex. RTN regulates breathing frequency, inspiratory amplitude, and active expiration. It is exquisitely responsive to acidosis in vivo and maintains breathing autorhythmicity during quiet waking, slow-wave sleep, and anesthesia. The RTN response to [H+] is partly an intrinsic neuronal property mediated by proton sensors TASK-2 and GPR4 and partly a paracrine effect mediated by astrocytes and the vasculature. The RTN also receives myriad excitatory or inhibitory synaptic inputs including from [H+]-responsive neurons (e.g., serotonergic). RTN is silenced by moderate hypoxia. RTN inactivity (periodic or sustained) contributes to periodic breathing and, likely, to central sleep apnea. RTN development relies on transcription factors Egr2, Phox2b, Lbx1, and Atoh1. PHOX2B mutations cause congenital central hypoventilation syndrome; they impair RTN development and consequently the central respiratory chemoreflex.

Effects of enriched-potassium diet on cardiorespiratory outcomes in experimental non-ischemic chronic heart failure

BackgroundChronic heart failure (CHF) is a global health problem. Increased sympathetic outflow, cardiac arrhythmogenesis and irregular breathing patterns have all been associated with poor outcomes in CHF. Several studies showed that activation of the renin-angiotensin system (RAS) play a key role in CHF pathophysiology. Interestingly, potassium (K+) supplemented diets showed promising results in normalizing RAS axis and autonomic dysfunction in vascular diseases, lowering cardiovascular risk. Whether subtle increases in dietary K+ consumption may exert similar effects in CHF has not been previously tested. Accordingly, we aimed to evaluate the effects of dietary K+ supplementation on cardiorespiratory alterations in rats with CHF.MethodsAdult male Sprague–Dawley rats underwent volume overload to induce non-ischemic CHF. Animals were randomly allocated to normal chow diet (CHF group) or supplemented K+ diet (CHF+K+ group) for 6 weeks. Cardiac arrhythmogenesis, sympathetic outflow, baroreflex sensitivity, breathing disorders, chemoreflex function, respiratory–cardiovascular coupling and cardiac function were evaluated.ResultsCompared to normal chow diet, K+ supplemented diet in CHF significantly reduced arrhythmia incidence (67.8 ± 15.1 vs. 31.0 ± 3.7 events/hour, CHF vs. CHF+K+), decreased cardiac sympathetic tone (ΔHR to propranolol: − 97.4 ± 9.4 vs. − 60.8 ± 8.3 bpm, CHF vs. CHF+K+), restored baroreflex function and attenuated irregular breathing patterns. Additionally, supplementation of the diet with K+ restores normal central respiratory chemoreflex drive and abrogates pathological cardio-respiratory coupling in CHF rats being the outcome an improved cardiac function.ConclusionOur findings support that dietary K+ supplementation in non-ischemic CHF alleviate cardiorespiratory dysfunction.

Acute hyperglycemia does not affect central respiratory chemoreflex responsiveness to CO2 in healthy humans

The central respiratory chemoreceptor complex (CCRC) is comprised of brainstem neurons and surrounding interoceptors, which collectively increase ventilation in response to elevated brainstem tissue CO2/[H+] (i.e., central chemoreflex; CCR). The extent that the CCRC detects/responds to other metabolically related chemostimuli is unknown. We aimed to test the effects of acute oral glucose ingestion on CCR reactivity in heathy human participants (n = 38). We instrumented participants with a pneumotachometer (minute ventilation) and a gas sample line connected to a dual gas analyzer (pressure of end-tidal CO2). Following a baseline (BL) period and capillary blood [glucose] (BG) sample, fasted (F) participants underwent a modified hyperoxic rebreathing test to assess CCR reactivity. Participants then consumed a 75 g standard glucose beverage (glucose loaded; GL). Following 30-min, they underwent a second BL, BG sample and hyperoxic rebreathing test. BG and metabolic rate were higher in GL, confirming the metabolic stimulus. However, the ventilatory recruitment threshold and the CCR responses were unchanged between F and GL states.

