Persistent inward currents (PICs) and persistent outward currents (POCs) regulate the excitability and firing behaviours of spinal motoneurons (MNs). Given their potential role in MN excitability dysfunction in amyotrophic lateral sclerosis (ALS), PICs have been previously studied in superoxide dismutase 1 (SOD1)-G93A mice (the standard animal model of ALS); however, conflicting results have been reported on how the net PIC changes during disease progression. Also, individual PICs and POCs have never been examined before in symptomatic ALS. To fill this gap, we measured the net and individual PIC and POC components of wild-type (WT) and SOD MNs in current clamp and voltage clamp during disease progression (assessed by neuroscores). We show that SOD MNs of symptomatic mice experience a much larger net PIC, relative to WT cells from age-matched littermates. Specifically, the Na+ and Ca2+ PICs are larger, whereas the lasting SK-mediated (SKL) POC is smaller than WT (Na+ PIC is the largest and SKL POC is the smallest components in SOD MNs). We also show that PIC dysregulation is present at symptom onset, is sustained throughout advanced disease stages and is proportional to SOD MN cell size (largest dysregulation is in the largest SOD cells, the most vulnerable in ALS). Additionally, we show that studying disease progression using neuroscores is more accurate than using SOD mouse age, which could lead to misleading statistics and age-based trends. Collectively, this study contributes novel PIC and POC data, reveals ionic mechanisms contributing to the vulnerability differential among MN types/sizes, and provides insights on the roles PIC and POC mechanisms play in MN excitability dysfunction in ALS. KEY POINTS: Individual persistent inward currents (PICs) and persistent outward currents (POCs) have never been examined before in spinal motoneurons (MNs) of symptomatic amyotrophic lateral sclerosis (ALS) mice. Thus, we contribute novel PIC and POC data to the ALS literature. Male SOD MNs of symptomatic mice have elevated net PIC, with larger Na+ and Ca2+ PICs but reduced SKL POC vs. wild-type littermates. Na+ PIC is the largest and SKL POC is the smallest current in SOD cells. The PIC/POC dysregulation is present at symptom onset. PIC dysregulation is sustained throughout advanced disease, and is proportional to SOD MN size (largest dysregulation is in the largest cells, the most vulnerable in ALS). Thus, we reveal ionic mechanisms contributing to the vulnerability differential among MN types/sizes in ALS. Studying disease progression using SOD mice neuroscores is more accurate than using age, which could distort the statistical differences between SOD and WT PIC/POC data and the trends during disease progression.

Muscle force is modulated by sequential recruitment and firing rates of motor units (MUs). However, discrepancies exist in the literature regarding the relationship between MU firing rates and their recruitment, presenting two contrasting firing-recruitment schemes. The first firing scheme, known as \"onion skin,\" exhibits low-threshold MUs firing faster than high-threshold MUs, forming separate layers akin to an onion. This contradicts the other firing scheme, known as \"reverse onion skin\" or \"afterhyperpolarization (AHP),\" with low-threshold MUs firing slower than high-threshold MUs. To study this apparent dichotomy, we used a high-fidelity computational model that prioritizes physiological fidelity and heterogeneity, allowing versatility in the recruitment of different motoneuron types. Our simulations indicate that these two schemes are not mutually exclusive but rather coexist. The likelihood of observing each scheme depends on factors such as the motoneuron pool activation level, synaptic input activation rates, and MU type. The onion skin scheme does not universally govern the encoding rates of MUs but tends to emerge in unsaturated motoneurons (cells firing < their fusion frequency that generates peak force), whereas the AHP scheme prevails in saturated MUs (cells firing at their fusion frequency), which is highly probable for slow (S)-type MUs. When unsaturated, fast fatigable (FF)-type MUs always show the onion skin scheme, whereas S-type MUs do not show either one. Fast fatigue-resistant (FR)-type MUs are generally similar but show weaker onion skin behaviors than FF-type MUs. Our results offer an explanation for the longstanding dichotomy regarding MU firing patterns, shedding light on the factors influencing the firing-recruitment schemes.NEW & NOTEWORTHY The literature reports two contrasting schemes, namely the onion skin and the afterhyperpolarization (AHP) regarding the relationship between motor units (MUs) firing rates and recruitment order. Previous studies have examined these schemes phenomenologically, imposing one scheme on the firing-recruitment relationship. Here, we used a high-fidelity computational model that prioritizes biological fidelity and heterogeneity to investigate motoneuron firing schemes without bias toward either scheme. Our objective findings offer an explanation for the longstanding dichotomy on MU firing patterns.

