Jean-Charles Viemari

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 ∼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 ∼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.

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.

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.

Phox2a Gene, A6 Neurons, and Noradrenaline Are Essential for Development of Normal Respiratory Rhythm in Mice

Although respiration is vital to the survival of all mammals from the moment of birth, little is known about the genetic factors controlling the prenatal maturation of this physiological process. Here we investigated the role of the Phox2a gene that encodes for a homeodomain protein involved in the generation of noradrenergic A6 neurons in the maturation of the respiratory network. First, comparisons of the respiratory activity of fetuses delivered surgically from heterozygous Phox2a pregnant mice on gestational day 18 showed that the mutants had impaired in vivo ventilation, in vitro respiratory-like activity, and in vitro respiratory responses to central hypoxia and noradrenaline. Second, pharmacological studies on wild-type neonates showed that endogenous noradrenaline released from pontine A6 neurons potentiates rhythmic respiratory activity via α1 medullary adrenoceptors. Third, transynaptic tracing experiments in which rabies virus was injected into the diaphragm confirmed that A6 neurons were connected to the neonatal respiratory network. Fourth, blocking the α1 adrenoceptors in wild-type dams during late gestation with daily injections of the α1 adrenoceptor antagonist prazosin induced in vivo and in vitro neonatal respiratory deficits similar to those observed in Phox2a mutants. These results suggest that noradrenaline, A6 neurons, and the Phox2a gene, which is crucial for the generation of A6 neurons, are essential for development of normal respiratory rhythm in neonatal mice. Metabolic noradrenaline disorders occurring during gestation therefore may induce neonatal respiratory deficits, in agreement with the catecholamine anomalies reported in victims of sudden infant death syndrome.

Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies.

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm‐generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0–P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several‐fold during the next days. Hypoxia increased breathing frequency from E18–P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0–P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40–50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0–P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16–E18, in agreement with the concept of multiple rhythmogenic mechanisms.

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.

Developmental up-regulation of the potassium–chloride cotransporter type 2 in the rat lumbar spinal cord

The classical GABA/glycine hyperpolarizing inhibition is not observed in the immature spinal cord. GABA(A) and glycine receptors are anions channels and the efficacy of inhibitory transmission in the spinal cord is largely determined by the gradient between intracellular and extracellular chloride concentrations. The concentration of intracellular chloride in neurons is mainly regulated by two cation-chloride cotransporters, the potassium-chloride cotransporter 2 (KCC2) and the sodium-potassium-chloride co-transporter 1 (NKCC1). In this study, we measured the reversal potential of IPSPs (E(IPSP)) of lumbar motoneurons during the first postnatal week and we investigated the expression of KCC2 and NKCC1 in the ventral horn of the spinal cord from the embryonic day 17 to the postnatal day 20 in the rat. Our results suggest that the negative shift of E(IPSP) from above to below the resting membrane potential occurs during the first postnatal week when the expression of KCC2 increases significantly and the expression of NKCC1 decreases. KCC2 immunolabeling surrounded motoneurons, presumably in the plasma membrane and NKCC1 immunolabeling appeared outside this KCC2-labeled fine strip. Taken together, the present results indicate that maturation of chloride homeostasis is not completed at birth in the rat and that the upregulation of KCC2 plays a key role in the shift from depolarizing to hyperpolarizing IPSPs.

Ret deficiency in mice impairs the development of A5 and A6 neurons and the functional maturation of the respiratory rhythm.

Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret‐null mutants (Ret–/–) did not survive after birth, our experiments were performed in wild‐type (wt) and Ret–/– fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret–/– fetuses, high‐pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret–/– fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem ‘en bloc’ preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret–/– fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.

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.

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.