Integrated respiratory chemoreflex-mediated regulation of cerebral blood flow in hypoxia: Implications for oxygen delivery and acute mountain sickness.

What is the central question of this study? To what extent do hypoxia-induced changes in the peripheral and central respiratory chemoreflex modulate anterior and posterior cerebral oxygen delivery, with corresponding implications for susceptibility to acute mountain sickness? What is the main finding and its importance? We provide evidence for site-specific regulation of cerebral blood flow in hypoxia that preserves oxygen delivery in the posterior but not the anterior cerebral circulation, with minimal contribution from the central respiratory chemoreflex. External carotid artery vasodilatation might prove to be an alternative haemodynamic risk factor that predisposes to acute mountain sickness. The aim of the present study was to determine the extent to which hypoxia-induced changes in the peripheral and central respiratory chemoreflex modulate anterior and posterior cerebral blood flow (CBF) and oxygen delivery (CDO2 ), with corresponding implications for the pathophysiology of the neurological syndrome, acute mountain sickness (AMS). Eight healthy men were randomly assigned single blind to 7h of passive exposure to both normoxia (21% O2 ) and hypoxia (12% O2 ). The peripheral and central respiratory chemoreflex, internal carotid artery, external carotid artery (ECA) and vertebral artery blood flow (duplex ultrasound) and AMS scores (questionnaires) were measured throughout. A reduction in internal carotid artery CDO2 was observed during hypoxia despite a compensatory elevation in perfusion. In contrast, vertebral artery and ECA CDO2 were preserved, and the former was attributable to a more marked increase in perfusion. Hypoxia was associated with progressive activation of the peripheral respiratory chemoreflex (P<0.001), whereas the central respiratory chemoreflex remained unchanged (P>0.05). Symptom severity in participants who developed clinical AMS was positively related to ECA blood flow (Lake Louise score, r=0.546-0.709, P=0.004-0.043; Environmental Symptoms Questionnaires-Cerebral symptoms score, r=0.587-0.771, P=0.001-0.027, n=4). Collectively, these findings highlight the site-specific regulation of CBF in hypoxia that maintains CDO2 selectively in the posterior but not the anterior cerebral circulation, with minimal contribution from the central respiratory chemoreflex. Furthermore, ECA vasodilatation might represent a hitherto unexplored haemodynamic risk factor implicated in the pathophysiology of AMS.

Dynamic characteristics of cerebrovascular reactivity or ventilatory response to change in carbon dioxide.

What is the central question of this study? What are the dynamic characteristics of cerebrovascular carbon dioxide reactivity and the central respiratory chemoreflex? What is the main finding and its importance? The transfer function gain from the end-tidal partial pressure of carbon dioxide to cerebral blood flow or ventilation decreased in the high frequency range at rest and during exercise. These findings indicate that the dynamic characteristics of both systems were not constant in all frequency ranges, and this trend was not modified by exercise. The purpose of this study was to examine the dynamic characteristics of cerebrovascular reactivity and ventilatory response to change in arterial CO2 in all frequency ranges at rest using frequency domain analysis, and also to examine whether this is modified by dynamic exercise as with the traditionally determined cerebrovascular CO2 reactivity. In nine healthy young subjects, at rest and during exercise (cycling exercise at constant predetermined work rate corresponding to a level of 0.90lmin-1 ), the dynamic characteristics of cerebrovascular CO2 reactivity and the central respiratory chemoreflex were assessed by transfer function analysis using a binary white-noise sequence (0-7% inspired CO2 fraction) from the end-tidal partial pressure of CO2 ( ) to the mean middle cerebral artery mean blood velocity (MCA Vm ) or minute ventilation ( ), respectively. In the high frequency range, both transfer function gains decreased but, interestingly, the cut-off frequency in the transfer function gain from to MCA Vm response was higher than that from to response at rest (0.024 vs. 0.015Hz) and during exercise (0.030 vs. 0.011Hz), indicating that cerebrovascular CO2 reactivity or central respiratory chemoreflex was not constant in all frequency ranges, and this trend was not modified by exercise. These findings suggest that dynamic characteristics of the cerebrovascular CO2 reactivity or central chemoreflex need to be assessed to identify the whole system because the traditional method cannot identify the property of time response of these systems.

