Activity-dependent formation and location of voltage-gated sodium channel clusters at a CNS nerve terminal during postnatal development.

In auditory pathways, the precision of action potential (AP) propagation depends on axon myelination and high densities of voltage-gated Na (Nav) channels clustered at nodes of Ranvier. Changes in Nav channel expression at the heminode, the final node before the nerve terminal, can alter AP invasion into the presynaptic terminal. We studied the activity-dependent formation of Nav channel clusters before and after hearing onset at postnatal day 12 in the rat and mouse auditory brain stem. In rats, the Nav channel cluster at the heminode formed progressively during the second postnatal week, around hearing onset, whereas the Nav channel cluster at the nodes was present before hearing onset. Initiation of heminodal Nav channel clustering was correlated with the expression of scaffolding protein ankyrinG and paranodal protein Caspr. However, in whirler mice with congenital deafness, heminodal Nav channels did not form clusters and maintained broad expression, but Nav channel clustering was normal at the nodes. In addition, a clear difference in the distance from the heminodal Nav channel to the calyx across the mediolateral axis of the medial nucleus of the trapezoid body (MNTB) developed after hearing onset. In the medial MNTB, where neurons respond best to high-frequency sounds, the heminodal Nav channel cluster was located closer to the terminal than in the lateral MNTB, where neurons respond best to low-frequency sounds. Thus sound-mediated neuronal activities are potentially associated with the refinement of the heminode adjacent to the presynaptic terminal in the auditory brain stem. NEW & NOTEWORTHY: Clustering of voltage-gated sodium (Nav) channels and their distribution along the axon, specifically at the unmyelinated axon segment next to the nerve terminal, are essential for tuning propagated action potentials. Nav channel clusters near the nerve terminal and their location as a function of neuronal position along the mediolateral axis are controlled by auditory inputs after hearing onset. Thus sound-mediated neuronal activity influences the tonotopic organization of Nav channels at the nerve terminal in the auditory brain stem.

Functional and structural properties of ion channels at the nerve terminal depends on compact myelin.

KEY POINTS: In the present study, we document the role of compact myelin in regulating the structural and functional properties of ion channels at the nerve terminals, using electrophysiology, dynamic Na(+) imaging and immunohistochemistry. The subcellular segregation of Na(+) channel expression and intracellular Na(+) dynamics at the heminode and terminal was lost in the dysmyelinated axon from Long-Evans shaker rats, which lack compact myelin. In Long-Evans shaker rats, loss of the Nav ß4 subunit specifically at the heminode reduced resurgent and persistent Na(+) currents, whereas K(+) channel expression and currents were increased. The results of the present study suggest that there is a specific role for compact myelin in dictating protein expression and function at the axon heminode and in regulating excitability of the nerve terminal. ABSTRACT: Axon myelination increases the conduction velocity and precision of action potential propagation. Although the negative effects of demyelination are generally attributed to conduction failure, accumulating evidence suggests that myelination also regulates the structural properties and molecular composition of the axonal membrane. In the present study, we investigated how myelination affects ion channel expression and function, particularly at the last axon heminode before the nerve terminal, which regulates the presynaptic excitability of the nerve terminal. We compared the structure and physiology of normal axons and those of the Long-Evans shaker (LES) rat, which lacks compact myelin. The normal segregation of Na(+) channel expression and dynamics at the heminode and terminal was lost in the LES rat. Specifically, NaV -α subunits were dispersed and NaV ß4 subunit was absent, whereas the density of K(+) channels was increased at the heminode. Correspondingly, resurgent and persistent Na(+) currents were reduced and K(+) current was increased. Taken together, these data suggest a specific role for compact myelin in the orchestration of ion channel expression and function at the axon heminode and in regulating excitability of the nerve terminal.

Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population.

The median preoptic nucleus (MnPO) holds a strategic position in the hypothalamus. It is adjacent to the third ventricle; hence, it can directly access the ionic composition of the CSF. MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as central sensors of extracellular Na(+) concentration ([Na(+)](ext)). The mechanism underlying Na(+) sensing involves the atypical Na(+) channel, Na(X). Here we sought to determine whether Na(+) influx in Na(+) sensors is actively regulated via interaction with other membrane proteins involved in cellular Na(+) homeostasis, such as Na(+)/K(+)-ATPase. The Na(+)/K(+)-ATPase role was investigated using patch-clamp recordings in rat MnPO dissociated neurons. Na(+) current evoked with hypernatriuric solution was diminished in the absence of ATP/GTP, indicating that Na(+)/K(+)-ATPase play a central role in [Na(+)](ext) detection. Specific blockers of α1 and α3 isoforms of Na(+)/K(+)-ATPase, ouabain or strophanthidin, inhibited this Na(+) current. However, strophanthidin, which selectively blocks the α1 isoform, was more effective in blocking Na(+) current, suggesting that the Na(+)/K(+)-ATPase-α1 isoform is specifically involved in [Na(+)](ext) detection. Although strophanthidin did not alter either the membrane resistance or the Na(+) reversal potential, the conductance and the permeability of the Na(X) channel decreased significantly. Our results suggest that Na(+)/K(+)-ATPase interacts with the Na(X) channel and regulates the high [Na(+)](ext)-evoked Na(+) current via influencing the Na(+) influx rate. This study describes a novel intracellular regulatory pathway of [Na(+)](ext) detection in MnPO neurons. The α1 isoform of Na(+)/K(+)-ATPase acts as a direct regulatory partner of the Na(X) channel and influences Na(+) influx via controlling the Na(+) permeability of the channel.

