Effects of Vocal Training on Thyroarytenoid Muscle Neuromuscular Junctions and Myofibers in Young and Older Rats.

The purpose of this investigation was to determine the effects of vocal training on neuromuscular junction (NMJ) morphology and muscle fiber size and composition in the thyroarytenoid muscle, the primary muscle in the vocal fold, in younger (9-month) and older (24-month) Fischer 344 × Brown Norway male rats. Over 4 or 8 weeks of vocal training, rats of both ages progressively increased their daily number of ultrasonic vocalizations (USVs) through operant conditioning and were then compared to an untrained control group. Neuromuscular junction morphology and myofiber size and composition were measured from the thyroarytenoid muscle. Acoustic analysis of USVs before and after training quantified the functional effect of training. Both 4- and 8-week training resulted in less NMJ motor endplate dispersion in the lateral portion of the thyroarytenoid muscle in rats of both ages. Vocal training and age had no significant effects on laryngeal myofiber size or type. Vocal training resulted in a greater number of USVs with longer duration and increased intensity. This study demonstrated that vocal training induces laryngeal NMJ morphology and acoustic changes. The lack of significant effects of vocal training on muscle fiber type and size suggests vocal training significantly improves neuromuscular efficiency but does not significantly influence muscle strength changes.

Morphological variability is greater at developing than mature mouse neuromuscular junctions.

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31-32) timepoints. The inter-muscular variability was greater in mature post-synaptic AChR morphology than that of the pre-synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post-synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre- and post-synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.

Comparative anatomy of the mammalian neuromuscular junction.

The neuromuscular junction (NMJ)-a synapse formed between lower motor neuron and skeletal muscle fibre-represents a major focus of both basic neuroscience research and clinical neuroscience research. Although the NMJ is known to play an important role in many neurodegenerative conditions affecting humans, the vast majority of anatomical and physiological data concerning the NMJ come from lower mammalian (e.g. rodent) animal models. However, recent findings have demonstrated major differences between the cellular anatomy and molecular anatomy of human and rodent NMJs. Therefore, we undertook a comparative morphometric analysis of the NMJ across several larger mammalian species in order to generate baseline inter-species anatomical reference data for the NMJ and to identify animal models that better represent the morphology of the human NMJ in vivo. Using a standardized morphometric platform ('NMJ-morph'), we analysed 5,385 individual NMJs from lower/pelvic limb muscles (EDL, soleus and peronei) of 6 mammalian species (mouse, cat, dog, sheep, pig and human). There was marked heterogeneity of NMJ morphology both within and between species, with no overall relationship found between NMJ morphology and muscle fibre diameter or body size. Mice had the largest NMJs on the smallest muscle fibres; cats had the smallest NMJs on the largest muscle fibres. Of all the species examined, the sheep NMJ had the most closely matched morphology to that found in humans. Taken together, we present a series of comprehensive baseline morphometric data for the mammalian NMJ and suggest that ovine models are likely to best represent the human NMJ in health and disease.

Age-related Changes in Motor Function (I). Mechanical and Neuromuscular Factors.

This two-part narrative review aims to provide an insight into the age-related mechanical and neuromuscular factors contributing to: (1) decreased maximal muscle strength and power; (2) decreased force control; and (3) increased fatigability. Structural and functional changes from the macro-level of the muscle-tendon unit to the micro-level of the single muscle fibre have been reviewed and are described. At the muscle-tendon unit level, muscle volume, thickness and cross-sectional area, as well as pennation angle and fascicle length all decrease as part of the natural ageing process. These changes negatively affect muscle quality, muscle and tendon stiffness and Young's modulus and account for impairment in motor performance. A progressive age-related alteration in neuromuscular function is also well-established, with reduction in number and firing rate of the motor unit, contractile velocity and specific tension of muscle fibres, and stability of neuromuscular junction. These could be the result of structural alterations in the: (i) motor neuron, with number reduced, size and collateral sprouting increased; (ii) neuromuscular junction, with decreased post-synaptic junctional fold and density of active zones and increased pre-synaptic branching and post-synaptic area; and (iii) muscle fibre, with decreased number and size and increased type I and co-expression of myosin heavy chain.

Intramuscular Nerve Distribution of the Inferior Oblique Muscle.