Interpretive Complications Underlying Cerebrovascular Response To Hypercapnia; Significance Of The Central Respiratory Chemoreflex Transient

Cerebral blood flow (CBF) is sensitive to changes in the arterial partial pressure of carbon dioxide (CO2) with cerebrovascular reactivity an established risk factor for stroke and neurodegenerative disease. However, its interpretation can be complicated given subtle differences in cerebrovascular and central respiratory chemoreflex response transients. PURPOSE: To examine to what extent exposure time to CO2 influences CBF response. METHOD: We measured CBF response to hypercapnia (FICO2 = 5%) in five healthy participants for 10 min in the supine position. End-tidal partial pressures of CO2 (PETCO2), minute ventilation (VE), and middle cerebral artery blood velocity (MCAv) were assessed during both the early (3~4 min) and late phases (9~10 min) of exposure. RESULT: We observed elevated VE larger during the late compared to the early phase of exposure (from 11 ± 2 to 29 ± 8 vs. 23 ± 4 L/min, P = 0.047) despite no differences in PETCO2 (P = 0.304). The corresponding increase in MCAv during the late phase was suppressed compared to the early phase (from 48 ± 11 to 58 ± 14 vs. 63 ± 13 cm/sec, P = 0.029). Thus, the response of CBF to change in PETCO2 at late phase was lower than that of early phase (1.1 ± 1 %/mmHg vs. 1.7 ± 1 %/mmHg, P=0.07). CONCLUSION: These findings highlight the importance of considering the central respiratory chemoreflex transient during the clinical assessment of cerebrovascular reactivity.

Adaptation of Respiratory-Related Brain Regions to Long-Term Hypercapnia: Focus on Neuropeptides in the RTN.

Long-term hypercapnia is associated with respiratory conditions including obstructive sleep apnea, chronic obstructive pulmonary disease and obesity hypoventilation syndrome. Animal studies have demonstrated an initial (within hours) increase in ventilatory drive followed by a decrease in this response over the long-term (days–weeks) in response hypercapnia. Little is known about whether changes in the central respiratory chemoreflex are involved. Here we investigated whether central respiratory chemoreceptor neurons of the retrotrapezoid nucleus (RTN), which project to the respiratory pattern generator within the ventral respiratory column (VRC) have a role in the mechanism of neuroplasticity associated with long-term hypercapnia. Adult male C57BL/6 mice (n = 5/group) were used. Our aims were (1) to determine if galanin, neuromedin B and gastrin-releasing peptide gene expression is altered in the RTN after long-term hypercapnia. This was achieved using qPCR to measure mRNA expression changes of neuropeptides in the RTN after short-term hypercapnia (6 or 8 h, 5 or 8% CO2) or long-term hypercapnia exposure (10 day, 5 or 8% CO2), (2) in the mouse brainstem, to determine the distribution of preprogalanin in chemoreceptors, and the co-occurrence of the galanin receptor 1 (GalR1:Gi-coupled receptor) with inhibitory GlyT2 ventral respiratory column neurons using in situ hybridization (ISH) to better characterize galaninergic RTN-VRC circuitry, (3) to investigate whether long-term hypercapnia causes changes to recruitment (detected by cFos immunohistochemistry) of respiratory related neural populations including the RTN neurons and their galaninergic subset, in vivo. Collectively, we found that hypercapnia decreases neuropeptide expression in the RTN in the short-term and has the opposite effect over the long-term. Following long term hypercapnia, the number of RTN galanin neurons remains unchanged, and their responsiveness to acute chemoreflex is sustained; in contrast, we identified multiple respiratory related sites that exhibit blunted chemoreflex activation. GalR1 was distributed in 11% of preBötC and 30% of BötC glycinergic neurons. Our working hypothesis is that during long-term hypercapnia, galanin co-release from RTN neurons may counterbalance glutamatergic inputs to respiratory centers to downscale energetically wasteful hyperventilation, thereby having a role in neuroplasticity by contributing to a decrease in ventilation, through the inhibitory effects of galanin.

