PHOX2B is a transcription factor essential for the development of different classes of neurons in the central and peripheral nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO2 chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naive rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but it was associated with reduced expression of both Task2 and Gpr4, two CO2/pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.

The central CO2 chemoreflex is a vital component of respiratory control networks, providing excitatory drive during resting conditions and challenges to blood gas homeostasis. The retrotrapezoid nucleus is a crucial hub for CO2 chemosensitivity; its ablation or inhibition attenuates CO2 chemoreflexes and diminishes restful breathing. Similar phenotypes characterize certain hypoventilation syndromes, suggesting underlying retrotrapezoid nucleus impairment in these disorders. Progesterone stimulates restful breathing and CO2 chemoreflexes. However, its mechanisms and sites of actions remain unknown and the experimental use of synthetic progestins in patients and animal models have been met with mixed respiratory outcomes.
We investigated whether acute or chronic administration of the progestinic drug, etonogestrel, could rescue respiratory chemoreflexes following selective lesion of the retrotrapezoid nucleus with saporin toxin. Adult female Sprague Dawley rats were grouped based on lesion size determined by the number of surviving chemosensitive neurons, and ventilatory responses were measured by whole body plethysmography.
Ventilatory responses to hypercapnia (but not hypoxia) were compromised in a lesion-dependent manner. Chronic etonogestrel treatment improved CO2 chemosensitivity selectively in rats with moderate lesion, suggesting that a residual number of chemosensitive neurons are required for etonogestrel-induced CO2 chemoreflex recovery.
This study provides new evidence for the use of progestins as respiratory stimulants under conditions of central hypoventilation and provides a new testable model for assessing the mechanism of action of progestins in the respiratory network.

At least four mechanisms have been proposed to elucidate how neurons in the retrotrapezoid (RTN) region sense changes in CO2 /H+ to regulate breathing (i.e., function as respiratory chemosensors). These mechanisms include: (1) intrinsic neuronal sensitivity to H+ mediated by TASK-2 and GPR4; (2) paracrine activation of RTN neurons by CO2 -responsive astrocytes (via a purinergic mechanism); (3) enhanced excitatory synaptic input or disinhibition; and (4) CO2 -induced vascular contraction. Although blood flow can influence tissue CO2 /H+ levels, there is limited understanding of how control of vascular tone in central CO2 chemosensitive regions might contribute to respiratory output. In this review, we focus on recent evidence that CO2 /H+ -induced purinergic-dependent vasoconstriction in the ventral parafacial region near RTN neurons supports respiratory chemoreception. This mechanism appears to be unique to the ventral parafacial region and opposite to other brain regions, including medullary chemosensor regions, where CO2 /H+ elicits vasodilatation. We speculate that this mechanism helps to maintain CO2 /H+ levels in the vicinity of RTN neurons, thereby maintaining the drive to breathe. Important next steps include determining whether disruption of CO2 /H+ vascular reactivity contributes to or can be targeted to improve breathing problems in disease states, such as Parkinson\'s disease.

OBJECTIVE: In mammals, central chemoreception plays a crucial role in the regulation of breathing function in both health and disease conditions. Recently, a correlation between high levels of superoxide anion (O2.-) in the Retrotrapezoid nucleus (RTN), a main brain chemoreceptor area, and enhanced central chemoreception has been found in rodents. Interestingly, deficiency in superoxide dismutase 2 (SOD2) expression, a pivotal antioxidant enzyme, has been linked to the development/progression of several diseases. Despite, the contribution of SOD2 on O2.- regulation on central chemoreceptor function is unknown. Accordingly, we sought to determine the impact of partial deletion of SOD2 expression on i) O2.-accumulation in the RTN, ii) central ventilatory chemoreflex function, and iii) disordered-breathing. Finally, we study cellular localization of SOD2 in the RTN of healthy mice.
METHODS: Central chemoreflex drive and breathing function were assessed in freely moving heterozygous SOD2 knockout mice (SOD2+/- mice) and age-matched control wild type (WT) mice by whole-body plethysmography. O2.- levels were determined in RTN brainstem sections and brain isolated mitochondria, while SOD2 protein expression and tissue localization were determined by immunoblot, RNAseq and immunofluorescent staining, respectively.
RESULTS: Our results showed that SOD2+/- mice displayed reductions in SOD2 levels and high O2.- formation and mitochondrial dysfunction within the RTN compared to WT. Additionally, SOD2+/- mice displayed a heightened ventilatory response to hypercapnia and exhibited overt signs of altered breathing patterns. Both, RNAseq analysis and immunofluorescence co-localization studies showed that SOD2 expression was confined to RTN astrocytes but not to RTN chemoreceptor neurons. Finally, we found that SOD2+/- mice displayed alterations in RTN astrocyte morphology compared to RTN astrocytes from WT mice.
UNASSIGNED: These findings provide first evidence of the role of SOD2 in the regulation of O2.- levels in the RTN and its potential contribution on the regulation of central chemoreflex function. Our results suggest that reductions in the expression of SOD2 in the brain may contribute to increase O2.- levels in the RTN being the outcome a chronic surge in central chemoreflex drive and the development/maintenance of altered breathing patterns. Overall, dysregulation of SOD2 and the resulting increase in O2.- levels in brainstem respiratory areas can disrupt normal respiratory control mechanisms and contribute to breathing dysfunction seen in certain disease conditions characterized by high oxidative stress.

