[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.

The vesicular glutamate transporter VGLUT3 contributes to protection against neonatal hypoxic stress.

Neonates respond to hypoxia initially by increasing ventilation, and then by markedly decreasing both ventilation (hypoxic ventilatory decline) and oxygen consumption (hypoxic hypometabolism). This latter process, which vanishes with age, reflects a tight coupling between ventilatory and thermogenic responses to hypoxia. The neurological substrate of hypoxic hypometabolism is unclear, but it is known to be centrally mediated, with a strong involvement of the 5-hydroxytryptamine (5-HT, serotonin) system. To clarify this issue, we investigated the possible role of VGLUT3, the third subtype of vesicular glutamate transporter. VGLUT3 contributes to glutamate signalling by 5-HT neurons, facilitates 5-HT transmission and is expressed in strategic regions for respiratory and thermogenic control. We therefore assumed that VGLUT3 might significantly contribute to the response to hypoxia. To test this possibility, we analysed this response in newborn mice lacking VGLUT3 using anatomical, biochemical, electrophysiological and integrative physiology approaches. We found that the lack of VGLUT3 did not affect the histological organization of brainstem respiratory networks or respiratory activity under basal conditions. However, it impaired respiratory responses to 5-HT and anoxia, showing a marked alteration of central respiratory control. These impairments were associated with altered 5-HT turnover at the brainstem level. Furthermore, under cold conditions, the lack of VGLUT3 disrupted the metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia. We conclude that VGLUT3 expression is dispensable under basal conditions but is required for optimal response to hypoxic stress in neonates.

Isoflurane anesthesia precipitates tauopathy and upper airways dysfunction in pre-symptomatic Tau.P301L mice: possible implication for neurodegenerative diseases.

The postoperative cognitive decline resulting from volatile anesthesia is gaining acceptance as a major health problem. The common anesthetic isoflurane is suspected to precipitate neurodegeneration in Alzheimer's disease by unknown mechanisms. We previously validated that 8month old Tau.P301L mice suffer upper airways defects related to tauopathy within the Kolliker-Fuse nucleus that controls upper airways function. We now report that isoflurane anesthesia in young, pre-symptomatic Tau.P301L mice triggered precocious upper airways defects and tauopathy in several brainstem nuclei, including the nucleus ambiguus that contains upper airways motor neurons and the Kolliker-Fuse. The prescription drug memantine, identified as an NMDA receptor antagonist, prevented the post-anesthesia upper airways dysfunction and alleviated tauopathy in the nucleus ambiguus and Kolliker-Fuse. We further identified protocols of anesthesia in young Tau.P301L mice that mitigated adverse effects of isoflurane anesthesia. Thus, our experimental findings in a validated mouse model for tauopathy demonstrate the link between isoflurane anesthesia, earlier onset of tauopathy and earlier onset of functional deficits, highlight the implication of NMDA-receptors in the mechanisms mediating the adverse effects of isoflurane, and potentially identify safer protocols for anesthesia in patients with tauopathy.

Teashirt 3 regulates development of neurons involved in both respiratory rhythm and airflow control.

Neonatal breathing in mammals involves multiple neuronal circuits, but its genetic basis remains unclear. Mice deficient for the zinc finger protein Teashirt 3 (TSHZ3) fail to breathe and die at birth. Tshz3 is expressed in multiple areas of the brainstem involved in respiration, including the pre-Bötzinger complex (preBötC), the embryonic parafacial respiratory group (e-pF), and cranial motoneurons that control the upper airways. Tshz3 inactivation led to pronounced cell death of motoneurons in the nucleus ambiguus and induced strong alterations of rhythmogenesis in the e-pF oscillator. In contrast, the preBötC oscillator appeared to be unaffected. These deficits result in impaired upper airway function, abnormal central respiratory rhythm generation, and altered responses to pH changes. Thus, a single gene, Tshz3, controls the development of diverse components of the circuitry required for breathing.

Upper airway dysfunction of Tau-P301L mice correlates with tauopathy in midbrain and ponto-medullary brainstem nuclei.

