Mouse models of Kcnq2 dysfunction.

Variants in the Kv7.2 channel subunit encoded by the KCNQ2 gene cause epileptic disorders ranging from a benign form with self-limited epileptic seizures and normal development to severe forms with intractable epileptic seizures and encephalopathy. The biological mechanisms involved in these neurological diseases are still unclear. The disease remains intractable in patients affected by the severe form. Over the past 20 years, KCNQ2 models have been developed to elucidate pathological mechanisms and to identify new therapeutic targets. The diversity of Kcnq2 mouse models has proven invaluable to access neuronal networks and evaluate the associated cognitive deficits. This review summarizes the available models and their contribution to our current understanding of KCNQ2 epileptic disorders.

Deficits of brainstem and spinal cord functions after neonatal hypoxia-ischemia in mice.

BACKGROUND: Perinatal cerebral hypoxia-ischemia (HI) can lead to severe neurodevelopmental disorders. Studies in humans and animal models mainly focused on cerebral outcomes, and little is known about the mechanisms that may affect the brainstem and the spinal cord. Dysfunctions of neuromodulatory systems, such as the serotonergic (5-HT) projections, critical for the development of neural networks, have been postulated to underlie behavioral and motor deficits, as well as metabolic changes. METHODS: The aim of this study was to investigate brainstem and spinal cord functions by means of plethysmography and sensorimotor tests in a neonatal Rice-Vanucci model of HI in mice. We also evaluated bioaminergic contents in central regions dedicated to the motor control of autonomic functions. RESULTS: Mice with cerebral infarct expressed motor disturbances and had a lower body weight and a decreased respiratory frequency than SHAM, suggesting defects of brainstem neural network involved in the motor control of feeding, suckling, swallowing, and respiration. Moreover, our study revealed changes of monoamine and amino acid contents in the brainstem and the spinal cord of HI mice. CONCLUSION: Our results suggest that monoaminergic neuromodulation plays an important role in the physiopathology of HI brain injury that may represent a good therapeutic target.

Isoproterenol modulates expiratory activities in the brainstem spinal cord preparation in neonatal mice in vitro.

Motor behaviors such as breathing required temporal coordination of different muscle groups to insured efficient ventilation and provide oxygen to the body. This action is the result of interactions between neural networks located within the brainstem. Inspiration and expiration depend at least in part on interactions between two separate oscillators: inspiration is driven by a neural network located in the preBötzinger complex (PreBötC) and active expiration is driven by a network in the parafacial respiratory group (pFRG). Neurons of the pFRG are silent at rest and become active when the respiratory drive increased. This study investigated the temporal coordination between the brainstem respiratory network and the lumbar spinal network that generates spontaneous activities that is different of the induced fictive locomotion. The remaining question is how these activities coordinate early during the development. Results of this study show that brainstem networks contribute to the temporal coordination of the lumbar spontaneous activity during inspiration since lumbar motor activity occurs exclusively during the expiratory time. This study also investigated the role of the ß-noradrenergic modulation on the respiratory activities. ß-noradrenergic receptors activation increased the frequency of the double bursts and increased expiratory activity at the lumbar level. These results suggest interactions between brainstem and spinal networks and reveal a descending drive that may contribute to the coordination of the respiratory and lumbar spontaneous activities.

Irregular Breathing in Mice following Genetic Ablation of V2a Neurons.

Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem-spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Bötzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.

Patterns of inspiratory phase-dependent activity in the in vitro respiratory network.

Mechanistic descriptions of rhythmogenic neural networks have often relied on ball-and-stick diagrams, which define interactions between functional classes of cells assumed to be reasonably homogenous. Application of this formalism to networks underlying respiratory rhythm generation in mammals has produced increasingly intricate models that have generated significant insight, but the underlying assumption that individual cells within these network fall into distinct functional classes has not been rigorously tested. In the present study we used multiunit extracellular recording in the in vitro pre-Bötzinger complex to identify and characterize the rhythmic activity of 951 cells. Inspiratory phase-dependent activity was estimated for all cells, and the data set as a whole was analyzed with principal component analysis, nonlinear dimensionality reduction, and hierarchical clustering techniques. None of these techniques revealed categorically distinct functional cell classes, indicating instead that the behavior of these cells within the network falls along several continua of spiking behavior.

[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.

Activation of alpha-2 noradrenergic receptors is critical for the generation of fictive eupnea and fictive gasping inspiratory activities in mammals in vitro.

