Dichotomy in phasic-tonic neuromuscular structure of crayfish inhibitory axons.

Crustacean muscles are unique in their innervation by both excitatory and inhibitory neurons; therefore, they exhibit polyneuronal and multiterminal innervation. Because excitatory motoneurons are broadly divided into phasic and tonic types, we hypothesized that inhibitory neurons would follow a similar dichotomy. The abdominal extensor muscles in crayfish are separated into parallel deep and superficial bundles; the former has fast muscle fibers innervated by phasic excitatory motoneurons, and the latter has slow fibers supplied by tonic excitatory motoneurons. Each muscle also is innervated by a single, separate inhibitory neuron that uses gamma-aminobutyric acid (GABA) as the inhibitory neurotransmitter. The pattern of axonal branching by the separate inhibitory axons in phasic and tonic abdominal extensor muscles was visualized with confocal microscopy in preparations labeled for GABA-like immunoreactivity. Initial observations indicated that the phasic muscle was covered by extensive GABAergic, filiform axon terminals, whereas innervation of the tonic muscle was comprised of more localized and varicose terminals. With quantitative analyses, we found that the phasic axon has a more highly branched nature than the tonic in first- and second-order branches. The phasic axon branches also were significantly longer than the tonic branches in the second- and third-order branches. Synaptic varicosities in the phasic branches were smaller and less frequent than those in the tonic branches. The fine structure of the inhibitory nerve terminals near synaptic contacts examined with thin-serial-section electron microscopy revealed distinct differences between the phasic system and the tonic system. The phasic terminals were smaller in cross-sectional area than the tonic terminals, and they had smaller synapses and fewer mitochondria. The presynaptic active zone dense bodies were similar in length and number between phasic and tonic synapses. However, their number per synaptic area was two-fold higher in phasic synapses compared with tonic synapses because of the smaller size of the phasic synapses. Thus, within the same neuromuscular system, inhibitory synaptic terminals revealed unique phasic and tonic identities similar to those observed for the excitatory axons.     

Thickness of the basal lamina at the frog neuromuscular junction

The Schwann cell basal lamina at the frog neuromuscular junction is much thicker than previously supposed. Its thickness has been demonstrated by modifying the fixation: the nerve muscle preparation is fixed in situ without prior exposure to Ringer solution and the osmolarity of the glutaraldehyde fixative is increased to 285 mOsm. With this procedure the basal lamina is seen to be up to 1 micron thick around the Schwann cells of the nerve terminal and the preterminal axon. The thickness of the basal lamina surrounding the Schwann cells of the myelinated portion of the axons within the perineural sheath (20 nm) is not changed by this modification of fixation. The present results indicate that in addition to the ability to form myelin, other properties of the Schwann cells differ inside and outside the perineurial sheath.     

Silent synapses in neuromuscular junction development

In the last few years, evidence has been found to suggest that some synaptic contacts become silent but can be functionally recruited before they completely retract during postnatal synapse elimination in muscle. The physiological mechanism of developmental synapse elimination may be better understood by studying this synapse recruitment. This Mini‐Review collects previously published data and new results to propose a molecular mechanism for axonal disconnection. The mechanism is based on protein kinase C (PKC)‐dependent inhibition of acetylcholine (ACh) release. PKC activity may be stimulated by a methoctramine‐sensitive M2‐type muscarinic receptor and by calcium inflow though P/Q‐ and L‐type voltage‐dependent calcium channels. In addition, tropomyosin‐related tyrosine kinase B (trkB) receptor‐mediated brain‐derived neurotrophic factor (BDNF) activity may oppose the PKC‐mediated ACh release depression. Thus, a balance between trkB and muscarinic pathways may contribute to the final functional suppression of some neuromuscular synapses during development. © 2010 Wiley‐Liss, Inc.     

Presynaptic architecture of the larval zebrafish neuromuscular junction

This article shows the ultrastructural architecture of larval zebrafish (Danio rerio) neuromuscular junctions in three dimensions. We compare classical electron microscopy fixation techniques with high‐pressure freezing followed by freeze substitution (HPF/FS) in combination with electron tomography. Furthermore, we compare the structure of neuromuscular junctions in 4‐ and 8‐dpf zebrafish larvae with HPF/FS because this allows for close‐to‐native ultrastructural preservation. We discovered that synaptic vesicles of 4‐dpf zebrafish larvae are larger than those of 8‐dpf larvae. Furthermore, we describe two types of dense‐core vesicles and quantify a filamentous network of small filaments interconnecting synaptic vesicles as well as tethers connecting synaptic vesicles to the presynaptic cell membrane. In the center of active zones, we found elaborate electron‐dense projections physically connecting vesicles of the synaptic vesicle pool to the presynaptic membrane. Overall this study establishes the basis for systematic comparisons of synaptic architecture at high resolution in three dimensions of an intact vertebrate in a close‐to‐native state. Furthermore, we provide quantitative information that builds the basis for diverse systems biology approaches in neuroscience, from comparative anatomy to cellular simulations. J. Comp. Neurol. 523:1984–1997, 2015 © 2015 Wiley Periodicals, Inc.     