Altered neuronal excitability and synaptic inputs to motoneurons are part of the pathophysiology of Amyotrophic Lateral Sclerosis. The cAMP/PKA pathway regulates both of them but therapeutic interventions at this level are limited by the lack of knowledge about suitable pharmacological entry points. Here we used transcriptomics on microdissected and in situ motoneurons to reveal the modulation of PKA-coupled receptorome in SOD1(G93A) ALS mice, vs WT, demonstrating the dysregulation of multiple PKA-coupled GPCRs, in particular on vulnerable MNs, and the relative sparing of β-adrenergic receptors. In vivo MN electrophysiology showed that β2/β3 agonists acutely increase excitability, in particular the input/output relationship, demonstrating a non-canonical adrenergic neuromodulation mediated by β2/β3 receptors both in WT and SOD1 mice. The excitability increase corresponds to the upregulation of immediate-early gene expression and dysregulation of ion channels transcriptome. However the β2/β3 neuromodulation is submitted to a strong homeostasis, since a ten days delivery of β2/β3 agonists results in an abolition of the excitability increase. The homeostatic response is largely caused by a substantial downregulation of PKA-coupled GPCRs in MNs from WT and SOD1 mice. Thus, β-adrenergic receptors are physiologically involved in the regulation of MN excitability and transcriptomics, but, intriguingly, a strong homeostatic response is triggered upon chronic pharmacologic intervention.

Muscle spasms are common in chronic spinal cord injury (SCI), posing challenges to rehabilitation and daily activities. Pharmacological management of spasms mostly targets suppression of excitatory inputs, an approach known to hinder motor recovery. To identify better targets, we investigated changes in inhibitory and excitatory synaptic inputs to motoneurons as well as motoneuron excitability in chronic SCI. We induced either a complete or incomplete SCI in adult mice of either sex and divided those with incomplete injury into low or high functional recovery groups. Their sacrocaudal spinal cords were then extracted and used to study plasticity below injury, with tissue from naive animals as a control. Electrical stimulation of the dorsal roots elicited spasm-like activity in preparations of chronic severe SCI but not in the control. To evaluate overall synaptic inhibition activated by sensory stimulation, we measured the rate-dependent depression of spinal root reflexes. We found inhibitory inputs to be impaired in chronic injury models. When synaptic inhibition was blocked pharmacologically, all preparations became clearly spastic, even the control. However, preparations with chronic injuries generated longer spasms than control. We then measured excitatory postsynaptic currents (EPSCs) in motoneurons during sensory-evoked spasms. The data showed no difference in the amplitude of EPSCs or their conductance among animal groups. Nonetheless, we found that motoneuron persistent inward currents activated by the EPSCs were increased in chronic SCI. These findings suggest that changes in motoneuron excitability and synaptic inhibition, rather than excitation, contribute to spasms and are better suited for more effective therapeutic interventions.Significance Statement Neural plasticity following spinal cord injury is crucial for recovery of motor function. Unfortunately, this process is blemished by maladaptive changes that can cause muscle spasms. Pharmacological alleviation of spasms without compromising the recovery of motor function has proven to be challenging. Here, we investigated changes in fundamental spinal mechanisms that can cause spasms post-injury. Our data suggest that the current management strategy for spasms is misdirected toward suppressing excitatory inputs, a mechanism that we found unaltered after injury, which can lead to further motor weakness. Instead, this study shows that more promising approaches might involve restoring synaptic inhibition or modulating motoneuron excitability.