Respiration is regulated by various types of neurons located in the pontine-medullary regions. The Kölliker-Fuse (KF)/A7 noradrenergic neurons play a role in modulating the inspiratory cycle by influencing the respiratory output. These neurons are interconnected and may also project to brainstem and spinal cord, potentially involved in regulating the post-inspiratory phase. In the present study, we hypothesize that the parafacial (pF) neurons, in conjunction with adrenergic mechanisms originating from the KF/A7 region, may provide the neurophysiological basis for breathing modulation. We conducted experiments using urethane-anesthetized, vagotomized, and artificially ventilated male Wistar rats. Injection of L-glutamate into the KF/A7 region resulted in inhibition of inspiratory activity, and a prolonged and high-amplitude genioglossal activity (GGEMG). Blockade of the α1 adrenergic receptors (α1-AR) or the ionotropic glutamatergic receptors in the pF region decrease the activity of the GGEMG without affecting inspiratory cessation. In contrast, blockade of α2-AR in the pF region extended the duration of GG activity. Notably, the inspiratory and GGEMG activities induced by KF/A7 stimulation were completely blocked by bilateral blockade of glutamatergic receptors in the Bötzinger complex (BötC). While our study found a limited role for α1 and α2 adrenergic receptors at the pF level in modulating the breathing response to KF/A7 stimulation, it became evident that BötC neurons are responsible for the respiratory effects induced by KF/A7 stimulation.

Respiratory chemosensitivity is an important mechanism by which the brain senses changes in blood partial pressure of CO2 (PCO2). It is proposed that special neurons (and astrocytes) in various brainstem regions play key roles as CO2 central respiratory chemosensors in rodents. Although common marmosets (Callithrix jacchus), New-World non-human primates, show similar respiratory responses to elevated inspired CO2 as rodents, the chemosensitive regions in marmoset brain have not been defined yet. Here, we used c-fos immunostainings to identify brain-wide CO2-activated brain regions in common marmosets. In addition, we mapped the location of the retrotrapezoid nucleus (RTN) and raphé nuclei in the marmoset brainstem based on colocalization of CO2-induced c-fos immunoreactivity with Phox2b, and TPH immunostaining, respectively. Our data also indicated that, similar to rodents, marmoset RTN astrocytes express Phox2b and have complex processes that create a meshwork structure at the ventral surface of medulla. Our data highlight some cellular and structural regional similarities in brainstem of the common marmosets and rodents.

暂无摘要。

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

Current models of respiratory CO2 chemosensitivity are centred around the function of a specific population of neurons residing in the medullary retrotrapezoid nucleus (RTN). However, there is significant evidence suggesting that chemosensitive neurons exist in other brainstem areas, including the rhythm-generating region of the medulla oblongata - the preBötzinger complex (preBötC). There is also evidence that astrocytes, non-neuronal brain cells, contribute to central CO2 chemosensitivity. In this study, we reevaluated the relative contributions of the RTN neurons, the preBötC astrocytes, and the carotid body chemoreceptors in mediating the respiratory responses to CO2 in experimental animals (adult laboratory rats). To block astroglial signalling via exocytotic release of transmitters, preBötC astrocytes were targeted to express the tetanus toxin light chain (TeLC). Bilateral expression of TeLC in preBötC astrocytes was associated with ∼20% and ∼30% reduction of the respiratory response to CO2 in conscious and anaesthetized animals, respectively. Carotid body denervation reduced the CO2 respiratory response by ∼25%. Bilateral inhibition of RTN neurons transduced to express Gi-coupled designer receptors exclusively activated by designer drug (DREADDGi ) by application of clozapine-N-oxide reduced the CO2 response by ∼20% and ∼40% in conscious and anaesthetized rats, respectively. Combined blockade of astroglial signalling in the preBötC, inhibition of RTN neurons and carotid body denervation reduced the CO2 -induced respiratory response by ∼70%. These data further support the hypothesis that the CO2 -sensitive drive to breathe requires inputs from the peripheral chemoreceptors and several central chemoreceptor sites. At the preBötC level, astrocytes modulate the activity of the respiratory network in response to CO2 , either by relaying chemosensory information (i.e. they act as CO2  sensors) or by enhancing the preBötC network excitability to chemosensory inputs. KEY POINTS: This study reevaluated the roles played by the carotid bodies, neurons of the retrotrapezoid nucleus (RTN) and astrocytes of the preBötC in mediating the CO2 -sensitive drive to breathe. The data obtained show that disruption of preBötC astroglial signalling, blockade of inputs from the peripheral chemoreceptors or inhibition of RTN neurons similarly reduce the respiratory response to hypercapnia. These data provide further support for the hypothesis that the CO2 -sensitive drive to breathe is mediated by the inputs from the peripheral chemoreceptors and several central chemoreceptor sites.

Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O2 and CO2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2bΔ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2bΔ8, Atoh1cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b+/TH--expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2bΔ8 mutation in Atoh1-expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.