Tauopathy comprises hyperphosphorylation of the microtubule-associated protein tau, causing intracellular aggregation and accumulation as neurofibrillary tangles and neuropil treads. Some primary tauopathies are linked to mutations in the MAPT gene coding for protein tau, but most are sporadic with unknown causes. Also, in Alzheimer's disease, the most frequent secondary tauopathy, neither the cause nor the pathological mechanisms and repercussions are understood. Transgenic mice expressing mutant Tau-P301L suffer cognitive and motor defects and die prematurely from unknown causes. Here, in situ electrophysiology in symptomatic Tau-P301L mice (7-8 months of age) revealed reduced postinspiratory discharges of laryngeal motor outputs that control laryngeal constrictor muscles. Under high chemical drive (hypercapnia), postinspiratory discharge was nearly abolished, whereas laryngeal inspiratory discharge was increased disproportionally. The latter may suggest a shift of postinspiratory laryngeal constrictor activity into inspiration. In vivo double-chamber plethysmography of Tau-P301L mice showed significantly reduced respiratory airflow but significantly increased chest movements during baseline breathing, but particularly in hypercapnia, confirming a significant increase in inspiratory resistive load. Histological analysis demonstrated hyperphosphorylated tau in brainstem nuclei, directly or indirectly involved in upper airway motor control (i.e., the Kölliker-Fuse, periaqueductal gray, and intermediate reticular nuclei). In contrast, young Tau-P301L mice did not show breathing disorders or brainstem tauopathy. Consequently, in aging Tau-P301L mice, progressive upper airway dysfunction is linked to progressive tauopathy in identified neural circuits. Because patients with tauopathy suffer from upper airway dysfunction, the Tau-P301L mice can serve as an experimental model to study disease-specific synaptic dysfunction in well defined functional neural circuits.

Necdin plays a role in the serotonergic modulation of the mouse respiratory network: implication for Prader-Willi syndrome.

Prader-Willi syndrome is a neurogenetic disease resulting from the absence of paternal expression of several imprinted genes, including NECDIN. Prader-Willi children and adults have severe breathing defects with irregular rhythm, frequent sleep apneas, and blunted respiratory regulations. For the first time, we show that Prader-Willi infants have sleep apneas already present at birth. In parallel, in wild-type and Necdin-deficient mice, we studied the respiratory system with in vivo plethysmography, in vitro electrophysiology, and pharmacology. Because serotonin is known to contribute to CNS development and to affect maturation and function of the brainstem respiratory network, we also investigated the serotonergic system with HPLC, immunohistochemistry, Rabies virus tracing approaches, and primary culture experiments. We report first that Necdin-deficiency in mice induces central respiratory deficits reminiscent of Prader-Willi syndrome (irregular rhythm, frequent apneas, and blunted respiratory regulations), second that Necdin is expressed by medullary serotonergic neurons, and third that Necdin deficiency alters the serotonergic metabolism, the morphology of serotonin vesicles in medullary serotonergic neurons but not the number of these cells. We also show that Necdin deficiency in neonatal mice alters the serotonergic modulation of the respiratory rhythm generator. Thus, we propose that the lack of Necdin expression induces perinatal serotonergic alterations that affect the maturation and function of the respiratory network, inducing breathing deficits in mice and probably in Prader-Willi patients.

Prenatal activation of 5-HT2A receptor induces expression of 5-HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice.

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

8-OH-DPAT suppresses spontaneous central apneas in the C57BL/6J mouse strain.

Apneas are common and prognostically relevant disorders of the central control of breathing, but pharmacological interventions are dissatisfying. The respiratory phenotype of C57BL/6J mice is characterized by the occurrence of spontaneous central apneas with laryngeal closure. In the present study we investigated the impact of the 5-HT(1A) receptor agonist 8-OH-DPAT on apneas in C57BL/6J mice, because of the important role of serotonin in the regulation of breathing and previous reports showing that serotonergic drugs can affect central apneas. Whole-body plethysmography in awake, unrestrained mice revealed that intraperitoneal application of 8-OH-DPAT (10microgkg(-1)) decreased the occurrence of spontaneous apneas from 1.91+/-0.25 to 1.05+/-0.05 apneas min(-1). The efficacy of 5-HT(1A) receptor activation was further verified in the in situ working heart-brainstem preparation. Here the apneas occurred at a frequency of 1.33+/-0.19min(-1). Intra-arterial perfusion with 1-2microM 8-OH-DPAT completely abolished spontaneous apneas. These results suggest that 5-HT(1A) receptor activation may be a potential treatment option for central apneas.

