Central Chemoreceptors Research Articles

Astrocytes within the retrotrapezoid nucleus maintain irregular breathing patterns in heart failure: role of P2X7 receptors

Irregular breathing (IB) patterns are a hallmark of heart failure (HF). The mechanisms underlying these alterations are not known. Interestingly, it has been described that purinergic signaling (PurS) in the retrotrapezoid nucleus (RTN), a main central chemoreceptor area, modulate ventilatory chemoreflex control in healthy conditions. However, nothing is known about the role of the RTN on IB in HF nor the precise molecular mechanisms underpinning the development/maintenance of IB in HF. Accordingly, we aimed to determine in HF rats: i) the contribution of the RTN on IB; ii) the alterations in PurS in the RTN; iii) the activation level of RTN chemoreceptor neurons/astrocytes; iv) the effects of chronic RTN chemogenetic manipulation using DREADDs on the maintenance of IB; and v) if astrocyte‐targeted expression of the P2X7 receptor (P2X7r) normalizes breathing. Sprague‐Dawley rats underwent volume overload to induce HF. IB was assessed by whole‐body plethysmography. RTN micropunches were used to determine neuron/astrocyte activation, P2X7r gene/protein expression and ATP levels. Adeno‐associated vector (AAV) expressing an excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) or an AAV carrying the P2X7r under the control of GFAP promoter were stereotaxically injected into the RTN of HF rats. Compared to control rats, HF rats (Sham vs. HF, p<.05) displayed increases in the apnea/hypoapnea index (AHI) (3.5±1 vs. 8.7±2 events/hr), breath‐to‐breath interval variability (SD1: 38.5±8 vs. 83.6±8 ms; SD2: 48.5±5 vs. 107.4±10 ms) and in the coefficient of variation (CV) of VTamplitudes (8.7±2 vs. 14.3±3 %). In addition, both ATP levels (78±2 vs. 21±10 pmoles/100μg of protein) and P2X7r gene/protein expression were reduced in the RTN of HF rats. Importantly, we found that P2X7r expression was restricted only to astrocytes within the RTN. No chronic activation of RTN neurons were found in HF as evidenced by no changes in fosB expression compared to control rats. Contrarily, GFAP expression was significantly lower in the RTN from HF rats compared to controls. Chronic chemogenetic activation of RTN astrocytes in HF increases ATP levels (28±3 vs. 71±4 pmoles/100μg of protein; HF vs. HFDREADD; p<.05, respectively) and improve breathing regularity (Irregularity score: 25±3 vs. 12±1%; HF vs. HFDREADD; p<.05, respectively). Finally, increasing the expression of P2X7r in RTN astrocytes markedly reduced AHI (8.7±2 vs. 3,7±1 events/hr; HF vs. HFP2X7r; p<.05, respectively) and decreases breath‐to‐breath (SD1 83.6±8 vs. 53.6±4 ms; SD2 107.4±10.1 vs 68.5±5.0 ms; HF vs. HFP2X7r; p<.05, respectively) and VTvariability (CV: 25±1 vs. 19±2; HF vs. HFP2X7r; p<.05, respectively). Our results show that the RTN is necessary for the maintenance of IB in HF and that RTN astrocytes play a pivotal role on breathing rhythm regulation by a mechanism encompassing ATP release and the P2X7r.Support or Funding InformationSupported by FONDECYT 1180172 and the basal Center of Excellence in Aging and Regeneration (AFB 170005).

Orexin in Respiratory and Autonomic Regulation, Health and Diseases.

Orexin neurons, located in the hypothalamus, produce orexin-A and orexin-B neuropeptides and send widespread projections throughout the central nervous system, including many nuclei that are critically involved in sleep-wake, cardiorespiratory, and autonomic regulation. Significant progress has been made to better understand the roles of orexins in the control of breathing and autonomic functions since the discovery of orexins in 1998. Orexin neurons are CO2 /pH chemosensitive and blockade of orexin receptors with orexin receptor antagonists can significantly attenuate ventilatory response to hypercapnia or CO2 chemoreflex. Animal models with orexin abnormalities, for example, too little or too much, have all been reported to have significant alterations in breathing, central chemoreception (hypercapnic chemoreflex), blood pressure, thermoregulation, and cardiorespiratory responses to stress. More recent studies further show that abnormalities of the orexin system are linked to many neurological disorders in addition to narcolepsy, for example, sleep disorders, neurodegenerative disorders, neurogenic hypertension, and sudden infant death syndrome. These new findings have significantly advanced the knowledge in understanding the underlying mechanism of orexin-associated health and diseases while providing a new pathway for possible treatments. In this article, we will discuss some of the progresses in basic research and in health and diseases. © 2020 American Physiological Society. Compr Physiol 10:345-363, 2020.