Purpose: The intramuscular nerve distribution in the extraocular muscles is important for understanding their function. This study aimed to determine the intramuscular nerve distribution of the oculomotor nerve within the inferior oblique muscle (IO) using Sihler's staining.Method: Seventy-two IOs from 50 formalin-embalmed cadavers were investigated. The IO including its branch of the oculomotor nerve was finely dissected from its origin to its insertion point into the sclera. The total length of the muscle and its width were measured. The intramuscular nerve course was investigated after performing Sihler's staining, which is a whole-mount nerve-staining technique that stains the nerves while rendering other soft tissues either translucent or transparent.Results: The total length of the muscle and muscle width were 30.0 ± 2.8 mm (mean±standard deviation), 8.8 ± 1.2 mm, respectively. The oculomotor nerve enters the IO around the middle of the muscle and then divides into multiple smaller branches without distinct subdivisions. The intramuscular nerve distribution within the IO has a root-like arborization and supplies the entire width of the muscle. The Sihler's stained intramuscular nerve course (covering a length of 7.6 ± 1.2 mm) finishes around the distal one-third of the IO in gross observations.Conclusion: Sihler's staining is a useful technique for visualizing the gross nerve distribution of the IO. This new information about the nerve distribution and morphological features will improve the understanding of the biomechanics of the IO.

Location of the myoneural junction of the inferior oblique muscle: An anatomic study.

PURPOSE: To examine both the horizontal and vertical locations of the myoneural junction of the inferior oblique muscle (IOM) in reference to the ala nasi, IOM origin, and inferior orbital rim. MATERIALS AND METHODS: Fifty-six orbits from 56 Japanese cadavers (15 male and 41 female cadavers; average age at time of death, 86.5±9.4 years) were used in this experimental anatomical study. The inter-alae-nasi distance and the horizontal distances from the ipsilateral ala nasi to the IOM origin (ala-nasi-origin distance) and to the myoneural junction (ala-nasi-junction distance) were measured. The horizontal distance from the IOM origin to the myoneural junction (origin-junction distance) was calculated by subtraction of the ala-nasi-origin distance from the ala-nasi-junction distance. The vertical distance from the inferior orbital rim to the myoneural junction (rim-junction distance) was also measured. RESULTS: The ala-nasi-junction, origin-junction, and rim-junction distances were 12.2±3.2mm, 10.6±3.2mm, and 3.4±1.0mm, respectively. Males had a longer inter-alae-nasi distance than females (P<0.001), although the other distances did not show statistically significant sex-related difference (P>0.050). The ala-nasi-junction distance tended to be negatively correlated with the inter-alae-nasi distance (r=-0.222, P=0.050). CONCLUSIONS: The ala-nasi-junction distance can be affected by the inter-alae-nasi distance, which was found to be longer in males. Therefore, the IOM origin and the inferior orbital rim can be considered as more practical and reliable reference points to predict the location of the myoneural junction during the posterior inferior orbitotomy, irrespective of sex.

Improved Human Muscle Biopsy Method To Study Neuromuscular Junction Structure and Functions with Aging.

Reduced mobility and physical independence of elders has emerged as a major clinical and public health priority with extended life expectancy. The impact of the neuromuscular function on muscle activity and properties has emerged as a critical factor influencing the progress and outcome of muscle changes with aging. However, very little is known about the neuromuscular junctions (NMJs) in humans, in part due to technical constraints limiting the access to healthy, fresh neuromuscular tissue. Here, we describe a method, called Biopsy using Electrostimulation for Enhanced NMJ Sampling (BeeNMJs) that improves the outcome of muscle biopsies. We used local cutaneous stimulation to identify the area enriched with NMJs for each participant at the right Vastus lateralis (VL). The needle biopsy was then performed in proximity of that point. The BeeNMJs procedure was safe for the participants. We observed NMJs in 53.3% of biopsies in comparison with only 16.7% using the traditional method. Furthermore, we observed an average of 30.13 NMJs per sample compared to only 2.33 for the traditional method. Importantly, high-quality neuromuscular material was obtained whereby pre-, postsynaptic, and glial elements were routinely labeled, simultaneously with myosin heavy chain type I. The BeeNMJs approach will facilitate studies of NMJs, particularly in human disease or aging process.

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus.

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February-March) when these toads are active, to toads obtained from dry season (October-November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila.

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ∼50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.

Practical Anatomy of the Neuromuscular Junction in Health and Disease.

Neuromuscular junctions (NMJs) form between nerve terminals of spinal cord motor neurons and skeletal muscles, and perisynaptic Schwann cells and kranocytes cap NMJs. One muscle fiber has one NMJ, which is innervated by one motor nerve terminal. NMJs are excitatory synapses that use P/Q-type voltage-gated calcium channels to release the neurotransmitter acetylcholine. Acetylcholine receptors accumulate at the postsynaptic specialization called the end plate on the muscle fiber membrane, the sarcolemma. Proteins essential for the organization of end plates include agrin secreted from nerve terminals, Lrp4 and MuSK receptors for agrin, and Dok-7 and rapsyn cytosolic proteins in the muscle.