Spontaneous central apneas occur in the C57BL/6J mouse strain.

Despite the clinical significance of central apneas in a wide range of disorders little is known about their pathogenesis. Research in this field has been hindered by the lack of appropriate animal models. Our goal was to determine whether the C57BL/6J mouse strain, which has an inherited predisposition for dysrhythmic breathing, exhibits spontaneous apneas. In vivo plethysmography of unanesthetized, unrestrained adult C57BL/6J mice revealed a regular occurrence of spontaneous apneas. In situ recordings from respiratory outputs (phrenic, vagal, hypoglossal nerves) in the working heart-brainstem preparation (WHBP) also showed spontaneous central apneas accompanied by laryngeal closure as indicated by tonic vagal postinspiratory activity and increase in subglottal pressure. The apneas were further characterized by a hypoglossal discharge with delayed onset compared to the tonic vagal postinspiratory activity. We conclude that spontaneous central apneas with active laryngeal closure occur in C57BL/6J mice. This mouse strain is a useful animal model to study neuronal mechanisms that underlie the generation of spontaneous central apneas.

Oral treatment with desipramine improves breathing and life span in Rett syndrome mouse model.

Rett syndrome is a neurodevelopmental disease due to Mecp2 gene mutations that is associated to complex neurological symptoms, with bioaminergic deficits and life-threatening apneas related to sudden and unexpected death. In male mice, Mecp2-deficiency similarly induces medullary bioaminergic deficits, severe apneas and short life span. Here, we show that long-term oral treatment of Mecp2-deficient male mice with desipramine, an old drug of clinical use known to block norepinephrine uptake and to strengthen its synaptic effects, significantly alleviates their breathing symptoms and prolongs their life span. Although these mouse results identify desipramine as the first oral pharmacological treatment potentially able to alleviate breathing symptoms of Rett syndrome, we recommend further studies of desipramine effects in Mecp2-deficient mice before attempting any clinical trials in Rett patients.

Brain nuclei controlling the spinal respiratory motoneurons in the newborn mouse.

A retrograde and transneuronal infection with rabies virus was performed in mouse neonates to locate the central nervous structures involved in the motor command of the spinal respiratory motoneurons and to discriminate the location and hierarchical organization of the neurons in and between these infected central nervous structures.

Necdin gene, respiratory disturbances and Prader-Willi syndrome.

Prader-Willi Syndrome (PWS) is a complex neurogenetic disease with various symptoms, including breathing deficits and possible alteration of serotonin (5HT) metabolism. As PWS results from the absence of paternal expression of several imprinted genes among which NECDIN (Ndn), we examined whether Ndn deficiency in mice induced breathing and 5HT deficits. In vivo, Ndn-deficient mice (Ndn-/-) had irregular breathing, severe apneas and blunted respiratory response to hypoxia. In vitro, medullary preparations from Ndn-/- neonates produced a respiratory-like rhythm that was highly irregular, frequently interrupted and abnormally regulated by central hypoxia. In wild type (wt) and Ndn-/- neonates, immunohistofluorescence and biochemistry revealed that medullary 5HT neurons expressed Ndn in wt and that the medulla contained abnormally high levels of 5HT in Ndn-/-. Thus, our preliminary results fully confirm a primary role of Ndn in PWS, revealing that Ndn-deficiency in mice induces respiratory and 5HT alterations reminiscent of PWS.

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

Diazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network.

MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth.

The genetic basis for the development of brainstem neurons that generate respiratory rhythm is unknown. Here we show that mice deficient for the transcription factor MafB die from central apnea at birth and are defective for respiratory rhythmogenesis in vitro. MafB is expressed in a subpopulation of neurons in the preBötzinger complex (preBötC), a putative principal site of rhythmogenesis. Brainstems from Mafb(-/-) mice are insensitive to preBötC electrolytic lesion or stimulation and modulation of rhythmogenesis by hypoxia or peptidergic input. Furthermore, in Mafb(-/-) mice the preBötC, but not major neuromodulatory groups, presents severe anatomical defects with loss of cellularity. Our results show an essential role of MafB in central respiratory control, possibly involving the specification of rhythmogenic preBötC neurons.