Biogenic amines are not just 'modulators', they are often essential for the execution of behaviors. Here, we explored the role of biogenic amines acting on the pre-Bötzinger complex (pre-BötC), an area located in the ventrolateral medulla which is critical for the generation of different forms of breathing. Isolated in transverse slices from mice, this region continues to spontaneously generate rhythmic activities that resemble normal (eupneic) inspiratory activity in normoxia and gasping in hypoxia. We refer to these as 'fictive eupneic' and 'fictive gasping' activity. When exposed to hypoxia, the pre-BötC transitions from a network state relying on calcium-activated nonspecific cation currents (I(CAN)) and persistent sodium currents (I(Nap)) to one that primarily depends on the I(Nap) current. Here we show that in inspiratory neurons I(Nap)-dependent bursting, blocked by riluzole, but not I(CAN) -dependent bursting, required endogenously released norepinephrine acting on alpha2-noradrenergic receptors (α2-NR). At the network level, fictive eupneic activity persisted while fictive gasping ceased following the blockade of α2-NR. Blockade of α2-NR eliminated fictive gasping even in slice preparations as well as in inspiratory island preparations. Blockade of fictive gasping by α2-NR antagonists was prevented by activation of 5-hydroxytryptamine type 2A receptors (5-HT2A). Our data suggest that gasping depends on the converging aminergic activation of 5-HT2AR and α2-NR acting on riluzole-sensitive mechanisms that have been shown to be crucial for gasping.

Contribution of the potassium-chloride co-transporter KCC2 to the modulation of lumbar spinal networks in mice.

Spontaneous activity is observed in most developing neuronal circuits, such as the retina, hippocampus, brainstem and spinal cord. In the spinal cord, spontaneous activity is important for generating embryonic movements critical for the proper development of motor axons, muscles and synaptic connections. A spontaneous bursting activity can be recorded in vitro from ventral roots during perinatal development. The depolarizing action of the inhibitory amino acids γ-aminobutyric acid and glycine is widely proposed to contribute to spontaneous activity in several immature systems. During development, the intracellular chloride concentration decreases, leading to a shift of equilibrium potential for Cl(-) ions towards more negative values, and thereby to a change in glycine- and γ-aminobutyric acid-evoked potentials from depolarization/excitation to hyperpolarization/inhibition. The up-regulation of the outward-directed Cl(-) pump, the neuron-specific potassium-chloride co-transporter type 2 KCC2, has been shown to underlie this shift. Here, we investigated whether spontaneous and locomotor-like activities are altered in genetically modified mice that express only 8-20% of KCC2, compared with wild-type animals. We show that a reduced amount of KCC2 leads to a depolarized equilibrium potential for Cl(-) ions in lumbar motoneurons, an increased spontaneous activity and a faster locomotor-like activity. However, the left-right and flexor-extensor alternating pattern observed during fictive locomotion was not affected. We conclude that neuronal networks within the spinal cord are more excitable in KCC2 mutant mice, which suggests that KCC2 strongly modulates the excitability of spinal cord networks.

Neonatal apneic phenotype in a murine congenital central hypoventilation syndrome model is induced through non-cell autonomous developmental mechanisms.

Congenital central hypoventilation syndrome (CCHS) represents a rare genetic disorder usually caused by mutations in the homeodomain transcription factor PHOX2B. Some CCHS patients suffer mainly from deficiencies in CO2 and/or O2 respiratory chemoreflex, whereas other patients present with full apnea shortly after birth. Our goal was to identify the neuropathological mechanisms of apneic presentations in CCHS. In the developing murine neuroepithelium, Phox2b is expressed in three discrete progenitor domains across the dorsal-ventral axis, with different domains responsible for producing unique autonomic or visceral motor neurons. Restricting the expression of mutant Phox2b to the ventral visceral motor neuron domain induces marked newborn apnea together with a significant loss of visceral motor neurons, RTN ablation, and preBötzinger complex dysfunction. This finding suggests that the observed apnea develops through non-cell autonomous developmental mechanisms. Mutant Phox2b expression in dorsal rhombencephalic neurons did not generate significant respiratory dysfunction, but did result in subtle metabolic thermoregulatory deficiencies. We confirm the expression of a novel murine Phox2b splice variant which shares exons 1 and 2 with the more widely studied Phox2b splice variant, but which differs in exon 3 where most CCHS mutations occur. We also show that mutant Phox2b expression in the visceral motor neuron progenitor domain increases cell proliferation at the expense of visceral motor neuron development. We propose that visceral motor neurons may function as organizers of brainstem respiratory neuron development, and that disruptions in their development result in secondary/non-cell autonomous maldevelopment of key brainstem respiratory neurons.

