Expression and distribution of sodium channels in short- and long-term denervated rodent skeletal muscles.

1. Loose-patch voltage-clamp recordings were made from rat and mouse skeletal muscle fibres denervated for up to 6 weeks. Innervated muscles possessed a Na+ current density of 107 +/- 3.3 mA cm-2 in endplate membrane, and 6.3 +/- 0.6 mA cm-2 in extrajunctional membrane. This high concentration of Na+ channels at the endplate was gradually reduced following denervation. After 6 weeks of denervation, the endplate Na+ channel concentration was reduced by 40-50%, and the density of Na+ channels in extrajunctional membrane was increased by about 30%. 2. The tetrodotoxin (TTX)-resistant form of the Na+ channel appeared after 3 days of denervation and comprised approximately 43% of the endplate Na+ channels 5-6 days after denervation. Subsequently, TTX-resistant Na+ channels were reduced in density to approximately 25% of the postjunctional Na+ channels and remained at this level up to 6 weeks after denervation. 3. RNase protection analysis showed that mRNA encoding the TTX-resistant Na+ channel was virtually absent in innervated muscle, rose > 50-fold after 3 days of denervation, then decreased by 95% 6 weeks after denervation. The density of TTX-resistant Na+ channels correlated qualitatively with changes in mRNA levels. 4. These results suggest that the density of Na+ channels at neuromuscular junctions is maintained by two mechanisms, one influenced by the nerve terminal and the other independent of innervation.

Sodium channels aggregate at former synaptic sites in innervated and denervated regenerating muscles.

The role of innervation in the establishment and regulation of the synaptic density of voltage-activated Na channels (NaChs) was investigated at regenerating neuromuscular junctions. Rat muscles were induced to degenerate after injection of the Australian tiger snake toxin, notexin. The loose-patch voltage clamp technique was used to measure the density and distribution of NaChs on muscle fibers regenerating with or without innervation. In either case, new myofibers formed within the original basal lamina sheaths, and, NaChs became concentrated at regenerating endplates nearly as soon as they formed. The subsequent increase in synaptic NaCh density followed a time course similar to postnatal muscles. Neuromuscular endplates regenerating after denervation, with no nerve terminals present, had NaCh densities not significantly different from endplates regenerating in the presence of nerve terminals. The results show that the nerve terminal is not required for the development of an enriched NaCh density at regenerating neuromuscular synapses and implicate Schwann cells or basal lamina as the origin of the signal for NaCh aggregation. In contrast, the change in expression from the immature to the mature form of the NaCh isoform that normally accompanies development occurred only partially on muscles regenerating in the absence of innervation. This aspect of NaCh regulation is thus dependent upon innervation.

Aggregation of sodium channels during development and maturation of the neuromuscular junction.

The voltage-activated Na channel (NaCh) is an integral membrane protein that is enriched at the neuromuscular end plate. Using loose-patch voltage-clamp and immunofluorescence, we have found that the aggregation of NaChs occurs late, during maturation of the neuromuscular junction. A decline in expression of embryonic NaCh mRNA and increase in adult NaCh mRNA precedes the onset of aggregation, and the appearance of functional adult NaChs coincides with NaCh aggregation. We tested the possibility that only the adult NaCh subtype could aggregate during development and found that both the embryonic and adult isoforms become concentrated at the synapse. The NaCh is the first postsynaptic membrane protein shown to become clustered postnatally, and the mechanism producing this aggregation appears to be different from the process producing aggregation of other synaptic proteins.

Fast and slow twitch skeletal muscle fibres differ in their distribution of Na channels near the endplate.

Sodium channel distributions were measured in fast and slow twitch rodent skeletal muscle fibres using the loose patch voltage clamp technique. Large differences were found between these fibre types with respect to Na channel density in the perijunctional region. Fast twitch fibres exhibited a large increase in Na channel density near the endplate, while slow twitch fibres did not.

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture.

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.

A specific effect of muscle cells on the distribution of presynaptic proteins in neurites and its absence in a C2 muscle cell variant.

