Endplate Region Research Articles

High endocytotic and lysosomal activities in segments of rat myotubes differentiated in vitro

Endocytosis and the lysosome system have been studied in rat myotubes differentiated in vitro. Horseradish peroxidase was used as marker for endocytosis and was found to accumulate unevenly in the myotubes. Small segments of myotubes display very high endocytotic activity. Similar segments contained numerous lysosomes, as seen by the accumulation of neutral red or histochemical staining for acid phosphatase. The segments also contained accumulations of acetylcholine receptors as determined by binding of tetramethyl rhodamine-labelled alpha-bungarotoxin. Unstained segments in living cultures could be recognized by phase-contrast microscopy since they often appeared somewhat dilated and were not as well spread on the culture surface as the main parts of the myotubes. Ultrastructurally, the segments contained an intensely proliferating tubular system in communication with the extracellular space, which therefore probably represents the developing transverse tubular system. The segments also contained endocytosed marker within large phagosomes. Contractile filaments occurred in the segments but were frequently less well-organized than in other parts of the myotubes. The described characteristics of the segments in rat myotubes differentiated in vitro bear resemblance to some of the characteristics of the denervated endplate region of adult muscle.

The origin of secondary myotubes in mammalian skeletal muscles: ultrastructural studies

The distribution of secondary myotubes and undifferentiated mononucleated cells (presumed to be myoblasts) within foetal IVth lumbrical muscles of the rat was analyzed with serial section electron microscopy. In all myotube clusters for which the innervation zone was located, every secondary myotube overlapped the end-plate region of the primary myotube. No secondary myotubes were ever demonstrated to occur at a distance from the primary myotube innervation zone. This indicates that new secondary myotubes begin to form only in the innervation zone of the muscle. Some young secondary myotubes made direct contact with a nerve terminal, but we cannot say if this is true for all developing secondary myotubes. Myoblasts were not clustered near the innervation zone, but were uniformly distributed throughout the muscle. Myoblasts were frequently interposed between a primary and a secondary myotube, in equally close proximity to both cell membranes. We conclude that specificity in myoblast-myotube fusion does not depend on restrictions in the physical distribution of myoblasts within the muscle, and therefore must reflect more subtle mechanisms for intercellular recognition.

Selective increase of tetrameric (G4) acetylcholinesterase activity in rat hindlimb skeletal muscle following short-term denervation.

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.

Induction of active zones at ectopic neuromuscular junctions in the frog

To examine de novo differentiation of the active zone, ectopic neuromuscular junctions were studied in adult frog muscles. Ectopic junctions induced by excising the original endplate region and implanting the nerve to an endplate-free site were examined by light and electron microscopy 4 weeks-1 year after operations. The earliest time point at which ectopic junction formation was detected with freeze fracture was 6 weeks postoperation, when clusters of active zone particles were observed scattered across the nerve ending. Subsequently, short active zones (6-10 weeks postoperation, length mean = 0.36 +/- 0.24 microns) composed of the characteristic 2 double rows of particles are detected. Before junctional folds are observed with freeze fracture, many active zones are parallel to each other and to the long axis of the nerve. The average angle 6-10 weeks postoperation is 27 degrees +/- 23 degrees. Even during these early stages of formation, active zones are functional. As time passes, active zones attain a more typical, perpendicular orientation (12-18 weeks postoperation, mean = 62 degrees +/- 24 degrees) and also increase in length (mean = 0.69 +/- 0.45 microns). However, even after 1 year, the orientation (angle, mean = 70 degrees +/- 22 degrees) and the length (mean = 0.78 +/- 0.63 microns) of active zones at ectopic junctions are still not well correlated with active zones at normal junctions (normal active zone angle mean = 85 degrees +/- 5 degrees, length mean = 1.00 +/- 0.57 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of extracellular calcium at the endplate of mouse diaphragm after ecothiopate in vitro.

