Biomedical subjects
C R Slater
Publications and source records attributed to C R Slater.
Action potential generation in rat slow- and fast-twitch muscles.
1. In skeletal muscle fibres, voltage-gated sodium channels are concentrated at the neuromuscular junction. The effect of this accumulation of sodium channels on action potential generation was investigated in rat slow- and fast-twitch muscle fibres. 2. Intracellular microelectrodes were used to generate and record action potentials, from an imposed membrane potential of -75 and -90 mV, in junctional and extrajunctional regions of the muscle fibre. To identify junctional regions, preparations were incubated with 5 x 10(-7) M d-tubocurarine (dTC) to block muscle contraction in response to nerve stimulation whilst allowing endplate potentials (EPPs) to be recorded. Injection of rectangular depolarizing current pulses initiated action potentials at the endplate with threshold values several millivolts lower than those generated elsewhere in the fibre. In addition, the maximum rate of rise of the action potential was greater at the endplate than in extrajunctional regions. 3. In other muscles, neuromuscular transmission was partially blocked with dTC (2 x 10(-7) M), such that repetitive nerve stimulation evoked action potentials and EPPs in the same fibre. The threshold of these nerve-evoked action potentials was approximately 50% lower than values derived from action potentials generated by current injection. 4. It is concluded that the threshold for action potential generation is significantly lower at the neuromuscular junction than in extrajunctional regions of skeletal muscle fibres. Furthermore, nerve-evoked current is more effective at generating an action potential than is injected current.
Release and synthesis of acetylcholine at ectopic neuromuscular junctions in the rat.
1. The ability of axons in the superficial fibular nerve to synthesize and release acetylcholine (ACh) has been studied before and after the formation of ectopic neuromuscular junctions (NMJs) with denervated soleus muscles of adult rats. 2. The central end of the severed fibular nerve was transplanted to the surface of the soleus muscle. After 3.5-5 weeks the soleus muscle was denervated in one group of rats by cutting the tibial nerve, allowing the formation of functional ectopic NMJs within a few days. In other rats the tibial nerve remained intact, preventing the formation of functional ectopic NMJs. 3. A month later the content of ACh, the levels of activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and the amount of ACh released by depolarization by exposure to 50 mM KCl were measured in segments of isolated muscles that (i) contained normal or ectopic NMJs, (ii) were free of nerve or (iii) contained nerve that had not made NMJs. 4. Regions of muscles with ectopic nerve growth in which new NMJs had not formed contained substantial amounts of ACh and ChAT but no AChE. No detectable release of ACh could be evoked from these regions. 5. In muscles in which ectopic NMJs had formed after cutting the tibial nerve, the amounts of ACh and ChAT were about one-fifth of those in the regions of innervation of control muscles. ACh release could be evoked from the region of ectopic nerve growth in amounts nearly as great as those released from NMJs in normal and contralateral control muscles. 6. We conclude that the ability of the terminal parts of mature motor axons to synthesize and store ACh is largely independent of functional contact with muscle fibres. By contrast, the ability to release ACh in substantial amounts only develops when NMJs are formed. The possible significance of this situation for the development of synapses is discussed.
Different distributions of dystrophin and related proteins at nerve-muscle junctions.
The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.
Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.
To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.
Structure and function of neuromuscular junctions in the vastus lateralis of man. A motor point biopsy study of two groups of patients.
The properties of neuromuscular junctions (NMJs) in the vastus lateralis of man have been studied in motor point biopsy samples and compared with those reported for lower vertebrates (frogs and mice). The patients studied had no convincing evidence of a primary disturbance of neuromuscular transmission or other neurogenic component. Morphological studies were made using a variety of methods at the light- and electron-microscope levels. The size of the presynaptic nerve terminal and the area of postsynaptic specialization were smaller, relative to the size of the muscle fibres, than in the lower vertebrates. In contrast, the extent of postsynaptic folding was greater. Intracellular recordings from single muscle fibres showed that the duration of synaptic currents was longer than in most other vertebrates so far studied and that the number of transmitter 'quanta' released by a single nerve impulse, about 20, was lower, probably reflecting the small size of the presynaptic terminals. The hypothesis is discussed that in man, a relatively weak effect of transmitter on the muscle fibre surface is amplified by voltage-dependent sodium channels which have been shown in the rat to be concentrated in the depths of the synaptic folds. The implications of this hypothesis for the interpretation of pathological findings in myasthenic syndromes are also discussed.
