Synaptic segregation at the developing neuromuscular junction

Throughout the developing nervous system, competition between axons causes the permanent removal of some synaptic connections. In mouse neuromuscular junctions at birth, terminal branches of different axons are intermingled. However, during the several weeks after birth, these branches progressively segregated into nonoverlapping compartments before the complete withdrawal of all but one axon. Segregation was caused by selective branch atrophy, detachment, and withdrawal; the axon branches that were nearest to the competitor's branches were removed before the more distant branches were removed. This progression suggests that the signals that mediate the competitive removal of synapses must decrease in potency over short distances.     

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1--wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2--drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 microm to 1.05 microm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 microm to 0.75 microm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 microm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely.     

Neural activity affects distribution of glutamate receptors during neuromuscular junction formation in Drosophila embryos

The prevalence of diseases associated with obesity, such as cardiovascular disease and diabetes mellitus, is higher in the spinal cord injury (SCI) population. Specifically, the mortality rate for cardiovascular disease is 228% higher in the SCI population. In addition, 100% of SCI individuals have osteoporosis in the paralysed extremities. These diseases are related to physical activity level, the level of the spinal cord lesion, and time post injury. Physically active SCI men and women have above-average fat mass (16 to 24% and 24 to 32%, respectively, compared with 15% for able-bodied men and 23% for able-bodied women), while sedentary SCI individuals have 'at-risk' levels of body fat (above 25% and 32%, respectively). The proportions and densities of the 3 main constituents comprising the fat-free body (mineral, protein and water) are altered following SCI. Bone mineral content decreases by 25 to 50%, and the magnitude of reduction is dependent on the level, completeness and duration of SCI. Because of denervation resulting in skeletal muscle atrophy, total body protein reduces by 30%, and total body water relative to bodyweight decreases by 15% following SCI. Indirect methods based on 2-component body composition models assume constant proportions and densities of mineral, protein, and water in the fat-free body. As a result, prediction equations based on 2-component models yield invalid estimates of fat and fat-free mass in the SCI population. Therefore, future research needs to directly quantify the proportions and densities of the constituents of the fat-free body in the SCI population relative to age, sex, physical activity level, level of the spinal cord lesion and time post injury, and to develop equations based on multicomponent body composition models.     

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.     

Recycling of synaptic vesicles at the frog neuromuscular junction in the presence of strontium

In these experiments, we followed the exocytosis and endocytosis of synaptic vesicles with the vital dye FM1-43 and asked whether calcium is important for membrane retrieval at the frog neuromuscular junction. We replaced calcium with equimolar amounts of strontium and monitored the staining of recycling vesicles by inducing exocytosis with electrical stimulation. Trains of 2,400 (2 or 20 Hz) or 4,200 (20 Hz) pulses failed to induce FM1-43 internalization in the presence of strontium, but they did in the presence of calcium. This effect of strontium was not due to a decrease in exocytosis, because FM1-43 release was similar in the presence of calcium or strontium. The impairment in endocytosis, observed as inhibition of FM1-43 internalization, could be overcome by longer periods of stimulation (6,000 pulses at 2 or 20 Hz) in the presence of strontium (1.8 mM) or by increasing the extracellular concentration of strontium to 10 mM (2,400 action potentials at 20 Hz). It is suggested that endocytosis is dependent on calcium influx and that strontium is much less effective in replacing calcium for endocytosis than it is for exocytosis.     

Calcium-dependent desensitizing function of the postsynaptic neuronal-type nicotinic acetylcholine receptors at the neuromuscular junction

Several subunits that commonly have been regarded as neuronal-type nicotinic acetylcholine receptor (nAChR) subtypes, have been found in the postjunctional endplate membrane of adult skeletal muscle fibres. The postsynaptic function of these neuronal-type nAChR subtypes at the neuromuscular junction has been investigated by using aequorin luminescence and fluorescence confocal imaging. A biphasic elevation of intracellular Ca2+ is elicited by prolonged nicotinic action at the mouse muscle endplates. The fast and slow Ca2+ components are operated by a postsynaptic muscle- and colocalized neuronal-type nAChR, respectively. Neuromuscular functions may be regulated by a dual nAChR system to maintain the normal postsynaptic excitability. Certain neuronal-type nAChR may be endowed with the same functional role in the central nervous system also.     

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction. J. Neurophysiol. 80: 2893-2899, 1998. N-Methyl--aspartate (NMDA) reduces release from crayfish excitatory nerve terminals. We show here that polyclonal and monoclonal antibodies raised against the mammalian postsynaptic NMDA receptor subunit 1 stain specifically the presynaptic membrane of release boutons of the crayfish neuromuscular junction. In crayfish ganglionic membranes, the polyclonal antibody recognizes a single protein band that is somewhat larger (by approximately 30 kD) than the molecular weight of the rat receptor. Moreover, the monoclonal (but not the polyclonal) antibody abolishes the physiological effect of NMDA on glutamate release. The monoclonal antibody did not prevent the presynaptic effects of glutamate, which also reduces release by activation of quisqualate presynaptic receptors. Only when 6-cyano-7-nitroquinoxatine-2,3,dione (CNQX) was added together with the monoclonal antibody was the presynaptic effect of glutamate blocked. These results show that presynaptic glutamate receptors of the crayfish NMDA type are involved in the regulation of neurotransmitter release in crayfish axon terminals. Although the crayfish receptor differs in its properties from the mammalian NMDA receptor, the two receptors retained some structural similarity.     

Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.     

Association of adhesive macromolecules with terminal sprouts at the neuromuscular junction after botulinum treatment

Small quantities of botulinum toxin (BTX) are useful in the treatment of certain movement disorders, such as laryngeal spasmodic dysphonia, blepharospasm, and cervical dystonia. However, the corrective paralytic effects of BTX are only temporary, in part because of the formation of remodeled neuromuscular junctions. Here, we questioned whether various factors within and near the neuromuscular junction could contribute to the remodeling seen after BTX treatment. BTX was injected subcutaneously in the region of the levator auris longus muscle. At 1-week intervals, levator auris longus muscles were removed and examined histochemically. As previously described, BTX treatment results in a progressive elongation of end plates. The neural cell adhesion molecule was not associated with the elongated end plates but was associated with the BTX-induced nerve sprouts after long intervals (3 to 4 weeks). Similarly, after BTX, laminin-1 (composed of alpha 1, beta 1, and gamma 1 chains) reactivity was associated with the nerve sprouts, but not with the end plates. Laminin beta 2 reactivity at the end plate dispersed somewhat within 1 week but remained diffusely associated with the elongating end plates for up to 5 weeks. Together these results suggest that neural cell adhesion molecule and laminins may participate in the sprouting observed after BTX treatment and that alterations in laminin beta 2 expression may participate in initial loss of contacts.     

Evoked quantal currents at neuromuscular junctions of wild type Drosophila larvae

Malignant infantile osteopetrosis is a rare disease but can be clinically unequivocally diagnosed. Normal bone formation in the presence of decreased bone breakdown leads to the typical symptoms. The only proven curative approach, bone marrow transplantation, can reverse most of the symptoms and prevent progression to irreversible nerve damage when done early in infancy. Therefore, early diagnosis is decisive. We present a case report of an infant with osteopetrosis and discuss pathogenesis and therapeutical options.     

Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidase-labeled alpha-bungarotoxin

The cytochemical localization of acetylcholine (ACh) receptors at the neuromuscular junction was investigated with a procedure utilizing alpha-bungarotoxin (alpha-BtX) labeled directly with horseradish peroxidase (HRP). Following incubation of tissues in the conjugate and reaction for peroxidase, activity was observed on the junctional folds of the motor endplate. A uniform layer of reaction product approximately 15 nm thick occurred over the apical portions of the junctional folds. Membranes at the bases of the synaptic cleft showed only small amounts of reaction product. Non-junctional regions of the muscle fiber were unreactive. Activity was also observed in the membrane of the axon facing the muscle surface, often including the axolemma overlying the "active zones" of the nerve terminal. Such presynaptic activity was still evident on nerve terminals disjuncted from the synapse by enzymatic treatment prior to incubation in the conjugate. This localization indicates the possible presence of presynaptic ACh receptors within the axolemma. In muscle denervated for 7-12 days, motor endplates were reactive and parajunctional sarcolemma showed slight activity, but most extrajunctional regions contained no obvious accumulations of reaction product. Activity at all sites was prevented by preincubation of tissues in native alpha-BTX prior to incubation in the conjugate and reaction for HRP. This procedure represents a simple and convenient method for the high resolution localization of ACh receptors.     

Current-voltage relation and reversal potential at junctional and extrajunctional ACh-receptors of the frog neuromuscular junction

A tentative model is presented which is based on existing data and our own cell kinetic and morphological observations in mice. The model suggests that the epidermal Langerhans cell plays a role in proliferation control of keratinocytes and may also act as an epidermal stem cell.     

Isolation, sequence, and bioactivity of FMRFamide-related peptides from the locust ventral nerve cord

The electrochemical behaviour of the bioreductive redox active nitroimidazole drug metronidazole has been examined in the presence and absence of the DNA bases using three electrochemical techniques, all of which indicate the capacity for interaction between reduced products and DNA bases. The 4-electron metronidazole (RNO2) metronidazole-hydroxylamine (RNHOH) couple in an aqueous medium shows a positive shift in reduction potential upon addition of thymine, adenine and guanine, but a negative shift for cytosine. Interpretation of these results for an irreversible process is, however, inconclusive. In dimethylformamide/H2O the presence of DNA base on the one-electron addition product, the nitro radical anion, was examined by cyclic voltammetry. All except guanine resulted in interaction with the metronidazole nitro radical anion (RNO2-), as measured by the decrease in the return-to-forward peak current ratio, in the following order of increasing reactivity: cytosine, adenine and thymine (at a metronidazole: base ratio of 1:1). The increase in the stability of the radical anion by increasing the pH of the dimethylformamide/H2O medium resulted in a decreased reaction with thymine.     

Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron-neuron synapses in the peripheral as well as the central nervous system.     

ATP and adenosine inhibit transmitter release at the frog neuromuscular junction through distinct presynaptic receptors

1. The effects of exogenous ATP or adenosine on end-plate currents (e.p.cs; evoked by simultaneous action of a few hundred quanta of ACh) or on miniature e.p.cs (m.e.p.cs) were studied under voltage clamp conditions on frog sartorius muscle fibres. 2. ATP or adenosine (100 microM(-1) mM) reduced the e.p.c. amplitude but did not affect m.e.p.c. amplitude, decay time constant and voltage-dependence of m.e.p.c., suggesting that e.p.c. depression induced by these purines had presynaptic origin only. 3. The action of ATP, unlike that of adenosine, was prevented by the P2-purinoceptor antagonist suramin (100 microM). The stable ATP analogue alpha,beta-methylene ATP (100 microM), known to be desensitizing agent on P2X receptors, also abolished the depressant effect of ATP while sparing the action of adenosine. Concanavalin A, an inhibitor of ecto-5'-nucleotidase, did not affect the presynaptic action of exogenously applied ATP. 4. The presynaptic action of adenosine was prevented by theophylline (1 mM), a blocker of adenosine receptors, while the effect of ATP was not changed under these conditions. The selective blocker of A1 adenosine receptors, 8-cyclopentyl-1,3,dipropylxanthine (DPCPX; 0.1 microM), abolished the presynaptic action of adenosine but did not prevent the depressant effect of ATP. 5. The effects of ATP and adenosine (at nearly saturating concentration) were additive suggesting that these purines activated not only distinct receptors but also different intracellular signalling mechanisms. 6. In contrast to the hypothesis that at the neuromuscular junction ATP reduces transmitter release via enzymatic degradation to presynaptically active adenosine, our data suggest that ATP (through its own presynaptic receptors) directly inhibits ACh release. Thus, ATP and adenosine might be almost equipotent as endogenous prejunctional neuromodulators at the neuromuscular junction.     

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.     

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction

Vulvar vestibulitis, a subset of vulvodynia, is present in 15% of patients in a general gynecologic practice. Only a few studies have focused on pathologic features of vulvar vestibulitis and none have included a control group. Punch biopsies from the vulvar vestibule of 12 patients with an age range of 22 to 51 years (mean 28 years) and 12 age-matched controls were analyzed for histopathologic features and investigated for the role of probable etiologic factors including human papillomavirus (HPV). A chronic inflammatory infiltrate was present in all specimens from patients with vestibulitis, and was composed predominantly of T-lymphocytes with a small number of B cells and an admixture of plasma cells, mast cells, and occasional monocytes. T-helper suppressor ratio was normal. The infiltrate was mild in 5 patients, moderate in 1, and severe in 6. Minor vestibular glands were observed in 8 (66%) patients and were associated with a periglandular inflammatory infiltrate. Squamous metaplasia was observed in 4 (44%) patients. Epithelial hyperplasia was present in 10 (83%) patients with mild dysplasia in 2 (16%). Immunohistochemistry for immunoglobulins IgG, IgA, and IgM showed the presence of IgG-positive plasma cells in 75% of patients, suggesting chronic irritation, but an autoimmune etiology cannot be excluded or confirmed. Biopsies of control cases did not show any inflammatory infiltrate. In situ hybridization for HPV 6, 11, 16, and 18 was negative in the patient group as well as in the control group. We conclude that histopathologic abnormalities in patients with vulvar vestibulitis are the result of a chronic inflammatory reaction of the mucosa of the vulvar vestibule, for which the cause remains unclear.     

Prejunctional effect of quaternary derivatives of l-hyoscyamine at the rat neuromuscular junction. A structure-activity relationship study

1. The effects of phenthonium and related compounds on the spontaneous release of acetylcholine (ACh) were investigated with electrophysiological and radiolabelled techniques to correlate the prejunctional effect with their cholinolytic activities and to determine the structure-activity relationship. 2. Phenthonium and endophen are N-(4-phenyl)-phenacyl derivatives of l-hyoscyamine in "exo" and "endo" conformation, respectively. Tropol is N-(4-phenyl) phenacyl tropan-3-ol whereas ipratropium is 8-isopropyl-noratropine. 3. Only phenthonium increased the frequency of miniature endplate potentials and the resting efflux of spontaneous [3H]-ACh in rat diaphragm muscles. 4. The rank order of the antimuscarinic potency was: ipratropium > atropine > phenthonium = endophen > tropol. The rank order of the antinicotinic activity was: phenthonium = endophen > tropol > atropine > ipratropium. 5. It is concluded that the prejunctional facilitatory effect of phenthonium is associated with the N-phenyl-phenacyl group at "exo" conformation but the effect is unrelated to its cholinolytic properties.