Mafa-dependent GABAergic activity promotes mouse neonatal apneas.

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

Genetic identification of a hindbrain nucleus essential for innate vocalization.

Vocalization in young mice is an innate response to isolation or mechanical stimulation. Neuronal circuits that control vocalization and breathing overlap and rely on motor neurons that innervate laryngeal and expiratory muscles, but the brain center that coordinates these motor neurons has not been identified. Here, we show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization in mice. By generating genetically modified newborn mice that specifically lack excitatory NTS neurons, we show that they are both mute and unable to produce the expiratory drive required for vocalization. Furthermore, the muteness of these newborns results in maternal neglect. We also show that neurons of the NTS directly connect to and entrain the activity of spinal (L1) and nucleus ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control expiratory pressure and laryngeal tension, respectively, thereby establishing the essential biomechanical parameters used for vocalization. In summary, our work demonstrates that the NTS is an obligatory component of the neuronal circuitry that transforms breaths into calls.

Brainstem respiratory oscillators develop independently of neuronal migration defects in the Wnt/PCP mouse mutant looptail.

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

Prenatal development of central rhythm generation.

Foetal breathing in mice results from prenatal activity of the two coupled hindbrain oscillators considered to be responsible for respiratory rhythm generation after birth: the pre-Bötzinger complex (preBötC) is active shortly before the onset of foetal breathing; the parafacial respiratory group (e-pF in embryo) starts activity one day earlier. Transcription factors have been identified that are essential to specify neural progenitors and lineages forming each of these oscillators during early development of the neural tube: Hoxa1, Egr2 (Krox20), Phox2b, Lbx1 and Atoh1 for the e-pF; Dbx1 and Evx1 for the preBötC which eventually grow contralateral axons requiring expression of Robo3. Inactivation of the genes encoding these factors leads to mis-specification of these neurons and distinct breathing abnormalities: apneic patterns and loss of central chemosensitivity for the e-pF (central congenital hypoventilation syndrome, CCHS, in humans), complete loss of breathing for the preBötC, right-left desynchronized breathing in Robo3 mutants. Mutations affecting development in more rostral (pontine) respiratory territories change the shape of the inspiratory drive without affecting the rhythm. Other (primordial) embryonic oscillators start in the mouse three days before the e-pF, to generate low frequency (LF) rhythms that are probably required for activity-dependent development of neurones at embryonic stages; in the foetus, however, they are actively silenced to avoid detrimental interaction with the on-going respiratory rhythm. Altogether, these observations provide a strong support to the previously proposed hypothesis that the functional organization of the respiratory generator is specified at early stages of development and is dual in nature, comprising two serially non-homologous oscillators.

Hindbrain interneurons and axon guidance signaling critical for breathing.

Breathing is a bilaterally synchronous behavior that relies on a respiratory rhythm generator located in the brainstem. An essential component of this generator is the preBötzinger complex (preBötC), which paces inspirations. Little is known about the developmental origin of the interneuronal populations forming the preBötC oscillator network. We found that the homeobox gene Dbx1 controls the fate of glutamatergic interneurons required for preBötC rhythm generation in the mouse embryo. We also found that a conditional inactivation in Dbx1-derived cells of the roundabout homolog 3 (Robo3) gene, which is necessary for axonal midline crossing, resulted in left-right de-synchronization of the preBötC oscillator. Together, these findings identify Dbx1-derived interneurons as the core rhythmogenic elements of the preBötC oscillator and indicate that Robo3-dependent guidance signaling in these cells is required for bilaterally synchronous activity.

Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex.

The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

Differential involvement of projection neurons during emergence of spontaneous activity in the developing avian hindbrain.