Respiratory rhythm generation, hypoxia, and oxidative stress-Implications for development.

Encountered in a number of clinical conditions, repeated hypoxia/reoxygenation during the neonatal period can pose both a threat to immediate survival as well as a diminished quality of living later in life. This review focuses on our current understanding of central respiratory rhythm generation and the role that hypoxia and reoxygenation play in influencing rhythmogenesis. Here, we examine the stereotypical response of the inspiratory rhythm from the preBötzinger complex (preBötC), basic neuronal mechanisms that support rhythm generation during the peri-hypoxic interval, and the physiological consequences of inspiratory network responsivity to hypoxia and reoxygenation, acute and chronic intermittent hypoxia, and oxidative stress. These topics are examined in the context of Sudden Infant Death Syndrome, apneas of prematurity, and neonatal abstinence syndrome.

Noradrenergic modulation of the respiratory neural network.

Noradrenergic dysregulation has been reported in human pathologies affecting the control of breathing, such as sudden infant death syndrome, congenital central hypoventilation syndrome and Rett syndrome. Noradrenergic neurons, located predominantly in pontine nuclei, are among the earliest to arise within the hindbrain and play an essential role in the maturation of the respiratory network. Noradrenergic neurons also play a major role in the modulation of the respiratory motor pattern from birth through adulthood. The critical importance of this signaling system in respiratory control is illustrated by the severe respiratory disturbances associated with gene mutations affecting noradrenergic neurons (Phox2 and Mecp2). Here, the role of catecholaminergic pontine nuclei in the control of breathing, the cellular effects of norepinephrine on the respiratory network and the pathological consequence to breathing of abnormalities in this signaling system will be discussed.

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.

Disturbed sensorimotor and electrophysiological patterns in lead intoxicated rats during development are restored by curcumin I.

Lead poisoning is one of the most significant health problem of environmental origin. It is known to cause different damages in the central and peripheral nervous system which could be represented by several neurophysiological and behavioral symptoms. In this study we firstly investigated the effect of lead prenatal exposure in rats to (3g/L), from neonatal to young age, on the motor/sensory performances, excitability of the spinal cord and gaits during development. Then we evaluated neuroprotective effects of curcumin I (Cur I) against lead neurotoxicity, by means of grasping and cliff avoidance tests to reveal the impairment of the sensorimotor functions in neonatal rats exposed prenatally to lead. In addition, extracellular recordings of motor output in spinal cord revealed an hyper-excitability of spinal networks in lead treated rats. The frequency of induced fictive locomotion was also increased in treated rats. At the young age, rats exhibited an impaired locomotor gait. All those abnormalities were attenuated by Cur I treatment at a dose of 16g/kg. Based on our finding, Cur I has shown features of a potent chemical compound able to restore the neuronal and the relative locomotor behaviors disturbances induced by lead intoxication. Therefore, this chemical can be recommended as a new therapeutic trial against lead induced neurotoxicity.

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.

Determinants of inspiratory activity.

In vitro and in vivo studies have identified the pre-Bötzinger complex as an important kernel for the generation of inspiratory activity. The mechanisms underlying inspiratory rhythm generation involve pacemaker as well as synaptic mechanisms. In slice preparations, blockade of pacemaker properties with blockers for the persistent Na+ current, and the Ca2+-activated inward cationic current, abolishes respiratory activity. Here we show that blockade of the persistent Na+ current alone is sufficient to abolish respiratory activity in the in situ preparation. Although pacemaker neurons may be critical for establishing the basic respiratory rhythm, their rhythmic output is modulated by many elements of the respiratory network. For example, levels of synaptic inhibition control whether they burst or not, and endogenously released neuromodulators, such as serotonin and substance P modulate their intrinsic membrane currents. We hypothesize that the balance between synaptic and intrinsic pacemaker properties in the respiratory network is plastic, and that alterations of this balance may lead to dynamic reconfigurations of the respiratory network, which ultimately give rise to different activity patterns.

Monoamine oxidase A-deficiency and noradrenergic respiratory regulations in neonatal mice.