What bothers severe asthma patients most? A paired patient-clinician study across seven European countries.

Introduction: Severe asthma is a complex, multidimensional disease. Optimal treatment, adherence and outcomes require shared decision-making, rooted in mutual understanding between patient and clinician. This study used a novel, patient-centred approach to examine the most bothersome aspects of severe asthma to patients, as seen from both perspectives in asthma registries. Methods: Across seven countries, 126 patients with severe asthma completed an open-ended survey regarding most the bothersome aspect(s) of their asthma. Patients' responses were linked with their treating clinician who also completed a free-text survey about each patient's most bothersome aspect(s). Responses were coded using content analysis, and patient and clinician responses were compared. Finally, asthma registries that are part of the SHARP (Severe Heterogeneous Asthma Research collaboration, Patient-centred) Clinical Research Collaboration were examined to see the extent to which they reflected the most bothersome aspects reported by patients. Results: 88 codes and 10 themes were identified. Clinicians were more focused on direct physical symptoms and were less focused on "holistic" aspects such as the effort required to self-manage the disease. Clinicians accurately identified a most bothersome symptom for 29% of patients. Agreement was particularly low with younger patients and those using oral corticosteroids infrequently. In asthma registries, patient aspects were predominantly represented in questionnaires. Conclusions: Results demonstrated different perspectives and priorities between patients and clinicians, with clinicians more focused on physical aspects. These differences must be considered when treating individual patients, and within multidisciplinary treatment teams. The use of questionnaires that include multifaceted aspects of disease may result in improved asthma research.

Evaluation of real-world mepolizumab use in severe asthma across Europe: the SHARP experience with privacy-preserving federated analysis.

Background: An objective of the Severe Heterogeneous Asthma Registry, Patient-centered (SHARP) is to produce real-world evidence on a pan-European scale by linking nonstandardised, patient-level registry data. Mepolizumab has shown clinical efficacy in randomised controlled trials and prospective real-world studies and could therefore serve as a proof of principle for this novel approach. The aim of the present study was to harmonise data from 10 national severe asthma registries and characterise patients receiving mepolizumab, assess its effectiveness on annual exacerbations and maintenance oral glucocorticoid (OCS) use, and evaluate treatment patterns. Methods: In this observational cohort study, registry data (5871 patients) were extracted for harmonisation. Where harmonisation was possible, patients who initiated mepolizumab between 1 January 2016 and 31 December 2021 were examined. Changes of a 12-month (range 11-18 months) period in frequent (two or more) exacerbations, maintenance OCS use and dose were analysed in a privacy-preserving manner using meta-analysis of generalised estimating equation parameters. Periods before and during the coronavirus disease 2019 pandemic were analysed separately. Results: In 912 patients who fulfilled selection criteria, mepolizumab significantly reduced frequent exacerbations (OR 0.18, 95% CI 0.13-0.25), maintenance OCS use (OR 0.75, 95% CI 0.61-0.92) and dose (mean -3.93 mg·day-1, 95% CI -5.24-2.62 mg·day-1) in the pre-pandemic group, with similar trends in the pandemic group. Marked heterogeneity was observed between registries in patient characteristics and mepolizumab treatment patterns. Conclusions: By harmonising patient-level registry data and applying federated analysis, SHARP demonstrated the real-world effectiveness of mepolizumab on asthma exacerbations and maintenance OCS use in severe asthma patients across Europe, consistent with previous evidence. This paves the way for future pan-European real-world severe asthma studies using patient-level data in a privacy-proof manner.

Blueprint for harmonising unstandardised disease registries to allow federated data analysis: prepare for the future.

Real-world evidence from multinational disease registries is becoming increasingly important not only for confirming the results of randomised controlled trials, but also for identifying phenotypes, monitoring disease progression, predicting response to new drugs and early detection of rare side-effects. With new open-access technologies, it has become feasible to harmonise patient data from different disease registries and use it for data analysis without compromising privacy rules. Here, we provide a blueprint for how a clinical research collaboration can successfully use real-world data from existing disease registries to perform federated analyses. We describe how the European severe asthma clinical research collaboration SHARP (Severe Heterogeneous Asthma Research collaboration, Patient-centred) fulfilled the harmonisation process from nonstandardised clinical registry data to the Observational Medical Outcomes Partnership Common Data Model and built a strong network of collaborators from multiple disciplines and countries. The blueprint covers organisational, financial, conceptual, technical, analytical and research aspects, and discusses both the challenges and the lessons learned. All in all, setting up a federated data network is a complex process that requires thorough preparation, but above all, it is a worthwhile investment for all clinical research collaborations, especially in view of the emerging applications of artificial intelligence and federated learning.