1. Experiments were carried out to investigate the accumulation from the extracellular medium of 45Ca2+ by the endplate region of skeletal muscle. 2. Mouse diaphragm muscle was incubated in physiological saline labelled with 45Ca at 37 degrees C for periods of up to 1.5 h. 3. The muscle was divided into junctional and non-junctional portions and the Ca from the extracellular fluid accumulated at the endplate determined from the 45Ca content of the portions. 4. The accumulation of extracellular Ca at the endplate region of muscles incubated in pysiological saline alone was nil, but there was accumulation in the presence of the anticholinesterase ecothiopate iodide 0.5 x 10(-6) M (ECO). Stimulation of the phrenic nerve at 0.02 Hz caused no further increase in accumulation but reduced the amount of spontaneous fasciculation. In tetrodotoxin (TTX) 10(-6) M, the accumulation was halved, and in 3.5 mM Mg2+ the accumulation was nil. Carbachol 10(-4) M resulted in an accumulation of Ca similar to that in ECO. 5. It is concluded that there was an accumulation of extracellular Ca following excitation of the nerve by stimulation at a low frequency and during the spontaneous fasciculations, and about half of the accumulation of extracellular Ca after ECO in the experiments was due to the postsynaptic action of ACh released non-quantally from the nerve terminals.

Focal adhesions to the synaptic matrix at mouse neuromuscular junctions

Studies of identified synapses in living animals have revealed ongoing synaptic remodelling in the mature nervous system. We recently demonstrated the existence of filopodia and lamellipodia (structures associated with nerve outgrowth in developing or tissue culture systems) at the mature mouse neuromuscular junction (NMJ) . Evidence of motor nerve terminal retraction, regeneration and perisynaptic outgrowth indicated that even established synaptic contacts were not permanent. It now appears that variation in the stability of synaptic contacts, not the amount of nerve terminal outgrowth,is the basis for synaptic remodelling and extension. Although it has been documented that regenerating motor nerve terminals adhere to former synaptic matrix,and that the matrix contains information for inducing and aligning presynaptic and postsynaptic specializations,it was not clear whether the synaptic matrix was uniformly adhesive, or formed specialized adhesive foci with the preterminal membrane. To determine if there are specific sites of synaptic adhesion we utilized hypertonic fixatives to induce shrinkage at the NMJ.Male mice were anaesthetized, the soleus muscle was exposed, and the overlying skin was retracted to create a pool into which either 2% glutaraldehyde in HEPES Krebs (normal fixative) or 2% glutaraldehyde in HEPES Krebs with a 2X NaCl concentration (hypertonic fixative) was added continually for 30 minutes. The surface layer of each muscle was then removed and submerged in the same fixative for an additional 1.5 hours. Tissue was washed in buffer (pH 7.2) and immersed in cholinesterase stain for 5 minutes to localize endplate regions . Endplate regions were cut into 1 mm blocks, washed in buffer (pH 7.2) and post-fixed for 1 hour in HEPES Krebs buffered 2% osmium tetroxide (pH 7.2). Following fixation, specimens were dehydrated in a graded ethanol series and embedded in Polybed 812 (Polysciences). Mouse soleus muscle was stained for immunocytochemical localization of actin with a rabbit polyclonal antiserum against chicken gizzard actin (from Dr. James Lessard, University of Cincinnati) as previously described .

Synaptic localization of a 66-kDa soluble protein from skeletal muscle: Evidence for its developmental and neural regulation

Soluble proteins from normal, adult denervated, and developing rat muscles were studied in order to identify common molecular species undergoing developmental regulation and nerve dependence. Significant increases in 66- and 30-kDa proteins were found as a consequence of 14 days of denervation. Subsequent reinnervation restores normal adult levels. During development, high levels of the 66-kDa protein were found in neonatal muscles but slowly decreased concomitant with the following postnatal maturation period; the adult levels were reached at Postnatal Day (P) 21. From the immunocytochemical studies it is deduced that both proteins were concentrated mainly at the end-plate region in adult normal muscle. Following denervation, the proteins were found distributed over the entire cell. For the 66-kDa protein, a similar pattern of extensive distribution was seen in immature muscle. Although no data for functional implications for these proteins are available at present, the properties described here make them of interest in understanding nerve-muscle interactions.

