Endplate Region Research Articles

Biochemical and histochemical evidence of 16S acetylcholinesterase in salivary glands.

Three forms of acetylcholinesterase (AChE) have been reported: the 4S, 10S, and 16S forms. It was suggested previously that 16S AChE is characteristic of the end-plate region; subsequently its presence has also been demonstrated in sciatic nerve, vagus nerve, several nerve trunks, cardiac atria, and distal ileum of rat. The purpose of the present study was to investigate further the occurrence of 16S AChE. We found that not only is it present in motor axons but that it may also occur in the secretory postganglionic parasympathetic fibers in the synapse-free parotid and submandibular glands.

The origin of spontaneous electrical activity at the end-plate zone.

Two types of spontaneous electrical activity are present at the end-plate zone: low-voltage negative potentials that correspond to miniature end-plate potentials, and larger voltage negative-positive potentials. The electrogenic origin of the latter has been uncertain. The origin of these larger potentials was investigated in the rat phrenic nerve diaphragm preparation and in human gastrocnemius muscle just prior to intubation during administration of preoperative anesthesia. In the hemidiaphragm the larger voltage negative-positive potentials were rarely triggered by intracellular or tungsten microelectrodes. The negative-positive potentials, however, were clearly triggered by contact of the concentric needle electrode with muscle hemidiaphragm at the end-plate region. The potentials were abolished by curare. Likewise, the equivalent potentials observed at the human gastrocnemius end-plate zone were blocked by neuromuscular blocking agents. Therefore, these positive-negative discharges represent postsynaptic muscle fiber action potentials and not nerve fiber activity. They were probably presynaptically activated by mechanical irritation of the motor axon terminal and preterminal branches.

Effect of Spinal Cord Transection on Neuromuscular Function in the Rat

The effect of T6 spinal cord transection on neuromuscular physiology, pharmacology, and histochemistry as well as succinylcholine-induced changes in serum potassium were studied in 88 Sprague-Dawley rats and compared to 19 control animals. Resting membrane potential of spinal cord transected animals decreased by a maximum of 15 mV. This change was significant at days 3, 7, and 30 posttransection. Cell membrane input resistance was significantly decreased at posttransection days 10 and 15. Succinylcholine-induced contracture increased 3- to 4-fold and was significantly greater than control values on days 5 and 10 posttransection. Concomitant with the development of contracture there was a spread of cholinergic receptor from the end plate region. This differed from control acetylcholine sensitivity on days 7, 15, and 30 when receptor could be detected in excess of 1000 micrometer from the endplate. Serum K+ levels 3 min after administration of succinylcholine (1 mg/kg) was significantly elevated on days 10 and 30. Spinal cord transection causes denervation-like changes in the involved skeletal muscle.

Satellite cell distribution within the soleus muscle of the adult mouse.

The frequency of satellite cells was quantitated by electron microscopy in five proximal to distal regions of the soleus muscle of adult mice. In all, 236 satellite cell nuclei and 4, 475 myonuclei were counted on 51 transverse thin sections. The mean percentage of satellite cells, as a ratio of satellite cells to myonuclei, per region was found to be 5.4%, 5.3%, 5.0%, 5.2% and 4.9% for the most proximal to distal areas, respectively. Analysis of variance revealed no significant differences between either the regions or the animals studied. The number of satellite cell nuclei per cross-sectional area of muscle was also calculated for each of the five regions, and these values did not vary significantly from the proximal to distal ends of the muscle. Despite the fact that satellite cells were frequently noted in close association with cross-sectional profiles of myoneural junctions, this study establishes that the number of such perisynaptic satellite cells was not large enough to affect significantly the mean percentages of all satellite cells counted within the motor endplate regions(areas 3 and 4) of the soleus muscle. It is concluded from this study that satellite cells are uniformly distributed throughout the whole muscle.

Recovery of acetylcholinesterase in the diaphragm, brain, and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate.

Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in he end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.

Nerve growth from nodes of Ranvier in inactive muscle

Paralysis of mouse gluteus maximus muscle with botulinum toxin not only evoked the expected sprouting from nerve terminals but also induced growth from some nodes of Ranvier close to the endplate region. This finding shows that nerve degeneration is not essential for nodal sprouting and supports the hypothesis that inactive muscle liberates a motor nerve growth factor.

