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.

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.

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.

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.

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3-5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.

Enhanced central chemoreflex drive and irregular breathing are both hallmarks in heart failure (HF) and closely related to disease progression. Central chemoreceptor neurons located within the retrotrapezoid nucleus (RTN) are known to play a role in breathing alterations in HF. It has been shown that exercise (EX) effectively reduced reactive oxygen species (ROS) in HF rats. However, the link between EX and ROS, particularly at the RTN, with breathing alterations in HF has not been previously addressed. Accordingly, we aimed to determine: i) ROS levels in the RTN in HF and its association with chemoreflex drive, ii) whether EX improves chemoreflex/breathing function by reducing ROS levels, and iii) determine molecular alterations associated with ROS generation within the RTN of HF rats and study EX effects on these pathways. Adult male Sprague-Dawley rats were allocated into 3 experimental groups: Sham (n = 5), volume overloaded HF (n = 6) and HF (n = 8) rats that underwent EX training for 6 weeks (60 min/day, 25 m/min, 10% inclination). At 8 weeks post-HF induction, breathing patterns and chemoreflex function were analyzed by unrestrained plethysmography. ROS levels and anti/pro-oxidant enzymes gene expression were analyzed in the RTN. Our results showed that HF rats have high ROS levels in the RTN which were closely linked to the enhanced central chemoreflex and breathing disorders. Also, HF rats displayed decreased expression of antioxidant genes in the RTN compared with control rats. EX training increases antioxidant defense in the RTN, reduces ROS formation and restores normal central chemoreflex drive and breathing regularity in HF rats. This study provides evidence for a role of ROS in central chemoreception in the setting of HF and support the use of EX to reduce ROS in the brainstem of HF animals and reveal its potential as an effective mean to normalize chemoreflex and breathing function in HF.

Glutamatergic neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating breathing in response to tissue CO2/H+. The RTN and greater parafacial region may also function as a chemosensing network composed of CO2/H+-sensitive excitatory and inhibitory synaptic interactions. In the context of disease, we showed that loss of inhibitory neural activity in a mouse model of Dravet syndrome disinhibited RTN chemoreceptors and destabilized breathing (Kuo et al., 2019). Despite this, contributions of parafacial inhibitory neurons to control of breathing are unknown, and synaptic properties of RTN neurons have not been characterized. Here, we show the parafacial region contains a limited diversity of inhibitory neurons including somatostatin (Sst)-, parvalbumin (Pvalb)-, and cholecystokinin (Cck)-expressing neurons. Of these, Sst-expressing interneurons appear uniquely inhibited by CO2/H+. We also show RTN chemoreceptors receive inhibitory input that is withdrawn in a CO2/H+-dependent manner, and chemogenetic suppression of Sst+ parafacial neurons, but not Pvalb+ or Cck+ neurons, increases baseline breathing. These results suggest Sst-expressing parafacial neurons contribute to RTN chemoreception and respiratory activity.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Parkinson\'s disease (PD) is a neurodegenerative disorder anatomically characterized by a progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). Much less known, yet clinically very important, are the detrimental effects on breathing associated with this disease. Consistent with the human pathophysiology, the 6-hydroxydopamine hydrochloride (6-OHDA) rodent model of PD shows reduced respiratory frequency (fR) and NK1r-immunoreactivity in the pre-Bötzinger complex (preBötC) and PHOX2B+ neurons in the retrotrapezoid nucleus (RTN). To unravel mechanisms that underlie bradypnea in PD, we employed a transgenic approach to label or stimulate specific neuron populations in various respiratory-related brainstem regions. PD mice were characterized by a pronounced decreased number of putatively rhythmically active excitatory neurons in the preBötC and adjacent ventral respiratory column (VRC). Specifically, the number of Dbx1 and Vglut2 neurons was reduced by 47.6% and 17.3%, respectively. By contrast, inhibitory Vgat+ neurons in the VRC, as well as neurons in other respiratory-related brainstem regions, showed relatively minimal or no signs of neuronal loss. Consistent with these anatomic observations, optogenetic experiments identified deficits in respiratory function that were specific to manipulations of excitatory (Dbx1/Vglut2) neurons in the preBötC. We conclude that the decreased number of this critical population of respiratory neurons is an important contributor to the development of irregularities in inspiratory rhythm generation in this mouse model of PD.SIGNIFICANCE STATEMENT We found a decreased number of a specific population of medullary neurons which contributes to breathing abnormalities in a mouse model of Parkinson\'s disease (PD).

Brainstem networks that control regular tidal breathing depend on excitatory drive, including from tonically active, CO2/H+-sensitive neurons of the retrotrapezoid nucleus (RTN). Here, we examine intrinsic ionic mechanisms underlying the metronomic firing activity characteristic of RTN neurons. In mouse brainstem slices, large-amplitude membrane potential oscillations are evident in synaptically isolated RTN neurons after blocking action potentials. The voltage-dependent oscillations are abolished by sodium replacement; blocking calcium channels (primarily L-type); chelating intracellular Ca2+; and inhibiting TRPM4, a Ca2+-dependent cationic channel. Likewise, oscillation voltage waveform currents are sensitive to calcium and TRPM4 channel blockers. Extracellular acidification and serotonin (5-HT) evoke membrane depolarization that augments TRPM4-dependent oscillatory activity and action potential discharge. Finally, inhibition of TRPM4 channels in the RTN of anesthetized mice reduces central respiratory output. These data implicate TRPM4 in a subthreshold oscillation that supports the pacemaker-like firing of RTN neurons required for basal, CO2-stimulated, and state-dependent breathing.