[Electrophysiological, molecular and genetic identifications of the pre-Bötzinger complex]. / Complexe de pré-Bötzinger et automatisme respiratoire: identification électrophysiologique, moléculaire et génétique d'une structure cruciale pour la respiration.

From birth onwards, rhythmic breathing is required for blood oxygenation and survival in mammals. During their lifespan, human or mouse or elephant will spontaneously produce several hundreds of millions of respiratory movements. The central nervous command responsible for these spontaneous rhythmic movements is elaborated by a complex neural network extending within the brainstem. In the medulla, a special part of this network contains respiratory pacemaker neurons that play a crucial role in respiratory rhythmogenesis: the pre-Bötzinger complex. This review summarizes and discusses the main electrophysiological, molecular and genetic mechanisms contributing to the function and the perinatal maturation of the pre-Bötzinger complex.

Rnx deficiency results in congenital central hypoventilation.

The genes Tlx1 (Hox11), Enx (Hox11L2, Tlx-2) and Rnx (Hox11L2, Tlx-3) constitute a family of orphan homeobox genes. In situ hybridization has revealed considerable overlap in their expression within the nervous system, but Rnx is singularly expressed in the developing dorsal and ventral region of the medulla oblongata. Tlx1-deficient and Enx-deficient mice display phenotypes in tissues where the mutated gene is singularly expressed, resulting in asplenogenesis and hyperganglionic megacolon, respectively. To determine the developmental role of Rnx, we disrupted the locus in mouse embryonic stem (ES) cells. Rnx deficient mice developed to term, but all died within 24 hours after birth from a central respiratory failure. The electromyographic activity of intercostal muscles coupled with the C4 ventral root activity assessed in a medulla-spinal cord preparation revealed a high respiratory rate with short inspiratory duration and frequent apnea. Furthermore, a coordinate pattern existed between the abnormal activity of inspiratory neurons in the ventrolateral medulla and C4 motorneuron output, indicating a central respiratory defect in Rnx mice. Thus, Rnx is critical for the development of the ventral medullary respiratory centre and its deficiency results in a syndrome resembling congenital central hypoventilation.

Ablation of Zfhx4 results in early postnatal lethality by disrupting the respiratory center in mice.

Breathing is an integrated motor behavior that is driven and controlled by a network of brainstem neurons. Zfhx4 is a zinc finger transcription factor and our results showed that it was specifically expressed in several regions of the mouse brainstem. Mice lacking Zfhx4 died shortly after birth from an apparent inability to initiate respiration. We also found that the electrical rhythm of brainstem‒spinal cord preparations was significantly depressed in Zfhx4-null mice compared to wild-type mice. Immunofluorescence staining revealed that Zfhx4 was coexpressed with Phox2b and Math1 in the brainstem and that Zfhx4 ablation greatly decreased the expression of these proteins, especially in the retrotrapezoid nucleus. Combined ChIP‒seq and mRNA expression microarray analysis identified Phox2b as the direct downstream target gene of Zfhx4, and this finding was validated by ChIP‒qPCR. Previous studies have reported that both Phox2b and Math1 play key roles in the development of the respiratory center, and Phox2b and Math1 knockout mice are neonatal lethal due to severe central apnea. On top of this, our study revealed that Zfhx4 is a critical regulator of Phox2b expression and essential for perinatal breathing.

Defective respiratory rhythmogenesis and loss of central chemosensitivity in Phox2b mutants targeting retrotrapezoid nucleus neurons.

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

Extracellular calcium induces quiescence of the low-frequency embryonic motor rhythm in the mouse isolated brainstem.

Although extracellular calcium ionic concentration ([Ca](o) ) is known to increase during late gestation and to drop after parturition, little is known about the influence of [Ca](o) on fetal brain function. We have investigated the influence of [Ca](o) , calcium-sensing receptors/nonselective cation currents (CaSR/NSCC), and GABAergic inhibitions on maturation of brainstem-spinal motor activities: the primary low-frequency embryonic rhythm [LF; silent since embryonic day (E)16] and the fetal respiratory rhythm (RR; emerging at E14-E15). Using in vitro isolated brainstem-spinal cord preparations of mice at different fetal and postnatal (P) stages (E16-P1), we demonstrate that reducing fetal [Ca](o) from 1.2 mM to 0.7 mM at E16-E18 or blocking GABA(A) receptors at E16-P0 reactivates LF and reveals LF-related disturbance of RR at E16-E18. This LF is stopped by adding gadolinium or spermidine (CaSR/NSCC agonists) at E18-P0 or GABA(A) receptor agonists at E16-E18. In contrast, [Ca](o) -induced slowing of RR at E16-E18 is not reproduced by gadolinium and spermidine. We conclude that perinatal CaSR/NSCC and GABA(A) inhibition allow quiescence of the LF, thereby improving functional maturation of the RR.

