Christian Gestreau

Task2 potassium channels set central respiratory CO2 and O2 sensitivity

Task2 K+ channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2−/− mice showed disturbed chemosensory function with hypersensitivity to low CO2 concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2−/− mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem–spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O2 chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.

Medullary respiratory neurones and control of laryngeal motoneurones during fictive eupnoea and cough in the cat

1 This study addressed the hypothesis that ventrolateral medullary respiratory neurones participate in the control of laryngeal motoneurones during both eupnoea and coughing. 2 Data were obtained from 28 mid‐collicular decerebrated, artificially ventilated cats. Cough‐like motor patterns (fictive cough) in phrenic, lumbar and recurrent laryngeal nerves were elicited by mechanical stimulation of the intrathoracic trachea. Microelectrode arrays were used to monitor simultaneously several neurones in the ventral respiratory group, including the Bötzinger and pre‐Bötzinger complexes. Spike trains were evaluated for responses during fictive cough and evidence of functional connectivity with spike‐triggered averages of efferent recurrent laryngeal nerve activity. 3 Primary features were observed in averages triggered by 94 of 332 (28 %) neurones. An offset biphasic wave with a positive time lag was present in the unrectified average for 10 inspiratory and 13 expiratory neurones. These trigger neurones were respectively identified as inspiratory laryngeal motoneurones with augmenting, decrementing, plateau and ‘other’ discharge patterns, and expiratory laryngeal motoneurones with decrementing firing patterns. 4 Rectified averages triggered by inspiratory neurones included 37 offset peaks, 11 central peaks and one offset trough. Averages triggered by expiratory neurones had 12 offset peaks, six central peaks and four offset troughs. Relationships inferred from these features included premotor actions of inspiratory neurones with augmenting, decrementing, plateau and ‘other’ patterns on inspiratory laryngeal motoneurones, and premotor actions of decrementing and ‘other’ expiratory neurones on expiratory laryngeal motoneurones. Corresponding changes in neuronal firing patterns during fictive cough supported these inferences. 5 The data confirm and extend previous results on the control of laryngeal motoneurones during eupnoea and support the hypothesis that the same premotor neurones help to shape motoneurone firing patterns during both eupnoea and coughing.

Activation of XII motoneurons and premotor neurons during various oropharyngeal behaviors

Neural control of tongue muscles plays a crucial role in a broad range of oropharyngeal behaviors. Tongue movements must be rapidly and accurately adjusted in response to the demands of multiple complex motor tasks including licking/mastication, swallowing, vocalization, breathing and protective reflexes such as coughing. Yet, central mechanisms responsible for motor and premotor control of hypoglossal (XII) activity during these behaviors are still largely unknown. The aim of this article is to review the functional organization of the XII motor nucleus with particular emphasis on breathing, coughing and swallowing. Anatomical localization of XII premotor neurons is also considered. We discuss results concerned with multifunctional activity of medullary and pontine populations of XII premotor neurons, representing a single network that can be reconfigured to produce different oromotor response patterns. In this context, we introduce new data on swallowing-related activity of XII (and trigeminal) motoneurons, and finally suggest a prominent role for the pontine Kölliker-Fuse nucleus in the control of inspiratory-related activity of XII motoneurons supplying tongue protrusor and retrusor muscles.

Activity of dorsal respiratory group inspiratory neurons during laryngeal-induced fictive coughing and swallowing in decerebrate cats

