Timeline of hypoglossal motor neuron death and intrinsic tongue muscle denervation in high-copy number SOD1G93A mice.

In amyotrophic lateral sclerosis (ALS) postmortem tissue and the SOD1 mouse model at mid-disease, death of hypoglossal motor neurons (XII MNs) is evident. These XII MNs innervate the intrinsic and extrinsic tongue muscles, and despite their importance in many oral and lingual motor behaviours that are affected by ALS (e.g., swallowing, speech, and respiratory functions), little is known about the timing and extent of tongue muscle denervation. Here in the well-characterised SOD1G93A (high-copy) mouse model, we evaluated XII MN numbers and intrinsic tongue muscle innervation using standard histopathological approaches, which included stereological evaluation of Nissl-stained brainstem, and the presynaptic and postsynaptic evaluation of neuromuscular junctions (NMJs), using synapsin, neurofilament, and α-bungarotoxin immunolabelling, at presymptomatic, onset, mid-disease, and endstage timepoints. We found that reduction in XII MN size at onset preceded reduced XII MN survival, while the denervation of tongue muscle did not appear until the endstage. Our study suggests that denervation-induced weakness may not be the most pertinent feature of orolingual deficits in ALS. Efforts to preserve oral and respiratory functions of XII MNs are incredibly important if we are to influence patient outcomes.

The morphological discrepancy of neuromuscular junctions between bilateral paraspinal muscles in patients with adolescent idiopathic scoliosis: A quantitative immunofluorescence assay.

Introduction: Prior studies suggested that neuromuscular factors might be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS). The neuromuscular junction (NMJ) is the important pivot where the nervous system interacts with muscle fibers, but it has not been well characterized in the paraspinal muscles of AIS. This study aims to perform the quantitative morphological analysis of NMJs from paraspinal muscles of AIS. Methods: AIS patients who received surgery in our center were prospectively enrolled. Meanwhile, age-matched congenital scoliosis (CS) and non-scoliosis patients were also included as controls. Fresh samples of paraspinal muscles were harvested intraoperatively. NMJs were immunolabeled using different antibodies to reveal pre-synaptic neuronal architecture and post-synaptic motor endplates. A confocal microscope was used to acquire z-stack projections of NMJs images. Then, NMJs images were analyzed on maximum intensity projections using ImageJ software. The morphology of NMJs was quantitatively measured by a standardized 'NMJ-morph' workflow. A total of 21 variables were measured and compared between different groups. Results: A total of 15 AIS patients, 10 CS patients and 5 normal controls were enrolled initially. For AIS group, NMJs in the convex side of paraspinal muscles demonstrated obviously decreased overlap when compared with the concave side (34.27% ± 8.09% vs. 48.11% ± 10.31%, p = 0.0036). However, no variables showed statistical difference between both sides of paraspinal muscles in CS patients. In contrast with non-scoliosis controls, both sides of paraspinal muscles in AIS patients demonstrated significantly smaller muscle bundle diameters. Conclusions: This study first elucidated the morphological features of NMJs from paraspinal muscles of AIS patients. The NMJs in the convex side showed smaller overlap for AIS patients, but no difference was found in CS. This proved further evidence that neuromuscular factors might contribute to the mechanisms of AIS and could be considered as a novel potential therapeutic target for the treatment of progressive AIS.

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43 toxicity.

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

Muscular and neuronal control of voice production - forgotten findings, current concepts, and new developments.

Voice production has been an area of interest in science since ancient times, and although advancing research has improved our understanding of the anatomy and function of the larynx, there is still little general consensus on these two topics. This review aims to outline the main developments in this field and highlight the areas where further research is needed. The most important hypotheses are presented and discussed highlighting the four main lines of research in the anatomy of the human larynx and their most important findings: (1) the arrangement of the muscle fibers of the thyroarytenoid muscle is not parallel to the vocal folds in the internal part (vocalis muscle), leading to altered properties during contraction; (2) the histological structure of the human vocal cords differs from other striated muscles; (3) there is a specialized type of heavy myosin chains in the larynx; and (4) the neuromuscular system of the larynx has specific structures that form the basis of an intrinsic laryngeal nervous system. These approaches are discussed in the context of current physiological models of vocal fold vibration, and new avenues of investigation are proposed.

CaMKIIß deregulation contributes to neuromuscular junction destabilization in Myotonic Dystrophy type I.

BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.

Induced Pluripotent Stem Cells for Modeling Physiological and Pathological Striated Muscle Complexity.

Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.

