Figure 3 - available via license: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International
Content may be subject to copyright.
Nmb cells in rostral parafacial region and dorsal pons are not catecholaminergic. A, Transcripts for Pnmt (green) and Nmb (magenta) in dorsal medial medulla at level of C2/C3 adrenergic cell groups. B, Transcripts for Th (green) and Nmb (magenta) in more rostral pontine brain regions. LC, locus coeruleus. C, Enlargement of the white box (rostral RTN) in A. Scale bars: A, 100 m; B, 500 m; C, 50 m.
Source publication
The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells...
Contexts in source publication
Context 1
... to determine a total average number of cells containing Nmb transcripts (Table 2). Using this method, we counted 266. A sparse collection of Nmb cells was present dorsomedial to the prepositus nucleus. These Nmb neurons were located generally medial to the C2 neurons and dorsal to the C3 neurons and, unlike the latter, they did not express Pnmt (Fig. 3A). A second and much larger cluster of Nmb cells was located in the region of the central gray and posterodorsal tegmental nucleus medial to the locus ceruleus (Fig. 3B), from 5.63 to 5.41 mm caudal to bregma (coordinates after the corresponding plates in the atlas of Paxinos and Franklin, 2013) and did not express ...
Context 2
... to the prepositus nucleus. These Nmb neurons were located generally medial to the C2 neurons and dorsal to the C3 neurons and, unlike the latter, they did not express Pnmt (Fig. 3A). A second and much larger cluster of Nmb cells was located in the region of the central gray and posterodorsal tegmental nucleus medial to the locus ceruleus (Fig. 3B), from 5.63 to 5.41 mm caudal to bregma (coordinates after the corresponding plates in the atlas of Paxinos and Franklin, 2013) and did not express ...
Citations
... Neuromedin B is currently considered the most selective anatomical marker for chemosensitive RTN neurons (Shi et al., 2017) and was used in this study to identify RTN location, cell numbers and relative PHOX2B expression. Since changes in the level of the transcription factor PHOX2B could regulate the expression of its target genes, we investigated whether Nmb is a target gene of PHOX2B and it is affected by PHOX2B knockdown. ...
... Although the PHOX2B shRNA approach was not selective for Nmb neurons, we used small volumes and localized injections to target the RTN. We identified RTN neurons by the mRNA expression of the neuropeptide NMB (Shi et al., 2017) and observed a reduced number in both total Nmb and Nmb + / PHOX2B + neurons in PHOX2B shRNA-treated rats compared to naive and NT-shRNA rats two weeks after viral infection. Interestingly, despite the small reduction in total Nmb cells of the RTN in both NT-shRNA and PHOX2B-shRNA injected rats, possibly due to some off-target effects due to activation of innate immunity or saturation of the microRNA pathway (McBride et al., 2008;van Gestel et al., 2014), no significant impairment in ventilation was observed. ...
... Sections were mounted on slides for combined RNAScope in situ hybridization (Advanced Cell Diagnostics-ACD Bio, Newark CA, USA) and immunofluorescence assay and processed as previously detailed (Biancardi et al., 2021;Cardani et al., 2022). The mRNA expression for Neuromedin B (Nmb) was used as marker of RTN CO 2 -sensing neurons (Shi et al., 2017). Slides were incubated with probes for Nmb (Rn-NMB-C2 #494791-C2, ACDBio, Newark, CA, USA), Phox2b (Rn-Phox2b-O1-C1 #1064121-C1, ACDBio), G-protein-coupled receptor 4 (Gpr4) (Mm-Gpr4-C1, #427941, ACDBio), and potassium channel, subfamily K, member 5 (Kcnk5 or Task-2) (Mm-Kcnk5-C3, #427951-C3, ACDBio) for 2 hr at 40 °C. ...
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 CO 2 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 CO 2 /pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO 2 chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.
