The use of gold chloride to stain developing and adult neuromuscular junctions in the insect

Individual insect muscle fibers, whose neuromuscular junctions have been stained with a modification of Ranvier's gold chloride method, can be dissected free and mounted whole if the muscle is prefixed in aldehydes. The neuromuscular junctions along the length of the individual fibers are well delineated and can be measured and counted. Effective procedures include fixation with glutaraldehyde buffered to low pH with sodium citrate, or glutaraldehyde and paraformaldehyde combined in phosphate buffer at neutral pH, followed by exposure to citric acid and to gold chloride. The method is convenient, and could be useful for the study of arthropod neuromuscular junctions in general, since their nerve terminals do not release acetylcholine as a transmitter and cannot be stained by the more commonly used cholinesterase methods.     

Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.     

A Method for Myoglobin in Cryostat Sections of Muscle by Precipitation with Sulfosalicylic Acid

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixation prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

A method for myoglobin in cryostat sections of muscle by precipitation with sulfosalicylic acid.

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixations prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

Role of Activity in Determining Properties of the Neuromuscular System in Crustaceans

SYNOPSIS. Crustacean muscle fibers, like those of higher vertebrates,are diversified in physiology, morphology, and biochemical attributes.However, unlike motor units of mammals, those of crustaceansusually do not contain fibers of uniform type. Motor neuronactivity acts as a unifying force for the motor units of mammalianmuscles, but its role in determining properties of crustaceanmotor units is less well defined. In certain crustacean muscles,differential activity of sensory-motor systems is importantfor establishing muscle fiber properties during early development.In freshwater crayfish, neuromuscular junctions of a phasicmotor neuron are altered physiologically and morphologicallyby chronic stimulation; the adapted junctions release less transmitterper impulse and are more fatigue-resistant than naive junctions.The muscle fibers may also adapt to chronic stimulation, butless dramatically and at a slower rate. The adaptive responsesof the neuromuscular junction can be achieved through manipulationof sensory input and with little increase in motor impulse activity.This suggests that altered protein synthesis is triggered centrallyby synaptic input to the motor neuron. In general, present evidencesuggests that long-term adaptation of neuromuscular junctionsand muscle fibers of crustaceans can occur in response to alteredactivity in the nervous system, in spite of the fact that certainmuscle fiber properties appear to be genetically predetermined.Some aspects of matching between neuromuscular junction andmuscle fiber appear to be determined in response to growth ofthe muscle fiber; other features are activity-dependent; andsome may result from expression of inherent neuronal properties.     

Numerical analysis of the voltage-clamp technique applied to frog neuromuscular junctions.

The nonlinear cable equation was solved numerically by means of an implicit procedure. The correlation between end-plate length and fiber diameter was determined in frog (Rana pipiens) sartorius muscles stained with gold chloride (Löwit, 1875). The diameter of the fibers stained by the Löwit method was 80 (74-85) micron (median and its 95% confidence interval for 52 fibers), the length of the end plates in the same fibers was 382 (353-417) micron. The fibers simulated were 80 micron in diameter. To solve the equation the muscle fibers were represented by 500 segments 20 micron long, and the equation was solved in steps of 10 microseconds; a double exponential function was incorporated to the first seven segments to represent the neuromuscular junction. The potential of the first segment of the cable was set to the clamping level and the membrane potential of the remaining segments calculated. The current needed to hold the first segment was estimated by adding the current flowing through the first segment to the current flowing from it to the second segment. Our results indicate that the lack of space clamp in the point voltage-clamp studies of the frog neuromuscular junction introduces serious errors in the estimates of the end-plate conductance value, the kinetics of the conductance changes, and the reversal potential of the end-plate currents. The possibility of an efficient voltage-clamp technique is also explored. Our calculations suggest that the study of end-plate current and conductance is possible with little error if the end-plate potential is controlled at both ends of the synaptic area simultaneously.     

Structure of neuromuscular junctions and differentiation of striated muscle fibers in mdx mice after bone-marrow stem cell therapy

Mdx mice are an experimental model of Duchenne muscular dystrophy caused by mutations in the dystrophin gene. Repeated cycles of muscle degeneration-regeneration are common for mdx mice. Disrupted neuromuscular junctions also characterize mdx mice. The structure of mdx mice neuromuscular junctions and the differentiation of striated muscle fibers were investigated 4, 8, 16, and 24 weeks after transplantation of C57BL/6 Lin(−) bone-marrow stem cells. We found that the death of striated muscle fibers decreased 4 weeks after the transplantation of bone-marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began in 8 weeks and dystrophin synthesis increased in 16–24 weeks after the bone-marrow stem cells transplantation. On the longitudinal sections of quadriceps muscle of mdx mice 4 weeks after transplantation, we observed a reduced quantity of acetylcholine receptor clusters and an increase in their area in neuromuscular junctions. Sixteen weeks after the transplantation, the total area of neuromuscular junctions increased due to an enlarged number of acethylcholine receptors and their extended area. The single intramuscular transplantation of C57BL/6 Lin(−) bone-marrow stem cells induces the differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

The composite structure of quail pectoralis muscle.

