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.

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.

The dual role of the orexin/hypocretin system in modulating wakefulness and respiratory drive.

PURPOSE OF REVIEW: Today, numerous studies show that orexin peptides act as regulators of many functions including the control of sleep-wake states, breathing, and central chemosensitivity. However, little is known on neuronal mechanisms by which orexin regulates breathing in a state-dependent manner. This review summarizes recent data on the control of neuronal circuits by orexin, with a special emphasis on breathing, central chemosensitivity, and obstructive sleep apneas. RECENT FINDINGS: Activity of hypothalamic orexinergic neurons is subjected to maturation and is mandatory to maintain long bouts of wakefulness in adults. At wake onset, this activity progressively builds up as a result of synaptic interactions and reinforces the awake state. Orexin deficiency attenuates the hypercapnic reflex only during wakefulness and is correlated with an increase in sleep apneas. Intrinsic sensitivity to CO2/pH of orexin neurons may impact on brainstem chemosensitive neurons, and this effect likely involves TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid sensitive K+ (TASK)-like potassium currents. SUMMARY: Orexin signaling is directly involved in the control of upper airway patency in particular during wakefulness, whereas decreasing activity of orexinergic neurons may contribute to upper airway collapse during sleep causing obstructive sleep apnea. Future research should focus on the role of orexin in upper airway control, which may lead to new clinical strategies for treating breathing disorders associated with sleep.

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

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

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.

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.

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.

Gamma-Aminobutyric Acid-Inhibitory Motor Innervation of Leg Muscles of the Shore Crab.

The inhibitory motor innervation of a crustacean leg was studied in the crab, Carcinus maenas. In in vitro preparations of the central nervous system and the proximal leg nerves, motor nerve recordings demonstrate the presence of a single common inhibitory motor neuron which elicits picrotoxin-sensitive inhibitory junction potentials in a distal leg muscle, the accessory flexor. This inhibitor is the common inhibitor (CI). Immunohistochemical detection of the inhibitory motor neuron neurotransmitter, gamma-aminobutyric acid (GABA), allows us to identify three immunoreactive motor neuron axons in sections of the distal leg nerves and of proximal leg nerves. One corresponds to the CI whereas the other two are the specific inhibitors, one to the stretcher and one to the opener muscles. After nickel chloride backfills of the CI in proximal leg nerves, GABA immunodetection fails and thus confirms that CI is the single inhibitor having branches in proximal leg nerves. These results demonstrate that the inhibitory motor innervation of a crab leg comprises three and only three inhibitors: the common inhibitor innervating all leg muscles and the two specific inhibitors, each innervating a single distal leg muscle. Further conclusions can be drawn: first, a muscle innervated by more than one excitatory axon has no specific inhibitor; second, sensory afferents are not mediated by GABA. Finally, during locomotion, the leg muscles receive two very distinct types of motor input: (1) one common to all the muscles coming from the common inhibitor which was previously shown by other authors to prevent build-up of tension in the muscles, thus allowing each muscle to contract according to (2) the specific motor input it receives from its own excitors.

Identification of central nervous system neurons innervating the respiratory muscles of the mouse: a transneuronal tracing study.

In recent years, the central control of breathing in mammals has been the subject of numerous studies. The aim of the present one was to characterize the neuronal network projecting to the main respiratory motoneurons, in adult mice. To this end, the morphology and location of the respiratory motoneurons and their sequential connections with other neurons were revealed using a transneuronal tracing technique by means of the rabies virus infection. The injections of the rabies virus in the respiratory muscles resulted in labeling the motoneurons and their serially connected interneurons at multiple levels of the mouse central nervous system: spinal cord, pons and medulla, cerebellum, mesencephalon, diencephalon, and telencephalon. Most of these labeled areas have been previously identified in the control of cardiorespiratory regulation, as well as in other autonomic functions. These anatomical data provide support for the integration of respiratory-related activities in complex behavioral responses. Furthermore, these data suggest similarities in the evolution of central respiratory networks in mammals.

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.

The H3K27 demethylase JMJD3 is required for maintenance of the embryonic respiratory neuronal network, neonatal breathing, and survival.

JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks.

Fluoxetine treatment abolishes the in vitro respiratory response to acidosis in neonatal mice.