... The transport of these molecules would be favored in acidic extracellular conditions, in which H + ions would be readily available, provided Na + is also available intracellularly 41 . Considering Phox2b neurons in the RTN are glutamatergic 26,42 , the increased firing frequency in acidic conditions would require elevated levels of free glycine for the conversion to glutamate, which could be provided by this subset of astrocytes. Coupled with Kir4.1 inhibition via elevated CO 2 /H + , this could provide evidence for maintenance of elevated firing frequency of RTN neurons in an acidic environment. ...
Astrocytes in the retrotrapezoid nucleus (RTN) stimulate breathing in response to CO2/H⁺, however, it is not clear how these cells detect changes in CO2/H⁺. Considering Kir4.1/5.1 channels are CO2/H⁺-sensitive and important for several astrocyte-dependent processes, we consider Kir4.1/5.1 a leading candidate CO2/H⁺ sensor in RTN astrocytes. To address this, we show that RTN astrocytes express Kir4.1 and Kir5.1 transcripts. We also characterized respiratory function in astrocyte-specific inducible Kir4.1 knockout mice (Kir4.1 cKO); these mice breathe normally under room air conditions but show a blunted ventilatory response to high levels of CO2, which could be partly rescued by viral mediated re-expression of Kir4.1 in RTN astrocytes. At the cellular level, astrocytes in slices from astrocyte-specific inducible Kir4.1 knockout mice are less responsive to CO2/H⁺ and show a diminished capacity for paracrine modulation of respiratory neurons. These results suggest Kir4.1/5.1 channels in RTN astrocytes contribute to respiratory behavior.
... In 2017, single-cell RNA sequencing and in situ hybridization were used to systematically characterize the transcriptome of individual PHOX2B+ RTN neurons (Shi et al., 2017). This study offered several key insights into the genes expressed by RTN neurons. ...
... First, it confirmed expression of GPR4 and TASK-2 in PHOX2B+ RTN neurons; these membrane proteins mediate the activation of these cells by H + and CO 2 (Gestreau et al., 2010;Kumar et al., 2015). Second, it demonstrated that PHOX2B+ RTN neurons with chemoreceptor properties selectively express the mRNA for the neuropeptide Neuromedin B (Nmb) (Shi et al., 2017). This observation extended an earlier study by Li et al. (2016) that showed an enrichment of Nmb and the co-expression of Nmb with PHOX2B in the RTN. ...
... Shi et al. observed that Nmb-expressing neurons in the RTN (RTN Nmb ) are pH/CO 2 sensitive, glutamatergic and do not express markers genes for catecholaminergic other nearby neurons. Importantly, at least 80% of RTN Nmb neurons express one or both acid-sensitive proteins TASK-2 and GPR4, and all neurons expressing these proton sensors in the RTN express Nmb (Shi et al., 2017). Clearly, there is an impressive alignment between the presence of Nmb and the expression of proteins that are proposed to confer intrinsic H + sensitivity in the RTN neurons. ...
... These neurons exhibit robust responses to hypercapnia, both in vivo and in vitro (Mulkey et al., 2004;Takakura et al., 2006Takakura et al., , 2013. Moreover, we possess a detailed understanding of their phenotype (Shi et al., 2017;Stornetta et al., 2006). The RTN has significant disease relevance, particularly in congenital central hypoventilation syndrome, and the cellular and molecular mechanisms involved in CO 2 detection have been studied extensively in comparison to other CO 2 chemosensors (Amiel et al., 2009;Ferreira et al., 2022;Kumar et al., 2015). ...
At least four mechanisms have been proposed to elucidate how neurons in the retrotrapezoid (RTN) region sense changes in CO 2 /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 CO 2 ‐responsive astrocytes (via a purinergic mechanism); (3) enhanced excitatory synaptic input or disinhibition; and (4) CO 2 ‐induced vascular contraction. Although blood flow can influence tissue CO 2 /H ⁺ levels, there is limited understanding of how control of vascular tone in central CO 2 chemosensitive regions might contribute to respiratory output. In this review, we focus on recent evidence that CO 2 /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 CO 2 /H ⁺ elicits vasodilatation. We speculate that this mechanism helps to maintain CO 2 /H ⁺ levels in the vicinity of RTN neurons, thereby maintaining the drive to breathe. Important next steps include determining whether disruption of CO 2 /H ⁺ vascular reactivity contributes to or can be targeted to improve breathing problems in disease states, such as Parkinson's disease.