Decreased excitability of locus coeruleus neurons during hypercapnia is exaggerated in the streptozotocin-model of Alzheimer's disease

The locus coeruleus (LC) is a pontine nucleus important for respiratory control and central chemoreception. It is affected in Alzheimer's disease (AD) and alteration of LC cell function may account for respiratory problems observed in AD patients. In the current study, we tested the electrophysiological properties and CO2/pH sensitivity of LC neurons in a model for AD. Sporadic AD was induced in rats by intracerebroventricular injection of 2 mg/kg streptozotocin (STZ), which induces behavioral and molecular impairments found in AD. LC neurons were recorded using the patch clamp technique and tested for responses to CO2 (10% CO2, pH = 7.0). The majority (~60%) of noradrenergic LC neurons in adult rats were inhibited by CO2 exposure as indicated by a significant decrease in action potential (AP) discharge to step depolarizations. The STZ-AD rat model had a greater sensitivity to CO2 than controls. The increased CO2-sensitivity was demonstrated by a significantly stronger inhibition of activity during hypercapnia that was in part due to hyperpolarization of the resting membrane potential. Reduction of AP discharge in both groups was generally accompanied by lower LC network activity, depolarized AP threshold, increased AP repolarization, and increased current through a subpopulation of voltage-gated K+ channels (KV). The latter was indicated by enhanced transient KV currents particularly in the STZ-AD group. Interestingly, steady-state KV currents were reduced under hypercapnia, a change that would favor enhanced AP discharge. However, the collective response of most LC neurons in adult rats, and particularly those in the STZ-AD group, was inhibited by CO2.

What Is the Meaning of Fetal Heart Rate Patterns According to Physiopathology?

What is the meaning of Fetal Heart Rate patterns according to physiopathology? The analysis and interpretation of the Fetal Heart Rate in the parturition watch have shown their limits in the identification of the fetuses risking neonatal asphyxia, which consequences are death or Cerebral Palsy. The weak specificity of the continuous electronic heart rate monitoring generates an unjustified C-section excess, associated to an increasing of the immediate and ulterior materno-fetal morbi-mortality. Presently, the visual analysis of the Fetal Heart Rate exclusively lays on chrono-morphological patterns. However, there is another way that calls on the knowledge of the physiological processes of the fetus’ cardiovascular control: - In situation of normoxemia, the peripheral baroreceptors of the near term fetus are responsible for the control of the arterial pressure with both systems in action: the permanent accelerating sympathetic system and the intermittent braking parasympathetic system, which causes short-term variability. - In situation of acute hypoxemia, the peripheral chemoreceptors are stimulated and induce a rapid vagal mediated fall in heart rate (i.e. deceleration), and a peripheral vasoconstriction mediated by sympathetic nervous system. - In situation of acidosis, the central chemoreceptor is activated. It then stimulates the sympathetic system generating a fetal tachycardia, and inhibits the functioning of the peripheral baroreceptors, producing minimal to absent variability. From that comes out that the variability and the heart baseline are the only two patterns that reflect the fetal prognosis.