Muscarinic receptors and alpha2-adrenoceptors interact to modulate the respiratory rhythm in mouse neonates.

The respiratory rhythm generator (RRG) is modulated by several endogenous substances, including acetylcholine (ACh) and noradrenaline (NA) that interact in several modulatory processes. To know whether ACh and NA interacted to modulate the RRG activity, we used medullary "en bloc" and slice preparations from neonatal mice where the RRG has been shown to receive a facilitatory modulation from A1/C1 neurons, via a continuous release of endogenous NA and activation of alpha2 adrenoceptors. Applying ACh at 25 microM activated the RRG but ACh had no effects at 50 microM. Applying the ACh receptor agonists nicotine and muscarine facilitated and depressed the RRG, respectively. After yohimbine pre-treatment that blocked the alpha2 facilitation, the nicotinic facilitation was not altered, the muscarinic depression was reversed and ACh 50 microM significantly facilitated the RRG. After L-tyrosine pre-treatment that potentiated the alpha2 facilitation, the muscarinic depression was enhanced. Thus, ACh regulates the RRG activity via nicotinic and muscarinic receptors, the muscarinic receptors interacting with alpha2 adrenoceptors.

Necdin shapes serotonergic development and SERT activity modulating breathing in a mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder that presents with hypotonia and respiratory distress in neonates. The Necdin-deficient mouse is the only model that reproduces the respiratory phenotype of PWS (central apnea and blunted response to respiratory challenges). Here, we report that Necdin deletion disturbs the migration of serotonin (5-HT) neuronal precursors, leading to altered global serotonergic neuroarchitecture and increased spontaneous firing of 5-HT neurons. We show an increased expression and activity of 5-HT Transporter (SERT/Slc6a4) in 5-HT neurons leading to an increase of 5-HT uptake. In Necdin-KO pups, the genetic deletion of Slc6a4 or treatment with Fluoxetine, a 5-HT reuptake inhibitor, restored normal breathing. Unexpectedly, Fluoxetine administration was associated with respiratory side effects in wild-type animals. Overall, our results demonstrate that an increase of SERT activity is sufficient to cause the apneas in Necdin-KO pups, and that fluoxetine may offer therapeutic benefits to PWS patients with respiratory complications.

Mecp2 deficiency disrupts norepinephrine and respiratory systems in mice.

Rett syndrome is a severe X-linked neurological disorder in which most patients have mutations in the methyl-CpG binding protein 2 (MECP2) gene and suffer from bioaminergic deficiencies and life-threatening breathing disturbances. We used in vivo plethysmography, in vitro electrophysiology, neuropharmacology, immunohistochemistry, and biochemistry to characterize the consequences of the MECP2 mutation on breathing in wild-type (wt) and Mecp2-deficient (Mecp2-/y) mice. At birth, Mecp2-/y mice showed normal breathing and a normal number of medullary neurons that express tyrosine hydroxylase (TH neurons). At approximately 1 month of age, most Mecp2-/y mice showed respiratory cycles of variable duration; meanwhile, their medulla contained a significantly reduced number of TH neurons and norepinephrine (NE) content, even in Mecp2-/y mice that showed a normal breathing pattern. Between 1 and 2 months of age, all unanesthetized Mecp2-/y mice showed breathing disturbances that worsened until fatal respiratory arrest at approximately 2 months of age. During their last week of life, Mecp2-/y mice had a slow and erratic breathing pattern with a highly variable cycle period and frequent apneas. In addition, their medulla had a drastically reduced number of TH neurons, NE content, and serotonin (5-HT) content. In vitro experiments using transverse brainstem slices of mice between 2 and 3 weeks of age revealed that the rhythm produced by the isolated respiratory network was irregular in Mecp2-/y mice but could be stabilized with exogenous NE. We hypothesize that breathing disturbances in Mecp2-/y mice, and probably Rett patients, originate in part from a deficiency in noradrenergic and serotonergic modulation of the medullary respiratory network.

