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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease with progressive motor neuron death, where patients usually die within 5 years of diagnosis. Previously, we showed that the C-boutons, which are large cholinergic synapses to motor neurons that modulate motor neuron activity, are necessary for behavioral compensation in mSOD1G93A mice, a mouse model for ALS. We reasoned that, since the C-boutons likely increase the excitability of surviving motor neurons to compensate for motor neuron loss during ALS disease progression, then amplitude modulation through the C-boutons likely increases motor neuron stress and worsens disease progression. By comparing male and female mSOD1G93A mice to mSOD1G93A mice with genetically silenced C-boutons [mSOD1G93A; Dbx1::cre; ChATfl/fl (mSOD1G93A/Coff)], we show that the Cboutons do not influence the humane end point of mSOD1G93A mice; however, our histologic analysis shows that C-bouton silencing significantly improves fast-twitch muscle innervation over time. Using immunohistology, we also show that the Cboutons are active in a task-dependent manner, and that symptomatic mSOD1G93A mice show significantly higher C-bouton activity than wild-type mice during low-intensity walking. Last, by using behavioral analysis, we provide evidence that C-bouton silencing in combination with swimming is beneficial for the behavioral capabilities of mSOD1G93A mice. Our observations suggest that manipulating the C-boutons in combination with a modulatory-targeted training program may therefore be beneficial for ALS patients and could result in improved mobility and quality of life. [ABSTRACT FROM AUTHOR]

The spatial and temporal balance of spinal α‐motoneuron (αMN) intrinsic membrane conductances underlies the neural output of the final common pathway for motor commands. Although the complete set and precise localization of αMN K+ channels and their respective outward conductances remain unsettled, important K+ channel subtypes have now been documented, including Kv1, Kv2, Kv7, TASK, HCN and SK isoforms. Unique kinetics and gating parameters allow these channels to differentially shape and/or modify αMN firing properties, and recent immunohistochemical localization of K+‐channel complexes reveals a framework in which their spatial distribution and/or focal clustering within different surface membrane compartments is highly tuned to their physiological function. Moreover, highly evolved regulatory mechanisms enable specific channels to operate over variable levels of αMN activity and contribute to either state‐dependent enhancement or diminution of firing. While recent data suggest an additional, non‐conducting role for clustered Kv2.1 channels in the formation of endoplasmic reticulum–plasma membrane junctions postsynaptic to C‐bouton synapses, electrophysiological evidence demonstrates that conducting Kv2.1 channels effectively regulate αMN firing, especially during periods of high activity in which the cholinergic C‐boutons are engaged. Intense αMN activity or cell injury rapidly disrupts the clustered organization of Kv2.1 channels in αMNs and further impacts their physiological role. Thus, αMN K+ channels play a critical regulatory role in motor processing and are potential therapeutic targets for diseases affecting αMN excitability and motor output, including amyotrophic lateral sclerosis. [ABSTRACT FROM AUTHOR]

Key points: Kv2 currents maintain and regulate motoneuron (MN) repetitive firing properties.Kv2.1 channel clustering properties are dynamic and respond to both high and low activity conditions.The enzyme calcineurin regulates Kv2.1 ion channel declustering.In patholophysiological conditions of high activity, Kv2.1 channels homeostatically reduce MN repetitive firing.Modulation of Kv2.1 channel kinetics and clustering allows these channels to act in a variable way across a spectrum of MN activity states. Kv2.1 channels are widely expressed in the central nervous system, including in spinal motoneurons (MNs) where they aggregate as distinct membrane clusters associated with highly regulated signalling ensembles at specific postsynaptic sites. Multiple roles for Kv2 channels have been proposed but the physiological role of Kv2.1 ion channels in mammalian spinal MNs is unknown. To determine the contribution of Kv2.1 channels to rat α‐motoneuron activity, the Kv2 inhibitor stromatoxin was used to block Kv2 currents in whole‐cell current clamp electrophysiological recordings in rat lumbar MNs. The results indicate that Kv2 currents permit shorter interspike intervals and higher repetitive firing rates, possibly by relieving Na+ channel inactivation, and thus contribute to maintenance of repetitive firing properties. We also demonstrate that Kv2.1 clustering properties in motoneurons are dynamic and respond to both high and low activity conditions. Furthermore, we show that the enzyme calcineurin regulates Kv2.1 ion channel clustering status. Finally, in a high activity state, Kv2.1 channels homeostatically reduce motoneuron repetitive firing. These results suggest that the activity‐dependent regulation of Kv2.1 channel kinetics allows these channels to modulate repetitive firing properties across a spectrum of motoneuron activity states. Key points: Kv2 currents maintain and regulate motoneuron (MN) repetitive firing properties.Kv2.1 channel clustering properties are dynamic and respond to both high and low activity conditions.The enzyme calcineurin regulates Kv2.1 ion channel declustering.In patholophysiological conditions of high activity, Kv2.1 channels homeostatically reduce MN repetitive firing.Modulation of Kv2.1 channel kinetics and clustering allows these channels to act in a variable way across a spectrum of MN activity states. [ABSTRACT FROM AUTHOR]