Temperature Effects on Membrane Chloride Conductance and Electrical Excitability of Lizard Skeletal Muscle Fibres

ABSTRACT Intracellular recording techniques were used to study the effects of temperature on resting membrane conductances, electrical excitability and synaptic efficacy in fast-glycolytic (FG) skeletal muscle fibres from the lizard Dipsosaurus dorsalis. The conductance of the resting muscle membrane to chloride ions (gci) increased from 488 μS cm−2 at 15°C (pH7-8) to 730 μS cm−2 at 45°C (pH7·4), yielding a temperature coefficient (thermal ratio, Rio) of 1·14. Resting potassium conductance (gK) increased from 84μS cm−2 at 15 °C to 236 μS cm−2 at 45 °C (R10=1·41). Fibres bathed in Cl−-free Ringer’s solution were hyperexcitable, and produced repetitive action potentials both during and following intracellular current injection. At the preferred body temperature of Dipsosaurus (near 40°C) the fibres also fired repetitively in response to single nerve shock. The electrical excitability of Dipsosaurus fibres decreased with increasing temperature. Threshold current, measured at endplate regions of fibres bathed in normal Ringer’s solution, was 146 nA at 15°C and 353 nA at 45°C (R10= 1·34). Despite the temperature-dependent change in threshold current, at both 15 and 45 °C all fibres examined had suprathreshold neuromuscular transmission in response to single nerve shock. The relative thermal independence of gCl in Dipsosaurus fibres may be an adaptation that contributes to a large safety factor for neuromuscular transmission at the high body temperatures preferred by this lizard species.

Sensitivity of rodent skeletal muscles to dicholines: dependence on innervation and age.

Depolarizations provoked by acetylcholine (ACh) and three dicholines, succinylcholine (SCh), glutarylcholine (GCh) and azelainylcholine (AzCh) were measured in normal and 3-7 days denervated muscles of adult, and in normal muscles of 0-27 days old mice and rats. Soleus and flexor digitorum superficialis muscles were mainly used. To prevent movement during measurement of the membrane potential by microelectrodes, the muscles were bathed in solutions containing 30 mM K+. At the adult endplate region of muscles of the mouse, the -log concentrations required for 7 mV depolarization were 6.5 for AzCh and approximately 5.9 for SCh, GCh and ACh. In extrajunctional areas of denervated muscles the values were 7.1, 6.4, 5.9 and 5.6 for AzCh, ACh, GCh and SCh, respectively. In non-denervated soleus muscles of 1-3 days-old mice, the average difference in sensitivity (estimated as mentioned above) to AzCh and SCh was approximately 2.0 log units, not significantly different from that found in denervated adult muscles. Between days 3 and 24 this difference decreased to about 0.8 at the endplate region but it remained greater in the periphery of soleus muscles. The AzCh-SCh sensitivity differences were smaller in muscles from juvenile rats than in juvenile mice. Sensitivity to ACh and AzCh, but not that to SCh, was by 0.6-0.8 log units greater in the presence than in the absence of anticholinesterases in endplates at any age of the animals. The blocking effectiveness of d-tubocurarine was low in newborn animals (similar to that observed in denervated adult muscles) and it increased with age.

Quantitative investigation of the neuromuscular junction of rat skeletal muscle fibres after double innervation.

