Motoneuronal persistent inward currents (PICs) are facilitated by neuromodulatory inputs but are highly sensitive to local inhibitory circuits. Estimates of PICs are reduced by group Ia reciprocal inhibition, and increased with the diffuse actions of neuromodulators released during remote muscle contraction. However, it remains unknown how motoneurons function in the presence of simultaneous excitatory and inhibitory commands. To probe this topic, we investigated motor unit discharge patterns and estimated PICs during voluntary co-contraction of ankle muscles, which simultaneously demands the contraction of agonist-antagonist pairs. Twenty participants performed triangular ramps of both co-contraction (simultaneous dorsiflexion and plantar flexion) and isometric dorsiflexion to a peak of 30% of their maximum muscle activity from a maximal voluntary contraction. Motor unit spike trains were decomposed from high-density surface EMG activity recorded from tibialis anterior using blind source separation algorithms. Voluntary co-contraction altered motor unit discharge rate characteristics. Discharge rate at recruitment and peak discharge rate were modestly reduced (∼6% change; P < 0.001; d = 0.22) and increased (∼2% change; P = 0.001, d = -0.19), respectively, in the entire dataset but no changes were observed when motor units were tracked across conditions. The largest effects during co-contraction were that estimates of PICs (ΔF) were reduced by ∼20% (4.47 vs. 5.57 pulses per second during isometric dorsiflexion; P < 0.001, d = 0.641). These findings suggest that, during voluntary co-contraction, the inhibitory input from the antagonist muscle overcomes the additional excitatory and neuromodulatory drive that may occur due to the co-contraction of the antagonist muscle, which constrains PIC behaviour. KEY POINTS: Voluntary co-contraction is a unique motor behaviour that concurrently provides excitatory and inhibitory synaptic input to motoneurons. Co-contraction of agonist-antagonist pairs alters agonist motor unit discharge characteristics, consistent with reductions in persistent inward current magnitude.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease characterized by motor neuron death and distal axonopathy. Despite its clinical severity and profound impact in the patients and their families, many questions about its pathogenesis remain still unclear, including the role of Schwann cells and axon-glial signaling in disease progression. Upon axonal injury, upregulation of JUN transcription factor promotes Schwann cell reprogramming into a repair phenotype that favors axon regrowth and neuronal survival. To study the potential role of repair Schwann cells on motoneuron survival in amyotrophic lateral sclerosis, we generated a mouse line that over-expresses JUN in the Schwann cells of the SOD1G93A mutant, a mouse model of this disease. Then, we explored disease progression by evaluating survival, motor performance and histology of peripheral nerves and spinal cord of these mice. We found that Schwann cell JUN overexpression does not prevent axon degeneration neither motor neuron death in the SOD1G93A mice. Instead, it induces a partial demyelination of medium and large size axons, worsening motor performance and resulting in more aggressive disease phenotype.

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

This study investigated the effects of cilostazol on motor dysfunction, spinal motor neuron abnormalities, and schwannopathy in rats with diabetes. Diabetes mellitus (DM) was induced in rats via femoral intravenous streptozotocin (STZ) injection (60 mg/kg). After successful DM induction, cilostazol was administered on day 15 via oral gavage (100 mg/kg/day) for 6 weeks until sacrifice. Behavioral assays, including motor function, were performed weekly. The sciatic nerve, L5 spinal cord, and spinal ventral root were collected to evaluate the expression of the glial fibrillary acidic protein (GFAP), myelin protein zero (P0), and choline acetyltransferase (ChAT) by immunofluorescence and Western blotting. DM rats displayed decreased running speeds, running distances, and toe spread but increased foot pressure. In addition, loss of non-myelinating Schwann cells and myelin sheaths was observed in the sciatic nerve and L5 spinal ventral root. Reduced numbers of motor neurons were also found in the L5 spinal ventral horn. Cilostazol administration significantly potentiated running speed and distance; increased hind paw toe spread; and decreased foot pressure. In the sciatic nerve and L5 spinal ventral root, cilostazol treatment significantly improved non-myelinated Schwann cells and increased myelin mass. ChAT expression in motor neurons in the spinal ventral horn was improved, but not significantly. Cilostazol administration may protect sensorimotor function in diabetic rats.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS.
METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice.
RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice.
CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits.

OBJECTIVE: Conventional F wave analysis involves a relatively uniform physiological environment induced by supramaximal stimulations. The F wave characteristics in a dynamic physiological condition, however, are rarely investigated. This study aimed to improve understanding of F wave properties in the more dynamic process by introducing a novel method to analyze F waves based on the compound muscle action potential (CMAP) scan technique.
METHODS: Twenty four healthy subjects participated in the study. The CMAP scan was applied to record muscle responses in the abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles, respectively. F wave characteristics including mean F wave amplitude and latency (F-M latency), persistence and activating threshold were quantified.
RESULTS: An average of 200 F waves per muscle were obtained from the CMAP scan recording. Weak to moderate correlations between F wave amplitude and stimulating intensity were observed in most of the APB (19 muscles; r = 0.33 ± 0.14, all p < .05) and ADM (23 muscles, r = 0.46 ± 0.16, all p < .05) muscles. Significantly longer mean F latency and lower activating F-threshold were found in the ADM muscles (F-M latency: APB: 25.43 ± 2.39 ms, ADM: 26.15 ± 2.32 ms, p < .05; F-threshold: APB: 7.68 ± 8.96% CMAP, ADM: 2.35 ± 2.42% CMAP, p < .05).
CONCLUSIONS: This study introduces new features of F waves using the CMAP scan technique and identifies differences of F wave characteristics between the hand muscles. The CMAP scan based F waves analysis can be combined with the motor unit number estimation to assess functional alterations in motor neurons in neurological disorders.

Carpal tunnel syndrome (CTS) is the most common cause of peripheral compressive neuropathy and consists of compression of the median nerve in the wrist. Although there are several etiologies, idiopathic is the most prevalent origin, and among the forms of treatment for CTS, conservative is the most indicated. However, despite the high prevalence in and impact of this syndrome on the healthcare system, there are still controversies regarding the best therapeutic approach for patients. Therefore, noting that some studies point to vitamin D deficiency as an independent risk factor, which increases the symptoms of the syndrome, this study evaluated the role of vitamin D supplementation and its influence on pain control, physical examination and response electroneuromyography to conservative treatment of carpal tunnel syndrome. For this, the sample consisted of 14 patients diagnosed with CTS and hypovitaminosis D, who were allocated into two groups. The control group received corticosteroid treatment, while the experimental group received corticosteroid treatment associated with vitamin D. Thus, from this study, it can be concluded that patients who received vitamin D, when compared to those who did not receive it, showed improvement in the degree of pain intensity, a reduction in symptom severity and an improvement in some electroneuromyographic parameters.