The twitch fibers of the quail pectoralis muscle were found to have one neuromuscular junction each, located in the middle third of the fiber. The length of isolated fibers varied between 8.8 and 33.2 mm, with mean and median values of 16 and 15.6 mm, respectively. The lengths of the fascicles from which the fibers were isolated varied between 30 and 51 mm. The muscle fibers taper at both ends. The neuromuscular junctions, revealed after histochemically reacting the intact muscle for acetyl cholinesterase activity, were arranged in discrete bands, separated by intervals of between 0.94 and 6.70 mm, with a mean value of 3.14 mm. The quail pectoralis muscle is thus composed of discontinuous, tapered muscle fibers, arranged in an overlapping series. It is therefore a muscle in which tension is transmitted laterally between muscle fibers.     

Alterations in the permeability of dystrophic fibers during neuromuscular junction development

In the mdx mice, lack of dystrophin leads to increases in calcium influx and myonecrosis, followed by muscle regeneration. Synapse elimination is faster in mdx than in controls, suggesting that increases in calcium influx during development could be involved. In the present study, we evaluated whether dystrophic fibers display changes in permeability to Evans Blue Dye (EBD) during development of the neuromuscular junction. EBD is a sensitive label for the early detection of increased myofiber permeability and sarcolemmal damage. After intraperitoneal injection of EBD, sternomastoid (STN) and tibialis anterior (T. anterior) muscles were analyzed with fluorescence microscopy. At 01, 07 and 14 days of age, STN and TA mdx myofibers were not stained with EBD. At 21 days of age, positive labeling of TA and STN mdx myofibers was seen, suggesting permeability modification and myonecrosis. Adult muscles showed a decrease (T. anterior) or no changes (STN) in the amount of EBD-positive fibers. These results suggest that there is no sarcolemmal damage detected by EBD during development of dystrophic neuromuscular junctions and other factors may contribute to the earlier synapse elimination seen in dystrophic muscle.     

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.     

Ultrastructural features of neuromuscular junctions in the stapedius muscle of Gallus gallus

The ultrastructure of neuromuscular junctions in the twitch fibers of the stapedius muscle of Gallus gallus (domesticus) was investigated as part of a series of neurophysiological studies. Among the morphological features observed were elongated end-plates with numerous large and clear synaptic vesicles mixed with larger dense core vesicles and irregular or aperiodic “active sites” in the presynaptic membrane where synaptic vesicles were focused. The most remarkable features of these junctions were large synaptic clefts (50-80 nm) and the absence of junctional folds in the sarcolemmal surface. Unlike the large periodic junctional folds seen in the neuromuscular junctions of frogs and in the fast twitch fibers of the mammalian stapedius, the preparations studied only show small aperiodic invaginations (primitive folds) in the postsynaptic membranes. This morphological feature remains essentially constant from newly hatched to adult chickens. While these smooth junctions are consistent with earlier findings of inconspicuous junctional folds in the twitch fibers of the chicken posterior latissimus dorsi they are unlike those seen in the fast twitch fibers of the mammalian stapedius muscle, or other twitch fibers in general. The morphological findings of the present study may also suggest that the simple, unmodified neuromuscular junctions in the stapedius of Gallus may be a useful preparation for studies of synaptic membrane structures that employ the freeze-fracture technique.     

The fine structure of a multiterminal innervation of an insect muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

The Fine Structure of a Multiterminal Innervation of an Insect Muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

Immunofluorescence comparisons of anti-actin specificity

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.     

Motor Nerve Terminal Staining Combined with Catecholamine Histofluorescence or Immunocytochemistry

A number of excellent techniques are available to stain and characterize different types of neurons and nerve terminals. However, because these different techniques are frequently not compatible, their usefulness in determining the relationships between specific axons and neuromuscular junctions is often limited. The goal was to develop specific procedures for simultaneous visualization of different types of unmyelinated axons and motor nerve terminals in the same preparation. First we modified the formal-dehyde/glutaraldehyde staining solutions of the aqueous aldehyde fluorescence technique (Faglu) to observe catecholamine containing axons in whole mount amphibian skeletal muscle. The compatibility of this modified staining solution with other histological procedures made it possible to stain both motor nerve terminals with tetrazolium salts and, in the same preparation, to observe unmyelinated axons with aldehyde-induced catecholamine histofluorescence. This same general formaldehyde/glutaraldehyde staining procedure was also used with immunocytochemical techniques to visualize fluorescent antibody stained nerves and motor nerve terminals in the same whole mount preparation.     

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.     

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.     

Agrin isoforms and their role in synaptogenesis.

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.