Heart to breathe: partial ablation of rostral ventrolateral medulla catecholaminergic neurons mediates disordered breathing in volume overload heart failure rats

Heart to breathe: partial ablation of rostral ventrolateral medulla catecholaminergic neurons mediates disordered breathing in volume overload heart failure rats

Maternal cigarette smoke exposure disturbs glutamate/GABA balance in pFRG of neonatal rats

We previously found that maternal cigarette smoke (CS) exposure resulted in impairment of central chemoreception and oxidative stress and mitochondrial dysfunction of parafacial respiratory group (pFRG, a critical site for mammalian central chemoreception) in neonatal rats. The present work was carried out to identify if maternal CS exposure could disturb the glutamate (GLU)-ergic and γ-aminobutyric acid (GABA)-ergic balance in pFRG of neonatal rats. We found that maternal CS exposure induced a decrease in GLU content and consequently in GLU/GABA ratio in pFRG of neonatal rats. Maternal CS exposure also decreased glutamine content and glutaminase and glutamine synthetase activity in offspring pFRG. In addition, expression of vesicular glutamate transporter 2 was depressed, and those of glutamate transporter 1 and GABA transporter 3 were elevated by maternal CS exposure. These results indicate that maternal CS exposure leads to a disturbance of GLU/GABA balance in pFRG of the neonatal rats, which might contribute to the suppression of central chemoreception in maternal CS-exposed offspring.

The pH-Sensitive Potassium Channel TASK-1 Is a Chemosensor for Central Respiratory Regulation in Rats

TWIK-related acid-sensitive potassium channel-1 (TASK-1) is a "leak" potassium channel sensitive to extracellular protons. It contributes to setting the resting potential in mammalian neurons. TASK-1 channels are widely expressed in respiratory-related neurons in the central nervous system. Inhibition of TASK-1 by extracellular acidosis can depolarize and increase the excitability of these cells. Here we describe the distribution of TASK-1 in the rat brainstem and show that TASK-1 mRNAs are present in respiratory-related nuclei in the ventrolateral medulla, which have been proposed as neural substrates for central chemo-reception in rats. After inhalation of 8% CO2 for 30 and 60 min, TASK-1 mRNA levels in positive-expression neurons were remarkably upregulated. Injection of the TASK-1 blocker anandamide (AEA) into the rat lateral cerebral ventricle, showed a significant excitement of respiratory at 10 min posttreatment, with a marked decrease in inspiratory and expiratory durations and an increased frequency of respiration. We suggest that TASK-1 channel may serve as a chemosensor for in central respiration and may contribute to pH-sensitive respiratory effects. TASK-1 channel might be an attractive candidate for sensing H^(+)/CO2 in several respiratory-related nuclei in the brainstem. It is likely that TASK-1 participates in pH-sensitive chemical regulation in the respiratory center under physiological and pathological conditions.

Mechanisms underlying the generation of autonomorespiratory coupling amongst the respiratory central pattern generator, sympathetic oscillators, and cardiovagal premotoneurons.

The respiratory rhythm and pattern and sympathetic and parasympathetic outflows are generated by distinct, though overlapping, propriobulbar arrays of neuronal microcircuit oscillators constituting networks utilizing mutual excitatory and inhibitory neuronal interactions, residing principally within the metencephalon and myelencephalon, and modulated by synaptic influences from the cerebrum, thalamus, hypothalamus, cerebellum, and mesencephalon and ascending influences deriving from peripheral stimuli relayed by cranial nerve afferent axons. Though the respiratory and cardiovascular regulatory effector mechanisms utilize distinct generators, there exists significant overlap and interconnectivity amongst and between these oscillators and pathways, evidenced reciprocally by breathing modulation of sympathetic oscillations and sympathetic modulation of neural breathing. These coupling mechanisms are well-demonstrated coordinately in sympathetic- and respiratory-related central neuronal and efferent neurogram recordings and quantified by the findings of cross-correlation, spectra, and coherence analyses, combined with empirical interventions including lesioning and pharmacological agonist and antagonist microinjection studies, baroloading, barounloading, and hypoxic and/or hypercapnic peripheral and/or central chemoreceptor stimulation. Sympathetic and parasympathetic central neuronal and efferent neural discharge recordings evidence classic fast rhythms produced by propriobulbar neuronal networks located within the medullary division of the lateral tegmental field, coherent with cardiac sympathetic nerve discharge. These neural efferent nerve discharges coordinately evidence slow synchronous oscillations, constituted by Traube Hering (i.e., high frequency), Mayer wave (i.e., medium or low frequency), and vasogenic autorhythmicity (i.e., very low frequency) wave spectral bands. These oscillations contribute to coupling neural breathing, sympathetic oscillations, and parasympathetic cardiovagal premotoneuronal activity. The mechanisms underlying the origins of and coupling amongst, these waves remains to be unresolved.