Structure and function of the neuromuscular junction in young adult mdx mice.
Dystrophin, the protein product of the gene responsible for X-linked muscular dystrophies, shares structural features with the cytoskeletal proteins spectrin and alpha-actinin. Like spectrin, it is localized at the cytoplasmic surface of the sarcolemma and is particularly concentrated in the subsynaptic region of the neuromuscular junction. Mdx mice have a profound deficiency of dystrophin and develop a necrotizing myopathy in the first weeks of life. Abnormalities of the neuromuscular junction, including a redistribution of postsynaptic molecules and reduction in synaptic folding, are also observed. We have studied these mice to see whether the lack of dystrophin has a specific effect on the structure and function of their neuromuscular junctions. Using a fore-limb muscle from 8 week old mdx mice we confirm the previously described postsynaptic structural changes and in addition show that many nerve terminals are abnormally complex. We demonstrate that these structural abnormalities are found exclusively at neuromuscular junctions on regenerated muscle fibres. Despite these structural abnormalities, miniature endplate potential frequency, the quantal content of endplate potentials, the amplitude and time course of miniature endplate currents and the number of acetylcholine receptors at the postsynaptic membrane are normal in mdx mice of this age. We conclude that in the mdx mouse the absence of dystrophin from the postsynaptic membrane has little direct effect on the function of the neuromuscular junction but that degeneration and regeneration of muscle fibres leads to remodelling of both its pre- and postsynaptic components.
Is dystrophin labelling always discontinuous in Becker muscular dystrophy?
It has been reported that immunofluorescent labelling of dystrophin in muscle from patients with Becker muscular dystrophy (BMD) is invariably patchy or discontinuous. This observation has led to the suggestion that BMD dystrophin molecules, which are usually smaller than normal due to the presence of "in frame" gene deletions, cannot be assembled into a complete lattice network under the plasma membrane and instead form isolated patches. Our experience with immunoperoxidase labelling of BMD muscle indicates that complete gaps in the reaction around fibres are uncommon. We have therefore compared immunofluorescence and immunoperoxidase labelling patterns on sets of serial sections from 6 BMD patients using a monoclonal antibody to dystrophin. No difference was detected between the two types of label used: the incidence of discontinuous labelling was rare in both cases. We suggest that significantly different patterns of dystrophin labelling may be obtained using different primary antibodies, and that caution needs to be exercised in extrapolating models of structure/function relationships from observations of antibody binding patterns.
The effect of age on motor neurone death following axotomy in the mouse.
The ability of mouse motor neurones to survive axotomy during the first month of life was studied. The motor neurones that lie in the dorsolateral columns of spinal segments C7 and C8 and supply the flexor muscles of the forepaw were axotomized by cutting and removing part of the median and ulnar nerves above the elbow. The number and position of cell bodies with axons in these nerves were confirmed by retrograde labelling of the cut axons with horseradish peroxidase. The ability of these neurones to survive axotomy varies with the age of the animal at the time of axotomy. When the axons are sectioned within the first four postnatal days, 80-90% of the cell bodies will die, more than half of this death occurring in less than one week after axotomy. If the animals are one week old at the time the nerves are cut, a significantly smaller number (50%) die (P = 0.013), and the time-course of death is different, with eight to ten days elapsing before half the death has occurred. 40% of the neurones will die if sectioned at two weeks of age, and it is not until four weeks of age that more than 90% of the cells can survive axotomy. We conclude, therefore, that the kinetics of motor neurone death, as well as the final extent of neuronal loss, are affected by the age at which the animal is axotomized.
Loss of motor neurons from the median nerve motor nucleus of the mutant mouse 'wobbler'.