... Central chemoreception is essential for regulating breathing in mammals by sensing carbon dioxide (CO 2 ) and pH levels in the cerebrospinal fluid [20,22,32,39]. Enhanced central chemoreflex drive has been associated with irregular breathing and increased mortality in various disease conditions [11,24,30,31,36,41]. Indeed, potentiation of the ventilatory chemoreflex has been shown in heart failure, hypertension, chronic obstructive pulmonary disease, stroke, diabetes, sleep apnea and asthma. ...
... Central chemoreceptors are primarily located on the ventral surface of the medulla, but they can also be found in other regions of the brainstem, hypothalamus, cerebellum, and midbrain, albeit in smaller numbers [15,20,32]. In rodents, the Retrotrapezoid nucleus (RTN) is a major brainstem region that regulates multiple aspects of breathing, such as inspiratory amplitude, breathing frequency, and active expiration in response to changes in brain CO 2 (PCO 2 ) levels [21,22,26,29,36]. Recent research has shown that removing some RTN chemoreceptor neurons using substance P-conjugated saporin toxin can normalize central chemoreflex and respiratory disorders in animals with heart disease, highlighting the significance of RTN neurons in the pathological process of central chemoreflex potentiation [11]. ...
Aims
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.
Innovation & conclusion
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.
... This helps elucidate similarities in RTN size, density, and potential location as the rodent model is a similar physical size to the marmoset. This is comparable to a larger non-human primate such as a macaque where the tentative outline of the RTN matches reasonably well with the marmoset map of the RTN region based on Phox2b and c-fos expression patterns, however the number of Phox2B positive neurons in the RTN is much greater (Shi et al. 2017;Feldman and Del Negro 2006;Guyenet et al. 2010;Smith et al. 1989;Levy et al. 2019). ...
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 raphe 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 the medulla. Our data highlight some cellular and structural regional similarities in the brainstem of the common marmosets and rodents.
... Of the transcription factors associated with RTN development, only Phox2b expression persists at appreciable levels in postnatal RTN neurons; however, Phox2b is also found in other neurons, including the nearby C1 adrenergic neurons and facial motoneurons (Stornetta et al., 2006). Additional work using immunochemical and single cell molecular approaches has produced a more precise and limited phenotypic definition for RTN neurons (Figure 1Aiii) (Shi et al., 2017;Cleary et al., 2021). In addition to Phox2b expression, all RTN neurons express Slc17a6 (VGlut2); they can be differentiated from other nearby Phox2bexpressing populations, like C1 neurons and motoneurons, by the absence of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) expression (Stornetta et al., 2006;Guyenet et al., 2019). ...
... In addition to Phox2b expression, all RTN neurons express Slc17a6 (VGlut2); they can be differentiated from other nearby Phox2bexpressing populations, like C1 neurons and motoneurons, by the absence of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) expression (Stornetta et al., 2006;Guyenet et al., 2019). All RTN neurons express the excitatory neuropeptide PACAP (pituitary adenylate cyclase activating peptide), and subsets also express variable levels of the inhibitory neuropeptides enkephalin and galanin, but these are not specific for the RTN (Stornetta et al., 2009;Shi et al., 2017;Cleary et al., 2021). Of particular note, RTN neurons can be most definitively identified in this region of the rostroventrolateral medulla by their unique and universal expression of the neuropeptide, Neuromedin B (NMB) (Stornetta et al., 2006;Shi et al., 2017). ...