Near field non-invasive electrophysiology of retrotrapezoid nucleus using amperometric cation sensor

Central chemoreception is the process whereby the brainstem senses blood gas levels and adjusts homeostatic functions such as breathing and cardiovascular tone accordingly. Rodent evidence suggests that the retrotrapezoid nucleus (RTN) is a master regulator of central chemoreception, in particular, through direct sensation of acidosis induced by CO2 levels. The oscillatory dynamics caused by pH changes as sensed by the RTN surface and its relationship to the fluctuations in cation flux is not clearly understood due to the current limitations of electrophysiology tools and this article presents our investigations to address this need. A cation selective sensor fabricated from polypyrrole doped with dodecyl benzenesulfonate (PPy (DBS)) is placed over RTN in an ex-vivo en bloc brain and changes in cation concentration in the diffusion limited region above the RTN is measured due to changes in externally imposed basal pH. The novelty of this technique lies in its feasibility to detect cation fluxes from the cells in the RTN region without having to access either sides of the cell membrane. Owing to the placement of the sensor in close proximity to the tissue, we refer to this technique as near-field electrophysiology. It is observed that lowering the pH in the physiological range (7.4–7.2) results in a significant increase in cation concentration in the vicinity of RTN with a median value of ~5 μM. The utilization of such quantifiable measurement techniques to detect sub-threshold brain activity may help provide a platform for future neural network architectures. Findings from this paper present a quantifiable, sensitive, and robust electrophysiology technique with minimal damage to the underlying tissue.

CYCLOOXYGENASE MECHANISMS IN THE REGULATION OF RESPIRATORY EFFECTS OF TUMOR NECROSIS FACTOR

Introduction. It is known that the systemic level of the major pro-inflammatory cytokine increases in many respiratory diseases such as asthma, COPD and sleep apnea [1, 2]. The lung ventilation changes and the pathological types of breathing are typical in these diseases. By the reason, the research of the respiratory effects of cytokine is actual. The aim of this study was to compare the respiratory effects of tumor necrosis factor - α (TNF-α) before and after pretreatment with diclofenac, a nonspecific cyclooxygenase (COX) inhibitor. Materials and methods. Тhe experiments were performed in tracheostomized anaesthetized with urethane rats. A respiratory flow head connected to a pneumotachometer (AD Instruments ML141 Spirometer, Dunedin, New Zealand) was used to measure peak airflow and respiratory rate. The hypoxic ventilatory response was measured by using rebreathing with hypoxic gas mixture before and after the tail vein injection of TNF-α (10 μg/rat). In order to determine the role of the cyclooxygenase pathway in the ventilatory effects of TNF-α, introperitoneal administration of diclofenac, a nonspecific COX inhibitor, was used (0.5 mg/rat). Results and discussion. We have shown that the increase in level of TNF-α in blood increased the parameters of respiration such as minute ventilation (by 40%), tidal volume (by 18%), and the mean inspiratory flow (by 33%). The slope of the hypoxic ventilatory response decreased from 6.06 ± 0.91 to 3.48 ± 0.38 ml/min-1 mmHg-1 (by 40%) 40 min after administration of TNF-α (p < 0.05), the slope of tidal volume and mean inspiratory flow also decreased (by 27%) (p < 0.05). After pretreatment with diclofenac, the influence of TNF-α on breathing was dampened, as no significant changes were observed. Conclusion. We concluded that the elevation of inflammatory cytokine level in blood intensifies ventilation during the resting breathing that may be associated with increased central inspiratory activity. At the same time TNF-α reduces the chemoreflex sensitivity to hypoxia, thereby worsening the compensatory capabilities of the respiratory system. Thus, the results of our study suggest participation of inflammatory cytokines in mechanisms of central breathing control and chemoreception. Diclofenac pretreatment eliminated the respiratory effects of TNF-α. The data indicate that the ability of TNF-α to enhance basal ventilation and to reduce the ventilatory hypoxic response is mediated by the COX pathway.

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.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

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.

On the Use of Linear-Modelling-based Algorithms for Physiological Noise Correction in fMRI Studies of the Central Breathing Control.