This paper describes the location and number of motor neurons in the median nerve pool of wobbler mice and normal littermates as determined by retrograde labelling of the cut median nerve with horseradish peroxidase (HRP) in animals from 3 weeks to 1-year-old. The median nerve motor nucleus is located in spinal segments C5-T1, and in normal animals contains 199 (6) (mean (SEM] motor neurons. Three-week-old wobbler mice have the same number of labelled neurons as control animals, and this number falls to 75% of normal values by 4 weeks of age, and to approximately 60% by 6 weeks of age and older. Numerous swollen, pale and frequently vacuolated perikarya are present in the same 3-6-week-old mice. In the 3-week-old mutants these comprise on average 17% of the total large (greater than 20 microns) neuronal cell bodies counted in segments C5-T1. By 6 weeks this figure has fallen to 10%, and to less than 4% in adult wobblers. We conclude that the most active period in the expression of the wobbler phenotype is from 3 to 6 weeks of age.
The basal lamina and stability of the mammalian neuromuscular junction.
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Dystrophin in skeletal muscle. I. Western blot analysis using a monoclonal antibody.
The value of analysing dystrophin on Western blots of skeletal muscle for the differential diagnosis of Xp21 muscular dystrophies is now fairly well established. Here we describe a sensitive system based on monoclonal antibodies to dystrophin. The specificity of the antibodies was established and experiments were undertaken to identify the source of dystrophin-related protein bands which were detected on blots of normal skeletal muscle. These investigations formed a necessary preliminary study to the application of the assay to samples of muscle obtained at biopsy from patients with Duchenne and Becker muscular dystrophy.
Dystrophin in skeletal muscle. II. Immunoreactivity in patients with Xp21 muscular dystrophy.
In the preceding paper a sensitive Western blotting analysis system based on the use of a monoclonal antibody to dystrophin was described. Here we report the immunoreactivity on blots and on unfixed frozen sections of muscle from patients with Duchenne (DMD) and Becker (BMD) muscular dystrophy. Muscle from 3 BMD patients showed variation both in the band pattern observed on blots and in the immunocytochemical labelling of dystrophin on frozen sections. In contrast to previous reports, we were able to detect some minor dystrophin bands on blots from 6 of 9 DMD biopsy samples. Tissue sections from 8 of the 9 contained isolated fibres with dystrophin-positive labelling. We conclude that the majority of DMD patients have muscle fibres which can synthesize dystrophin in a limited manner.
Muscle proteins and muscular dystrophy.
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Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions.
When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.
The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle.
If skeletal muscles are damaged in ways that 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 fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.
Spatial distribution of acetylcholine receptors at developing chick neuromuscular junctions.
The development of high-density clusters of acetylcholine receptors (AChRs) and the relationship of these clusters to nerve contacts on embryonic chick wing muscle fibres has been studied. Fluorescent labelling of AChRs with rhodamine-conjugated alpha-bungarotoxin (R-Bgt) revealed the presence of irregularly shaped AChR clusters in wing buds at 4 1/2-5 days of incubation. This is within a day of when myotubes first appear in the wing bud, and close to the time when functional innervation becomes established. At 10 days of incubation AChR clusters present on muscle cells in anterior and posterior latissimus dorsi appear as round or oval, uniformly labelled plaques. At about the time of hatching, however, these plaques break into numerous smaller clusters. Similar changes in the morphology of AChR clusters have been observed previously in mammalian skeletal muscle during development. Using horseradish peroxidase labelled alpha-bungarotoxin (HRP-Bgt), the relationship between AChR clusters and motor nerve terminals was studied at the ultrastructural level. At all stages of development nerve-muscle contacts were labelled with HRP-Bgt. In wing buds, however, the majority (90%) of labelled clusters observed were not in contact with a motor nerve terminal. The incidence of AChR clusters with axon contacts increased sharply during development such that by 10 days more than 50% and by hatching more than 90% of all sections through labelled AChR clusters contained nerve terminal profiles. At all times studied nerve-contacted receptor clusters were longer (about 5 micron) than non-contacted clusters (about 2 micron).
Postnatal maturation of nerve-muscle junctions in hindlimb muscles of the mouse.
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