An altered neuronal excitability of spinal motoneurones has consistently been implicated in Amyotrophic Lateral Sclerosis (ALS) leading to several investigations of synaptic input to these motoneurones. One such input that has repeatedly been shown to be affected is a population of large cholinergic synapses terminating mainly on the soma of the motoneurones referred to as C-boutons. Most research on these synapses during disease progression has used transgenic Superoxide Dismutase 1 (SOD1) mouse models of the disease which have not only produced conflicting findings, but also fail to recapitulate the key pathological feature seen in ALS; cytoplasmic accumulations of TAR DNA-binding protein 43 (TDP-43). Additionally, they fail to distinguish between slow and fast motoneurones, the latter of which have more C-boutons, but are lost earlier in the disease. To circumvent these issues, we quantified the frequency and volume of C-boutons on traced soleus and gastrocnemius motoneurones, representing predominantly slow and fast motor pools respectively. Experiments were performed using the TDP-43ΔNLS mouse model that carries a transgenic construct of TDP-43 devoid of its nuclear localization signal, preventing its nuclear import. This results in the emergence of pathological TDP-43 inclusions in the cytoplasm, modelling the main pathology seen in this disorder, accompanied by a severe and lethal ALS phenotype. Our results confirmed changes in both the number and volume of C-boutons with a decrease in number on the more vulnerable, predominantly fast gastrocnemius motoneurones and an increase in number on the less vulnerable, predominantly slow soleus motoneurones. Importantly, these changes were only found in male mice. However, both sexes and motor pools showed a decrease in C-bouton volume. Our experiments confirm that cytoplasmic TDP-43 accumulation is sufficient to drive C-bouton changes. • Cytoplasmic relocation/aggregation of TDP-43 is sufficient to drive C-bouton changes. • We show changes in both the number and volume of C-boutons on spinal motoneurones. • A decrease in number is seen on the predominantly fast gastrocnemius motoneurones. • An increase in number is seen on the predominantly slow soleus motoneurones. • Both motor pools showed a decrease in C-bouton volume. [ABSTRACT FROM AUTHOR]

Homeostatic plasticity occurs through diverse cellular and synaptic mechanisms, and extensive investigations over the preceding decade have established Kv2.1 ion channels as key homeostatic regulatory elements in several central neuronal systems. As in these cellular systems, Kv2.1 channels in spinal motoneurons (MNs) localize within large somatic membrane clusters. However, their role in regulating motoneuron activity is not fully established in vivo. We have previously demonstrated marked Kv2.1 channel redistribution in MNs following in vitro glutamate application and in vivo peripheral nerve injury (Romer et al., 2014, Brain Research, 1547:1-15). Here, we extend these findings through the novel use of a fully intact, in vivo rat preparation to show that Kv2.1 ion channels in lumbar MNs rapidly and reversibly redistribute throughout the somatic membrane following 10 min of electrophysiological sensory and/or motor nerve stimulation. These data establish that Kv2.1 channels are remarkably responsive in vivo to electrically evoked and synaptically driven action potentials in MNs, and strongly implicate motoneuron Kv2.1 channels in the rapid homeostatic response to altered neuronal activity. [ABSTRACT FROM AUTHOR]

C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a "signaling ensemble") for cholinergic regulation of outward K+ currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems. [ABSTRACT FROM AUTHOR]

Abstract: The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterised by loss of motoneurons of the brainstem and spinal cord, and corticospinal neurons of the motor cortex. There is also increasing evidence of involvement of glial cells and interneurons, with non-cell autonomous disease mechanisms now thought to contribute to motoneuron degeneration in ALS. Given the apparent involvement of altered motoneuron excitability in ALS and the recent demonstration that motoneuron excitability is controlled by C-boutons, a specific class of synaptic input recently shown to originate from a small cluster of spinal interneurons, we hypothesised that perturbations in C-bouton inputs to motoneurons may contribute to altered excitability and the eventual degeneration of motoneurons in ALS. To begin to assess this we performed a detailed, developmental study of the anatomy of C-boutons in a mouse model of ALS (G93A SOD1 mutant). We found that C-bouton number is unchanged in ALS mice compared to wildtype littermates at any age. In contrast, we found that the size of C-boutons increases in ALS mice between postnatal day (P)8 and P30, with boutons remaining larger throughout symptomatic stages (P120–P140). Interestingly, we found that C-boutons are only enlarged in male mice. We found no evidence of concomitant changes in clusters of postsynaptic proteins known to align with C-boutons (Kv2.1 K+ channels and m2-type muscarinic receptors). In conclusion, these data support the involvement of pre-symptomatic changes in C-bouton anatomy in ALS pathogenesis and in particular mechanisms underlying the male bias of this disease. [Copyright &y& Elsevier]

Neuronal Ca 2+ entry elicited by electrical activity contributes to information coding via activation of K + and Cl - channels. While Ca 2+ -dependent K + channels have been extensively studied, the molecular identity and role of Ca 2+ -activated Cl - channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca 2+ -activated Cl - conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS., Competing Interests: Declaration of Interests Authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)