Contribution of the Retrotrapezoid Nucleus and Carotid Bodies to Hypercapnia- and Hypoxia-induced Arousal from Sleep.

The combination of hypoxia and hypercapnia during sleep produces arousal, which helps restore breathing and normalizes blood gases. Hypercapnia and hypoxia produce arousal in mammals by activating central (pH-sensitive) and peripheral (primarily O2-sensitive) chemoreceptors. The relevant chemoreceptors and the neuronal circuits responsible for arousal are largely unknown. Here we examined the contribution of two lower brainstem nuclei that could be implicated in CO2 and hypoxia-induced arousal: the retrotrapezoid nucleus (RTN), a CO2-responsive nucleus, which mediates the central respiratory chemoreflex; and the C1 neurons, which are hypoxia activated and produce arousal and blood pressure increases when directly stimulated. Additionally, we assessed the contribution of the carotid bodies (CBs), the main peripheral chemoreceptors in mammals, to hypoxia and CO2-induced arousal. In unanesthetized male rats, we tested whether ablation of the RTN, CBs, or C1 neurons affects arousal from sleep and respiratory responses to hypercapnia or hypoxia. The sleep-wake pattern was monitored by EEG and neck EMG recordings and breathing by whole-body plethysmography. The latency to arousal in response to hypoxia or hypercapnia was determined along with changes in ventilation coincident with the arousal. RTN lesions impaired CO2-induced arousal but had no effect on hypoxia-induced arousal. CB ablation impaired arousal to hypoxia and, to a lesser extent, hypercapnia. C1 neuron ablation had no effect on arousal. Thus, the RTN contributes to CO2-induced arousal, whereas the CBs contribute to both hypoxia and CO2-induced arousal. Asphyxia-induced arousal likely requires the combined activation of RTN, CBs and other central chemoreceptors.SIGNIFICANCE STATEMENT Hypercapnia and hypoxia during sleep elicit arousal, which facilitates airway clearing in the case of obstruction and reinstates normal breathing in the case of hypoventilation or apnea. Arousal can also be detrimental to health by interrupting sleep. We sought to clarify how CO2 and hypoxia cause arousal. We show that the retrotrapezoid nucleus, a brainstem nucleus that mediates the effect of brain acidification on breathing, also contributes to arousal elicited by CO2 but not hypoxia. We also show that the carotid bodies contribute predominantly to hypoxia-induced arousal. Lesions of the retrotrapezoid nucleus or carotid bodies attenuate, but do not eliminate, arousal to CO2 or hypoxia; therefore, we conclude that these structures are not the sole trigger of CO2 or hypoxia-induced arousal.

Acute hyperglycemia does not alter central respiratory CO2 chemoreflex responsiveness