Origin of acetylcholinesterase in the neuromuscular junction formed in the in vitro innervated human muscle

Synaptic basal lamina is interposed between the pre- and postsynaptic membrane of the neuromuscular junction (NMJ). This position permits deposition of basal lamina-bound NMJ components of both neuronal and muscle fibre origin. One such molecule is acetylcholinesterase (AChE). The origin of NMJ AChE has been investigated previously as the answer would elucidate the relative contributions of muscle fibers and motor neurons to NMJ formation. However, in the experimental models used in prior investigations either the neuronal or muscular components of the NMJs were removed, or the NMJs were poorly differentiated. Therefore, the question of AChE origin in the intact and functional NMJ remains open. Here, we have approached this question using an in vitro model in which motor neurons, growing from embryonic rat spinal cord explants, form well differentiated NMJs with cultured human myotubes. By immunocytochemical staining with species-specific anti-AChE antibodies, we are able to differentiate between human (muscular) and rat (neuronal) AChE at the NMJ. We observed strong signal at the NMJ after staining with human AChE antibodies, which suggests a significant muscular AChE contribution. However, a weaker, but still clearly recognizable signal is observed after staining with rat AChE antibodies, suggesting a smaller fraction of AChE was derived from motor neurons. This is the first report demonstrating that both motor neuron and myotube contribute synaptic AChE under conditions where they interact with each other in the formation of an intact and functional NMJ.     

Morphology and molecular organization of the adult neuromuscular junction of Drosophila

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5-1.5 microm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8-5.5 microm) to an elongate terminal (80-100 microm long) with few branches and contiguous swellings (3-15 microm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II-immunoreactivity is not detectable at type I terminals, while DLG-immunoreactivity is observed at the synapse; 3) a Shaker-GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle-specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs.     

Recovery of neuromuscular junction morphology following 16 days of spaceflight.

It has previously been established that spaceflight elicits alterations in the morphology of the neuromuscular system that includes expansion of the neuromuscular junction (NMJ) and myofiber atrophy. The purpose of this study was to determine the capacity of the neuromuscular system to recover from spaceflight-induced modifications upon return to normal gravity. Soleus muscles were obtained from rats participating in the 16-day Neurolab space shuttle mission at 1 day and 14 days after returning to Earth: solei were also taken at the same time points from ground-based control rats. Cytofluorescent techniques, coupled with confocal microscopy, were used to assess NMJ morphology. Histochemistry, in conjunction with phase contrast microscopy, was employed to examine myofiber size and type. Results indicate that 1 day after landing both pre- and postsynaptic stained areas of the NMJ were significantly (P < or = 0.05) larger in the spaceflight group than in controls. Moreover, significant myofiber atrophy was demonstrated in animals subjected to 0 gravity. By 14 days following return to the Earth, however, NMJ stained areas and muscle fiber size were no longer different from control values at that same interval. These results suggest that the neuromuscular system possesses a robust capacity to recover from spaceflight-induced perturbations upon return to normal gravitational influences.     

The relationship between exercise participation and well-being of the retired elderly

The objective of this study is to identify the relationship between physical exercise and the feelings of well-being of the retired elderly. Face-to-face questionnaire survey was adopted, and quota sampling was chosen to select the respondents. A total of 352 valid questionnaires were collected in selected parks in Taipei. The survey found that the Taiwanese retired elderly participated in a wide range of sports, from light exercise such as walking to vigorous exercise such as tennis and aerobics, and their participation frequency was very high. Most of the respondents (87.2%) were defined as having positive well-being. The results showed that exercise frequency and well-being were positively correlated, but a negative correlation was found between exercise intensity and well-being. The survey found that the intensity of exercise was self-evaluated by as being low to moderate, but most of the activities were in the categories of moderate or vigorous intensity according to the metabolic equivalents suggested by American College of Sports Medicine. The study suggest that the elderly felt more comfortable and gained more pleasure psychologically while participating in exercises less intensive. As a result, the retired elderly are recommended to take exercise as frequently as possible. As to exercise intensity, self-evaluated low-to-moderate intensity exercise might be better for the psychological well-being of the elderly.     