The distribution of neurofilament (NF) and synaptic vesicle (SV) proteins in neurites cultured in vitro was visualized with immunocytochemical methods. NF and SV proteins were detected in neurites from both embryonic mouse spinal cord and chick ciliary ganglion neurons. NF proteins generally occupied more proximal, unbranched neurite segments while SV proteins were most often found in highly branched terminal segments. Neurites from mouse spinal cord cells showed a striking segregation of the NF and SV proteins into distinct domains; neurites from chick ciliary ganglion cells exhibited a similar, though less pronounced segregation. In cocultures of neurons and muscle cells, the neurite segments in contact with myotubes more often stained for SV than for NF while the opposite was true for neurites not in contact with myotubes. The preferential association of SV neurites with myotubes was also observed when the myotubes were previously fixed with paraformaldehyde. This association was absent in neurites growing over Chinese hamster ovary cells, suggesting that the effect is specific for muscle cells. Coculture of neurons with variant strains of C2 myotubes that are deficient in AChR (1R-) or proteoglycans (S27) revealed a preferential association of SV neurites with 1R- myotubes but not with S27 myotubes. Thus, proteoglycans on the surface of C2 myotubes may influence the growth and/or differentiation of presynaptic neurons.

Progressive restriction of synaptic vesicle protein to the nerve terminal during development of the neuromuscular junction.

Antibodies to synaptic vesicle (SV) proteins and to neurofilament (NF) proteins were used to investigate presynaptic differentiation and its relation to the formation of acetylcholine receptor (AChR) clusters at developing mouse neuromuscular junctions. At all times during development, SV proteins and NF proteins were segregated into neighboring, but separate regions of the presynaptic neurite. At embryonic day (ED) 14 SV proteins were present throughout preterminal neurites but at later ages became progressively restricted to the distal parts of the neurites. NF proteins occupied a more proximal region that extended distally during development until NF proteins occupied the entire axon up to the terminal. The restriction of SV proteins exclusively to the terminal did not occur until the second postnatal week. At the time of their first appearance (ED 14), up to 50% of AChR clusters were not associated with neurites; precise colocalization required 12-36 hr to develop. These findings demonstrate a progressive restriction of both pre- and postsynaptic components to the synapse during development.

Effects of an inhibitor of the synaptic vesicle acetylcholine transport system on quantal neurotransmitter release: an electrophysiological study.

The drug 2-(4-phenylpiperidino)cyclohexanol (AH5183), which potently inhibits the active transport of acetylcholine (ACh) into synaptic vesicles, was used as a pharmacological tool to study the functional role of synaptic vesicles in quantal transmitter release. Using microelectrode recording techniques, miniature endplate potentials (mepps) and nerve-evoked endplate potentials (epps) were recorded from frog cutaneous pectoris neuromuscular junctions in low Ca2+/high Mg2+ Ringer solution, and in normal Ringer with added D-tubocurarine (D-TC). Stimulation in the presence of AH5183 caused a 40% reduction in quantal size (mepp amplitude), depressed tetanic potentiation, and decreased the number of quanta released with each nerve impulse in the presence of D-TC. All of these effects appeared gradually and only after extended stimulation of the nerve, during which several hundred thousand quanta were released. Consequently, these findings suggest a serial one-time usage of vesicles, with little or no re-entry of recycled vesicles until after a large fraction of the original vesicles has been exhausted. The results primarily show that filling of synaptic vesicles with ACh is crucial for sustaining synaptic transmission, and gives further evidence that the ACh released by nerve impulses originates from these organelles.

Calcium-insensitive miniature endplate potentials at the neuromuscular junction.