Membrane potential changes and/or discharges from 36 inspiratory neurons were recorded intracellularly in the dorsal respiratory group (DRG; i.e., the ventrolateral subdivision of the nucleus tractus solitarii) in decerebrate, paralyzed, and ventilated cats. Electrical activities were recorded from both somata (n=10) and axons (n=26). Activities during quiet breathing were compared with those observed during fictive coughing and swallowing evoked by repetitive electrical stimulation of afferent fibers of the superior laryngeal nerve (SLN). These nonrespiratory behaviors were evident in paralyzed animals as characteristic discharge patterns of the phrenic, abdominal, and hypoglossal nerves. Twenty-six neurons exhibiting antidromic action potentials in response to electrical stimuli applied to the cervical (C3–5) spinal cord were classified as inspiratory bulbospinal neurons (IBSNs). These neurons were considered as premotoneurons. The remaining 10 inspiratory neurons (INAA) were not antidromically activated by electrical stimuli applied to either cervical spinal cord or ipsilateral cervical vagus. These neurons are thought to be propriobulbar neurons. We recorded the activity of 31 DRG inspiratory neurons (24 IBSNs and 7 I-NAA) during coughing. All but one (a late-recruited IBSN) discharged a burst of action potentials during the coughing-related phrenic nerve activity. Typically, ramp-like membrane depolarization trajectories and discharge frequencies during coughing were similar to those observed during inspiration. We recorded the activity of 33 DRG inspiratory neurons (23 IBSNs and 10 I-NAA) during swallowing. Most (28/33) neurons were briefly activated, i.e., discharged a burst of action potentials during swallowing, but peak discharge frequency decreased compared with that measured during inspiration. The membrane potentials of nine somata exhibited a brief bell-shaped depolarization during swallowing, the amplitude of which was similar to that observed during inspiration. These results suggest that some inspiratory premotoneurons and propriobulbar neurons of the DRG might be involved in nonrespiratory motor activities, even if clearly antagonistic to breathing (e.g., swallowing). We postulate the existence in the medulla oblongata of adult mammals of neurons exhibiting a “functional flexibility”.

Fos expression in the rat brain after exposure to gravito-inertial force changes

The immediate-early genes constitute useful neurobiological tools for mapping brain functional activity after sensory stimulation. We immunohistochemically investigated Fos protein expression in the brain of rats exposed to gravito-inertial force changes. Experiments were performed in hypergravity rats born and housed for 60 days in terrestrian gravity (1xg) and thereafter exposed for 90 min to 2xg or 4xg in a centrifuge, and in hypogravity rats born and housed for 60 days at 2xg and submitted for 90 min to 1xg. Data from these two experimental groups were quantified by light microscopy and compared to those from two groups of control rats born and permanently housed in either 1xg or 2xg environments that never had to adapt to novel gravito-inertial environments. Results showed a low basal Fos expression in the controls and a strong Fos staining in the experimental rats. Only the hypergravity rats displayed Fos-positive cells in vestibular-related brainstem regions (medial, inferior, and superior vestibular nuclei (VN); group y; dorsomedial cell column (DMCC) of the inferior olive (IO)). By contrast, many suprabulbar areas were strongly labeled in both the hyper- and hypogravity rats, as shown by the numerous Fos-positive cells in mesencephalic (colliculus, laterodorsal periaqueductal gray, autonomic nuclei), diencephalic (hypothalamic and thalamic nuclei), and telencephalic (parietal, temporal, entorhinal and visual cortices) structures. These spatial patterns of Fos expression suggest that an increase in gravito-inertial force activates otolith-vestibulo-olivar pathways and various suprabulbar structures underlying the corticovestibular interactions, which govern the multiple representations of vestibular information in the cortex. A decrease in gravito-inertial force has the opposite effects on the vestibulo-olivar structures as a result of otolith system disfacilitation which, in turn, modifies the activity of complex neural pathways. Exposure to both hyper- and hypogravity environments likely induces neurovegetative and/or stress effects that could account for Fos labeling in autonomic nuclei and in nervous structures involved in the hypothalamo-pituitary-adrenal axis.

Activity of respiratory laryngeal motoneurons during fictive coughing and swallowing

