Persistent Nav1.1 and Nav1.6 currents drive spinal locomotor functions through nonlinear dynamics.

Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identify the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The inhibition of Nav1.6, but not of Nav1.1, in motoneurons impairs INaP, bistability, postural tone, and locomotor performance. In interneurons of the rhythmogenic CPG region, both Nav1.6 and Nav1.1 equally mediate INaP. Inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG.

Chronic Intermittent Hypoxia Alters Local Respiratory Circuit Function at the Level of the preBötzinger Complex.

Chronic intermittent hypoxia (CIH) is a common state experienced in several breathing disorders, including obstructive sleep apnea (OSA) and apneas of prematurity. Unraveling how CIH affects the CNS, and in turn how the CNS contributes to apneas is perhaps the most challenging task. The preBötzinger complex (preBötC) is a pre-motor respiratory network critical for inspiratory rhythm generation. Here, we test the hypothesis that CIH increases irregular output from the isolated preBötC, which can be mitigated by antioxidant treatment. Electrophysiological recordings from brainstem slices revealed that CIH enhanced burst-to-burst irregularity in period and/or amplitude. Irregularities represented a change in individual fidelity among preBötC neurons, and changed transmission from preBötC to the hypoglossal motor nucleus (XIIn), which resulted in increased transmission failure to XIIn. CIH increased the degree of lipid peroxidation in the preBötC and treatment with the antioxidant, 5,10,15,20-Tetrakis (1-methylpyridinium-4-yl)-21H,23H-porphyrin manganese(III) pentachloride (MnTMPyP), reduced CIH-mediated irregularities on the network rhythm and improved transmission of preBötC to the XIIn. These findings suggest that CIH promotes a pro-oxidant state that destabilizes rhythmogenesis originating from the preBötC and changes the local rhythm generating circuit which in turn, can lead to intermittent transmission failure to the XIIn. We propose that these CIH-mediated effects represent a part of the central mechanism that may perpetuate apneas and respiratory instability, which are hallmark traits in several dysautonomic conditions.

KCNK5 channels mostly expressed in cochlear outer sulcus cells are indispensable for hearing.

In the cochlea, K(+) is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K(+) channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5(-/-) mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5(-/-) mice have a normal endolymphatic [K(+)] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K(+) secretion in stria vascularis have normal expression in Kcnk5(-/-) mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K(+) recycling along the outer sulcus lateral route.

Prostaglandin E2 differentially modulates the central control of eupnoea, sighs and gasping in mice.

Prostaglandin E2 (PGE2) augments distinct inspiratory motor patterns, generated within the preBötzinger complex (preBötC), in a dose-dependent way. The frequency of sighs and gasping are stimulated at low concentrations, while the frequency of eupnoea increases only at high concentrations. We used in vivo microinjections into the preBötC and in vitro isolated brainstem slice preparations to investigate the dose-dependent effects of PGE2 on the preBötC activity. Synaptic measurements in whole cell voltage clamp recordings of inspiratory neurons revealed no changes in inhibitory or excitatory synaptic transmission in response to PGE2 exposure. In current clamp recordings obtained from inspiratory neurons of the preBötC, we found an increase in the frequency and amplitude of bursting activity in neurons with intrinsic bursting properties after exposure to PGE2. Riluzole, a blocker of the persistent sodium current, abolished the effect of PGE2 on sigh activity, while flufenamic acid, a blocker of the calcium-activated non-selective cation conductance, abolished the effect on eupnoeic activity caused by PGE2. Prostaglandins are important regulators of autonomic functions in the mammalian organism. Here we demonstrate in vivo that prostaglandin E2 (PGE2) can differentially increase the frequency of eupnoea (normal breathing) and sighs (augmented breaths) when injected into the preBötzinger complex (preBötC), a medullary area that is critical for breathing. Low concentrations of PGE2 (100-300 nm) increased the sigh frequency, while higher concentrations (1-2 µm) were required to increase the eupnoeic frequency. The concentration-dependent effects were similarly observed in the isolated preBötC. This in vitro preparation also revealed that riluzole, a blocker of the persistent sodium current (INap), abolished the modulatory effect on sighs, while flufenamic acid, an antagonist for the calcium-activated non-selective cation conductance (ICAN ) abolished the effect of PGE2 on fictive eupnoea at higher concentrations. At the cellular level PGE2 significantly increased the amplitude and frequency of intrinsic bursting in inspiratory neurons. By contrast PGE2 affected neither excitatory nor inhibitory synaptic transmission. We conclude that PGE2 differentially modulates sigh, gasping and eupnoeic activity by differentially increasing INap and ICAN currents in preBötC neurons.