Development of the brainstem respiratory circuit.

The respiratory circuit is comprised of over a dozen functionally and anatomically segregated brainstem nuclei that work together to control respiratory rhythms. These respiratory rhythms emerge prenatally but only acquire vital importance at birth, which is the first time the respiratory circuit faces the sole responsibility for O2 /CO2 homeostasis. Hence, the respiratory circuit has little room for trial-and-error-dependent fine tuning and relies on a detailed genetic blueprint for development. This blueprint is provided by transcription factors that have specific spatiotemporal expression patterns along the rostral-caudal or dorsal-ventral axis of the developing brainstem, in proliferating precursor cells and postmitotic neurons. Studying these transcription factors in mice has provided key insights into the functional segregation of respiratory control and the vital importance of specific respiratory nuclei. Many studies converge on just two respiratory nuclei that each have rhythmogenic properties during the prenatal period: the preBötzinger complex (preBötC) and retrotrapezoid nucleus/parafacial nucleus (RTN/pF). Here, we discuss the transcriptional regulation that guides the development of these nuclei. We also summarize evidence showing that normal preBötC development is necessary for neonatal survival, and that neither the preBötC nor the RTN/pF alone is sufficient to sustain normal postnatal respiratory rhythms. Last, we highlight several studies that use intersectional genetics to assess the necessity of transcription factors only in subregions of their expression domain. These studies independently demonstrate that lack of RTN/pF neurons weakens the respiratory circuit, yet these neurons are not necessary for neonatal survival because developmentally related populations can compensate for abnormal RTN/pF function at birth. This article is categorized under: Nervous System Development > Vertebrates: Regional Development.

Developmental changes of serotonin 4(a) receptor expression in the rat pre-Bötzinger complex.

Serotonin receptors (5-HTRs) are known to be involved in the regulation of breathing behavior and to mediate neurotrophic actions that exert a significant function in network formation during development. We studied neuronal 5-HT(4(a))R-immunoreactivity (-IR) at developmental ages from E14 to P10. Within the pre-Bötzinger complex (pre-BötC), a part of the respiratory network important for rhythmogenesis, 5-HT(4(a))R-IR was most extensive in rats at an age of E18. The 5-HT(4(a))-IR was found predominantly in the neuropil, whereas somatic staining was sporadic at late embryonic (E18-E20) stages. At birth, we observed a dramatic change to a predominantly somatic staining, and neuropil staining was greatly reduced and disappeared at an age of P4. In all developmental stages, 5-HT(4(a)) and mu-opioid receptors were strongly coexpressed in neurons of the pre-BötC, whereas 5-HT(4(a))R expression was absent in neurons within the dorsal horn. Nestin, a marker for CNS progenitor cells, was used to obtain information about the degree of pre-BötC differentiation. Nestin-positive cells did not appear within the pre-BötC before age E20. At E16, nestin-expressing cells were absent in the nucleus ambiguus (NA) and its ventral periphery. The number of nestin-positive cells increased after birth within and outside the pre-BötC, the majority of cells being glial. Coexpression of nestin and 5-HT(4(a))R was localized predominantly within the NA and appeared only sporadically within the pre-BötC. We conclude that 5-HT(4(a))Rs are important not only for neuromodulation of cellular excitability but also for respiratory network formation.

Development of vagal afferent projections circumflex to the obex in the embryonic chick brainstem visualized with voltage-sensitive dye recording.