Morphological changes in neuromuscular junctions during exercise

Long lasting exercise produces several morphological changes in teleostean neuromuscular junctions (NJs), consisting of progressive synaptic vesicles (SVs) depletion and lamellar branching of the nerve endings. Exercised fishes kept swimming during 1 hr against a 3.5 1/min flow of oxygenated water in spite of the fact that the number of SVs was reduced in about 70% after 10 min of exercise. This observation indicates that the SVs formation fails to restore their original number and consequently, under such circumstances, the transmitter release may occur by a different mechanism.     

Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti‐PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc‐specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse. J. Neurosci. Res. 55:261–267, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Acetylcholine receptor distribution and synapse elimination at the developing neuromuscular junction of mdx mice

The pattern of innervation of the vertebrate neuromuscular junction is established during early development, when junctions go from multiple to single innervation in the phenomenon of synapse elimination, suggesting that changes at the molecular level in the postsynaptic cell lead to the removal of nerve terminals. The mdx mouse is deficient in dystrophin and associated proteins that are part of the postsynaptic cytoskeleton. We used rhodamine-alpha-bungarotoxin and anti-neurofilament IgG-FITC to stain acetylcholine receptors and nerve terminals of the sternomastoid muscle during postnatal development in mdx and control C57BL/10 mice. Using fluorescence confocal microscopy, we observed that, 7 days after birth, 86.7% of the endplates of mdx mice were monoinnervated (n = 200) compared with 41.4% in control mice (n = 200). By the end of the second postnatal week, all endplates were innervated singly (100% mdx and 94.7% controls, n = 200 per group). These results show that dystrophic fibers achieve single innervation earlier, perhaps because dystrophin or a normal cytoskeletal complex is implicated in this phenomenon.     

The neuromuscular junction: Anatomical features and adaptations to various forms of increased,or decreased neuromuscular activity

The neuromuscular junction (NMJ) allows communication between motor neurons and muscle fibers. During development, marked morphological changes occur as the functional NMJ is formed. During the postnatal period of rapid growth and muscle enlargement, endplate size concurrently increases. Even beyond this period of pronounced plasticity, the NMJ undergoes subtle morphological remodeling—expansion and retraction—although its overall dimensions remain stable. This natural, continual NMJ remodeling is amplified with alterations in neuromuscular activity. Increased activity, presented by exercise training, typically results in expansion of NMJ size. Disuse, brought about by neurotoxins, denervation, or spaceflight, also elicits substantial reconfiguring of the endplate.     

Capsaicin delays regeneration of the neuromuscular junction of rat extensor digitorum longus muscle after ischemia

Trauma or the tourniquet used in orthopedic surgery is often associated with ischemia-reperfusion (I/R) injury with a consequent decrease of muscle power. To explore whether components of the neuromuscular junction (NMJ) are involved in this muscle dysfunction, NMJs were ultrastructurally characterized in the extensor digitorum longus muscle of rats at reperfusion times of 1, 24, 72, and 168 h after a 120-min arterial occlusion. Disorganization of the presynaptic membrane and mitochondrial injury was noted at 1 h, followed by fragmentation and partial engulfment of nerve terminals by Schwann cells at 24 and 72 h. The magnitude of degenerative changes declined at 168 h, suggesting the commencement of regeneration. The postsynaptic membrane remained intact throughout the whole period. In our previous study, deafferentation with pretreatment of the sciatic nerve with capsaicin, which reduces neurogenic inflammation and has a selective effect on nociceptive fibers, improved functional recovery of the muscle after I/R. The present results document a significantly delayed structural regeneration of the motor nerve terminals after combined capsaicin and I/R treatment. Since capsaicin treatment alone had no discernible effect on the structure of NMJs, the findings point to a possibly indirect effect of capsaicin on the motor nerves, which may predispose them to increased susceptibility unmasked only by a subsequent injury. The mismatch between the enhanced functional improvement of the muscle and delayed regeneration of the nerve after capsaicin pretreatment questions the efficient use of such deafferentation to protect the integrity of neuromuscular junctions in I/R injury.     