To better characterize the emergence of spontaneous neuronal activity in the developing hindbrain, spontaneous activity was recorded optically from defined projection neuron populations in isolated preparations of the brain stem of the chicken embryo. Ipsilaterally projecting reticulospinal (RS) neurons and several groups of vestibuloocular (VO) neurons were labeled retrogradely with Calcium Green-1 dextran amine and spontaneous calcium transients were recorded using a charge-coupled-device camera mounted on a fluorescence microscope. Simultaneous extracellular recordings were made from one of the trigeminal motor nerves (nV) to register the occurrence of spontaneous synchronous bursts of activity. Two types of spontaneous activity were observed: synchronous events (SEs), which occurred in register with spontaneous bursts in nV once every few minutes and were tetrodotoxin (TTX) dependent, and asynchronous events (AEs), which occurred in the intervals between SEs and were TTX resistant. AEs occurred developmentally before SEs and were in general smaller and more variable in amplitude than SEs. SEs appeared at the same stage as nV bursts early on embryonic day 4, first in RS neurons and then in VO neurons. All RS neurons participated equally in SEs from the outset, whereas different subpopulations of VO neurons participated differentially, both in terms of the proportion of neurons that exhibited SEs, the fidelity with which the SEs in individual neurons followed the nV bursts, and the developmental stage at which SEs appeared and matured. The results show that spontaneous activity is expressed heterogeneously among hindbrain projection neuron populations, suggesting its differential involvement in the formation of different functional neuronal circuits.

Influence of differential expression of acetylcholinesterase in brain and muscle on respiration.

A mouse strain with a deleted acetylcholinesterase (AChE) gene (AChE knockout) shows a decreased inspiration time and increased tidal volume and ventilation .To investigate the respective roles of AChE in brain and muscle, we recorded respiration by means of whole-body plethysmography in knockout mice with tissue selective deletions in AChE expression. A mouse strain with the anchoring domains of AChE deleted (del E5+6 knockout mice) has very low activity in the brain and neuromuscular junction, but increased monomeric AChE in serum. A mouse strain with deletion of the muscle specific region of AChE (del i1RR knockout mice) exhibits no expression in muscle, but unaltered expression in the central nervous system. Neither strain exhibits the pronounced phenotypic traits observed in the complete AChE knockout strain. A third strain lacking the anchor molecule PRiMA, has no functional AChE and butyrylcholinesterase (BChE) in brain and an unaltered respiratory function. BChE inhibition by bambuterol decreases tidal volume and body temperature in del E5+6 and i1RR knockout strains, but not in PRiMA deletion or wild-type controls. We find that: (1) deletion of the full AChE gene is required for a pronounced alteration in respiratory phenotype, (2) BChE is involved in respiratory muscles contraction and temperature control in del E5+6 and i1RR knockout mice, and (3) AChE expression requiring a gene product splice to either exons 5 and 6 or regulated by intron1 influences temperature control.

Brain-derived neurotrophic factor enhances fetal respiratory rhythm frequency in the mouse preBötzinger complex in vitro.

Brain-derived neurotrophic factor (BDNF) is required during the prenatal period for normal development of the respiratory central command; however, the underlying mechanisms remain unknown. To approach this issue, the present study examined BDNF regulation of fetal respiratory rhythm generation in the preBötzinger complex (preBötC) of the mouse, using transverse brainstem slices obtained from prenatal day 16.5 animals. BDNF application (100 ng/mL, 15 min) increased the frequency of rhythmic population activity in the preBötC by 43%. This effect was not observed when preparations were exposed to nerve growth factor (100 ng/mL, 30 min) or pretreated with the tyrosine kinase inhibitor K252a (1 h, 200 nm), suggesting that BDNF regulation of preBötC activity requires activation of its cognate tyrosine receptor kinase, TrkB. Consistent with this finding, single-cell reverse transcription-polymerase chain reaction experiments showed that one third of the rhythmically active preBötC neurons analysed expressed TrkB mRNA. Moreover, 20% expressed BDNF mRNA, suggesting that the preBötC is both a target and a source of BDNF. At the network level, BDNF augmented activity of preBötC glutamatergic neurons and potentiated glutamatergic synaptic drives in respiratory neurons by 34%. At the cellular level, BDNF increased the activity frequency of endogenously bursting neurons by 53.3% but had no effect on basal membrane properties of respiratory follower neurons, including the Ih current. Our data indicate that BDNF signalling through TrkB can acutely modulate fetal respiratory rhythm in association with increased glutamatergic drive and bursting activity in the preBötC.