Neuronal sodium leak channel is responsible for the detection of sodium in the rat median preoptic nucleus.

Sodium (Na(+)) ions are of primary importance for hydromineral and cardiovascular homeostasis, and the level of Na(+) in the body fluid compartments [plasma and cerebrospinal fluid (CSF)] is precisely monitored in the hypothalamus. Glial cells seem to play a critical role in the mechanism of Na(+) detection. However, the precise role of neurons in the detection of extracellular Na(+) concentration ([Na(+)](out)) remains unclear. Here we demonstrate that neurons of the median preoptic nucleus (MnPO), a structure in close contact with the CSF, are specific Na(+) sensors. Electrophysiological recordings were performed on dissociated rat MnPO neurons under isotonic [Na(+)] (100 mM NaCl) with local application of hypernatriuric (150, 180 mM NaCl) or hyponatriuric (50 mM NaCl) external solution. The hyper- and hyponatriuric conditions triggered an in- and an outward current, respectively. The reversal potential of the current matched the equilibrium potential of Na(+), indicating that a change in [Na(+)](out) modified the influx of Na(+) in the MnPO neurons. The conductance of the Na(+) current was not affected by either the membrane potential or the [Na(+)](out). Moreover, the channel was highly selective for lithium over guanidinium. Together, these data identified the channel as a Na(+) leak channel. A high correlation between the electrophysiological recordings and immunofluorescent labeling for the Na(X) channel in dissociated MnPO neurons strongly supports this channel as a candidate for the Na(+) leak channel responsible for the Na(+)-sensing ability of rat MnPO neurons. The absence of Na(X) labeling and of a specific current evoked by a change in [Na(+)](out) in mouse MnPO neurons suggests species specificity in the hypothalamus structures participating in central Na(+) detection.

RETRACTED: Insular cortex processes aversive somatosensory information and is crucial for threat learning.

Learning about threats is essential for survival. During threat learning, an innocuous sensory percept such as a tone acquires an emotional meaning when paired with an aversive stimulus such as a mild footshock. The amygdala is critical for threat memory formation, but little is known about upstream brain areas that process aversive somatosensory information. Using optogenetic techniques in mice, we found that silencing of the posterior insula during footshock reduced acute fear behavior and impaired 1-day threat memory. Insular cortex neurons respond to footshocks, acquire responses to tones during threat learning, and project to distinct amygdala divisions to drive acute fear versus threat memory formation. Thus, the posterior insula conveys aversive footshock information to the amygdala and is crucial for learning about potential dangers in the environment.

Oligodendroglial excitability mediated by glutamatergic inputs and Nav1.2 activation.

Oligodendrocyte (OL) maturation and axon-glial communication are required for proper myelination in the developing brain. However, physiological properties of OLs remain largely uncharacterized in different brain regions. The roles of oligodendroglial voltage-activated Na+ channels (Nav) and electrical excitability in relation to maturation to the myelinating stage are controversial, although oligodendroglial excitability is potentially important for promoting axon myelination. Here we show spiking properties of OLs and their role in axon-glial communication in the auditory brainstem. A subpopulation of pre-myelinating OLs (pre-OLs) can generate Nav1.2-driven action potentials throughout postnatal development to early adulthood. In addition, excitable pre-OLs receive glutamatergic inputs from neighboring neurons that trigger pre-OL spikes. Knockdown of Nav1.2 channels in pre-OLs alters their morphology, reduces axon-OL interactions and impairs myelination. Our results suggest that Nav1.2-driven spiking of pre-OLs is an integral component of axon-glial communication and is required for the function and maturation of OLs to promote myelination.Axon-glial communication is important for myelination. Here the authors show that during postnatal development in rats, a subpopulation of pre-myelinating oligodendrocytes in the auditory brainstem receive excitatory inputs and can generate Nav 1.2-driven action potentials, and that such process promotes myelination.

Extracellular Na(+) levels regulate formation and activity of the NaX/alpha1-Na(+)/K(+)-ATPase complex in neuronal cells.

MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na(+)]out). The mechanism underlying Na(+)-sensing involves Na(+)-flow through the NaX channel, directly regulated by the Na(+)/K(+)-ATPase α1-isoform which controls Na(+)-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na(+)]in). Here we aim to determine whether environmental changes in Na(+) could actively modulate the NaX/Na(+)/K(+)-ATPase complex activity. We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na(+)] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na(+)] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na(+)/K(+)-ATPase remained unaltered. This unbalance between NaX and Na(+)/K(+)-ATPase pump proportion would induce a bigger portion of Na(+)/K(+)-ATPase-control-free NaX channel. Thus, we suggest that hypernatremic environment increases NaX/Na(+)/K(+)-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.