Using voltage-sensitive dye recording, we surveyed neural responses related to the vagus nerve in the embryonic chick brainstem. In our previous studies, we identified four vagus nerve-related response areas in the brainstem. On the stimulated side, they included (1) the nucleus of the tractus solitarius (NTS: the primary sensory nucleus) and (2) the dorsal motor nucleus of the vagus nerve (DMNV), whereas on the contralateral side, they corresponded to (3) the parabrachial nucleus (PBN: the second/higher-ordered nucleus) and (4) the medullary non-NTS region. In the present study, in addition to these areas, we identified another response area circumflex to the obex. The intensity of the optical signal in the response area was much smaller than that in the NTS/DMNV, and the spatio-temporal pattern could be discerned after signal averaging. The conduction rate to the response area was slower than that to the other four areas. Ontogenetically, the response area was distributed on the stimulated side at the 6-day embryonic stage, and it spread into the contralateral side in 7- and 8-day embryonic stages. These distribution patterns were consistent with projection patterns of vagal afferent fibers stained with a fluorescent tracer, suggesting that the response area included a primary sensory nucleus. In comparison with the functional development of the other four response areas, we traced the functional organization of vagus nerve-related nuclei in the embryonic brainstem.

The embryonic development of hindbrain respiratory networks is unaffected by mutation of the planar polarity protein Scribble.

The central command for breathing arises mainly from two interconnected rhythmogenic hindbrain networks, the parafacial respiratory group (pFRG or epF at embryonic stages) and the preBötzinger complex (preBötC), which are comprised of a limited number of neurons located in confined regions of the ventral medulla. In rodents, both networks become active toward the end of gestation but little is known about the signaling pathways involved in their anatomical and functional establishment during embryogenesis. During embryonic development, epF and preBötC neurons migrate from their territories of origin to their final positions in ventral brainstem areas. Planar Cell Polarity (PCP) signaling, including the molecule Scrib, is known to control the developmental migration of several hindbrain neuronal groups. Accordingly, a homozygous mutation of Scrib leads to severe disruption of hindbrain anatomy and function. Here, we aimed to determine whether Scrib is also involved in the prenatal development of the hindbrain nuclei controlling breathing. We combined immunostaining, calcium imaging and electrophysiological recordings of neuronal activity in isolated in vitro preparations. In the Scrib mutant, despite severe neural tube defects, epF and preBötC neurons settled at their expected hindbrain positions. Furthermore, both networks remained capable of generating rhythmically organized, respiratory-related activities and exhibited normal sensitivity to pharmacological agents known to modify respiratory circuit function. Thus Scrib is not required for the proper migration of epF and preBötC neurons during mouse embryogenesis. Our findings thus further illustrate the robustness and specificity of the developmental processes involved in the establishment of hindbrain respiratory circuits.

Emergence of the pre-Bötzinger respiratory rhythm generator in the mouse embryo.

To obtain insights into the emergence of rhythmogenic circuits supporting respiration, we monitored spontaneous activities in isolated brainstem and medullary transverse slice preparations of mouse embryos, combining electrophysiological and calcium imaging techniques. At embryonic day 15 (E15), in a restricted region ventral to the nucleus ambiguus, we observed the onset of a sustained high-frequency (HF) respiratory-like activity in addition to a preexisting low-frequency activity having a distinct initiation site, spatial extension, and susceptibility to gap junction blockers. At the time of its onset, the HF generator starts to express the neurokinin 1 receptor, is connected bilaterally, requires active AMPA/kainate glutamatergic synapses, and is modulated by substance P and the mu-opioid agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin. We conclude that a rhythm generator sharing the properties of the neonatal pre-Bötzinger complex becomes active during E15 in mice.

Adrenaline contributes to prenatal respiratory maturation in rat medulla-spinal cord preparation.

Adrenaline is a potent respiratory regulator. However, adrenergic contribution to the developing respiratory center has not been studied extensively. Adrenaline application on embryonic day 17 medulla-spinal cord block preparations abolished non-respiratory activity and enhanced respiratory frequency. Phentolamine application on neonatal blocks that produced stable neonatal respiration resulted in respiratory destabilization. These results suggest that central adrenergic modulation is involved in fetal respiratory development and maintenance of stable respiration.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network.

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons.

Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice.