Axotomy-induced synaptic stripping modulates survival and axon regeneration of injured motoneurons. Celsr2 is supposed to mediate homophilic interactions of neighboring cells during development, and its role in synaptic stripping remains unknow. In a model of brachial plexus avulsion, Celsr2 knockout improved functional recovery, motoneuron survival, and axon regeneration. Celsr2 was indicated to express in spinal motoneurons, excitatory and inhibitory interneurons, astrocytes, and a subset of oligodendrocytes using Celsr2LacZ mice. Double immunostaining showed that the coverage of inhibitory and excitatory vesicles on injured motoneurons were remarkably reduced after injury, whereas more inhibitory vesicles were maintained in Celsr2-/- mutants than control mice. In the ultrastructure, the density of inhibitory F-boutons on injured motoneurons was higher in Celsr2-/- mutants than controls. Conditional knockout of Celsr2 in astrocytes or oligodendrocytes showed the similar axotomy-induced synaptic withdrawal to the control. RNAseq of injured spinal samples identified 12 MHC I molecules with significant changes between Celsr2-/- and control mice. After injury, expression of MHC I surrounding injured motoneurons was increased, particularly high in Celsr2-/- mutants. In conclusion, Celsr2 knockout enhances MHC I signaling, alleviates inhibitory synaptic stripping cell-autonomously, and contributes to motoneuron survival and regeneration, and Celsr2 is a potential target for neural repair.

Non-invasive techniques are scarce with which human (motor) cortical mechanisms can be investigated. In a series of previous experiments, we have applied an advanced form of conditioning technique with transcranial magnetic stimulation (TMS) and peripheral nerve stimulation by which excitability changes at the laminar level in the primary motor cortex can be estimated. This method builds on the assumption that the first of subsequent corticospinal waves from TMS which is assessed with H-reflexes (called early facilitation) results from indirect excitation of corticospinal neurons in motor cortex (I-wave) and not direct excitation of corticospinal axons (D-wave). So far, we have not provided strong experimental evidence that this is actually the case. In the present study, we therefore compared temporal differences of the early facilitation between transcranial magnetic and electrical stimulation (TES). TES is known to excite the axons of corticospinal neurons. TES in our study caused a temporal shift of the early facilitation of H-reflexes in all subjects compared to TMS, which indicates that the early facilitation with TMS is indeed produced by an I-wave. Additionally, we investigated temporal shifts of the early facilitation with different TMS intensities and two TMS coils. It has long been known that TMS with higher intensities can induce a D-wave. Accordingly, we found that TMS with an intensity of 150% of resting motor threshold compared to 130%/110% results in a temporal shift of the early facilitation, indicating the presence of a D-wave. This effect was dependent on the coil type.