Several different types of acetylcholine secretion have been shown to coexist at the neuromuscular junction along with the Ca2+-dependent quantal release producing miniature endplate potentials (mepps) and endplate potentials. One of these, the Ca2+-insensitive, slow-rising mepps (slow mepps), is present in normal untreated muscles but is most prominent in many conditions where the Ca2+-dependent quantal release mechanism is not functioning properly. Slow mepps occur at a frequency of less than 0.1 Hz in normal muscles, with large variability between fibres and muscles, and can reach frequencies of 1-2 Hz in several pathological conditions. The potentials are also highly variable in size and shape, being generally of high amplitude (0.1-15 mV) and prolonged time course (1-15 ms rise time). Most importantly, slow mepps are not affected by procedures which increase the intraterminal Ca2+ concentration, including nerve stimulation, thus being unable to contribute to the function of synaptic transmission. The cellular source of the Ca2+-insensitive mepps has been determined to be the nerve terminal and not the Schwann cells or nerve sprouts. The release process producing slow mepps is generally insensitive to many drugs, ions, and procedures, stimulation being observed with vinblastine, cytochalasin B, and caffeine. Depression of this secretion is effected by uncouplers of oxidative phosphorylation and by a drug (AH5183) which inhibits the vesicular active acetylcholine transport system. It is concluded that the slow mepps are due to an exocytic fusion of unique synaptic vesicles with the plasma membrane near the active zones, in a process insensitive to many intracellular ions and regulators. Since slow mepps are prominent in many pathological conditions of nerve and muscle, it is speculated that they play some role in the recovery or development of synaptic function.

The nature and origin of calcium-insensitive miniature end-plate potentials at rodent neuromuscular junctions.

1. To study the nature and origin of slow-rising, Ca2+-insensitive miniature end-plate potentials (m.e.p.p.s) in mammalian muscle we used intracellular recording techniques and drugs which block acetylcholine (ACh) synthesis or the uptake of ACh into synaptic vesicles. Slow m.e.p.p.s were induced in vivo by paralysing the extensor digitorum longus muscle of the rat with botulinum toxin type A or in vitro by the application of 4-aminoquinoline to the mouse diaphragm nerve-muscle preparation. 2. Hemicholinium-3, which blocks ACh synthesis, reduced the amplitude of all synaptic potentials including slow m.e.p.p.s, but only if the nerve was stimulated. 3. 2(4-phenylpiperidino)cyclohexanol (AH-5183), which blocks the active uptake of ACh into synaptic vesicles, reduced both the frequency and the amplitude of slow m.e.p.p.s and did so without requiring nerve stimulation. 4. No correlation was observed between the molecular leakage of ACh from the motor nerve and the frequency and amplitude of slow m.e.p.p.s. 5. We conclude that slow m.e.p.p.s are caused by the release of ACh from the nerve terminal, possibly from a small pool of synaptic vesicle-like structures.

A comparison of miniature end-plate potentials at normal, denervated, and long-term botulinum toxin type A poisoned frog neuromuscular junctions.

The effects of denervation and long-term botulinum toxin type A (BoTx) poisoning on miniature end-plate potentials (m.e.p.p.s) in the frog were studied with intracellular microelectrode recording. BoTx reduced the frequency of m.e.p.p.s to less than 1% of the level seen in untreated frogs, leaving a large percentage of tiny m.e.p.p.s and slow-rising m.e.p.p.s (slow m.e.p.p.s). Unlike what is observed in the rat, the frequency of slow m.e.p.p.s never increased above the low rate measured in the untreated controls, and in fact slightly but significantly decreased after BoTx. A comparison of the m.e.p.p.s seen after BoTx poisoning (BoTx m.e.p.p.s) and m.e.p.p.s seen after denervation (Schwann m.e.p.p.s) revealed many similarities between the two including amplitude and time-to-peak distributions, temperature Q10 values and responses to several drugs and procedures. However, it was concluded that BoTx m.e.p.p.s do not originate from the Schwann cells because denervation of BoTx-paralysed frogs abolishes all m.e.p.p.s and the drug 4-aminoquinoline affects BoTx m.e.p.p.s and Schwann m.e.p.p.s in opposite ways, increasing the frequency of the former while almost eliminating the latter. BoTx m.e.p.p.s and Schwann m.e.p.p.s probably represent similar processes of secretion which are non-specific in nature, having a lower energy barrier than for normal release and not originating from specialized areas of transmitter release.

Pharmacological characterization of the calcium-insensitive, intermittent acetylcholine release at the rat neuromuscular junction.