A full characterization of the physiological behavior of human central chemoreceptors through fMRI is crucial to understand the pathophysiology of central abnormal breathing patterns. In this scenario, physiological noise and activity of interest may be naturally correlated. Here, we examined the adequacy of linear-modelling-based retrospective physiological noise correction for studies of the central breathing control. We focused on the relationship between a nonlinear model of BOLD response, hypothesized to describe neuronal specific activity, and noise modelled by correction algorithms. Analyses were performed on fMRI acquisitions from healthy subjects during a breath hold task. A general linear model including static nonlinearities in the response to end-tidal CO2 was applied to data preprocessed both with and without physiological noise correction. Relations between physiological noise and PETCO2 were explored both with linear and nonlinear measures. Lastly, parametric maps of noise spatial distribution were extracted. Our results evidenced that correction algorithms based on linear modelling remove components that are both linearly and nonlinearly related to end-tidal CO2, whereas uncorrected data showed spurious activations in regions outside gray matter. Thus, despite a correction step is fundamental, these algorithms are shown to be over-conservative approaches to noise correction and need to be adapted to the specific purpose.

Exposure to mild intermittent hypoxia increases loop gain and the arousal threshold in participants with obstructive sleep apnoea

Exposure to mild intermittent hypoxia increases loop gain and the arousal threshold in participants with obstructive sleep apnoea

Simultaneous assessment of central and peripheral chemoreflex regulation of muscle sympathetic nerve activity and ventilation in healthy young men.

Central chemoreceptor stimulation, by hypercapnia (acidosis), and peripheral, by hypoxia plus hypercapnia, evoke reflex increases in ventilation and sympathetic outflow. The assumption that central or peripheral chemoreceptor-mediated sympathetic activation elicited when increases parallels concurrent ventilatory responses is unproven. Applying a modified rebreathing protocol that equilibrates central and peripheral chemoreceptor whilst clamping O2 tension at either hypoxic or hyperoxic concentrations, the independent ventilatory and muscle sympathetic stimulus-response properties of the central and peripheral chemoreflexes were quantified and compared in young men. The novel findings were that ventilatory and sympathetic responses to central and peripheral chemoreflex stimulation are initiated at similar recruitment thresholds but individual specific sympathetic responsiveness cannot be predicted from the ventilatory sensitivities of either chemoreceptor reflex. Such findings in young men, if replicated in heart failure or hypertension, should temper present enthusiasm for trials targeting the peripheral chemoreflex based solely on ventilatory responsiveness to non-specific chemoreceptor stimulation. In humans, stimulation of peripheral or central chemoreceptor reflexes is assumed to evoke equivalent ventilatory and sympathetic responses. We evaluated whether central or peripheral chemoreceptor-mediated sympathetic activation elicited by increases in CO2 tension ( ) parallels concurrent ventilatory responses. Twelve healthy young men performed a modified rebreathing protocol designed to equilibrate central and peripheral chemoreceptor tensions with end-tidal ( ) at two isoxic end-tidal ( ) such that central responses can be segregated, by hyperoxia, from the net response (hypoxia minus hyperoxia). Ventilation and muscle sympathetic nerve activity (MSNA) were recorded continuously during rebreathing at isoxic of 150 and 50mmHg. During rebreathing, the values at which ventilation (Lmin-1 ) and total MSNA (units) began to rise were identified ( recruitment thresholds) and their slopes above the recruitment threshold were determined (sensitivity). The central chemoreflex recruitment threshold for ventilation (46±3mmHg) and MSNA (45±4mmHg) did not differ (P=0.55) and slopes were 2.3±0.9Lmin-1 mmHg-1 and 2.1±1.5unitsmmHg-1 , respectively. The peripheral chemoreflex recruitment thresholds, at 41±3mmHg for both ventilation and MSNA were lower (P<0.05) compared to the central chemoreflex recruitment thresholds. Peripheral chemoreflex sensitivity was 1.7±0.1Lmin-1 mmHg-1 for ventilation and 2.9±2.6unitsmmHg-1 for MSNA. There was no relationship between the ventilatory and MSNA sensitivity for either the central (r2 =0.01, P=0.76) or peripheral (r2 =0.01, P=0.73) chemoreflex. In healthy young men, ventilatory and sympathetic responses to central and peripheral chemoreceptor reflex stimulation are initiated at similar recruitment thresholds but individual ventilatory responsiveness does not predict sympathetic sensitivities of either chemoreflex.