In vitro experiments were performed on brainstem-spinal cord preparations from mouse neonates to compare the noradrenergic regulations of the respiratory network in the control C3H/HeJ strain and the transgenic Tg8 strain which has been created from the C3H/HeJ strain by deletion of the gene encoding monoamine oxidase A (MAOA), the main enzyme for serotonin degradation. In both control and MAOA-deficient strains, we show: (i). that the pontine A5 area exerts a potent inhibitory modulation on the respiratory rhythm generator; (ii). that noradrenaline application induces a tonic phrenic activity; and (iii). that noradrenaline increases the respiratory rhythm. The latter effect is however delayed and weak in the Tg8 strain. Therefore, MAOA-deficiency has only slightly altered the noradrenergic regulations of the respiratory network.

Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents.

The aim of the present review is to summarise available studies dealing with the respiratory control exerted by pontine noradrenergic neurones in neonatal and adult mammals. During the perinatal period, in vitro studies on neonatal rodents have shown that A5 and A6 neurones exert opposite modulations onto the respiratory rhythm generator, inhibitory and facilitatory respectively, that the anatomical support for these modulations already exists at birth, and that genetically induced alterations in the formation of A5 and A6 neurones affect the maturation of the respiratory rhythm generator, leading to lethal respiratory deficits at birth. The A5-A6 modulation of the respiratory rhythm generator is not transient, occurring solely during the perinatal period but it persists throughout life: A5 and A6 neurones display a respiratory-related activity, receive inputs from and send information to the medullary respiratory centres and contribute to the adaptation of adult breathing to physiological needs.

ß-Noradrenergic receptor activation specifically modulates the generation of sighs in vivo and in vitro.

The pre-Bötzinger complex (preBötC), an area that is critical for generating breathing (eupnea), gasps and sighs is continuously modulated by catecholamines. These amines and the generation of sighs have also been implicated in the regulation of arousal. Here we studied the catecholaminergic modulation of sighs not only in anesthetized freely breathing mice (in vivo), but also in medullary slice preparations that contain the preBötC and that generate fictive eupneic and sigh rhythms in vitro. We demonstrate that activating ß-noradrenergic receptors (ß-NR) specifically increases the frequency of sighs, while eupnea remains unaffected both in vitro and in vivo. ß-NR activation specifically increased the frequency of intrinsically bursting pacemaker neurons that rely on persistent sodium current (I(Nap)). By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (I(CAN)) remained unaffected. Moreover, riluzole, which blocks bursting in I(Nap) pacemakers abolished sighs altogether, while flufenamic acid (FFA) which blocks the I(CAN) current did not alter the sigh-increasing effect caused by ß-NR. Our results suggest that the selective ß-NR action of sighs may result from the modulation of I(Nap) pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response. The ß-NR action on the preBötC may be an important mechanism in modulating behaviors that are specifically associated with sighs, such as the regulation of the early events leading to the arousal response.

Prenatal exposure to fenugreek impairs sensorimotor development and the operation of spinal cord networks in mice.

Fenugreek is a medicinal plant whose seeds are widely used in traditional medicine, mainly for its laxative, galactagogue and antidiabetic effects. However, consumption of fenugreek seeds during pregnancy has been associated with a range of congenital malformations, including hydrocephalus, anencephaly and spina bifida in humans. The present study was conducted to evaluate the effects of prenatal treatment of fenugreek seeds on the development of sensorimotor functions from birth to young adults. Pregnant mice were treated by gavage with 1 g/kg/day of lyophilized fenugreek seeds aqueous extract (FSAE) or distilled water during the gestational period. Behavioral tests revealed in prenatally treated mice a significant delay in righting, cliff avoidance, negative geotaxis responses and the swimming development. In addition, extracellular recording of motor output in spinal cord isolated from neonatal mice showed that the frequency of spontaneous activity and fictive locomotion was reduced in FSAE-exposed mice. On the other hand, the cross-correlation coefficient in control mice was significantly more negative than in treated animals indicating that alternating patterns are deteriorated in FSAE-treated animals. At advanced age, prenatally treated mice displayed altered locomotor coordination in the rotarod test and also changes in static and dynamic parameters assessed by the CatWalk automated gait analysis system. We conclude that FSAE impairs sensorimotor and coordination functions not only in neonates but also in adult mice. Moreover, spinal neuronal networks are less excitable in prenatally FSAE-exposed mice suggesting that modifications within the central nervous system are responsible, at least in part, for the motor impairments.