Distinct roles of Hoxa2 and Krox20 in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening.

BACKGROUND: Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of Hoxa2, the only Hox gene expressed up to r2, and of Krox20, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of Hoxa2 by Krox20 in r3. RESULTS: We found that Hoxa2 mutants displayed an impaired oro-buccal reflex, similarly to Krox20 mutants. In contrast, while Krox20 is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency, Hoxa2 inactivation did not affect neonatal breathing frequency. Instead, we found that Hoxa2-/- but not Krox20-/- mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of Hoxa2 expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory. CONCLUSION: Thus, inspiratory shaping and respiratory frequency are under the control of distinct Hox-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.

Exposure to retinoic acid at the onset of hindbrain segmentation induces episodic breathing in mice.

Hyperpnoeic episodic breathing (HEB), a cyclic waxing and waning of breathing, has been widely reported in pre-term neonates, patients with Joubert syndrome and adults (Cheyne-Stokes respiration) with congestive heart failure and brainstem infarction. We now provide a developmental mouse model of neonatal HEB. We used retinoic acid (RA) (0.5-10 mg/kg of maternal weight) to alter embryonic development of the respiratory neuronal network at the onset of hindbrain segmentation (7.5 days post-coitum). HEB was observed in vivo after RA treatment during post-natal days 1-7 but not in control animals. HEB persisted after reduction of the chemoafferent input by hypocapnic hyperoxia (100% O(2)). A large increase and decrease of the rhythm resembling an HEB episode was induced in vitro by stimulating the parafacial respiratory oscillator in treated but not in control neonates. Post-natal localization of the superior cerebellar peduncle and adjacent dorsal tegmentum was found to be abnormal in the pons of RA-treated juvenile mice. Thus, early developmental specifications in the rostral hindbrain are required for the development of neurones that stabilize the function of the respiratory rhythm generator, thereby preventing HEB during post-natal maturation.

Acute role of the brain-derived neurotrophic factor (BDNF) on the respiratory neural network activity in mice in vitro.

In humans, several pathologies are associated with disturbances of the respiratory control, some of them including alteration in the brain-derived neurotrophic factor (BDNF) signalling pathway. BDNF has long been known as a neurotrophic factor involved in survival, differentiation and maintenance of neuronal populations in the peripheral and central nervous system. More recently BDNF has also been discovered to be a potent neuromodulator with acute effects on neuronal excitability and synaptic plasticity. Animals deleted for the gene encoding BDNF exhibit respiratory alteration suggesting an important but yet undefined role of the neurotrophin in respiratory rhythmogenesis either by a trophic and/or an acute action. The possibility that BDNF might exert an acute regulatory role on the rhythmic activity of the respiratory generator of the pre-Bötzinger complex has been recently examined in newborn mice in vitro. Results obtained, reviewed in the present paper, will help getting insights in respiratory rhythm regulatory mechanisms that involve BDNF signalling.

Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius.

The transmembrane receptor Patched (Ptc) mediates the action of the diffusing factor Sonic hedgehog (Shh), which is implicated in establishing morphogenetic gradients during embryonic development. Whereas alteration of Ptc function is associated with developmental abnormalities and brain tumors, its functional activity and roles in the adult brain have yet to be elucidated. Here we describe the complementary pattern of Shh and Ptc expression in the rat dorsal vagal motor nucleus and the ventrolateral nucleus tractus solitarius (vNTS), respectively. Those two interconnected structures regulate the cardiorespiratory function during hypoxia. Bath application of a subnanomolar concentration of aminoterminal Shh protein (ShhN) to a slice preparation of the vNTS induces a rapid decrease in neuronal firing followed by a bursting activity that propagates in the neuronal network. Intracellular current injections show that bursts result from an action on the neuronal membrane electro-responsiveness. Both inhibiting and bursting effects are blocked by the monoclonal Shh antibody 5E1 and may require the Ptc binding site of ShhN. Thus, ShhN acting on specific neuronal sites controls electrophysiological properties of differentiated neurons of the vNTS. We speculate on a retrocontrol of cardiorespiratory signals in the vNTS, by Shh generated in dorsal vagal motoneurons.