... All RTN neurons express the excitatory neuropeptide PACAP (pituitary adenylate cyclase activating peptide), and subsets also express variable levels of the inhibitory neuropeptides enkephalin and galanin, but these are not specific for the RTN (Stornetta et al., 2009;Shi et al., 2017;Cleary et al., 2021). Of particular note, RTN neurons can be most definitively identified in this region of the rostroventrolateral medulla by their unique and universal expression of the neuropeptide, Neuromedin B (NMB) (Stornetta et al., 2006;Shi et al., 2017). NMB-positive RTN neurons express a variety of receptors for other neuromodulators, including serotonin (primarily 5-HT2C), substance P (NK1R), orexin (Hcrt1/Hcrt2), and ATP (P2Y12) . ...
An interoceptive homeostatic system monitors levels of CO 2 /H ⁺ and provides a proportionate drive to respiratory control networks that adjust lung ventilation to maintain physiologically appropriate levels of CO 2 and rapidly regulate tissue acid-base balance. It has long been suspected that the sensory cells responsible for the major CNS contribution to this so-called respiratory CO 2 /H ⁺ chemoreception are located in the brainstem—but there is still substantial debate in the field as to which specific cells subserve the sensory function. Indeed, at the present time, several cell types have been championed as potential respiratory chemoreceptors, including neurons and astrocytes. In this review, we advance a set of criteria that are necessary and sufficient for definitive acceptance of any cell type as a respiratory chemoreceptor. We examine the extant evidence supporting consideration of the different putative chemoreceptor candidate cell types in the context of these criteria and also note for each where the criteria have not yet been fulfilled. By enumerating these specific criteria we hope to provide a useful heuristic that can be employed both to evaluate the various existing respiratory chemoreceptor candidates, and also to focus effort on specific experimental tests that can satisfy the remaining requirements for definitive acceptance.
... Activation of RTN neurons and astrocytes by elevated CO 2 or O 2 was assessed by Fos expression (Shi et al., 2016(Shi et al., , 2017. In short, adult mice were habituated to the plethysmography chamber for 1 h in hyperoxia (60% O 2 , balance N 2 ) before an additional 35 min exposure to hyperoxia (60% O 2 , balance N 2 ) or hypercapnia (12% CO 2 , 60% O 2 , balance N 2 ). ...
... Brains and kidneys were removed, immersed in the same fixative for 16−18 h at 4°C, cut in the transverse plane (30 μm) and placed in cryoprotectant (30% ethylene glycol, 20% glycerol, 50 mm sodium phosphate buffer, pH 7.4) at −20°C until further processing. Tissue preparation and staining procedure utilized the RNAscope Multiplex Fluorescent Assay (Advanced Cell Diagnostics, Hayward, CA, USA; RRID: SCR_012481), according to the manufacturer's instructions and as previously described (Shi et al., 2017). Catalogue probes were used for Aldh1l1, Nmb, Gfap, Slc4a5, and Fos, whereas a custom Slc4a4 probe was designed specifically to encompass the floxed exon 12 that was deleted by Cre-mediated excision (Cat. ...
... Serial coronal sections (1:3 series) through the rostrocaudal extent of the RTN were mounted on glass slides, and images were acquired using an epifluorescence microscope (Zeiss Axioimager Z1) equipped with Neurolucida software (MBF Bioscience, Williston, VT, USA). One section from each mouse was used for cell counts and was chosen from the rostrocaudal level where maximal numbers of RTN neurons are located (6.48 mm caudal to bregma; Shi et al., 2017). In the two mouse lines, there were no Cre-dependent and/or tamoxifen-dependent differences in the average number of RTN neurons counted from these sections (44.2 ± 12.7 Nmb + neurons, range 21-76, from N = 42 mice). ...
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. image
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.
... The retrotrapezoid nucleus consists in mice of around 700 Phox2b-positive cells located ventrolateral to the facial nucleus. The locations of these neurons partially overlap with those of the lateral parafacial nucleus (Smith et al., 1989;Onimaru and Homma, 2003;Ramanantsoa et al., 2011;Shi et al., 2017). The retrotrapezoid nucleus houses also around 200 biochemically and morphologically different neurons that lack CO 2 -sensitivity, and that could be related to sighing via their direct projection to the pre-Bötzinger complex Shi et al., 2017). ...