The control of breathing is modulated by two distinct but interacting respiratory chemoreceptor compartments: peripheral chemoreceptors (PCRs; carotid bodies) and central chemoreceptors (CCRs; brainstem). The PCRs respond to variety of chemostimuli, including arterial oxygen, arterial carbon dioxide, glucose, insulin and temperature, and are now thought to act as metabolic sensors. However, CCRs have only been characterized as carbon dioxide/acid sensors (PCO2/[H+]). PCRs respond to chemostimulation faster than CCRs, which may mask CCR responses to stimuli like acute hyperglycemia. The aim of this study was to characterize the relationship between alterations in blood glucose concentration and central respiratory chemoreflex responsiveness via hyperoxic rebreathing in healthy humans. We hypothesized that experimentally‐induced relative hyperglycemia following an oral glucose tolerance test (OGTT; 75g, 300 ml) would (a) increase the rate of CO2 accumulation during rebreathing and (b) augment central respiratory chemoreflex responsiveness due to a synergistic effect of hyperglycemia on the CO2 sensitivity of CCR neurons. In 10 healthy participants, we measured minute ventilation (VI; L/min; pneumotachometer) and end‐tidal carbon dioxide (PETCO2; Torr; gas analyzer). We used the modified hyperoxic rebreathing test to isolate CCRs and quantify the central chemoreflex responsiveness to increases in metabolically‐derived CO2. Blood glucose levels were higher 30‐min following the OGTT beverage (i.e., glucose‐loaded) compared to fasted (7.8±1.1 vs. 4.7±0.4 mmol/L; P&lt;0.0001). During rebreathing, the rate of end‐tidal (PET)CO2 accumulation was greater in the glucose loaded trial compared to the fasted trial (0.086±0.007 vs. 0.07±0.008 vs. Torr/sec; P=0.0002). These findings confirm the hyperglycemic stimulation between trials. However, the central respiratory chemoreflex responsiveness during rebreathing was not different between glucose loaded and fasted trials (2.3±1.4 vs. 2.5±1.8 vs. L/min/Torr CO2; P=0.59). These preliminary findings suggest that relative hyperglycemia does not affect the central respiratory chemoreflex responsive to hyperoxic rebreathing. The CCRs may be a more specialized respiratory chemoreceptor, with a phenotype to detect only metabolically‐derived accumulations in CO2.Support or Funding InformationNSERC Discovery and MRU Faculty of Science and TechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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; &gt; 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, &gt;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.

Quantifying cerebrovascular reactivity in anterior and posterior cerebral circulations during voluntary breath holding.

What is the central question of this study? We developed and validated a 'stimulus index' (SI; ratio of end-tidal partial pressures of CO2 and O2 ) method to quantify cerebrovascular reactivity (CVR) in anterior and posterior cerebral circulations during breath holding. We aimed to determine whether the magnitude of CVR is correlated with breath-hold duration. What is the main finding and its importance? Using the SI method and transcranial Doppler ultrasound, we found that the magnitude of CVR of the anterior and posterior cerebral circulations is not positively correlated with physiological or psychological break-point during end-inspiratory breath holding. Our study expands the ability to quantify CVR during breath holding and elucidates factors that affect break-point. The central respiratory chemoreflex contributes to blood gas homeostasis, particularly in response to accumulation of brainstem CO2 . Cerebrovascular reactivity (CVR) affects chemoreceptor stimulation inversely through CO2 washout from brainstem tissue. Voluntary breath holding imposes alterations in blood gases, eliciting respiratory chemoreflexes, potentially contributing to breath-hold duration (i.e. break-point). However, the effects of cerebrovascular reactivity on break-point have yet to be determined. We tested the hypothesis that the magnitude of CVR contributes directly to breath-hold duration in 23 healthy human participants. We developed and validated a cerebrovascular stimulus index methodology [SI; ratio of end-tidal partial pressures of CO2 and O2 (P ET ,CO2/P ET ,O2)] to quantify CVR by correlating measured and interpolated values of P ET ,CO2 (r=0.95, P<0.0001), P ET ,O2 (r=0.98, P<0.0001) and SI (r=0.94, P<0.0001) during rebreathing. Using transcranial Doppler ultrasound, we then quantified the CVR of the middle (MCAv) and posterior (PCAv) cerebral arteries by plotting cerebral blood velocity against interpolated SI during a maximal end-inspiratory breath hold. The MCAv CVR magnitude was larger than PCAv (P=0.001; +70%) during breath holding. We then correlated MCAv and PCAv CVR with the physiological (involuntary diaphragmatic contractions) and psychological (end-point) break-point, within individuals. There were significant inverse but modest relationships between both MCAv and PCAv CVR and both physiological and psychological break-points (r<-0.53, P<0.03). However, these relationships were absent when MCAv and PCAv cerebrovascular conductance reactivity was correlated with both physiological and psychological break-points (r>-0.42; P>0.06). Although central chemoreceptor activation is likely to be contributing to break-point, our data suggest that CVR-mediated CO2 washout from central chemoreceptors plays no role in determining break-point, probably because of a reduced arterial-to-tissue CO2 gradient during breath holding.

