Motor Nerve Terminal Branches Research Articles

The formation and function of single transmitter release sites at mature amphibian motor-nerve terminals.

The extent of quantal transmitter release from single sites of synaptic vesicle accumulations along the length of motor-nerve terminal branches at the amphibian neuromuscular junction has been investigated. Such a determination involves development of a model for the generation of quantal potential fields at single styryl-dye stained sites along the length of a branch. Successful testing and application of this model indicates that the extent of quantal release at a dye-stained site is proportional to the total length of active zone at the site. The stability of these sites and of their ensheathing terminal Schwann cell processes was also investigated. Following simultaneous injection of the terminal Schwann cell and nerve terminal with different fluorescent dyes, terminal branches were observed to show dynamic changes in their length, with these occurring in relatively short periods of hours or less. Redistribution of styryl dye stained sites at the ends of branches also occurred in such short periods of time. These were accompanied by changes in the configuration of terminal Schwann cells, which generally occurred prior to changes in the length of nerve terminal branches.

Schwann cell dynamics with respect to newly formed motor-nerve terminal branches on mature (Bufo marinus) muscle fibers.

A study has been made of the formation of synaptic terminals from long processes formed at the end of motor nerve branches of endplates in mature amphibian (Bufo marinus) muscle. Injection of fluorescent dyes into individual motor axons showed the full extent of their branches at single endplates. Synaptic vesicle clusters at these branches were identified with styryl dyes. Some terminal branches consisted of well separated varicosities, each possessing a cluster of functioning synaptic vesicles whilst others formed by the same axon consisted of closely spaced clusters of vesicles in a branch of approximately uniform diameter. All the varicosities gave rise to calcium transients on stimulation of their parent axon. Both types of branches sometimes possessed short processes (<5 microm long) or very long thin processes (>10 microm long) which ended in a bulb that possessed a functional synaptic vesicle cluster. These thin processes could move and form a varicosity along their length in less than 30 min. Injection of a fluorescent dye into terminal Schwann cells (TSCs) at an endplate showed that they also possessed very long thin processes (>10 microm long) which could move over relatively short times (<30 min). Injecting fluorescent dyes into both axons and their associated TSCs showed that on some occasions long TSC processes were accompanied by a long nerve terminal process and at other times they were not. It is suggested that the mature motor-nerve terminal is a dynamic structure in which the formation of processes by TSCs guides nerve terminal sprouting.

Distribution of neurotrophin receptors in the mouse neuromuscular system.

The neurotrophins are a family of secreted proteins with critical roles in regulation of many aspects of neural development, survival and maintenance. Their actions on neural tissue are thought to be mediated by interaction with high affinity (trk family members) or low affinity (p75NTR) cell surface receptors. In general, neurotrophins are considered to be supplied in limiting quantity by cells of a target tissue or synaptic partner. To date, alpha motoneurons have been shown surprisingly indifferent to loss of neurotrophic factors. Direct evidence for supply of a critical motoneuron factor(s) by skeletal muscle and a specific uptake mechanism in vivo remains elusive. We wished to directly establish whether targets in the periphery might be potential sources of neurotrophic support for motoneurons by examining whether neurotrophin receptors are present on motoneuron nerve terminals. We have used immunofluorescence techniques with a panel of antibodies against known neurotrophin receptors (trk A, trk B, trk C, p75NTR) to map the locations of these receptors in the developing neuromuscular system of mice from our neurotrophin-3 (NT-3) knockout colony. To our surprise, we failed to locate immunoreactivity for any of these receptors in association with motor nerve endplates or terminal intramuscular axon branches, although they were found in association with a population of unidentified cells. We believe this result indicates that the neurotrophic relationship between alpha motoneurons and their target cells is not a simple one of neurotrophin supply by skeletal muscle cells and its uptake at the neuromuscular junction.

Formation and Function of Synapses with Respect to Schwann Cells at the End of Motor Nerve Terminal Branches on Mature Amphibian (Bufo marinus) Muscle

A study has been made of the formation and regression of synapses with respect to Schwann cells at the ends of motor nerve terminal branches in mature toad (Bufo marinus) muscle. Synapse formation and regression, as inferred from the appearance and loss of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43)-stained vesicle clusters, occurred at the ends of terminal branches over a 16 hr period. Multiple microelectrodes placed in an array about FM1-43 blobs at the ends of terminal branches detected the electrical signs of neurotransmitter being released onto receptors. Injection of a calcium indicator (Oregon Green 488 BAPTA-1) into the motor nerve with subsequent imaging of the calcium transients, in response to stimulation, often showed a reduced calcium influx in the ends of terminal branches. Injection of a fluorescent dye into motor nerves revealed the full extent of their terminal branches and growing processes. Injection of the terminal Schwann cells (TSCs) often revealed pseudopodial TSC processes up to 10-microm-long. Imaging of these TSC processes over minutes or hours showed that they were highly labile and capable of extending several micrometers in a few minutes. Injection of motor nerve terminals with a different dye to that injected into their TSCs revealed that terminal processes sometimes followed the TSC processes over a few hours. It is suggested that the ends of motor nerve terminals in vivo are in a constant state of remodeling through the formation and regression of processes, that TSC processes guide the remodeling, and that it can occur over a relatively short period of time.