Cellular and Molecular Anatomy of the Human Neuromuscular Junction.

The neuromuscular junction (NMJ) plays a fundamental role in transferring information from lower motor neuron to skeletal muscle to generate movement. It is also an experimentally accessible model synapse routinely studied in animal models to explore fundamental aspects of synaptic form and function. Here, we combined morphological techniques, super-resolution imaging, and proteomic profiling to reveal the detailed cellular and molecular architecture of the human NMJ. Human NMJs were significantly smaller, less complex, and more fragmented than mouse NMJs. In contrast to mice, human NMJs were also remarkably stable across the entire adult lifespan, showing no signs of age-related degeneration or remodeling. Super-resolution imaging and proteomic profiling revealed distinctive distribution of active zone proteins and differential expression of core synaptic proteins and molecular pathways at the human NMJ. Taken together, these findings reveal human-specific cellular and molecular features of the NMJ that distinguish them from comparable synapses in other mammalian species.

Imaging three-dimensional innervation zone distribution in muscles from M-wave recordings.

OBJECTIVE: To localize neuromuscular junctions in skeletal muscles in vivo which is of great importance in understanding, diagnosing and managing of neuromuscular disorders. APPROACH: A three-dimensional global innervation zone imaging technique was developed to characterize the global distribution of innervation zones, as an indication of the location and features of neuromuscular junctions, using electrically evoked high-density surface electromyogram recordings. MAIN RESULTS: The performance of the technique was evaluated in the biceps brachii of six intact human subjects. The geometric centers of the distributions of the reconstructed innervation zones were determined with a mean distance of 9.4 ± 1.4 cm from the reference plane, situated at the medial epicondyle of the humerus. A mean depth was calculated as 1.5 ± 0.3 cm from the geometric centers to the closed points over the skin. The results are consistent with those reported in previous histology studies. It was also found that the volumes and distributions of the reconstructed innervation zones changed as the stimulation intensities increased until the supramaximal muscle response was achieved. SIGNIFICANCE: Results have demonstrated the high performance of the proposed imaging technique in noninvasively imaging global distributions of the innervation zones in the three-dimensional muscle space in vivo, and the feasibility of its clinical applications, such as guiding botulinum toxin injections in spasticity management, or in early diagnosis of neurodegenerative progression of amyotrophic lateral sclerosis.

The anatomy and prevalence of the juncturae tendinum in the hands. A systematic review and meta-analysis

The movement of the fingers of the hand is thought to be limited in extension due in part to the presence of the juncturae tendinum (JT) in the dorsal webspaces. Clinically, these structures may help surgeons in identifying the extensor slips of the extensor digitorum communis (edc) and constitute an additional resource for tendon repair. These connections are highly variable in relation to their frequency and shape. Based on von Schroeder’s classification, an evidence synthesis of those structures was performed; nineteen cadaveric studies were located with a total of 2060 hands. Using meta-analytical methods, the pooled prevalence values were computed for each webspace and each type of JT; the JT type 1 (JT-1) and 3 (JT-3) were by far the most common in the 2nd and 4th webspaces, respectively. In the 3rd webspace, the JT type 2 (JT-2) had the highest prevalence value, followed by JT-3 and JT-1. The pooled means of JT size revealed that JT-3 was significantly longer and thicker than JT-1 and JT-2, while JT-1 was significantly wider than JT-2 and JT-3. Ancestry-based prevalence values showed that Middle Eastern populations had the lowest overall JT prevalence values, and Indian and Turkish ancestries had the highest values. Side-based prevalence values demonstrate significance only for the JT-3y type in the 4th webspace, where it was twice more frequent on the left side. While JT-3 was not infrequent between edc to the ring finger and extensor digiti minimi (16.5%), it occurred 13 times more when edc to the little finger was absent. This anatomical meta-analysis is likely to generate more accurate prevalence and mean size values of the juncturae tendinum in human hands

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Some reminiscences on studies of age-dependent and activity-dependent degeneration of sensory and motor endings in mammalian skeletal muscle.

I present here an overview of research on the biology of neuromuscular sensory and motor endings that was inspired and influenced partly by my educational experience in the Department of Zoology at the University of Durham, from 1971 to 1974. I allude briefly to neuromuscular synaptic structure and function in dystrophic mice, influences of activity on synapse elimination in development and regeneration, and activity-dependent protection and degeneration of neuromuscular junctions in Wld(S) mice.

Electrophysiological analysis of neuromuscular synaptic function in myasthenia gravis patients and animal models.