Impaired central respiratory chemoreflex in an experimental genetic model of epilepsy.

It is recognized that seizures commonly cause apnoea and oxygen desaturation, but there is still a lack in the literature about the respiratory impairments observed ictally and in the post-ictal period. Respiratory disorders may involve changes in serotonergic transmission at the level of the retrotrapezoid nucleus (RTN). In this study, we evaluated breathing activity and the role of serotonergic transmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We conclude that the respiratory impairment in the WAR could be correlated to an overall decrease in the number of neurons located in the respiratory column. Respiratory disorders may involve changes in serotonergic neurotransmission at the level of the chemosensitive neurons located in the retrotrapezoid nucleus (RTN). Here, we investigated the central respiratory chemoreflex and the role of serotonergic neurotransmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We found that naive or kindled WARs have reduced resting ventilation and ventilatory response to hypercapnia (7% CO2 ). The number of chemically coded (Phox2b+ /TH- ) RTN neurons, as well as the serotonergic innervation to the RTN, was reduced in WARs. We detected that the ventilatory response to serotonin (1mm, 50nl) within the RTN region was significantly reduced in WARs. Our results uniquely demonstrated a respiratory impairment in a genetic model of tonic-clonic seizures, the WAR strain. More importantly, we demonstrated an overall decrease in the number of neurons located in the ventral respiratory column (VRC), as well as a reduction in serotonergic neurons in the midline medulla. This is an important step forward to demonstrate marked changes in neuronal activity and breathing impairment in the WAR strain, a genetic model of epilepsy.

Influence of prior hyperventilation duration on respiratory chemosensitivity and cerebrovascular reactivity during modified hyperoxic rebreathing

What is the central question of this study? We characterized and compared the cardiorespiratory and cerebrovascular responses to the 'Duffin' modified hyperoxic CO2 rebreathing test by randomly altering the prior hyperventilation duration. What is the main finding and its importance? Our main finding was that prior hyperventilation duration (1, 3 or 5min) had no effect on cardiorespiratory and cerebrovascular responses to the hyperoxic rebreathing test, within individuals. These findings suggest that the standard 5min prior hyperventilation duration used to clear body CO2 stores is unnecessary and can reasonably be shortened to 1min, reducing protocol times and improving participant comfort. The 'Duffin' modified hyperoxic rebreathing test allows investigators to characterize and quantify the ventilatory and cerebrovascular responses to CO2 across a large physiological range, allowing quantification of basal ventilation and the ventilatory recruitment threshold (VRT). Although the standard protocol includes 5min of prior hyperventilation to clear body CO2 stores, there is no experimental evidence that a full 5min is required. We hypothesized that there would be no within-individual differences in the cardiorespiratory or cerebrovascular responses to rebreathing with shortened hyperventilation duration prior to hyperoxic rebreathing. Using a rebreathing apparatus, transcranial Doppler ultrasound and beat-to-beat blood pressure monitoring, we tested 19 participants in the supine position using three randomly assigned hyperoxic rebreathing tests with 1, 3 or 5min of prior hyperventilation. We measured VRT (in Torr CO2 ), time to VRT (in seconds), central respiratory chemoreflex (breathing frequency, tidal volume and minute ventilation), cerebrovascular (middle and posterior cerebral artery velocity) and cardiovascular (heart rate and mean arterial pressure) responses to CO2 during hyperoxic rebreathing. Using linear regression and repeated-measures ANOVAs, we found no differences in any of the cardiorespiratory or cerebrovascular response magnitudes between trials (P>0.05). The only difference observed was in the time to VRT (in seconds), whereby 1min prior hyperventilation duration was shorter (135.4±19.7s) than with 3 or 5min prior hyperventilation (176.3±15.1 and 187.2±11.6s, respectively; P<0.001). Our findings indicate that 5min of prior hyperventilation is unnecessary during modified rebreathing when using it to quantify respiratory or cerebrovascular responses and can be reasonably shortened to 1min, reducing protocol times and improving participant comfort.