BACKGROUND: To secure pH homeostasis, the central respiratory network must permanently adapt its rhythmic motor drive to environment and behaviour. In neonates, it is commonly admitted that the retrotrapezoid/parafacial respiratory group of neurons of the ventral medulla plays the primary role in the respiratory response to acidosis, although the serotonergic system may also contribute to this response. METHODOLOGY/PRINCIPAL FINDINGS: Using en bloc medullary preparations from neonatal mice, we have shown for the first time that the respiratory response to acidosis is abolished after pre-treatment with the serotonin-transporter blocker fluoxetine (25-50 µM, 20 min), a commonly used antidepressant. Using mRNA in situ hybridization and immunohistology, we have also shown the expression of the serotonin transporter mRNA and serotonin-containing neurons in the vicinity of the RTN/pFRG of neonatal mice. CONCLUSIONS: These results reveal that the serotonergic system plays a pivotal role in pH homeostasis. Although obtained in vitro in neonatal mice, they suggest that drugs targeting the serotonergic system should be used with caution in infants, pregnant women and breastfeeding mothers.

Effects of antidromic discharges in crayfish primary afferents.

Contrary to orthodromic spikes that are generated in sensory organs and conveyed to CNS, antidromic spikes are generated in the axon terminals of the sensory neurons within the CNS and are conveyed to the peripheral sensory organ. Antidromic discharges are observed in primary afferent neurons of both vertebrates and invertebrates and seem to be related to the rhythmic activity of central neural networks. In this study, we analyzed the effect of antidromic discharges on the sensory activity of a leg proprioceptor in in vitro preparations of the crayfish CNS. Intracellular microelectrodes were used both to record the orthodromic spikes and to elicit antidromic spikes by injecting squares pulses of depolarizing current at various frequencies. Experiments were performed on the three types of identified sensory afferents (tonic, phasotonic, and phasic). The main results showed a reduction of the firing frequency of the orthodromic activity in 82% of the tested afferents. In tonic afferents, during their occurrences and according to their frequency, antidromic spikes or bursts reduced or suppressed the orthodromic activity. Following their terminations, they also induced a silent period and a gradual recovery of the orthodromic activity, both of which increased as the duration and the frequency of the antidromic bursts increased. In phasotonic and phasic afferents, antidromic bursts reduced or suppressed the phasic responses as their frequency and durations increased. In phasotonic afferents, if elicited prior to the movements, long-duration bursts with increasing frequency reduced more rapidly the tonic background activity than the phasic one whereas short-duration bursts at high frequency produced strong decreases of both. The effect of antidromic bursts accumulated when they are repetitively elicited. Antidromic bursts induced a much larger decrease of the sensory activity than adaptation alone. The occurrences of antidromic spikes or bursts may have a functional role in modulating the incoming sensory messages during locomotion. The mechanisms by which antidromic spikes modulate the firing sensitivity of the primary afferents may well lie in modifications of the properties of either mecanotransduction and/or spike initiation.

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.

Nasal trigeminal inputs release the A5 inhibition received by the respiratory rhythm generator of the mouse neonate.

Experiments were performed on neonatal mice to analyze why, in vitro, the respiratory rhythm generator (RRG) was silent and how it could be activated. We demonstrated that in vitro the RRG in intact brain stems is silenced by a powerful inhibition arising from the pontine A5 neurons through medullary alpha(2) adrenoceptors and that in vivo nasal trigeminal inputs facilitate the RRG as nasal continuous positive airway pressure increases the breathing frequency, whereas nasal occlusion and nasal afferent anesthesia depress it. Because nasal trigeminal afferents project to the A5 nuclei, we applied single trains of negative electric shocks to the trigeminal nerve in inactive ponto-medullary preparations. They induced rhythmic phrenic bursts during the stimulation and for 2-3 min afterward, whereas repetitive trains produced on-going rhythmic activity up to the end of the experiments. Electrolytic lesion or pharmacological inactivation of the ipsilateral A5 neurons altered both the phrenic burst frequency and occurrence after the stimulation. Extracellular unitary recordings and trans-neuronal tracing experiments with the rabies virus show that the medullary lateral reticular area contains respiratory-modulated neurons, not necessary for respiratory rhythmogenesis, but that may provide an excitatory pathway from the trigeminal inputs to the RRG as their electrolytic lesion suppresses any phrenic activity induced by the trigeminal nerve stimulation. The results lead to the hypothesis that the trigeminal afferents in the mouse neonate involve at least two pathways to activate the RRG, one that may act through the medullary lateral reticular area and one that releases the A5 inhibition received by the RRG.