Although respiration is vital to the survival of all mammals from the moment of birth, little is known about the genetic factors controlling the prenatal maturation of this physiological process. Here we investigated the role of the Phox2a gene that encodes for a homeodomain protein involved in the generation of noradrenergic A6 neurons in the maturation of the respiratory network. First, comparisons of the respiratory activity of fetuses delivered surgically from heterozygous Phox2a pregnant mice on gestational day 18 showed that the mutants had impaired in vivo ventilation, in vitro respiratory-like activity, and in vitro respiratory responses to central hypoxia and noradrenaline. Second, pharmacological studies on wild-type neonates showed that endogenous noradrenaline released from pontine A6 neurons potentiates rhythmic respiratory activity via alpha1 medullary adrenoceptors. Third, transynaptic tracing experiments in which rabies virus was injected into the diaphragm confirmed that A6 neurons were connected to the neonatal respiratory network. Fourth, blocking the alpha1 adrenoceptors in wild-type dams during late gestation with daily injections of the alpha1 adrenoceptor antagonist prazosin induced in vivo and in vitro neonatal respiratory deficits similar to those observed in Phox2a mutants. These results suggest that noradrenaline, A6 neurons, and the Phox2a gene, which is crucial for the generation of A6 neurons, are essential for development of normal respiratory rhythm in neonatal mice. Metabolic noradrenaline disorders occurring during gestation therefore may induce neonatal respiratory deficits, in agreement with the catecholamine anomalies reported in victims of sudden infant death syndrome.

Absence of Ndn, encoding the Prader-Willi syndrome-deleted gene necdin, results in congenital deficiency of central respiratory drive in neonatal mice.

necdin (Ndn) is one of a cluster of genes deleted in the neurodevelopmental disorder Prader-Willi syndrome. necdin is upregulated during neuronal differentiation and is thought to play a role in cell cycle arrest in terminally differentiated neurons. Most necdin-deficient Ndn(tm2Stw) mutant pups carrying a targeted replacement of Ndn with a lacZ reporter gene die in the neonatal period of apparent respiratory insufficiency. We now demonstrate that the defect can be explained by abnormal neuronal activity within the putative respiratory rhythm-generating center, the pre-Bötzinger complex. Specifically, the rhythm is unstable with prolonged periods of depression of respiratory rhythmogenesis. These observations suggest that the developing respiratory center is particularly sensitive to loss of necdin activity and may reflect abnormalities of respiratory rhythm-generating neurons or conditioning neuromodulatory drive. We propose that necdin deficiency may contribute to observed respiratory abnormalities in individuals with Prader-Willi syndrome through a similar suppression of central respiratory drive.

Ontogeny of the pre-Bötzinger complex in perinatal rats.

The aim of this study was to provide a systematic examination of the ontogenesis of the mammalian respiratory rhythm generating center, the pre-Bötzinger complex (pre-BötC). A combination of immunohistochemical markers and electrophysiological recordings was used to determine the time of inception of the pre-BötC and the developmental changes during the perinatal period in rats spanning from embryonic day 15 (E15) to postnatal day 7. The first clear indication of neurons immunopositive for neurokinin-1 receptors (NK1Rs) and somatostatin expression, two proposed markers for pre-BötC neurons, was at approximately E17. Birth dating of neurons in the ventrolateral medulla using 5-bromo-2'-deoxyuridine demonstrated that NK1R-positive neurons populating the area of the pre-BötC during late E16-E18 are born at E12.5-E13.5, approximately 2 d later than adjacent NK1R-positive neurons in the ventrolateral medulla. Extracellular recordings of neuronal populations within the pre-BötC of perinatal medullary slice preparations demonstrated that the onset of rhythmical respiratory discharge commences at approximately E17. Application of substance P, a ligand for NK1R receptors, to the media bathing E17 medullary slice and brainstem-spinal cord preparations resulted in a marked increase in respiratory frequency. These data provide insights into the ontogeny of the pre-BötC, giving fundamental information on the genesis, settlement, and inception of rhythmic activity within the group of neurons proposed to be responsible for the respiratory rhythm generation. Furthermore, this provides the foundation for further analyses of cell lineage, the transcriptional control of respiratory neuronal development, and electrophysiological and pharmacological properties of the pre-BötC during the prenatal period.

Malfunction of respiratory-related neuronal activity in Na+, K+-ATPase alpha2 subunit-deficient mice is attributable to abnormal Cl- homeostasis in brainstem neurons.

Na+, K+-ATPase 2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2-/-) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2-/- was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2-/- fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2-/- than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2-/-. Lack of inhibitory neural activities in VLM of Atp1a2-/- was observed by optical recordings. High intracellular Cl- concentrations in neurons of the VLM of Atp1a2-/- were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Na+, K+-ATPase alpha2 subunit and KCC2, which excludes Cl- from the cytosol in respiratory center neurons.

Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.