Accumulation of Dystrophin-Positive Muscle Fibers and Improvement of Neuromuscular Junctions in mdx Mouse Muscles after Bone Marrow Transplantation under Different Conditions.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder caused by mutations in the dystrophin gene. It leads to respiratory and cardiac failure and premature death at a young age. Although recent studies have greatly deepened the understanding of the primary and secondary pathogenetic mechanisms of DMD, an effective treatment remains elusive. In recent decades, stem cells have emerged as a novel therapeutic product for a variety of diseases. In this study, we investigated nonmyeloablative bone marrow cell (BMC) transplantation as a method of cell therapy for DMD in an mdx mouse model. By using BMC transplantation from GFP-positive mice, we confirmed that BMCs participate in the muscle restoration of mdx mice. We analyzed both syngeneic and allogeneic BMC transplantation under different conditions. Our data indicated that 3 Gy X-ray irradiation with subsequent BMC transplantation improved dystrophin synthesis and the structure of striated muscle fibers (SMFs) in mdx mice as well as decreasing the death rate of SMFs. In addition, we observed the normalization of neuromuscular junctions (NMJs) in mdx mice after nonmyeloablative BMC transplantation. In conclusion, we demonstrated that nonmyeloablative BMC transplantation could be considered a method for DMD treatment.

Advances in In Vitro Models of Neuromuscular Junction: Focusing on Organ-on-a-Chip, Organoids, and Biohybrid Robotics.

The neuromuscular junction (NMJ) is a peripheral synaptic connection between presynaptic motor neurons and postsynaptic skeletal muscle fibers that enables muscle contraction and voluntary motor movement. Many traumatic, neurodegenerative, and neuroimmunological diseases are classically believed to mainly affect either the neuronal or the muscle side of the NMJ, and treatment options are lacking. Recent advances in novel techniques have helped develop in vitro physiological and pathophysiological models of the NMJ as well as enable precise control and evaluation of its functions. This paper reviews the recent developments in in vitro NMJ models with 2D or 3D cultures, from organ-on-a-chip and organoids to biohybrid robotics. Related derivative techniques are introduced for functional analysis of the NMJ, such as the patch-clamp technique, microelectrode arrays, calcium imaging, and stimulus methods, particularly optogenetic-mediated light stimulation, microelectrode-mediated electrical stimulation, and biochemical stimulation. Finally, the applications of the in vitro NMJ models as disease models or for drug screening related to suitable neuromuscular diseases are summarized and their future development trends and challenges are discussed.

Emerging role of TAK1 in the regulation of skeletal muscle mass.

Maintenance of skeletal muscle mass and strength throughout life is crucial for heathy living and longevity. Several signaling pathways have been implicated in the regulation of skeletal muscle mass in adults. TGF-ß-activated kinase 1 (TAK1) is a key protein, which coordinates the activation of multiple signaling pathways. Recently, it was discovered that TAK1 is essential for the maintenance of skeletal muscle mass and myofiber hypertrophy following mechanical overload. Forced activation of TAK1 in skeletal muscle causes hypertrophy and attenuates denervation-induced muscle atrophy. TAK1-mediated signaling in skeletal muscle promotes protein synthesis, redox homeostasis, mitochondrial health, and integrity of neuromuscular junctions. In this article, we have reviewed the role and potential mechanisms through which TAK1 regulates skeletal muscle mass and growth. We have also proposed future areas of research that could be instrumental in exploring TAK1 as therapeutic target for improving muscle mass in various catabolic conditions and diseases.

Sphingomyelinase modulates synaptic vesicle mobilization at the mice neuromuscular junctions.

AIMS: Sphingomyelin is an abundant component of the presynaptic membrane and an organizer of lipid rafts. In several pathological conditions, sphingomyelin is hydrolyzed due to an upregulation and release of secretory sphingomyelinases (SMases). Herein, the effects of SMase on exocytotic neurotransmitter release were studied in the diaphragm neuromuscular junctions of mice. MAIN METHODS: Microelectrode recordings of postsynaptic potentials and styryl (FM) dyes were used to estimate neuromuscular transmission. Membrane properties were assessed with fluorescent techniques. KEY FINDINGS: Application of SMase at a low concentration (0.01 U ml-1) led to a disruption of lipid-packing in the synaptic membranes. Neither spontaneous exocytosis nor evoked neurotransmitter release (in response to single stimuli) were affected by SMase treatment. However, SMase significantly increased neurotransmitter release and the rate of fluorescent FM-dye loss from the synaptic vesicles at 10, 20 and 70 Hz stimulation of the motor nerve. In addition, SMase treatment prevented a shift of the exocytotic mode from "full-collapse" fusion to "kiss-and-run" during high-frequency (70 Hz) activity. The potentiating effects of SMase on neurotransmitter release and FM-dye unloading were suppressed when synaptic vesicle membranes were also exposed to this enzyme (i.e., stimulation occurred during SMase treatment). SIGNIFICANCE: Thus, hydrolysis of the plasma membrane sphingomyelin can enhance mobilization of synaptic vesicles and facilitate full fusion mode of exocytosis, but SMase acting on vesicular membrane had a depressant effect on the neurotransmission. Partially, the effects of SMase can be related with the changes in synaptic membrane properties and intracellular signaling.