Study of the electrophysiological function of the neuromuscular junction (NMJ) is instrumental in the understanding of the symptoms and pathophysiology of myasthenia gravis (MG), an autoimmune disorder characterized by fluctuating and fatigable muscle weakness. Most patients have autoantibodies to the acetylcholine receptor at the NMJ. However, in recent years autoantibodies to other crucial postsynaptic membrane proteins have been found in previously 'seronegative' MG patients. Electromyographical recording of compound and single-fibre muscle action potentials provides a crucial in vivo method to determine neuromuscular transmission failure while ex vivo (miniature) endplate potential recordings can reveal the precise synaptic impairment. Here we will review these electrophysiological methods used to assess NMJ function and discuss their application and typical results found in the diagnostic and experimental study of patients and animal models of the several forms of MG.

Nerve branches to the posterior cricoarytenoid muscle may complicate the laryngeal reinnervation procedure.

OBJECTIVES/HYPOTHESIS: To better understand the reason for the low success rate of posterior cricoarytenoid (PCA) muscle reinnervation, we attempted to identify the communicating branches that may exist between the nerve branch to the PCA muscle and the other laryngeal adductors in addition to the interarytenoid (IA) muscle. STUDY DESIGN: Quantitative histological assessment. METHODS: Twenty human hemilarynges from patients with laryngeal or hypopharyngeal cancer were obtained after surgery and stained with Sihler's stain, which rendered the muscle translucent while counterstaining the neuroanatomy of the recurrent laryngeal nerve (RLN) inside the larynges. RESULTS: The nerve supply to the PCA muscle was separated into two main branches. One upper branch supplied the horizontal compartment, and the lower branch supplied the vertical/oblique compartment. In 14 specimens, two nerve branches to the PCA muscle arose separately from the RLN. In six specimens, one single main branch arose from the RLN and immediately ramified into two secondary branches. In all specimens except one, the nerve branch to the horizontal compartment was connected to the IA muscle. However, no communicating branches were observed between the nerve to the PCA muscle and the other laryngeal adductors. No anastomosis between nerve branches to the horizontal and vertical/oblique compartments or other variations of nerve distribution were observed. CONCLUSIONS: The communicating branches between the nerve branches to the PCA muscle and the IA muscle may be the only branch, complicating the reinnervation procedure. More investigations are needed to identify variations in the terminal branch distribution from the RLN. LEVEL OF EVIDENCE: NA.

Morphological and functional remodelling of the neuromuscular junction by skeletal muscle PGC-1α.

The neuromuscular junction (NMJ) exhibits high morphological and functional plasticity. In the mature muscle, the relative levels of physical activity are the major determinants of NMJ function. Classically, motor neuron-mediated activation patterns of skeletal muscle have been thought of as the major drivers of NMJ plasticity and the ensuing fibre-type determination in muscle. Here we use muscle-specific transgenic animals for the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) as a genetic model for trained mice to elucidate the contribution of skeletal muscle to activity-induced adaptation of the NMJ. We find that muscle-specific expression of PGC-1α promotes a remodelling of the NMJ, even in the absence of increased physical activity. Importantly, these plastic changes are not restricted to post-synaptic structures, but extended to modulation of presynaptic cell morphology and function. Therefore, our data indicate that skeletal muscle significantly contributes to the adaptation of the NMJ subsequent to physical activity.

Dissection of the transversus abdominis muscle for whole-mount neuromuscular junction analysis.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

The neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

Evidence for the functional compartmentalization of the temporalis muscle: a 3-dimensional study of innervation.

PURPOSE: The temporalis muscle is commonly used for functional transfer. It is architecturally complex, but few studies have examined its intramuscular innervation and none has used 3-dimensional modeling techniques. Understanding neuromuscular compartmentalization may allow the design of local muscle transfers to minimize donor-site morbidity. The purpose of the present study was to document the intramuscular innervation patterns throughout the volume of the temporalis muscle and define functional units within the muscle. MATERIALS AND METHODS: In 10 formalin-embalmed cadaveric specimens, the foramen ovale was exposed and the branches of the mandibular nerve were identified. Each branch was digitized in short segments extramuscularly and intramuscularly. Three-dimensional models were reconstructed from the digitized data using Maya software, and the innervation patterns were documented. RESULTS: The temporalis muscle was found to have superior and inferior parts that were further grouped by innervation into regions, with each receiving its innervation from 1 primary nerve. The nerves originated directly from the mandibular nerve, except in 3 specimens, where the posterior deep temporal nerve arose from the masseteric nerve. CONCLUSION: These results provide a detailed mapping of innervation patterns and suggest there are at least 5 functional compartments. Each of these has the capacity for selective activation, 3 of which have clinical value. These findings may allow for decreased donor-site morbidity and more functionally sophisticated designs in clinical practice.