A variety of pharmacologically active compounds was surveyed for effects on the Ca2+-insensitive miniature end-plate potentials (slow mepps) induced by botulinum toxin type A (Botx) poisoning in rat muscle. The purpose was to gain insight into the release process responsible for this type of acetylcholine secretion. It was found that caffeine and dibutyryl cyclic adenosine 3',5'-monophosphate increased significantly the frequency of slow mepps in Botx-poisoned muscles, but had no effect on slow mepps in control muscles. Vinblastine and cytochalasin B significantly increased the slow mepp frequency in Botx-poisoned as well as in normal control muscles. Inhibitors of oxidative metabolism reduced the frequency of slow mepps by 90%, indicating a high energy requirement for this type of release. No agent was found to augment the slow mepp frequency above 1-2 Hz, suggesting that an upper limit exists for this type of packaging and release of acetylcholine.

Facilitation, augmentation and potentiation of transmitter release at frog neuromuscular junctions poisoned with botulinum toxin.

Botulinum toxin type A (Botx) is a potent neurotoxin which inhibits specifically cholinergic synaptic transmission by an unknown mechanism. In order to gain further insight into the mode of action of this toxin, the effect of conditioning nerve stimuli on neuromuscular transmission was studied at endplates of Botx-poisoned and unpoisoned control cutaneous pectoris muscles in the frog. Effects of single conditioning stimuli (facilitation) and multiple high-frequency stimuli (augmentation and potentiation) on epp amplitude and mepp frequency were studied. The main results were that initial facilitation was significantly increased and its decay time constant significantly decreased in Botx-poisoned muscles, while augmentation was unchanged and potentiation was abolished. These changes could be detected before the muscle became completely paralysed, suggesting that they reflect a primary disturbance in the Ca2+-dependent release process.

Miniature end-plate potentials in rat skeletal muscle poisoned with botulinum toxin.

Spontaneous transmitter release, recorded as miniature end-plate potentials (m.e.p.p.s), was studied in rat extensor digitorum longus (e.d.l.) and soleus muscles partially or completely paralysed by botulinum toxin type A (BoTx). Normal unpoisoned muscles were examined for comparison. Analysis of m.e.p.p.s in both normal and BoTx-poisoned muscles confirmed the presence of two populations of potentials. One population, which comprised about 96% of the m.e.p.p.s recorded at non-poisoned end-plates, was characterized by a uniform time course and a mean time-to-peak of 0.5-0.7 ms. These potentials had a shape and time-to-peak similar to that of quantal end-plate potentials (e.p.p.s) evoked by nerve stimuli. These were designated 'fast m.e.p.p.s'. The other population of m.e.p.p.s was characterized by a slower, more variable rise-time, the time-to-peak exceeding 1.1 ms, and generally a larger amplitude. These were designated 'slow m.e.p.p.s'. In both partial and complete paralysis by BoTx the frequency of fast m.e.p.p.s was reduced by more than 90% and the reduction lasted several weeks. After 6-10 days of poisoning the frequency of slow m.e.p.p.s gradually increased. The highest frequency of slow m.e.p.p.s (0.4 Hz) was recorded in the partially paralysed soleus muscle, the frequency being about ten times that at unpoisoned end-plates. In both partially paralysed muscles slow m.e.p.p. frequency returned towards normal 28 days after poisoning. A significant correlation (r = 0.67) was observed between the quantal content of e.p.p.s and the frequency of fast m.e.p.p.s in partially paralysed e.d.l. muscles. No significant correlation was observed between quantal content of e.p.p.s and the frequency of slow m.e.p.p.s. To further study if muscle activity influenced the appearance of slow m.e.p.p.s, partially paralysed soleus muscles were directly stimulated in vivo during the first 11-13 days following BoTx poisoning, using a stimulation pattern which inhibits nerve terminal sprouting and the appearance of denervation changes. This procedure did not alter the frequency of slow m.e.p.p.s as compared to unstimulated poisoned controls. It is concluded that enhancement of slow m.e.p.p. frequency in muscles poisoned with BoTx is related to the blockade of evoked Ca2+-dependent quantal transmitter release. However, additional factors influence this type of spontaneous and Ca2+-insensitive release of acetylcholine since there is a great variability between fibres and a time lag between the disappearance of fast m.e.p.p.s and the activation of slow m.e.p.p. frequency.(ABSTRACT TRUNCATED AT 400 WORDS)