GABAergic Neuromuscular Junction Suppresses Intestinal Defense of Caenorhabditis elegans by Attenuating Muscular Oxidative Phosphorylation.

Innate immunity is an ancient and evolutionarily conserved system that constitutes the first line of host defense against invading microbes. We previously determined that the GABAergic neuromuscular junction (NMJ) suppresses intestinal innate immunity via muscular insulin signaling. Here, we found that a muscular mitochondrial oxidative phosphorylation pathway of Caenorhabditis elegans is involved in GABAergic NMJs-mediated intestinal defense. Deficiency in GABAergic neurotransmission increases reactive oxygen species (ROS) abundance and inhibits the nuclear translocation of SKN-1, whereas exogenous GABA administration represses it. SKN-1 is an important transcription factor involved in oxidative stress and the innate immune response. Moreover, deficiency in GABAergic postsynaptic UNC-49/GABAAR robustly promotes the mitochondrial function of GABAergic postsynaptic muscle cells, which may contribute to the muscular ROS decrease and intestinal SKN-1 suppression, ultimately inhibiting the intestinal defense of C. elegans. Our findings reveal a potential role of muscle mitochondrial ROS in intestinal defense in vivo and expand our understanding of mechanisms of intestinal innate immunity.

KATP channels as ROS-dependent modulator of neurotransmitter release at the neuromuscular junctions.

AIMS: Neurotransmitter release requires high energy demands, making the nerve terminals metabolically fragile and susceptible to oxidative stress. ATP-sensitive potassium (KATP) channels can be an important regulator orchestrating the influence of metabolic-related signals on exocytosis. Here, the relevance of ROS in KATP channel-dependent control of neurotransmitter release at the frog neuromuscular junction was studied. METHODS: Microelectrode recordings of end plate potentials at the distal and proximal compartments of nerve terminals as well as fluorescent techniques were used. KEY FINDINGS: Activation of KATP channels in the proximal region suppressed evoked and spontaneous release in a lipid raft-dependent manner. Activation of KATP channels in the distal region reduced solely evoked release which was preserved after lipid raft disruption. Chelation of ROS potentiated the effects of KATP channel activation and unmasked the effects of KATP channel blocker on evoked exocytosis. Activation or inhibition of KATP channels suppressed or enhanced the depressant action of extracellular adenosine on evoked exocytosis. This was accompanied with an increase or decrease in adenosine-induced ROS production, respectively. KATP channel-dependent modulation of adenosine action was halted by antioxidant and NADPH-oxidase inhibitor. Also, activation of KATP channels led to an increase in ROS production suppressing the negative effects of extracellular ATP on evoked release in a ROS-dependent manner. SIGNIFICANCE: KATP channel-mediated modulation of release has specific features in distal and proximal compartments and depends on endogenous ROS levels and lipid raft integrity. Activation of KATP channels suppresses the action of extracellular adenosine and ATP on evoked release by increasing ROS production.

Diversity of Mammalian Motoneurons and Motor Units.

Although they share the common function of controlling muscle fiber contraction, spinal motoneurons display a remarkable diversity. Alpha-motoneurons are the "final common pathway", which relay all the information from spinal and supraspinal centers and allow the organism to interact with the outside world by controlling the contraction of muscle fibers in the muscles. On the other hand, gamma-motoneurons are specialized motoneurons that do not generate force and instead specifically innervate muscle fibers inside muscle spindles, which are proprioceptive organs embedded in the muscles. Beta-motoneurons are hybrid motoneurons that innervate both extrafusal and intrafusal muscle fibers. Even among alpha-motoneurons, there exists an exquisite diversity in terms of motoneuron electrical and molecular properties, physiological and structural properties of their neuromuscular junctions, and molecular and contractile properties of the innervated muscle fibers. This diversity, across species, across muscles, and across muscle fibers in a given muscle, underlie the vast repertoire of movements that one individual can perform.

Motoneurons innervation determines the distinct gene expressions in multinucleated myofibers.