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-bungarotoxin-induced sprouting.

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48-169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedure permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxin-induced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization.

Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.

A study was made to determine the disposition of vesicle-associated proteins (syntaxin, SV2, SNAP-25) and calcium channels with respect to the spatial extent of spontaneous and evoked quantal release within regions of amphibian motor-nerve terminal branches delineated by FM1-43 stained vesicle clusters (blobs). Discrete concentrations of vesicles revealed approximately 2 microm apart along the length of terminal branches through FM1-43 staining were identical in size and spacing to those identified along terminal branches with SV2 antibody (AbSV2). Fluorescent antibodies to syntaxin 1 (AbS), SNAP-25 (AbS25) and the calcium channel alpha1B subunit (Abalpha1B) were found in relatively high concentrations coincident with the AbSV2 blobs. Three extracellular recording electrodes were placed in the vicinity of individual FM1-43 blobs, and an algorithm was used to determine the spatial origin of miniature endplate potentials (MEPPs) and EPPs together with their relative amplitudes. MEPPs and EPPs originated throughout the region stained by FM1-43 but not elsewhere; amplitude-frequency distributions of MEPPs and EPPs were similar for all FM1-43 blobs with average coefficients of variation of no less than 0.28. A linear relationship existed between the size of an FM1-43 blob, measured as the integrated extent of FM1-43 staining of a blob, and the frequency of MEPPs as well as the probability of EPPs from the blob. There was a proximo-distal gradient in the size of FM1-43 blobs along the length of single terminal branches, suggesting a gradient in release probability along the branches. The frequency distribution of the distances between blobs was approximately Gaussian, whereas the frequency distribution of the size of blobs was highly skewed and was best fitted with a gamma distribution. It is concluded that there are correlations among the extent of labeling of SNAP-25, syntaxin and calcium channels at a release site, the store of vesicles to be found there, and the probability of spontaneous and evoked quantal release.

Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).

The effect of a conditioning depolarizing current pulse (80-200 micros) on quantal secretion evoked by a similar test pulse at another site was examined in visualized motor-nerve terminal branches of amphibian endplates (Bufo marinus). Tetrodotoxin (200 nM) and cadmium (50 microM) were used to block voltage-dependent sodium and calcium conductances. Quantal release at the test electrode was depressed at different distances (28-135 microm) from the conditioning electrode when the conditioning and test pulses were delivered simultaneously. This depression decreased when the interval between conditioning and test current pulses was increased, until, at an interval of approximately 0.25 ms, it was negligible. At no time during several thousand test-conditioning pairs, for electrodes at different distances apart (28-135 microm) on the same or contiguous terminal branches, did the electrotonic effects of quantal release at one electrode produce quantal release at the other. Analytic and numerical solutions were obtained for the distribution of transmembrane potential at different sites along terminal branches of different lengths for current injection at a point on a terminal branch wrapped in Schwann cell, in the absence of active membrane conductances. Solutions were also obtained for the combined effects of two sites of current injection separated by different time delays. This cable model shows that depolarizing current injections of a few hundred microseconds duration produce hyperpolarizations at approximately 30 microm beyond the site of current injection, with these becoming larger and occurring at shorter distances the shorter the terminal branch. Thus the effect of a conditioning depolarizing pulse at one site on a subsequent test pulse at another more than approximately 30 microm away is to substantially decrease the absolute depolarization produced by the latter, provided the interval between the pulses is less than a few hundred microseconds. It is concluded that the passive cable properties of motor nerve terminal branches are sufficient to explain the effects on quantal secretion by a test electrode depolarization of current injections from a spatially removed conditioning electrode.

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse--II. Proximal-distal measurements.

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal-distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 microm in length and with sufficient information to determine their proximal distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5-10 microm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure / microm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.

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.

Steroid‐responsive electromyographic abnormalities in polymyalgia rheumatica

Prior to definitive diagnosis, electrodiagnostic studies are often requested in patients with polymyalgia rheumatica to evaluate complaints of muscle aching, tenderness, and weakness. These studies are generally normal, although rare reports of electromyographic abnormalities in polymyalgia rheumatica exist. Two patients with polymyalgia rheumatica and electrodiagnostic findings consistent with diffuse denervation are described. Following steroid treatment, both patients experienced impressive clinical and electromyographic improvement. To explain this improvement, we hypothesize a steroid-responsive microvascular arteritis resulting in ischemic damage to axons of motor nerve terminal branches.

Motor nerve terminal loss from degenerating muscle fibers

The fate of nerve terminals following elimination of postsynaptic target cells was studied in living mouse muscle. Several days after muscle fiber damage, observations of previously identified neuromuscular junctions showed that motor nerve terminal branches had rapidly disappeared from degenerating muscle fibers. Following muscle fiber regeneration, loss of terminal branches ceased and nerve terminals regrew, reestablishing some of the original sites and adding new branches. The distribution of acetylcholine receptors reorganized in the regenerated muscle so that perfect alignment was reestablished with the newly configured nerve terminals. These results argue that the maintenance of the full complement of nerve terminal branches at a neuromuscular junction is dependent on the presence of a healthy muscle fiber. Similarly, regenerating muscle is dependent on the nerve terminal for the organization and maintenance of postsynaptic receptors.