Androgen receptor immunoreactivity in skeletal muscle: enrichment at the neuromuscular junction

Potential cellular targets of androgen action within skeletal muscle of the rat were determined by comparing the cellular distribution of androgen receptor (AR)-positive nuclei in the highly androgen-responsive levator ani (LA) muscle with that of the relatively androgen-unresponsive extensor digitorum longus (EDL) muscle. We found that androgen responsiveness correlates with AR expression in muscle fibers and not in fibroblasts. Results indicate that a much higher percentage of myonuclei in the LA are AR(+) than in the EDL (74% vs. 7%), correlating with differences in androgen responsiveness. Both muscles contain an equivalent proportion of AR(+) fibroblasts (approximately 62%). AR(+) nuclei were not observed in terminal Schwann cells in either muscle. These results suggest that ARs within LA muscle fibers mediate the androgen-dependent survival and growth of the LA muscle and its motoneurons. We also observed an unexpected enrichment of AR(+) myonuclei and fibroblasts proximate to neuromuscular junctions, suggesting that ARs at muscle synapses may selectively regulate synapse-specific genes important for the survival and growth of motoneurons. Although castration reduced the proportion of AR(+) fibroblasts in both muscles, the proportion of AR(+) myonuclei was reduced only in the LA. As expected, testosterone treatment prevented these effects of castration but, unexpectedly, increased the proportion of AR(+) myonuclei in the EDL to above normal. These results suggest that how AR expression in skeletal muscle is influenced by androgens depends not only on the particular muscle but on the particular cell type within that muscle.     

Factors affecting denervation-like changes at the neuromuscular junction during aging

The ability to sustain synaptic transmission diminishes with aging. To determine whether this is accompanied by alterations in the structure of the synapse, end-plate architecture was examined in EDL muscles from 10- to 25-month rats. There was a significant age-related increase in end-plate area and a decrease in the number of nerve terminals per end plate. Furthermore, the percentage of end plates with ultraterminal sprouts increased dramatically with age. To compare the morphological changes associated with aging to the changes in response to denervation, EDL muscles from 10- and 25-month animals were partially denervated. Both end-plate area and ultraterminal sprouting increased, while terminal number decreased following denervation in the 10-month muscles. To determine whether the age- and denervation-associated changes were accompanied by alterations in muscle-derived nerve-outgrowth factors, in vitro assays were performed. Neurite outgrowth was quantified from embryonic motoneurons incubated with muscle extract, or grown on cryostat-cut muscle cross sections from 10- and 25-month innervated and denervated EDL muscles. Both aged and denervated muscles induced greater degrees of neurite outgrowth compared with younger innervated muscles. Innervation to the EDL was then examined, and signs of axonal degeneration were observed. It is suggested that aging is associated with alterations in the motor axon to the EDL muscle. These changes are manifest at the neuromuscular junction. In turn, the muscle responds as if it were in a state of functional denervation.     

Restricted diffusion of extracellular potassium at the neuromuscular junction of aged rats

Changes in the extracellular K+ activity, aK, were recorded at the neuromuscular junction in aged and young rats. After the onset of stimulation, aK rose to a peak value which was maintained until stimulation was terminated. The peak value was greater in the older rats at any given distance. Also, it took longer for aK to attain one-half its peak value in the older animals. It is concluded that diffusion of K+ away from the end plate is probably slower in aged than in young rats.     

Effects of electrical stimulation on neuromuscular junction morphology in the aging rat tongue

Alterations in neuromuscular junction (NMJ) structure in cranial muscles may contribute to age-related deficits in critical sensorimotor actions such as swallowing. Neuromuscular electrical stimulation (NMES) is used in swallowing therapy, but it is unclear how NMJ structure is affected or if NMJ morphology is best measured in two or three dimensions. Two- and three-dimensional measurements of NMJ morphology in the genioglossus muscle were compared in rats that had undergone 8 weeks of hypoglossal nerve stimulation vs. untreated controls. The relationship between motor endplate volume and nerve terminal volume had a mean positive slope in 90% of the young adult controls, but it was positive in only 50% of the old controls; 89% of NMES old rats had a positive slope. NMJ measurements were more accurate when measured in three dimensions. In the NMJ, aging and NMES are associated with changes in the pre- and post-synaptic relationship.     

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda , Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu‐like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualate's inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N‐methyl‐D‐aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals. © 2016 Wiley Periodicals, Inc.     

Localization of neuronal calcium sensor-1 at the adult and developing rat neuromuscular junction

Neuronal calcium sensor (NCS-1; frequenin) is a calcium-binding protein involved in the regulation of neurotransmission in the central and peripheral nervous systems from insects to vertebrates. This study reports the localization of NCS-1 immunoreactivity, by Western blotting and immunohistochemistry, at the adult and developing postnatal rat neuromuscular junction. Our confocal immunofluorescence results on the whole-mount muscle and on semithin cross-sections are indicative of the localization of NCS-1 to motor axon terminals. There is no evidence of immunoreactivity in the postsynaptic side of the neuromuscular junctions or teloglial Schwann cells. These results suggest that NCS-1 is involved in the formation and function of presynaptic nerve terminal part of the neuromuscular junction during synaptogenesis and in adult mammals.