Reduced density of functional 5-HT1A receptors in the brain, medulla and spinal cord of monoamine oxidase-A knockout mouse neonates.

Abnormally high brain 5-HT levels in monoamine oxidase-A knockout (MAO-A KO) mouse neonates raise the question of whether the distribution and density of the 5-HT1A receptors (5-HT1AR) expressed in the brain by postnatal day P7 are affected and, if so, whether the 5-HT1A autoreceptors in the dorsal raphe are modified in the same way as the postsynaptic 5-HT1AR present in raphe target structures. [3H]8-OH-DPAT binding and quantitative autoradiography were performed to answer these questions. Binding specificity was first confirmed in adult wild-type mice and rat brain sections. 5-HT1AR binding was then analyzed in four MAO-A mutant vs. five wild-type neonatal brains, from olfactory bulb to cervical cord. Among 12 structures expressing postsynaptic 5-HT1AR in wild-type neonates, the highest densities involved the retrosplenial cortex, entorhinal cortex, and septum (52-46 fmol/mg tissue); low densities occurred in the hippocampus and spinal cord (24 fmol/mg tissue); in addition, the raphe autoreceptor density was only 20 fmol/mg tissue. In mutants, the distribution of postsynaptic 5-HT1AR was unchanged, but an overall decrease in density occurred (-32% to -63%); the raphe autoreceptors decreased in mutants by at least -79%. Data are discussed with reference to the ectopic 5-HT uptake and accumulation reported to occur during the first 10 postnatal days in wild-type and MAO-A KO mice. As previously suggested to explain the raphe autoreceptor loss in 2-month-old MAO-A KO mice, the overall 5-HT1AR down-regulation in mutant pups probably results from extracellular 5-HT excess in both raphe and target structures. The greater the 5-HT excess, the more the functional receptor density decreases.

Possible modulation of the mouse respiratory rhythm generator by A1/C1 neurones.

Although compelling evidence exist that the respiratory rhythm generator is modulated by endogenous noradrenaline released from pontine A5 and A6 neurones, we examined whether medullary catecholaminergic neurones also participated in respiratory rhythm modulation. Experiments were performed in neonatal (postnatal days 0-6, P0-P6) and young mice (P14-P18) using "en bloc" medullary preparations (pons resected) and transverse medullary slices. In "en bloc" preparations, blockade of medullary alpha2 adrenoceptors with yohimbine and activation of catecholamine biosynthesis with L-tyrosine significantly depresses and facilitates the respiratory rhythm, respectively. In slices from neonatal and young mice, blockade of medullary alpha2 adrenoceptors also depressed the respiratory rhythm. Yohimbine local applications and lesion-ablation experiments of the dorsal medulla revealed implication of A1/C1 neurones in the yohimbine depressing effect. Although the mechanisms responsible for the yohimbine-depressing effect remain to be elucidated, our in vitro results in neonatal and young mice suggest that endogenous catecholamines released from A1/C1 neurones participate in respiratory rhythm modulation via medullary alpha2 adrenoceptors.

Endogenous noradrenaline affects the maturation and function of the respiratory network: possible implication for SIDS.

Breathing is a vital, rhythmic motor act that is required for blood oxygenation and oxygen delivery to the whole body. Therefore, the brainstem network responsible for the elaboration of the respiratory rhythm must function from the very first moments of extrauterine life. In this review, it is shown that the brainstem noradrenergic system plays a pivotal role in both the modulation and the maturation of the respiratory rhythm generator. Compelling evidence are reported demonstrating that genetically induced alterations of the noradrenergic system in mice affect the prenatal maturation and the perinatal function of the respiratory rhythm generator and have drastic consequences on postnatal survival. Sudden Infant Death Syndrome (SIDS), the leader cause of infant death in industrialised countries, may result from cardiorespiratory disorders during sleep. As several cases of SIDS have been observed in infants having noradrenergic deficits, a possible link between prenatal alteration of the noradrenergic system, altered maturation and function of the respiratory network and SIDS is suggested.