Comparative Assessment of Central and Peripheral Chemoreceptor Reflex Regulation of Muscle Sympathetic Nerve Activity and Ventilation

We tested the hypothesis that central and peripheral chemoreceptor‐mediated sympathetic activation elicited by increases in CO2 tension (PCO2) mirror concurrent ventilatory responses. Twelve healthy young men (mean±SD; age: 24±4 yr; BMI: 24.5±1.3 kg·m−2) performed a modified rebreathing protocol designed to equilibrate central and peripheral chemoreceptor PCO2 tensions with end‐tidal PCO2 (PETCO2) at two isoxic end‐tidal PO2 (PETO2) tensions such that central chemoreceptor reflex responses (hyperoxia) can be isolated from additive peripheral responses (i.e., hypoxia minus hyperoxia). Subject's ventilation (volume turbine) and muscle sympathetic nerve activity (MSNA; microneurography, right fibular nerve) responses to rebreathing at isoxic PETO2 tensions of 150 mmHg (hyperoxia) and 50 mmHg (hypoxia) were recorded continuously. During rebreathing, the PETCO2 (mmHg) at which ventilation (L·min−1) and total MSNA (au) began to rise were identified as the PETCO2 recruitment thresholds (RT). Slopes of these responses above RT also were determined. In hyperoxic rebreathing (i.e., the central chemoreflex response), the mean RT for ventilation (46±3 mmHg) and MSNA (45±4 mmHg) did not differ (p&gt;0.05). Mean slopes were 2.3±0.9 L·min−1·mmHg−1 and 2.1±1.5 au·mmHg−1, respectively. In hypoxic rebreathing (i.e., the summed central and peripheral chemoreflex response), the RT for both ventilation and MSNA were not different (41±3 mmHg vs 41±3 mmHg, p&gt;0.05) but were lower (p&lt;0.05) compared to hyperoxia. Mean slopes for ventilation (4.0±1.8 L·min−1·mmHg−1) and MSNA (5.1±3.4 au·mmHg−1) in hypoxia were greater than in hyperoxia yielding a mean peripheral chemoreflex sensitivity (hypoxic slope minus hyperoxic slope) of 1.7±0.1 L·min−1·mmHg−1 for ventilation and 3.0±2.6 au·mmHg−1 for MSNA. The 95% limits of agreement of RT between ventilation and MSNA for the central chemoreflex were −7 to 6 mmHg (bias=−1 mmHg, p&gt;0.05) and −5 to 5 mmHg (bias=0 mmHg, p&gt;0.05) for the peripheral chemoreflex. There was no relationship (p&gt;0.05) between the ventilatory and MSNA sensitivity for either the central (r2=0.16) or peripheral (r2=0.01) chemoreceptor reflexes. These data highlight two novel findings: 1) ventilatory and sympathetic arms of the central and peripheral chemoreceptor reflexes are initiated at similar PETCO2 recruitment thresholds and 2) sympathetic responsiveness cannot be predicted from the sensitivities of the ventilatory central and peripheral chemoreceptor reflexes.Support or Funding InformationExperiments were funded by a Project Grant from the Canadian Institutes of Health Research (PJT 159491). Dr. Floras holds the Canada Research Chair in Integrative Cardiovascular Biology.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 CO2/pH Chemosensitivity of Locus Coeruleus Neurons Is Increased in the Streptozotocin‐Model of Alzheimer's Disease