BACKGROUND: Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor (AChR) proteins are clustered by the neuronal agrin signal. Meanwhile, several nuclei in each myofiber are specially enriched around the NMJ for postsynaptic gene transcription. It remains mysterious that how gene expressions in these synaptic nuclei are systematically regulated, especially by motoneurons. RESULTS: We found that synaptic nuclei have a distinctive chromatin structure and gene expression profiling. Synaptic nuclei are formed during NMJ development and maintained by motoneuron innervation. Transcriptome analysis revealed that motoneuron innervation determines the distinct expression patterns in the synaptic region and non-synaptic region in each multinucleated myofiber, probably through epigenetic regulation. Myonuclei in synaptic and non-synaptic regions have different responses to denervation. Weighted gene co-expression network analysis revealed that the histone lysine demethylases Kdm1a is a negative regulator of synaptic gene expression. Inhibition of Kdm1a promotes AChR expression but impairs motor functions. CONCLUSION: These results demonstrate that motoneurons innervation determines the distinct gene expressions in multinucleated myofibers. Thus, dysregulation of nerve-controlled chromatin structure and muscle gene expression might cause muscle weakness and atrophy in motoneuron degenerative disorders.

Glutamatergic Synapse Dysfunction in Drosophila Neuromuscular Junctions Can Be Rescued by Proteostasis Modulation.

Glutamate is the major excitatory neurotransmitter in the nervous system, and the Drosophila glutamatergic neuromuscular junctions (NMJs) offer a tractable platform to understand excitatory synapse biology both in health and disease. Synaptopathies are neurodegenerative diseases that are associated with synaptic dysfunction and often display compromised proteostasis. One such rare, progressive neurodegenerative condition, Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph Disease (MJD), is characterized by cerebellar ataxia, Parkinsonism, and degeneration of motor neuron synapses. While the polyQ repeat mutant protein ataxin-3 is implicated in MJD, it is unclear how it leads to impaired synaptic function. In this study, we indicated that a Drosophila model of MJD recapitulates characteristics of neurodegenerative disorders marked by motor neuron dysfunction. Expression of 78 polyQ repeats of mutant ataxin-3 protein in Drosophila motor neurons resulted in behavioral defects, such as impaired locomotion in both larval and adult stages. Furthermore, defects in eclosion and lifespan were observed in adult flies. Detailed characterization of larval glutamatergic neuromuscular junctions (NMJs) revealed defects in morphological features along with compromised NMJ functioning. Autophagy, one of the key proteostasis pathways, is known to be impaired in the case of several synaptopathies. Our study reveals that overexpression of the autophagy-related protein Atg8a rescued behavioral defects. Thus, we present a model for glutamatergic synapse dysfunction that recapitulates synaptic and behavioral deficits and show that it is an amenable system for carrying out genetic and chemical biology screens to identify potential therapeutic targets for synaptopathies.

Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration.

Terminal Schwann cells (TSCs) help regulate the formation, maintenance, function, and repair of neuromuscular junctions (NMJs) and axon guidance after muscle injury. Premature activation of muscle satellite cells (SCs), induced by isosorbide dinitrate (ISDN) before injury, accelerates myogenic regeneration, disrupts NMJ remodeling and maturation, decreases Sema3A protein-induced neuro-repulsion, and is accompanied by time-dependent changes in S100B protein levels. Here, to study the effects of premature SC activation on TSCs and SCs, both expressing P75 nerve growth-factor receptor, in situ hybridization was used to identify transcripts of S100B and Sema3A, and the number, intensity, and diameter of expression sites were analyzed. The number of sites/fields expressing S100B and Sema3A increased with regeneration time (both p < 0.001). Expression-site intensity (S100B) and diameter (S100B and Sema3A) decreased during regeneration (p = 0.005; p < 0.05, p = 0.006, respectively). P75 protein colocalized with a subset of S100B and Sema3A expression sites. Principal component analyses of gene expression, protein levels, and histological variables (fiber diameter, vascular density) in control and ISDN-pretreated groups explained 83% and 64% of the dataset variance, respectively. A very strong loading coefficient for colocalization of P75 protein with S100B and Sema3A mRNAs (0.91) in control regenerating muscle dropped markedly during regeneration disrupted by premature SC activation (-0.10 in Factor 1 to 0.55 in Factor 3). These findings strongly implicate the triple-expression profile by TSCs and/or SCs as a strong correlate of the important synchrony of muscle and nerve regeneration after muscle tissue injury. The results have the potential to focus future research on the complex interplay of TSCs and SCs in neuromuscular tissue repair and help promote effective function after traumatic muscle injury.

Brain-Derived Neurotrophic Factor Is an Important Therapeutic Factor in Mesenchymal Stem Cell Secretions for Treatment of Traumatic Peripheral Pelvic Injuries.

Traumatic neuromuscular injury to the pudendal nerve and urethra during childbirth does not regenerate well and contributes to stress urinary incontinence in women. Mesenchymal stem cells (MSCs) can improve neuroregeneration via their secretions, or secretome, which includes brain-derived neurotrophic factor (BDNF). In this study, we investigated whether BDNF is a key factor in the secretome of MSCs for the facilitation of functional recovery following a dual simulated childbirth injury. BDNF knockdown (KD) MSCs were created using an anti-BDNF shRNA lentivirus vector. A scrambled sequence was used as a transduction control (scrambled). Cells were cultured for 24 h before media was concentrated 50x to create concentrated conditioned media (CCM) containing MSC secretome. CCM of unmanipulated MSCs was screened for high BDNF expression (high BDNF CCM). Concentrated control media (CM) was created by concentrating media not conditioned by cells. Female Sprague-Dawley rats underwent bilateral pudendal nerve crush and vaginal distension (Injury) or sham injury. One hour and 1 week after injury, sham injured rats received CM, and injured rats received CM, high BDNF CCM, KD CCM, or scrambled CCM (300 µl intraperitoneally). Three weeks after injury, rats underwent leak point pressure (LPP) and pudendal nerve sensory branch potential (PNSBP) recordings. The urethra and pudendal nerve were harvested for anatomical assessment. ANOVA followed by the Student-Newman-Keuls test determined significant differences between groups (p < 0.05). BDNF KD CCM had significantly decreased BDNF concentration compared to scrambled CCM, while the concentration in high BDNF CCM was significantly increased. LPP was significantly decreased in CM and KD CCM treated animals compared to sham injury, but not with scrambled or high BDNF CCM. PNSBP firing rate showed a significant decrease with CM treatment compared to sham injury. Neuromuscular junctions in the urethral sphincter in KD CCM, scrambled CCM, and high BDNF CCM were healthier than CM treated rats. While anatomical and nerve function tests demonstrate regeneration of the pudendal nerve with any CCM treatment, LPP results suggest it takes longer to recover continence with reduced BDNF in CCM. BDNF in MSC CCM is an important factor for the acceleration of recovery from a dual nerve and muscle injury.

The distribution of neuromuscular junctions depends on muscle pennation, when botulinum neurotoxin receptors and SNAREs expression are uniform in the rat.

BACKGROUND: Botulinum neurotoxins (BoNTs) are used to treat spastic disorders. Depending on muscle size, one or multiple injections are recommended according to labels to target neuromuscular junctions (NMJ). However, information about NMJ distribution and number in muscles, as well as expression of receptors and molecular targets of toxins is scarce in human and animal models. METHODS: Seven muscles from adult rats were used to identify expression of BoNT receptors and SNAREs using immunohistochemistry (IHC), and fluorescent α-Bungarotoxin combined to light-sheet microscopy used to determine their distribution. RESULTS: The location, number, and density of NMJ were muscle specific and mostly dependent on the type of pennation (myofiber orientation). In the Flexor Digitorum Brevis (a very small muscle) NMJ were as numerous as in the Gastrocnemius lateralis. A strong expression of SV2C, Synaptotagmin 2, SNAP25 and VAMP1 were observed in all muscles, and SV2A, Synaptotagmin 1 and VAMP2 were never detected. CONCLUSION: This work highlights the specific distribution of NMJ in muscles which seems to depend on the type of pennation. Detailed observation of myofibers organization might help clinicians to better evaluate the location of NMJ in humans; the molecular phenotyping of NMJ will contribute to better integrate the rat model into research of BoNT therapeutics.

Optoionic Sensing.

The eye converts an optical signal to an ionic signal. This transduction is mimicked here using a photodiode in contact with ionic conductors, such as hydrogels. Photons generate electron-hole pairs in the photodiode. The photodiode/hydrogel interface forms capacitive coupling so that movements of electrons and holes in the photodiode induce movements of ions in the hydrogels. The hydrogels can be readily made stretchable and biocompatible to mimic the function of nerves. When light is turned on and off, the voltage between the hydrogels responds within 10 ms, comparable to the response in the human eye. A photosensitive skin is demonstrated to generate a voltage in response to light but not to stretch. Furthermore, a photosensitive actuation is demonstrated to mimic the light-triggered reflex, such as blinking of the eye and camouflage of the skin. Optoionic transduction has potential applications for wearable devices, implantable devices, and robotics.