Although slice recordings from spinal motoneurons (MNs) are being widely used, the effects of slicing on the measured MN electrical properties under normal and disease conditions have not been assessed. Using high-fidelity cell models of neonatal wild-type (WT) and superoxide dismutase-1 (SOD) cells, we examined the effects of slice thickness, soma position within the slice, and slice orientation to estimate the error induced in measured MN electrical properties from spinal slices. Our results show that most MN electrical properties are not adversely affected by slicing, except for cell time constant, cell capacitance, and Ca2+ persistent inward current (PIC), which all exhibited large errors, regardless of the slice condition. Among the examined factors, soma position within the slice appears to be the strongest factor in influencing the magnitude of error in measured MN electrical properties. Transverse slices appear to have the least impact on measured MN electrical properties. Surprisingly, and despite their anatomical enlargement, we found that G85R-SOD MNs experience similar error in their measured electrical properties to those of WT MNs, but their errors are more sensitive to the soma position within the slice than WT MNs. Unless in thick and symmetrical slices, slicing appears to reduce motoneuron type differences. Accordingly, slice studies should attempt to record from MNs at the slice center to avoid large and inconsistent errors in measured cell properties and have valid cell measurements\' comparisons. Our results, therefore, offer information that would enhance the rigor of MN electrophysiological data measured from the slice preparation under normal and disease conditions.NEW & NOTEWORTHY Although slice recordings from motoneurons are being widely used, the effects of slicing on the measured motoneuron electrical properties under normal and disease conditions have not been assessed. Using high-fidelity cell models of neonatal WT and SOD cells, we examined the effects of slice thickness, soma position within the slice, and slice orientation. Our results offer information that enhances the rigor of MN electrophysiological data measured from the slice preparation under normal and disease conditions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of largely unknown pathophysiology, characterized by the progressive loss of motoneurons (MNs). We review data showing that in presymptomatic ALS mice, MNs display reduced intrinsic excitability and impaired level of excitatory inputs. The loss of repetitive firing specifically affects the large MNs innervating fast contracting muscle fibers, which are the most vulnerable MNs in ALS. Interventions that aimed at restoring either the intrinsic excitability or the synaptic excitation result in a decrease of disease markers in MNs and delayed neuromuscular junction denervation. We then focus on trans-spinal direct current stimulation (tsDCS), a noninvasive tool, since it modulates the activity of spinal neurons and networks. Effects of tsDCS depend on the polarity of applied current. Recent work shows that anodal tsDCS induces long-lasting enhancement of MN excitability and synaptic excitation of spinal MNs. Moreover, we show preliminary results indicating that anodal tsDCS enhances the excitatory synaptic inputs to MNs in ALS mice. In conclusion, we suggest that chronic application of anodal tsDCS might be useful as a complementary method in the management of ALS patients.

Persistent inward currents are important to motoneuron excitability and firing behaviors and also have been implicated in excitotoxicity. In particular, L-type Ca2+ channels, usually located on motoneuron dendrites, play a primary role in amplification of synaptic inputs. However, recent experimental findings on L-type Ca2+ channel behaviors challenge some fundamental assumptions that have been used in interpreting experimental and computational modeling data. Thus, the objectives of this study were to incorporate recent experimental data into an updated, high-fidelity computational model in order to explain apparent inconsistencies and to better elucidate the spatial distributions, expression patterns, and functional roles of L-type Ca2+ and SKL channels. Specifically, the updated model incorporated asymmetric channel activation/deactivation kinetics, depolarization-dependent facilitation, randomness in channel gating, and coactivation of SKL channels. Our simulation results suggest that L-type Ca2+ and SKL channels colocalize primarily on distal dendrites of motoneurons in a punctate expression. Also, punctate expression, as opposed to a homogeneous expression, provides high synaptic current amplification, limits bistability and firing rates, and robustly regulates the Ca2+ persistent inward current, thereby reducing risk of excitotoxicity. The hysteresis and bistability observed experimentally in current-voltage and frequency-current relationships result from the L-type Ca2+ channels\' distal location and intrinsic warm-up. Accordingly, our results indicate that punctate expression of L-type Ca2+ and SKL channels is a potent mechanism for regulating excitability, which would provide a strong neuroprotective effect. Our results could provide broader insights into the functional significance of warm-up and punctate expression of ion channels to regulation of cell excitability.NEW & NOTEWORTHY Recent experimental findings on L-type Ca2+ channels challenge fundamental assumptions used in interpreting experimental and computational modeling data. Here, we incorporated recent experimental data into an updated, high-fidelity computational model to explain apparent inconsistencies and better elucidate the distributions, expression patterns, and functional roles of L-type Ca2+ and SKL channels. Our results indicate that punctate expression of L-type Ca2+ and SKL channels is a potent mechanism for regulating motoneuron excitability, providing a strong neuroprotective effect.

UNASSIGNED: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated.
UNASSIGNED: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control.
UNASSIGNED: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group.
UNASSIGNED: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration.