Neuromuscular Junction Size Research Articles

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.

Highwire Regulates Presynaptic BMP Signaling Essential for Synaptic Growth

Highwire (Hiw), a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Here we show that Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses.

Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity

BackgroundThe transcription factor AP1 mediates long-term plasticity in vertebrate and invertebrate central nervous systems. Recent studies of activity-induced synaptic change indicate that AP1 can function upstream of CREB to regulate both CREB-dependent enhancement of synaptic strength as well as CREB-independent increase in bouton number at the Drosophila neuromuscular junction (NMJ). However, it is not clear from this study if AP1 functions autonomously in motor neurons to directly modulate plasticity.ResultsHere, we show that Fos and Jun, the two components of AP1, are abundantly expressed in motor neurons. We further combine immunohistochemical and electrophysiological analyses with use of a collection of enhancers that tightly restrict AP1 transgene expression within the nervous system to show that AP1 induction or inhibition in, but not outside of, motor neurons is necessary and sufficient for its modulation of NMJ size and strength.ConclusionBy arguing against the possibility that AP1 effects at the NMJ occur via a polysynaptic mechanism, these observations support a model in which AP1 directly modulates NMJ plasticity processes through a cell autonomous pathway in the motor neuron. The approach described here may serve as a useful experimental paradigm for analyzing cell autonomy of genes found to influence structure and function of Drosophila motor neurons.

Testosterone Regulates Terminal Schwann Cell Number and Junctional Size during Developmental Synapse Elimination

Previous work has shown that exposure to exogenous testosterone during synapse elimination permanently stabilizes synapses that would normally be lost in the androgen-sensitive levator ani (LA) muscle, indicating that testosterone is a potent stabilizing factor for developing LA synapses. Terminal Schwann cells (TSCs), which cap the neuromuscular junction, have also been implicated in the control of synaptic stability and may play a decisive role in the selective stabilization of synapses during synapse elimination. In this study, we begin to investigate the possible role of TSCs in the effect of testosterone on synapse elimination by determining whether testosterone influences their number. As the number of TSCs generally correlates with the size of endplates, we also measured endplate size. Male rats were castrated or sham gonadectomized at postnatal day (P) 7 and given capsules containing either testosterone or nothing. Three weeks later (P27–28), LA neuromuscular junctions (NMJs) were stained using immune and nonimmune markers. As expected, testosterone treatment during synapse elimination maintained synapses that would ordinarily be eliminated. In addition, we find that the size of LA endplates and the number of TSCs per LA junction were also increased by the testosterone treatment. However, testosterone significantly increased the number of TSCs on both singly and multiply innervated fibers, indicating that the effect of testosterone on the number of TSCs is not specific to its effect on synapse maintenance. Rather the testosterone-induced increase in the number of TSCs appears related to the size of LA NMJs, a relationship that has precedence.

Sexual dimorphism in neuromuscular junction size on a muscle used in courtship by green anole lizards

The green anole lizard exhibits seasonal courtship behavior that is sexually dimorphic. This courtship consists of the extension of a bright red throat fan (dewlap) associated with head-bobbing display behavior. While males extend their dewlaps in aggressive encounters as well as in courtship, females use their considerably smaller dewlaps much less frequently and mainly in agonistic encounters. In parallel, a number of components of the neuromuscular system controlling dewlap extension are greater in males than in females during the breeding season, including dewlap motoneuron soma size and muscle fiber size and number. These features do not seem to change substantially in adulthood, despite a dramatic decline in dewlap use during the nonbreeding season. We explored the morphology of this neuromuscular system in more detail in the present experiment in males and females during both the breeding and nonbreeding seasons. Fiber and whole muscle length (approximately perpendicular to the fibers) were measured. Acetylcholinesterase histochemistry was used to visualize neuromuscular junctions (NMJs), and the surface area and density of NMJs were assessed for each animal. During the breeding season, NMJ size was larger in males than in females, but NMJ density along each fiber was equivalent between the sexes. In addition, whole muscle length and that of individual muscle fibers, was larger in males than in females. However, when corrected for body size, the sex difference in muscle fiber length disappeared. In the nonbreeding season, the sexual dimorphisms were maintained, suggesting that these features do not change substantially due to differences in circulating testosterone or a difference in use across seasons. Overall, these results are consistent with the idea that enhanced NMJ size is a relatively stable feature of the dewlap muscle in adulthood that either facilitates or is a consequence of using a larger muscle to extend a bigger dewlap in males compared to females.

Structural and Functional Alterations of Neuromuscular Junctions in NCAM-Deficient Mice

The role of neural cell adhesion molecule (NCAM) in the development and maturation of the neuromuscular junction (NMJ) was explored by characterizing structurally and functionally NMJs from postnatal day 11 (P11) to P30 +/+, +/-, and -/- NCAM null mutant mice. Differences in NCAM levels resulted in alterations in the size and shape of NMJs, with -/- NMJs being smaller. Additionally both the withdrawal of polyneuronal innervation and the selective accumulation of synaptic vesicle protein in the presynaptic terminal were delayed. These observations suggest that the bidirectional signaling responsible for these events is impaired at -/- NMJs. Functionally, miniature end plate potential size, end plate potential size, and quantal content did not differ from that of wild type under either normal or low release conditions. However at normal release conditions, -/- NMJs, unlike +/+ NMJs, lacked paired-pulse facilitation. The most striking abnormality was the inability of NCAM null junctions to maintain transmitter output with repetitive stimuli. Combined electrophysiological and FM1-43-labeling studies suggest that NCAM null junctions are unable either to dock or to mobilize a sufficient number of vesicles at high but physiological rates of transmitter release. Taken together our observations show that many aspects of transmission are normal and, thus, that many presynaptic and postsynaptic molecules have assembled properly in the absence of NCAM. However, the fact that NCAM was required for specific aspects of transmission, including paired-pulse facilitation and reliable transmission with repetitive stimuli, suggests that NCAM either is directly involved in these processes or is required for the proper organization and/or function of other molecules underlying these processes.

Alterations in neuromuscular junction morphology during fast-to-slow transformation of rabbit skeletal muscles

Chronic low frequency stimulation of motor nerves results in transformation of muscle fibre phenotype from fast- to slow-twitch. We examined the light and electron microscopic structure of neuromuscular junctions in normally fast twitch muscles, tibialis anterior and extensor digitorum longus of rabbit after 3 weeks of stimulation to determine whether synaptic structure is also modified during fibre type transformation. Neuromuscular junctions of stimulated and unstimulated (control) tibialis anterior and extensor digitorum longus muscles and unstimulated slow twitch soleus muscle were visualized with rhodamine-conjugated alpha-bungarotoxin. Video light microscopic images of neuromuscular junctions were digitized to allow quantification of their surface areas, perimeters, lengths and widths. Three weeks of stimulation resulted in a decrease in the maximal velocity of muscle fibre shortening and augmentation of mitochondrial volume in fast muscles, demonstrating the efficacy of the stimulation protocol employed in altering muscle fibre phenotype. Neuromuscular junctions of control tibialis anterior and extensor digitorum longus are thin, compact, and continuous, with complex branching patterns. In contrast, those of slow-twitch soleus are thicker and discontinuous. Neuromuscular junctions in control tibialis anterior and extensor digitorum longus are larger than those in soleus. Three weeks of stimulation causes a marked decrease in the size of neuromuscular junctions in tibialis anterior and extensor digitorum longus, as reflected in the significant reduction in neuromuscular junction surface area, length and width. Electron microscopy of these junctions suggests that secondary postsynaptic folds in stimulated muscles are more closely spaced. Also, axon terminals of stimulated muscles appear to contain more densely packed synaptic vesicles and mitochondria than controls. Decreases in neuromuscular junction dimensions can be partly explained by muscle fibre atrophy. However, the decrease in neuromuscular junction size is proportionately greater than that of muscle fibre diameter in both muscles, indicating that factors other than fibre atrophy may contribute to the reduced neuromuscular junction size in stimulated muscles. Neuromuscular junctions of stimulated tibialis anterior and extensor digitorum longus muscles exhibit some features characteristic of normal soleus neuromuscular junctions, indicating structural adaptations consistent with the altered muscle fibre phenotype. On the other hand, neuromuscular junctions of 3 week stimulated tibialis anterior and extensor digitorum longus and their synaptic branches remain as thin and continuous as those of unstimulated controls, suggesting that the transformation of neuromuscular junctions towards a morphology characteristic of slow muscle, is only partial. These results demonstrate that an altered pattern of impulse activity cause significant synaptic remodelling in adult rabbit skeletal muscles.

Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice

Neuromuscular synapses in an androgen-sensitive muscle of sexually mature male mice were repeatedly observed over several-month intervals in normal animals and in animals in which testosterone levels were manipulated. In normal bulbocavernosus muscles, pre- and postsynaptic regions of neuromuscular junctions enlarge as muscle fibers grow. After castration, junctional area decreased in parallel with muscle fiber atrophy. When testosterone was resupplied to castrated animals, junctions that previously decreased in size then enlarged in parallel with muscle fiber hypertrophy. Surprisingly, these size changes occurred without loss or addition of motor nerve terminal branches or acetylcholine (ACh) receptor regions. Rather, each nerve terminal branch and underlying receptor region became smaller following castration and reenlarged following testosterone treatment. Several lines of evidence argued that the size changes observed after castration and testosterone treatment were secondary to shrinkage and stretching of the postsynaptic muscle fiber membrane. Following castration, the spaces between synaptic regions decreased in size at the same time and to a similar extent as the regions themselves. Following testosterone replacement, the spaces between synaptic regions expanded and each existing ACh receptor region enlarged. Ultrastructural analysis showed that there was no loss or addition of postsynaptic secondary junctional folds in the muscle fiber membrane (where ACh receptors are located) as junctions shrank and expanded. Rather, folds became more densely packed as muscle fibers atrophied following castration and less densely packed as muscle fibers hypertrophied following testosterone replacement. From these studies of the bulbocavernosus muscle, as from our previous studies of the sternomastoid muscle, we conclude that neuromuscular junction size is directly coupled to muscle fiber size. Androgens modulate muscle fiber volume directly, leading to a change in the surface area of the muscle fiber membrane, which in turn causes the postsynaptic specializations to shrink or expand. The concomitant shrinkage and stretching of motor nerve terminals that we observed can only be accounted for by their adhesion to postsynaptic specializations that are also changing size. Thus adhesion, rather than an interchange of diffusible factors, trophic or otherwise, is likely to be the primary determinant of coordinated pre- and postsynaptic enlargement in growing mammalian skeletal muscles.

Nerve terminal morphology in C57BL/6NNia mice at different ages.

The mammalian neuromuscular junction (NMJ) appears to undergo a dynamic remodeling process with advancing age. Previous studies suggest that the NMJ becomes progressively larger with age, to a greater degree in fast-twitch muscles than slow-twitch muscles. To better understand this process, quantitative morphometry was conducted on soleus and extensor digitorum longus (EDL) nerve terminals from C57BL/6NNia mice aged 4, 8, 10, 12, 18, 22, 24, and 32 months. Computer-aided morphometry of camera lucida drawings of aging nerve terminals indicated significant differences in the size and complexity of NMJs in both muscles between different age groups; however, there were no significant net trends with age. The magnitude and direction of the morphometric changes depended on the particular ages compared, and occurred to a greater degree in the EDL. This study is the first comprehensive morphometric study of C57BL/6NNia nerve terminals at so many ages. The results are similar to those obtained for the Fischer 344 rat, and support the hypothesis that nerve terminal remodeling is a dynamic process, not a progressive elaboration.