The astrocytic Na+ -HCO3 - cotransporter, NBCe1, is dispensable for respiratory chemosensitivity.

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO2 /H+ is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+ -HCO3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.

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.

Adrenergic C1 neurons monitor arterial blood pressure and determine the sympathetic response to hemorrhage.

Hemorrhage initially triggers a rise in sympathetic nerve activity (SNA) that maintains blood pressure (BP); however, SNA is suppressed following severe blood loss causing hypotension. We hypothesized that adrenergic C1 neurons in the rostral ventrolateral medulla (C1RVLM) drive the increase in SNA during compensated hemorrhage, and a reduction in C1RVLM contributes to hypotension during decompensated hemorrhage. Using fiber photometry, we demonstrate that C1RVLM activity increases during compensated hemorrhage and falls at the onset of decompensated hemorrhage. Using optogenetics combined with direct recordings of SNA, we show that C1RVLM activation mediates the rise in SNA and contributes to BP stability during compensated hemorrhage, whereas a suppression of C1RVLM activity is associated with cardiovascular collapse during decompensated hemorrhage. Notably, re-activating C1RVLM during decompensated hemorrhage restores BP to normal levels. In conclusion, C1 neurons are a nodal point for the sympathetic response to blood loss.

Respiratory alkalosis provokes spike-wave discharges in seizure-prone rats.

Hyperventilation reliably provokes seizures in patients diagnosed with absence epilepsy. Despite this predictable patient response, the mechanisms that enable hyperventilation to powerfully activate absence seizure-generating circuits remain entirely unknown. By utilizing gas exchange manipulations and optogenetics in the WAG/Rij rat, an established rodent model of absence epilepsy, we demonstrate that absence seizures are highly sensitive to arterial carbon dioxide, suggesting that seizure-generating circuits are sensitive to pH. Moreover, hyperventilation consistently activated neurons within the intralaminar nuclei of the thalamus, a structure implicated in seizure generation. We show that intralaminar thalamus also contains pH-sensitive neurons. Collectively, these observations suggest that hyperventilation activates pH-sensitive neurons of the intralaminar nuclei to provoke absence seizures.

Rostral ventrolateral medulla, retropontine region and autonomic regulations.

The rostral half of the ventrolateral medulla (RVLM) and adjacent ventrolateral retropontine region (henceforth RVLMRP) have been divided into various sectors by neuroscientists interested in breathing or autonomic regulations. The RVLMRP regulates respiration, glycemia, vigilance and inflammation, in addition to blood pressure. It contains interoceptors that respond to acidification, hypoxia and intracranial pressure and its rostral end contains the retrotrapezoid nucleus (RTN) which is the main central respiratory chemoreceptor. Acid detection by the RTN is an intrinsic property of the principal neurons that is enhanced by paracrine influences from surrounding astrocytes and CO2-dependent vascular constriction. RTN mediates the hypercapnic ventilatory response via complex projections to the respiratory pattern generator (CPG). The RVLM contributes to autonomic response patterns via differential recruitment of several subtypes of adrenergic (C1) and non-adrenergic neurons that directly innervate sympathetic and parasympathetic preganglionic neurons. The RVLM also innervates many brainstem and hypothalamic nuclei that contribute, albeit less directly, to autonomic responses. All lower brainstem noradrenergic clusters including the locus coeruleus are among these targets. Sympathetic tone to the circulatory system is regulated by subsets of presympathetic RVLM neurons whose activity is continuously restrained by the baroreceptors and modulated by the respiratory CPG. The inhibitory input from baroreceptors and the excitatory input from the respiratory CPG originate from neurons located in or close to the rhythm generating region of the respiratory CPG (preBötzinger complex).

Vagus nerve stimulation activates two distinct neuroimmune circuits converging in the spleen to protect mice from kidney injury.

Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia-reperfusion injury. We identified the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.

Silent hypoxaemia in COVID-19 patients.

The clinical presentation of COVID-19 due to infection with SARS-CoV-2 is highly variable with the majority of patients having mild symptoms while others develop severe respiratory failure. The reason for this variability is unclear but is in critical need of investigation. Some COVID-19 patients have been labelled with 'happy hypoxia', in which patient complaints of dyspnoea and observable signs of respiratory distress are reported to be absent. Based on ongoing debate, we highlight key respiratory and neurological components that could underlie variation in the presentation of silent hypoxaemia and define priorities for subsequent investigation.

A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity1. The neural systems that regulate breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

Differential Contribution of the Retrotrapezoid Nucleus and C1 Neurons to Active Expiration and Arousal in Rats.

Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female Th-Cre rats to transduce selectively RTN (Phox2b+/Nmb+) or C1 neurons (Phox2b+/Th+) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP+ parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP+ processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses.SIGNIFICANCE STATEMENT The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco2 stability.

Neuronal Networks in Hypertension: Recent Advances.

Neurogenic hypertension is associated with excessive sympathetic nerve activity to the kidneys and portions of the cardiovascular system. Here we examine the brain regions that cause heightened sympathetic nerve activity in animal models of neurogenic hypertension, and we discuss the triggers responsible for the changes in neuronal activity within these regions. We highlight the limitations of the evidence and, whenever possible, we briefly address the pertinence of the findings to human hypertension. The arterial baroreflex reduces arterial blood pressure variability and contributes to the arterial blood pressure set point. This set point can also be elevated by a newly described cerebral blood flow-dependent and astrocyte-mediated sympathetic reflex. Both reflexes converge on the presympathetic neurons of the rostral medulla oblongata, and both are plausible causes of neurogenic hypertension. Sensory afferent dysfunction (reduced baroreceptor activity, increased renal, or carotid body afferent) contributes to many forms of neurogenic hypertension. Neurogenic hypertension can also result from activation of brain nuclei or sensory afferents by excess circulating hormones (leptin, insulin, Ang II [angiotensin II]) or sodium. Leptin raises blood vessel sympathetic nerve activity by activating the carotid bodies and subsets of arcuate neurons. Ang II works in the lamina terminalis and probably throughout the brain stem and hypothalamus. Sodium is sensed primarily in the lamina terminalis. Regardless of its cause, the excess sympathetic nerve activity is mediated to some extent by activation of presympathetic neurons located in the rostral ventrolateral medulla or the paraventricular nucleus of the hypothalamus. Increased activity of the orexinergic neurons also contributes to hypertension in selected models.

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.

The Retrotrapezoid Nucleus: Central Chemoreceptor and Regulator of Breathing Automaticity.

The ventral surface of the rostral medulla oblongata has been suspected since the 1960s to harbor central respiratory chemoreceptors [i.e., acid-activated neurons that regulate breathing to maintain a constant arterial PCO2 (PaCO2)]. The key neurons, a.k.a. the retrotrapezoid nucleus (RTN), have now been identified. In this review we describe their transcriptome, developmental lineage, and anatomical projections. We also review their contribution to CO2 homeostasis and to the regulation of breathing automaticity during sleep and wake. Finally, we discuss several mechanisms that contribute to the activation of RTN neurons by CO2in vivo: cell-autonomous effects of protons; paracrine effects of pH mediated by surrounding astrocytes and blood vessels; and excitatory inputs from other CO2-responsive CNS neurons.

Sodium Is Detected by the OVLT to Regulate Sympathetic Tone.

Hypernatremia is known to elicit a rise in sympathetic tone and blood pressure. In this issue of Neuron, Nomura et al. (2018) now show that this is mediated via the organum vasculosum laminae terminalis (OVLT). Na+ activates OVLT neurons via a paracrine mechanism involving sodium channel Nax expressed by astrocytes and the ependyma.

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

KEY POINTS: 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. ABSTRACT: 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 (-8 mmHg PaO2 ) and hypercapnic (+10 mmHg ). 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.

Interdependent feedback regulation of breathing by the carotid bodies and the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) regulates breathing in a CO2 - and state-dependent manner. RTN neurons are glutamatergic and innervate principally the respiratory pattern generator; they regulate multiple aspects of breathing, including active expiration, and maintain breathing automaticity during non-REM sleep. RTN neurons encode arterial PCO2 /pH via cell-autonomous and paracrine mechanisms, and via input from other CO2 -responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial PCO2 homeostasis. The carotid bodies stimulate the respiratory pattern generator directly and indirectly by activating RTN via a neuronal projection originating within the solitary tract nucleus. The indirect pathway operates under normo- or hypercapnic conditions; under respiratory alkalosis (e.g. hypoxia) RTN neurons are silent and the excitatory input from the carotid bodies is suppressed. Also, silencing RTN neurons optogenetically quickly triggers a compensatory increase in carotid body activity. Thus, in conscious mammals, breathing is subject to a dual and interdependent feedback regulation by chemoreceptors. Depending on the circumstance, the activity of the carotid bodies and that of RTN vary in the same or the opposite directions, producing additive or countervailing effects on breathing. These interactions are mediated either via changes in blood gases or by brainstem neuronal connections, but their ultimate effect is invariably to minimize arterial PCO2 fluctuations. We discuss the potential relevance of this dual chemoreceptor feedback to cardiorespiratory abnormalities present in diseases in which the carotid bodies are hyperactive at rest, e.g. essential hypertension, obstructive sleep apnoea and heart failure.