Exaggeration of epileptic-like patterns by nicotine receptor activation during the GABA withdrawal syndrome.

To understand how nicotinic cholinergic receptors may participate in epileptic seizures, we tested the effects of nicotine and of the competitive nicotinic antagonists dihydro-beta-erythroidine and alpha-bungarotoxin on synaptic paroxysmal depolarization shifts (PDSs) and intrinsic bursts of action potentials recorded in slices from rats presenting a cortical status epilepticus. This model named GABA-withdrawal syndrome (GWS) appears consecutive to the interruption of a prolonged intracortical GABA infusion. Effects of both nicotinic antagonists suggest a distinct involvement of alpha4-beta2 and alpha7 subunits in shaping individual PDSs and patterning repetitive bursts. On one hand, in GWS rats, an increase of PDS latency and prolongation of PDS and bursts were induced by nicotine and reduced by dihydro-beta-erythroidine, but not by alpha-bungarotoxin. The K+ blocker tetraethylammonium also increased duration without changing latency. Thus, dihydro-beta-erythroidine-sensitive receptors exert distinct controls on the presynaptic generation of PDS and on the process which terminates PDSs and bursts. On the other hand, alpha-bungarotoxin depolarized neurons and generated rhythmic discharges of clustered bursts. Clustered bursts were also observed in slices obtained from GWS rats treated with the acetylcholinesterase inhibitor eserine. We suggest that both dihydro-beta-erythroidine and alpha-bungarotoxin-sensitive sites control paroxysmic activities in GWS and could be involved in some human and animal epilepsies presenting mutations of nicotinic cholinergic receptors.

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.

Effects of acetylcholinesterase and butyrylcholinesterase inhibition on breathing in mice adapted or not to reduced acetylcholinesterase.

We investigated the contributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition to the respiratory dysfunction produced by organophosphates in mice which were adapted or not to low AChE activity. Effects of acute selective inhibition of AChE and BChE on ventilation measured by whole-body plethysmography were compared in mice with either normal AChE activity (wild-type), or mice adapted to a null AChE activity (homozygotes for AChE gene deletion) or adapted to an intermediate level of activity (heterozygotes). In wild-type mice acute reduction of AChE by Huperzine A (1 mg/kg) to the level found in asymptomatic heterozygotes, induced tremors but no respiratory depression, whereas the same dose of Huperzine in heterozygote animals further reduced AChE activity, increased tidal volume (V(T)) and decreased breathing frequency (f(R)). A lethal dose of Huperzine in wild-type mice augmented these respiratory effects, but was ineffective in homozygotes. BChE inhibition by bambuterol was ineffective in wild-type mice and heterozygotes, decreased V(T) in homozygotes adapted to null AChE activity but increased V(T) in wild-type mice acutely treated with Huperzine, also aggravating the cholinergic syndrome. We conclude that: (1) Huperzine does not perturb respiration at a dose inhibiting 40% of AChE, and at a lethal dose does not affect any other enzyme important for respiration; (2) Respiratory function is more sensitive to anticholinesterases in heterozygotes than in wild-type mice; (3) BChE may play distinct roles in respiratory function, because its inhibition has opposite effects on tidal volume depending on whether the mouse has adapted to null AChE or whether AChE has been lowered acutely; (4) BChE inhibition may contribute to the respiratory toxicity of organophosphates.