Abstract Membrane potential changes and discharges from 28 laryngeal motoneurons were recorded intracellularly in the caudal nucleus ambiguus of decerebrate, paralyzed and ventilated cats. Electrical activities were recorded from 17 expiratory laryngeal motoneurons (ELMs) with maximal depolarizing membrane potential in early expiration, and from 11 inspiratory laryngeal motoneurons (ILMs) with maximal depolarizing membrane potential in inspiration. Activities during breathing were compared with those observed during fictive coughing and swallowing evoked by electrical stimulation of the superior laryngeal nerves. These non-respiratory behaviors were evidenced in paralyzed animals by characteristic discharge patterns of the phrenic, abdominal nerves and pharyngeal branch of the vagus nerve. We recorded the activity of 11 ELMs and 5 ILMs during coughing in which ELMs, but not ILMs, exhibited increased membrane depolarization and discharge frequencies. Membrane depolarization and discharge frequencies of all ELMs were also significantly increased during swallowing. In addition, membrane depolarization of most ELMs (15/17) was preceded by a short-lasting hyperpolarization due to chloride-dependent inhibitory mechanisms occurring at the onset of swallowing. Out of 10 ILMs tested during swallowing, 7 exhibited membrane depolarization, preceded in 5 cases by a short-lasting hyperpolarization. Discharge frequencies of ILMs were significantly reduced during swallowing. The same pattern of phasic activities of ILMs and ELMs was observed during coughing and breathing, suggesting the involvement of similar excitatory pathways in both behaviors. These results imply that the duration of activation and the discharge frequency of neurons of the central generator for breathing that drive laryngeal motoneurons are enhanced during coughing. During swallowing, in addition to central excitatory mechanisms, laryngeal motoneurons are subjected to an initial inhibition of unknown origin. This inhibition probably contributes to the temporal organization of the swallowing motor sequence.

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 β-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 β-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.

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.

Upper Airway Dysfunction of Tau-P301L Mice Correlates with Tauopathy in Midbrain and Ponto-Medullary Brainstem Nuclei

Tauopathy comprises hyperphosphorylation of the microtubule-associated protein tau, causing intracellular aggregation and accumulation as neurofibrillary tangles and neuropil treads. Some primary tauopathies are linked to mutations in the MAPT gene coding for protein tau, but most are sporadic with unknown causes. Also, in Alzheimers disease, the most frequent secondary tauopathy, neither the cause nor the pathological mechanisms and repercussions are understood. Transgenic mice expressing mutant Tau-P301L suffer cognitive and motor defects and die prematurely from unknown causes. Here, in situ electrophysiology in symptomatic Tau-P301L mice (7–8 months of age) revealed reduced postinspiratory discharges of laryngeal motor outputs that control laryngeal constrictor muscles. Under high chemical drive (hypercapnia), postinspiratory discharge was nearly abolished, whereas laryngeal inspiratory discharge was increased disproportionally. The latter may suggest a shift of postinspiratory laryngeal constrictor activity into inspiration. In vivo double-chamber plethysmography of Tau-P301L mice showed significantly reduced respiratory airflow but significantly increased chest movements during baseline breathing, but particularly in hypercapnia, confirming a significant increase in inspiratory resistive load. Histological analysis demonstrated hyperphosphorylated tau in brainstem nuclei, directly or indirectly involved in upper airway motor control (i.e., the Kölliker–Fuse, periaqueductal gray, and intermediate reticular nuclei). In contrast, young Tau-P301L mice did not show breathing disorders or brainstem tauopathy. Consequently, in aging Tau-P301L mice, progressive upper airway dysfunction is linked to progressive tauopathy in identified neural circuits. Because patients with tauopathy suffer from upper airway dysfunction, the Tau-P301L mice can serve as an experimental model to study disease-specific synaptic dysfunction in well defined functional neural circuits.

Activation of Orexin B receptors in the pontine Kölliker-Fuse nucleus modulates pre-inspiratory hypoglossal motor activity in rat

Orexins (splice variants A and B) are hypothalamic neuropeptides that have essential functions in control of arousal and nutrition. Lack of Orexins is strongly associated with narcolepsy and sleep disordered breathing. However, the role of Orexins and particularly that of Orexin-B (OXB), in respiratory centres controlling upper-airway patency are less defined. In the present study we performed microinjections of OXB into the pontine Kölliker-Fuse nucleus (KF) of the dorsolateral pons, since this nucleus is particularly involved in the pre-motor control of upper airway muscles. The OXB mediated effects on heart, phrenic (PNA) and hypoglossal (XII-A) nerve activities were analysed in an in situ perfused brainstem preparation. Injection of OXB into the KF evoked significant augmentation of the respiratory frequency. Importantly, OXB provoked particularly prolonged pre-inspiratory discharge of the XII nerve, while no cardiovascular response was observed after KF microinjections. In summary, OXB in the KF exerts an excitatory effect on XII pre-motoneurones. Since pre-inspiratory activity of the XII is important for the decrease in upper airway resistance during inspiration, we conclude that OXB release in the KF has strong implications in the state-dependent control of upper airway patency under physiological and pathophysiological conditions.