Early organization of the vertebrate brainstem is characterized by cellular segmentation into compartments, the rhombomeres, which follow a metameric pattern of neuronal development. Expression of the homeobox genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they play an important role in the establishment of rhombomere-specific neuronal patterns. However, segmentation is a transient feature, and a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages. Thus, it is not clear whether the early rhombomeric pattern of Hox expression has any influence on the establishment of the neuronal circuitry of the mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of mouse Hoxa1(-/-) mutants has focused on early alterations of hindbrain segmentation and patterning. Here, we show that ectopic neuronal groups in the hindbrain of Hoxa1(-/-) mice establish a supernumerary neuronal circuit that escapes apoptosis and becomes functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2 identity, and integrates the rhythm-generating network controlling respiration at birth. This is the first demonstration that changes in Hox expression patterns allow the selection of novel neuronal circuits regulating vital adaptive behaviors. The implications for the evolution of brainstem neural networks are discussed.

Development of respiratory rhythm generation in ectothermic vertebrates.

Compared with birds and mammals, very little is known about the development and regulation of respiratory rhythm generation in ectothermic vertebrates. The development and regulation of respiratory rhythm generation in ectothermic vertebrates (fish, amphibians and reptiles) should provide insight into the evolution of these mechanisms. One useful model for examining the development of respiratory rhythm generation in ectothermic vertebrates has emerged from studies with the North American bullfrog (Rana catesbeiana). A major advantage of bullfrogs as a comparative model for respiratory rhythm generation is that respiratory output may be measured at all stages of development, both in vivo and in vitro. An emerging view of recent studies in developing bullfrogs is that many of the mechanisms of respiratory rhythm generation are very similar to those seen in birds and mammals. The overall conclusion from these studies is that respiratory rhythm generation during development may be highly conserved during evolution. The development of respiratory rhythm generation in mammals may, therefore, reflect the antecedent mechanisms seen in ectothermic vertebrates. The main focus of this brief review is to discuss recent data on the development of respiratory rhythm generation in ectothermic vertebrates, with particular emphasis on the North American bullfrog (R. catesbeiana) as a model.

Perinatal maturation of the respiratory rhythm generator in mammals: from experimental results to computational simulation.

The survival of neonatal mammals requires a correct function of the respiratory rhythm generator (RRG), and therefore, the processes that control its prenatal maturation are of vital importance. In humans, lambs and rodents, foetal breathing movements (FBMs) occur early during gestation, are episodic, sensitive to bioamines, central hypoxia and inputs from CNS upper structures, and evolve with developmental age. In vitro, the foetal rodent RRG studied in preparations where the upper CNS structures are lacking continuously produces a rhythmic command, which is sensitive to hypoxia and bioaminergic inputs. The rhythm is slow with variable periods 4 days before birth. It becomes faster 2 days before birth, similar to the postnatal rhythm. Compelling evidence suggests that a region of the RRG called the preBötzinger complex (PBC) contains respiratory pacemaker neurones which play a primary role in perinatal rhythmogenesis. Although the RRG functions during early gestation, no pacemakers are found in the putative PBC area and its electrical stimulation and lesion do not affect the early foetal rhythm. To know whether the early foetal and perinatal rhythms originate from either pacemaker neurones or network connection properties, and to know which maturational processes might explain the appearance of PBC pacemakers and the rhythm increase during perinatal development, we computationally modelled maturing RRG. Our model shows that both network noise and persistent sodium conductance are crucial for rhythmogenesis and that a slight increase in the persistent sodium conductance can solve the pacemaker versus network dilemma in a noisy network.

From hindbrain segmentation to breathing after birth: developmental patterning in rhombomeres 3 and 4.

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated within central pattern-generating neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the most understood part with respect to the cellular and molecular mechanisms that underlie its development. Hox paralogs and Hox-regulating genes kreisler/mafB and Krox20 are required for the normal formation of rhombomeres in vertebrate embryos. From studies of rhombomeres r3 and r4, the authors review mechanisms whereby these developmental genes may govern the early embryonic development of para-facial neuronal networks and specify patterns of motor activities operating throughout life. A model whereby the regional identity of progenitor cells can be abnormally specified in r3 and r4 after a mutation of these genes is proposed. Novel neuronal circuits may develop from some of these misspecified progenitors while others are eliminated, eventually affecting respiration and survival after birth.