When norepinephrine becomes a driver of breathing irregularities: how intermittent hypoxia fundamentally alters the modulatory response of the respiratory network.

Neuronal networks are endogenously modulated by aminergic and peptidergic substances. These modulatory processes are critical for maintaining normal activity and adapting networks to changes in metabolic, behavioral, and environmental conditions. However, disturbances in neuromodulation have also been associated with pathologies. Using whole animals (in vivo) and functional brainstem slices (in vitro) from mice, we demonstrate that exposure to acute intermittent hypoxia (AIH) leads to fundamental changes in the neuromodulatory response of the respiratory network located within the preBötzinger complex (preBötC), an area critical for breathing. Norepinephrine, which normally regularizes respiratory activity, renders respiratory activity irregular after AIH. Respiratory irregularities are caused both in vitro and in vivo by AIH, which increases synaptic inhibition within the preBötC when norepinephrine is endogenously or exogenously increased. These irregularities are prevented by blocking synaptic inhibition before AIH. However, regular breathing cannot be reestablished if synaptic inhibition is blocked after AIH. We conclude that subtle changes in synaptic transmission can have dramatic consequences at the network level as endogenously released neuromodulators that are normally adaptive become the drivers of irregularity. Moreover, irregularities in the preBötC result in irregularities in the motor output in vivo and in incomplete transmission of inspiratory activity to the hypoglossus motor nucleus. Our finding has basic science implications for understanding network functions in general, and it may be clinically relevant for understanding pathological disturbances associated with hypoxic episodes such as those associated with myocardial infarcts, obstructive sleep apneas, apneas of prematurity, Rett syndrome, and sudden infant death syndrome.

TASK-2 channels contribute to pH sensitivity of retrotrapezoid nucleus chemoreceptor neurons.

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H(+) via an unidentified pH-sensitive background K(+) channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K(+) channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or ß-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2(-/-) mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2(-/-) mice selected based on ß-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K(+) currents were reduced in amplitude in RTN neurons from TASK-2(-/-) mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2(-/-) mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.

Stable respiratory activity requires both P/Q-type and N-type voltage-gated calcium channels.

P/Q-type voltage-gated calcium channels (Ca(v)2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Ca(v)2.1 pore-forming α(1A) subunit (α(1A)⁻/⁻) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolated in vitro brainstem slice preparation containing the pre-Bötzinger complex. The loss of Ca(v)2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Ca(v)2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α(1A)⁻/⁻ mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α(1A)⁻/⁻ mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α(1A)⁻/⁻ mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α(1A)⁻/⁻ mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.

Fatal breathing dysfunction in a mouse model of Leigh syndrome.

Leigh syndrome (LS) is a subacute necrotizing encephalomyelopathy with gliosis in several brain regions that usually results in infantile death. Loss of murine Ndufs4, which encodes NADH dehydrogenase (ubiquinone) iron-sulfur protein 4, results in compromised activity of mitochondrial complex I as well as progressive neurodegenerative and behavioral changes that resemble LS. Here, we report the development of breathing abnormalities in a murine model of LS. Magnetic resonance imaging revealed hyperintense bilateral lesions in the dorsal brain stem vestibular nucleus (VN) and cerebellum of severely affected mice. The mutant mice manifested a progressive increase in apnea and had aberrant responses to hypoxia. Electrophysiological recordings within the ventral brain stem pre-Bötzinger respiratory complex were also abnormal. Selective inactivation of Ndufs4 in the VN, one of the principle sites of gliosis, also led to breathing abnormalities and premature death. Conversely, Ndufs4 restoration in the VN corrected breathing deficits and prolonged the life span of knockout mice. These data demonstrate that mitochondrial dysfunction within the VN results in aberrant regulation of respiration and contributes to the lethality of Ndufs4-knockout mice.

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.

Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

Chapter 3--networks within networks: the neuronal control of breathing.

Breathing emerges through complex network interactions involving neurons distributed throughout the nervous system. The respiratory rhythm generating network is composed of micro networks functioning within larger networks to generate distinct rhythms and patterns that characterize breathing. The pre-Bötzinger complex, a rhythm generating network located within the ventrolateral medulla assumes a core function without which respiratory rhythm generation and breathing cease altogether. It contains subnetworks with distinct synaptic and intrinsic membrane properties that give rise to different types of respiratory rhythmic activities including eupneic, sigh, and gasping activities. While critical aspects of these rhythmic activities are preserved when isolated in in vitro preparations, the pre-Bötzinger complex functions in the behaving animal as part of a larger network that receives important inputs from areas such as the pons and parafacial nucleus. The respiratory network is also an integrator of modulatory and sensory inputs that imbue the network with the important ability to adapt to changes in the behavioral, metabolic, and developmental conditions of the organism. This review summarizes our current understanding of these interactions and relates the emerging concepts to insights gained in other rhythm generating networks.

The role of spiking and bursting pacemakers in the neuronal control of breathing.

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I(NaP)) and the calcium-activated non-specific cation current (I(CAN)), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.

Graded reductions in oxygenation evoke graded reconfiguration of the isolated respiratory network.

Neurons depend on aerobic metabolism, yet are very sensitive to oxidative stress and, as a consequence, typically operate in a low O(2) environment. The balance between blood flow and metabolic activity, both of which can vary spatially and dynamically, suggests that local O(2) availability markedly influences network output. Yet the understanding of the underlying O(2)-sensing mechanisms is limited. Are network responses regulated by discrete O(2)-sensing mechanisms or, rather, are they the consequence of inherent O(2) sensitivities of mechanisms that generate the network activity? We hypothesized that a broad range of O(2) tensions progressively modulates network activity of the pre-Bötzinger complex (preBötC), a neuronal network critical to the central control of breathing. Rhythmogenesis was measured from the preBötC in transverse neonatal mouse brain stem slices that were exposed to graded reductions in O(2) between 0 and 95% O(2), producing tissue oxygenation values ranging from 20 ± 18 (mean ± SE) to 440 ± 56 Torr at the slice surface, respectively. The response of the preBötC to graded changes in O(2) is progressive for some metrics and abrupt for others, suggesting that different aspects of the respiratory network have different sensitivities to O(2).

N-methyl-D-aspartate-induced oscillatory properties in neocortical pyramidal neurons from patients with epilepsy.

N-Methyl-D-aspartate (NMDA) receptors have been implicated in epileptogenesis, but how these receptors contribute to epilepsy remains unknown. In particular, their role is likely to be complicated because of their voltage-dependent behavior. Here, the authors investigate how activation of NMDA receptors can affect the intrinsic production of oscillation and the resonance properties of neocortical pyramidal neurons from children with intractable epilepsy. Intracellular whole-cell patch clamp recordings in cortical slices from these patients revealed that pyramidal neurons do not produce spontaneous oscillation under control conditions. However, they did exhibit resonance around 1.5 Hz. On NMDA receptor activation, with bath-applied NMDA (10 µM), the majority of neurons produced voltage-dependent intrinsic oscillation associated with a change in the stability of the neuronal system as reflected by the whole-cell I-V curve. Furthermore, the degree of resonance was amplified while the frequency of resonance was shifted to lower frequencies (∼1 Hz) in NMDA. These results suggest that NMDA receptors may both promote the production of low-frequency oscillation and sharpen the response of the cell to lower frequencies. Both these behaviors may be amplified in tissue from patients with epilepsy, resulting in an increased propensity to generate seizures.

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.

Breathing deficits of the Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a multigenic disorder caused by the loss of paternal expression of genes in the 15q11-q13 region. It is a complex and progressive disease. From birth, patients present breathing disorders (apnea, rhythm instability, hypoventilation and blunted response to changes in CO(2) or O(2)). Recent advances allowing early diagnosis permitted to prevent obesity of PWS patients and to alleviate some symptoms mainly by growth hormone therapy but there is no therapy to alleviate all symptoms and respiratory distress in particular. To further understand PWS several mutant mice, in which each candidate gene has been separately inactivated, have been developed and shown variable symptoms depending on the genes inactivated. Among them the Necdin deficiency appears to be responsible for breathing disorders. In Necdin deficient mice, respiratory defects resembling PWS have been shown in vivo and in vitro. These defects are central from origin and are correlated with biochemical and anatomical anomalies of the respiratory regulatory systems including serotonergic alterations.

Early breathing defects after moderate hypoxia or hypercapnia in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disease caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2) and accompanied by complex symptoms, including erratic breathing and life-threatening apnoeas. In Mecp2-deficient male mice (Mecp2(-/y)), breathing is normal at birth but becomes altered after postnatal day 30 (P30), with erratic rhythm and apnoeas aggravating until death at around P60. Using plethysmography, we analyzed breathing of unrestrained wild type mice and Mecp2(-/y) at P15, P25 and P30 under air and under short-lasting exposure to moderate hypoxia or hypercapnia. In Mecp2(-/y) with normal resting ventilation, we report exacerbated respiratory responses to hypoxia at P30 and transient apnoeas with erratic rhythm after hypoxia and hypercapnia at P30, P25 and occasionally P15. Then environmental factors may induce breathing defects well before than expected in Mecp2(-/y) and possibly in RTT patients. We therefore suggest avoiding exposure of young RTT patients to environmental situations where they may encounter moderate hypoxia or hypercapnia.

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.

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.

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.

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.