In normal (untreated) rats the mean length ratio of postsynaptic to presynaptic membrane was 2.7 +/- 0.8 for neuromuscular junctions of slow-twitch soleus muscle fibres and 4.2 +/- 1.0 for neuromuscular junctions of fast-twitch extensor digitorum longus muscle fibres; this difference was significant (P less than 0.001). After experimental double innervation by fast and slow muscle nerves for four months, the ratio was (1) 2.9 +/- 0.8 for the original slow-twitch fibre end-plate and 2.8 +/- 0.8 for the newly established one, both not significantly different from that of the normal slow-twitch fibres; and (2) 2.2 +/- 0.5 for the original fast-twitch fibre end-plate and 2.2 +/- 0.7 for the newly established one, both significantly smaller than that of the normal fast-twitch fibres (P less than 0.001). This means that the double innervated slow-twitch muscle fibres retained their original neuromuscular junction type, whereas the doubly-innervated fast-twitch muscle fibres underwent a dramatic transformation of their neuromuscular junction from the fast-muscle to the slow-muscle type. In both doubly innervated fibres, the ultrastructural characteristics of neuromuscular junctions, whether altered or not, were identical at both end-plate regions.

Temperature and synaptic efficacy in frog skeletal muscle.

1. Intracellular recording and voltage-clamp techniques were used to measure synaptic efficacy and the safety factor for neuromuscular transmission in frog skeletal muscle. All measurements were made in normal Ringer solution, in the absence of presynaptic or postsynaptic blocking agents. 2. Over a broad temperature range (10-30 degrees C), a small percentage of sartorius fibres (about 6%) could be found which produced only subthreshold end-plate potentials and no action potential in response to single, supramaximal nerve shock. At lower temperatures the proportion of such fibres increased; 42% of the fibres had subthreshold transmission at 5 degrees C, and 59% were subthreshold at 2.5 degrees C. 3. Threshold current, measured by intracellularly injecting short pulses of depolarizing current at end-plate regions, was independent of temperature between 2.5 and 20 degrees C. Thus, the reduced synaptic efficacy observed at low temperatures was not due to decreased electrical excitability of the postsynaptic membrane. 4. The amplitude of evoked end-plate currents (EPCs) decreased with cooling. At temperatures below 10 degrees C, the evoked EPCs at many end-plates were too small to initiate action potentials. The decline in EPC amplitude was due to three factors: a decrease in the amplitude of single quantum currents (MEPCs), an increase in the temporal dispersion of transmitter release, and (below 5 degrees C) a decrease in quantal content. 5. The safety factor for neuromuscular transmission decreased dramatically as temperature was lowered. At 30 degrees C average safety factor was large and positive (540 nA), but at 2.5 degrees C it was negative (-78 nA). 6. The quantal content of evoked transmitter release was independent of temperature change between 5 and 30 degrees C, the average value over this range being 180. However, at temperatures below 5 degrees C, quantal content fell off sharply (average value = 37). 7. The thermal independence of transmitter release may be an important mechanism in allowing poikilothermic animals to maintain physiological function over a wide range of body temperatures.

Effect of 4-aminopyridine on the postsynaptic action of polymyxin B

Polymyxin B produces neuromuscular blockade by acting at pre- and postsynaptic sites. Its postsynaptic effect has been attributed either to blockade of open ionic channels or to the conversion of the end-plate receptor from a low to a high affinity state, i.e. to a state similar to the desensitized one caused by agonists. Since 4-aminopyridine inhibits agonist-induced end-plate receptor desensitization, we decided to investigate its effect on the postsynaptic action of polymyxin B. The experiments were carried out on the isolated rat diaphragm; miniature end-plate potentials (m.e.p.p.s) and carbachol-induced depolarization at the end-plate region were recorded with microelectrode techniques. Polymyxin B, 40 ω;g/ml reduced the amplitude of the m.e.p.p.s. and suppressed them in about 30 min. 4-Aminopyridine completely antagonized this effect. Neostigmine did not restore the m.e.p.p.s. suppressed by polymyxin B nor did 4-aminopyridine antagonize the effect of d-tubocurarine on these potentials. The carbachol-induced depolarization was blocked only partially by polymyxin B. 4-Aminopyridine B nearly completely antagonized this effect. The antagonistic effect of 4-aminopyridine on the postsynaptic action of polymyxin B favors the receptor desensitization' hypothesis.