Inhibition of the pontine Kölliker-Fuse nucleus reduces genioglossal activity elicited by stimulation of the retrotrapezoid chemoreceptor neurons

The Kölliker-Fuse (KF) region, located in the dorsolateral pons, projects to several brainstem areas involved in respiratory regulation, including the chemoreceptor neurons within the retrotrapezoid nucleus (RTN). Several lines of evidence indicate that the pontine KF region plays an important role in the control of the upper airways for the maintenance of appropriate airflow to and from the lungs. Specifically, we hypothesized that the KF region is involved in mediating the response of the hypoglossal motor activity to central respiratory chemoreflex activation and to stimulation of the chemoreceptor neurons within the RTN region. To test this hypothesis, we combined immunohistochemistry and physiological experiments. We found that in the KF, the majority of biotinylated dextran amine (BDA)-labeled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2), but few contained glutamic acid decarboxylase-67 (GAD67). The majority of the RTN neurons that were FluorGold (FG)-immunoreactive (i.e., projected to the KF) contained hypercapnia-induced Fos, but did not express tyrosine hydroxylase. In urethane-anesthetized sino-aortic denervated and vagotomized male Wistar rats, hypercapnia (10% CO2) or N-methyl-d-aspartate (NMDA) injection (0.1mM) in the RTN increased diaphragm (DiaEMG) and genioglossus muscle (GGEMG) activities and elicited abdominal (AbdEMG) activity. Bilateral injection of muscimol (GABA-A agonist; 2mM) into the KF region reduced the increase in DiaEMG and GGEMG produced by hypercapnia or NMDA into the RTN. Our data suggest that activation of chemoreceptor neurons in the RTN produces a significant increase in the genioglossus muscle activity and the excitatory pathway is dependent on the neurons located in the dorsolateral pontine KF region.

Proton detection and breathing regulation by the retrotrapezoid nucleus.

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H(+) ]. RTN neurons are glutamatergic. In vitro, their activation by [H(+) ] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome.

The role of pH-sensitive TASK channels in central respiratory chemoreception.

A number of the subunits within the family of K2P background K(+) channels are sensitive to changes in extracellular pH in the physiological range, making them likely candidates to mediate various pH-dependent processes. Based on expression patterns within several brainstem neuronal cell groups that are believed to function in CO2/H(+) regulation of breathing, three TASK subunits-TASK-1, TASK-2, and TASK-3-were specifically hypothesized to contribute to this central respiratory chemoreflex. For the acid-sensitive TASK-1 and TASK-3 channels, despite widespread expression at multiple levels within the brainstem respiratory control system (including presumptive chemoreceptor populations), experiments in knockout mice provided no evidence for their involvement in CO2 regulation of breathing. By contrast, the alkaline-activated TASK-2 channel has a more restricted brainstem distribution and was localized to the Phox2b-expressing chemoreceptor neurons of the retrotrapezoid nucleus (RTN). Remarkably, in a Phox2b(27Ala/+) mouse genetic model of congenital central hypoventilation syndrome (CCHS) that is characterized by reduced central respiratory chemosensitivity, selective ablation of Phox2b-expressing RTN neurons was accompanied by a corresponding loss of TASK-2 expression. Furthermore, genetic deletion of TASK-2 blunted RTN neuronal pH sensitivity in vitro, reduced alkaline-induced respiratory network inhibition in situ and diminished the ventilatory response to CO2/H(+) in vivo. Notably, a subpopulation of RTN neurons from TASK-2(-/-) mice retained their pH sensitivity, at least in part due to a residual pH-sensitive background K(+) current, suggesting that other mechanisms (and perhaps other K2P channels) for RTN neuronal pH sensitivity are yet to be identified.