Patients with Alzheimer's disease (AD) develop pathological changes in the Locus Coeruleus (LC), an important pontine nucleus involved in respiratory control and central chemoreception. In addition, AD patients have breathing disturbances that may be related to LC dysfunction. Here, we tested the electrophysiological properties of LC neurons in a model for AD. Sporadic AD was induced in rats (6–7 weeks) by intracerebroventricular injection of streptozotocin (STZ; 2 mg/kg). After 14 days following injection, LC neurons were recorded using the patch clamp technique and tested for CO2 chemosensitivity (10% CO2, pH = 7.0).Hypercapnic exposure lowered current‐evoked spike discharge in the majority of LC neurons (~60%) when compared to baseline responses. The remaining cells either increased spiking (~20%) or did not respond (~20%) to CO2. Within the cell group that was inhibited by hypercapnia, baseline spike discharge to current injection was similar between control and STZ rats. These responses decreased significantly in both groups when exposed to 10% CO2 (bsl vs. 10% CO2: CTL, p=0.003, n=8 and STZ, p=0.001, n=9). The CO2‐induced decrease in spike discharge of the STZ group was significantly lower when compared to control (CTL vs. STZ, p=0.038), indicating greater sensitivity to hypercapnia. However, we observed no difference in resting membrane potential and input resistance (Ri, resistance across the cell membrane) between groups at baseline and CO2. Despite lack of difference between groups, within the STZ group Ri decreased significantly with hypercapnia (bsl, 126.5 ± 14.9 MΩ vs. 10% CO2, 98.4 ± 8.2 MΩ; p=0.01), indicating ion channel opening when exposed to CO2. Analysis of the current‐voltage relationship only showed a significant decrease in the steady state current under CO2 (bsl vs. 10% CO2: CTL, p=0.002, and STZ, p=0.001,), which had a similar magnitude in both groups. Close analysis of action potential (AP) shape (spike threshold, peak, slope, peak to anti‐peak) showed no difference between groups. Within the STZ rats, however, spike threshold was significantly shifted to more positive potentials when exposed to CO2 (−39.6 ± 1.9 mV vs −34.8 ± 2.2 mV, p=0.01). This shift in spike threshold likely contributes to the pronounced decrease in spike discharge in the STZ group during hypercapnic conditions.In summary, our data suggest that the majority of LC neurons in adult rats are inhibited by CO2. Further, the STZ‐treated group exhibits a greater sensitivity to CO2, possibly due to an increased spike threshold and opening of additional, yet unidentified membrane channels. A decreased excitability of LC neurons may play a role in the respiratory dysfunction observed in patients with AD.Support or Funding InformationFAPESP 2017/21750‐9 (MCV); ATSU seed money (TDO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Ablation of PHOX2B‐Derived Astrocytes Results in Neuronal Dystrophy‐Like Neuropathology in the RTN

The transcription factor Phox2b is a regulator of the autonomic nervous system development and is especially expressed by respiratory circuits involved in chemosensory integration, as neurons of solitary tract nucleus (NTS) that relay information from peripheral chemoreceptors to the ventral respiratory column, and by central chemoreceptors located in the retrotrapezoid nucleus (RTN). In addition to neurons, recent studies show that astrocytes from ventral medullary surface also detect PCO2 increase, and contribute to respiratory drive. Since PHOX2B biased progenitor cells generate astrocytes that have a developmental connection with RTN neurons, the aim of this study is determine the extent to which selective ablation of these astrocytes affect synapse integrity in the retrotrapezoid nucleus. We utilized the astrocyte ablation technique described by Tsai et al., 2012. In this model, the astrocyte‐specific ALDH1L1 promoter induces GFP expression in the absence of cre recombinase, and in the presence of cre recombinase expresses diphtheria toxin A (DTA). We interbred ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA mice to PHOX2Bcre mice. Transmission electron photomicrographs were captured from the RTN/VLM area and morphometric analysis was performed using FIJI. We noted that axon terminals in the mutants (PHOX2Bcre, ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA) were on average larger in area and perimeter. Furthermore, we noted an increase in the percentage of axon terminals showing autophagic vacuoles/phagosomes. The proportion of axon terminals with other organelles, including mitochondria and dense core neurosecretory vesicles, was not different between groups. In summary, PHOX2B‐derived, astrocyte ablated mice show synaptic pathology characterized by dysmorphic neuroaxonal morphology, suggesting an underlying defect in the afferent input into the RTN. These data suggest that PHOX2B‐derived astrocytes may play a role in regulating the synaptic integrity of neuronal inputs into RTN neurons. Other experiments are being performed in order to elucidate if the cell body of these neurons is located in the commissural NTS, and also, if PHOX2b derived astrocytes depletion can imply local regulation of presynaptic autophagy, which can lead to neurodegeneration and breathing impair.Support or Funding InformationFAPESP and NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.