... The locations of these neurons partially overlap with those of the lateral parafacial nucleus (Smith et al., 1989;Onimaru and Homma, 2003;Ramanantsoa et al., 2011;Shi et al., 2017). The retrotrapezoid nucleus houses also around 200 biochemically and morphologically different neurons that lack CO 2 -sensitivity, and that could be related to sighing via their direct projection to the pre-Bötzinger complex Shi et al., 2017). ...
Respiration is a brain function on which our lives essentially depend. Control of respiration ensures that the frequency and depth of breathing adapt continuously to metabolic needs. In addition, the respiratory control network of the brain has to organize muscular synergies that integrate ventilation with posture and body movement. Finally, respiration is coupled to cardiovascular function and emotion. Here, we argue that the brain can handle this all by integrating a brainstem central pattern generator circuit in a larger network that also comprises the cerebellum. Although currently not generally recognized as a respiratory control center, the cerebellum is well known for its coordinating and modulating role in motor behavior, as well as for its role in the autonomic nervous system. In this review, we discuss the role of brain regions involved in the control of respiration, and their anatomical and functional interactions. We discuss how sensory feedback can result in adaptation of respiration, and how these mechanisms can be compromised by various neurological and psychological disorders. Finally, we demonstrate how the respiratory pattern generators are part of a larger and integrated network of respiratory brain regions.
... Under etonogestrel, acidosis-responder Phox2b mutants displayed a greater number of c-FOS cells in the raphe obscurus nucleus (ROb ; Table 1; Figures 4A, B, 5A). This increase concerned more particularly the part of the ROb at the rostro-caudal level delimited by the presence of inferior olives (Table 1; Figures 4A, B and also immediately to it in its caudal part (5,35,39), named respectively hereafter parafacial RTN (pf-RTN) and caudal RTN (cRTN). In the newborn, RTN is considered to be intertwined with the parafacial respiratory group (pFRG; expiratory generator) that is ventrolaterally below the facial nucleus and also contains PHOX2B cells (34,40,41). ...
Introduction:
Congenital Central Hypoventilation Syndrome, a rare disease caused by PHOX2B mutation, is associated with absent or blunted CO2/H+ chemosensitivity due to the dysfunction of PHOX2B neurons of the retrotrapezoid nucleus. No pharmacological treatment is available. Clinical observations have reported non-systematic CO2/H+ chemosensitivity recovery under desogestrel.
Methods:
Here, we used a preclinical model of Congenital Central Hypoventilation Syndrome, the retrotrapezoid nucleus conditional Phox2b mutant mouse, to investigate whether etonogestrel, the active metabolite of desogestrel, led to a restoration of chemosensitivity by acting on serotonin neurons known to be sensitive to etonogestrel, or retrotrapezoid nucleus PHOX2B residual cells that persist despite the mutation. The influence of etonogestrel on respiratory variables under hypercapnia was investigated using whole-body plethysmographic recording. The effect of etonogestrel, alone or combined with serotonin drugs, on the respiratory rhythm of medullary-spinal cord preparations from Phox2b mutants and wildtype mice was analyzed under metabolic acidosis. c-FOS, serotonin and PHOX2B were immunodetected. Serotonin metabolic pathways were characterized in the medulla oblongata by ultra-high-performance liquid chromatography.
Results:
We observed etonogestrel restored chemosensitivity in Phox2b mutants in a non-systematic way. Histological differences between Phox2b mutants with restored chemosensitivity and Phox2b mutant without restored chemosensitivity indicated greater activation of serotonin neurons of the raphe obscurus nucleus but no effect on retrotrapezoid nucleus PHOX2B residual cells. Finally, the increase in serotonergic signaling by the fluoxetine application modulated the respiratory effect of etonogestrel differently between Phox2b mutant mice and their WT littermates or WT OF1 mice, a result which parallels with differences in the functional state of serotonergic metabolic pathways between these different mice.
Discussion:
Our work thus highlights that serotonin systems were critically important for the occurrence of an etonogestrel-restoration, an element to consider in potential therapeutic intervention in Congenital Central Hypoventilation Syndrome patients.