Definition, at the Molecular Level, of a Thyroglobulin—Acetylcholinesterase Shared Epitope: Study of its Pathophysiological Significance in Patients with Graves' Ophthalmopathy

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.

The reversible carbamate, (−)physostigmine, reduces the size of synaptic end plate lesions induced by sarin, an irreversible organophosphate

Pretreatment of rats with atropine and the reversible esterase inhibitor physostigmine ((−)PHY), prior to injection of a lethal dose of the irreversible organophosphate sarin (0.13 mg/kg), protects 100% of the animals from lethality. We have used quantitative light and qualitative electron microscopy to show that damage to the end plate region of voluntary muscles is also strikingly limited by the same pretreatment. Drug effects on soleus motor end plates detectable 1 hr after treatment were (1) a single sublethal dose of sarin (0.08 mg/kg) produced large, blistered, and severely disrupted subjunctional regions. Damage extended from the end plate, in the form of myofiber necrosis and subsequent phagocytosis; (2) (−)PHY (0.1 mg/kg) itself had a selective effect in inducing irregularities of the subjunctional sarcomere band without any gross vacuolization; (3) the morphometric analysis done with light microscopy indicated that the combination of atropine (0.5 mg/kg) and (−)PHY (0.1 mg/kg) prior to a lethal dose of sarin (0.13 mg/kg) offered 86% reduction in the average area of the lesions, relative to the dimensions of damage induced by atropine/sarin alone. In most lesions induced by (−)PHY, recognizable changes were markedly less severe in degree and extent than those seen in sarin myopathy; there were few instances of extensive muscle damage and myofiber necrosis. The relationship of the (−)PHY dose to the level of protection against sarin suggested that (−)PHY pretreatment almost completely prevents the characteristic sarin-induced myopathy and, instead, imposes the characteristic PHY-induced subjunctional swelling. In all three experimental groups examined, the myopathic changes located extrajunctionally were reversible. The mechanism by which (−)PHY acts as a protective agent is discussed.

Localization of neural epitopes that bind to IgM monoclonal autoantibodies (M-proteins) from two patients with motor neuron disease

We investigated IgM monoclonal antibodies (M-proteins) specific for the carbohydrate epitopes Gal-(β1–3)GalNAc and Gal(β1–3)GlcNAc from two patients with motor neuron disease. The M-proteins from these patients immunostain central nervous system (CNS) and peripheral nervous system (PNS) tissue from human, monkey, dog and cat at greater dilutions than tissue from rabbit, guinea pig, rat and mouse, and immunostain gray matter at greater dilutions than white matter and nerve trunks. They also bind selectively to presynaptic structures at the motor endplate region, as denervation of muscle eliminates binding. Following in vivo injection of serum into the extracellular space of the spinal cord, the M-proteins appear to bind at the surface of cells and cell processes. These studies suggest that the M-proteins might act at any one of several anatomical sites in the nervous system. This information may be helpful in selecting an animal species for further investigation of the role of M-proteins in motor neuron disease.

Cross‐Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA‐Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.

On the role of the 200-kDa neurofilament protein at the developing neuromuscular junction

To examine whether the 200-kDa neurofilament protein (200K NFP) is involved in mechanically stabilizing axons, we studied the developmental appearance of immunoreactivity to nonphosphorylated and phosphorylated 200K NFP at the neuromuscular junction. Polyinnervated rat muscle fibers become singly innervated during the first 3 weeks of postnatal life through the process of synapse elimination. If production or post-translational modification of the 200K NFP is actively involved in imparting mechanical stability on neuromuscular synapses, then the selective presence of this protein in only one of several axons at each developing end plate region might make that one axon selectively resistant to elimination. The remaining axons would then be eliminated. Immunoreactivity to the 200K NFP is present on Gestational Day 14 and can be seen in more than one preterminal axon in the end plate region of a muscle fiber during the period of synapse elimination. These results suggest that the 200K NFP is present and phosphorylated early in development and, although the 200K NFP may increase the mechanical stability of axons, this increased stability does not determine the final outcome of synapse elimination.

Regional heterogeneity in the distal motor axon: three zones with distinctive intrinsic components.

In this study we examined the distribution of cytoskeletal and other intrinsic axonal elements in motor nerve terminals in vivo. Components of axons were visualized with immunocytochemical staining of frozen longitudinal sections of muscle. Using these methods we compared the distribution of neurofilaments, tubulin, MAP2, actin and synaptic elements in distal regions of axons at and near neuromuscular junctions in rat muscles. Our results show that three discrete regions can be defined based on the anatomy and intrinsic components of distal axons. The preterminal axon, extending from its exit from the intramuscular nerve toward the neuromuscular junction, has a cytoskeletal composition similar to the more proximal axon with abundant staining of neurofilaments, tubulin, MAP2 and actin. The terminal arborization, a branched region of the axon extending through the endplate region, contains neurofilaments but little tubulin, actin, MAP2 or synaptic elements. Finally, the synaptic zone, demonstrated with antibodies to the synaptic vesicle protein synaptophysin, contains few cytoskeletal elements. We conclude that there is considerable regional heterogeneity in the composition of distal motor axons. The distribution of neurofilaments, other cytoskeletal elements and synaptic vesicle proteins varies among different discrete zones of terminal motor axons.

Biochemical and physiological consequences of an age-related increase in acetylcholinesterase activity at the rat neuromuscular junction

Acetylcholinesterase (AChE) specific activity was assayed using diaphragm muscles obtained from mature adult (10 months) and aged (25-27 months) rats. Biochemical assays indicated significant age-related increases in the AChE specific activity of both noninnervated and innervated tissue. The different molecular forms of AChE were separated by velocity sedimentation and were further assayed. The age-related increase was manifest primarily in the 10S (G4) form in both noninnervated and innervated tissue and also the 16S (A12) form of the noninnervated samples. To ascertain more conclusively whether AChE activity in the end-plate junctional region of innervated tissue changed in the older rats, miniature end-plate currents (m.e.p.c.s) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited by 10 microM echothiopate or 1 mM methanesulfonyl fluoride, m.e.p.c. amplitudes and decay time constants increased in both age groups. The magnitude of these increases was larger in the older animals. However, calculations of the relative change in m.e.p.c. amplitudes after AChE inhibition indicated that less ACh was hydrolyzed by AChE in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh-induced membrane noise. These data indicate that the prolonged decay times in the older rats following AChE inhibition is quite likely due to an expanded field of postsynaptic ACh receptors and not exclusively to a change in junctional AChE.

Sodium channel distribution in normal and denervated rodent and snake skeletal muscle.

1. Sodium channel current density was measured using the loose-patch voltage clamp technique. Innervated rat, mouse and snake muscle had the highest density of Na+ channels in the end-plate region. These high Na+ channel densities were maintained in denervated muscle. 2. Perijunctional membrane had a Na+ current density 5- to 10-fold greater than the density several hundred micrometres from the end-plate. In all muscles this concentration of channels near the end-plate persisted following denervation. 3. At the tendon Na+ current density fell to low values (approximately 1 mA/cm2). The decrease in density began about 300-500 microns from the tendon. This pattern was found in all snake twitch fibres and fast-twitch (EDL) rat and mouse muscle fibres. This reduction in channel density near the tendon was not affected by denervation. 4. Sodium channels in all regions of innervated rat and snake muscle fibres were highly sensitive to tetrodotoxin (TTX). Sodium channels in snake muscle remained sensitive to TTX after denervation. Sodium channels that are relatively resistant to TTX appeared in rat muscle after denervation. TTX-resistant channels were even more concentrated near the end-plate than were TTX-sensitive channels in innervated muscle. At the tendon TTX-resistant Na+ channel density decreased. 5. We conclude that although the nerve presumably directs the localization of Na+ channels during development, the ability to maintain this distribution and to control the distribution of newly appearing channels persists long after the nerve has been removed.