Appearance of spontaneous fibrillation potentials after muscle fibre injury in man : V.J. Partanen and R. Danner (Kuopio, Finland)

<dm:abstracts xmlns:dm="http://www.elsevier.com/xml/dm/dtd"><ce:abstract xmlns:ce="http://www.elsevier.com/xml/common/dtd" class="author" xml:lang="en" id="ab005" view="all"><ce:section-title id="st005">Abstract</ce:section-title><ce:abstract-sec id="as005" view="all"><ce:simple-para id="sp0005" view="all">Spontaneous fibrillation potentials in EMG are a sign of denervation of a muscle fiber, as in motor axonal damage, or a part of a muscle fiber, as for example in necrotizing myopathy. Both rhythmic and irregular fibrillations have been described (<ce:cross-refs refid="b0005 b0010" id="r0005">Buchthal and Rosenfalck, 1966; Partanen and Danner, 1982</ce:cross-refs>). The underlying pathophysiology is different in rhythmic fibrillations and irregular fibrillations. Rhythmic fibrillations are a consequence of the slow rhythmic oscillations of an instable muscle membrane potential after denervation: Every depolarization wave reaching the firing threshold elicits a fibrillation potential. Irregular fibrillations are elicited by randomly occurring “fibrillatory origin potentials” (FOPs), arising in highly localized regions (&lt; 100 µm) of denervated muscle fibers, possibly associated with T-tubules (<ce:cross-ref refid="b0025" id="c0010">Purves and Sakmann, 1974</ce:cross-ref>). When a FOP reaches the threshold level and elicits a propagating fibrillation potential spreading to both ends of the denervated fiber, there is a long refractory period (<ce:cross-ref refid="b0035" id="c0015">Thesleff, 1982a</ce:cross-ref>), which prevents the early activation of a new fibrillation potential. Irregular spontaneous activity at the endplate region is a normal finding, not indicative of pathology, but it should be easily recognised and is the reason why fibrillation activity should be recorded from a number of sites (three) within a muscle before it is considered pathological.</ce:simple-para></ce:abstract-sec></ce:abstract></dm:abstracts>

A nerve stump dependent appearance of junctional and perijunctional acetylcholine receptors in organ culture

A rapid, nerve stump dependenl appearance of junctional and perijunctional acetylcholine receptors occurred after 42 h following the introduction of rat diaphragm into organ culture. Approximately2 × 10 6 or more acetylcholine receptors (assayed by [ 125I]alphabungarotoxin hinding) appeared at the endplate within a 2 h period, between 42 and 44 h, while only approximately2.5 × 10 3 acetylcholine receptors appeared in an equivalent area of extrajunctional membrane during the same time. Autoradiographic studies confirmed that most of the new acetylcholine receptor sites appeared at the endplate, although a small perijunctional component was also detected. The presence of a long nerve stump prevented the increase in the number of receptors specific to the endplate region, but had no effect on the appearance of extrajunctional acetylcholine receptors. Pharmacological studies showed that the nerve stump effect did not involve nerve impulse transmission, or the interaction of nicotinic acetylcholine receptors with acetylcholine. The findings suggest that junctional and extrajunctional acetylcholine receptors are controlled by different neuronal mechanisms and that junctional receptors are subject to

Prejunctional effects of anticholinesterase drugs at the endplate: mediated by presynaptic acetylcholine receptors or by postsynaptic potassium efflux?

Anticholinesterase drugs induce antidromic firing of motor axons in mammalian nerve-muscle preparations. Antidromic firing is observed in response to a conditioning stimulus applied to the nerve and hence is known as “back-firing”; the present investigation intends to clarify the mechanisms underlying this phenomenon. Acetylcholinesterase (AChE) was inhibited with neostigmine. Antidromic action potentials were recorded extracellularly from the nerve trunk. When the muscle was cut on one side of the main endplate region 0.1–0.2 cm away from the outermost myelinated nerve branches, back-firing gradually disappeared. This indicated that postsynaptic factors at least contribute to the generation of back-firing. One can conceive of a plausible mechanism whereby postsynaptic events influence presynaptic excitability: potassium ions leaking from extensively depolarized muscle fibers (block of AchE) might cause depolarization of nerve endings at some endplates (“potassium hypothesis”). Postsynaptic potassium currents were calculated according to a simple mathematical model of the muscle membrane. These simulations predict high postsynaptic potassium currents under the following experimental conditions: (1) low concentration of extracellular potassium, (2) exchange of extracellular chloride with nitrate, (3) small fiber diameter. Back-firing was found to increase under such conditions. The notion that postsynaptic potassium efflux may influence presynaptic membrane polarization was further substantiated: when carbamoylcholine chloride (Carbachol; 10 μmol · 1−1) was added to the bathing fluid, miniature endplate current frequency could be increased at some voltage clamped endplates by depolarizing the muscle fiber well below the potassium equilibrium potential.

Properties of 16S acetylcholinesterase from rat motor nerve skeletal muscle.

Rat obturator nerve 16S acetylcholinesterase (16S AChE) was separated by sucrose gradient velocity sedimentation and compared to the 16S form of AChE similarly derived from endplate regions of anterior gracilis muscles. The 16S AChE from both tissues could only be extracted in high ionic strength buffer; as it aggregated under low ionic strength conditions. Treatment of nerve and muscle 16S AChE with purified collagenase, in the presence of calcium, caused an identical "shift" in the enzyme's sedimentation coefficient to 17.5S. Other properties which were also equivalent for 16S AChE from both tissue sources included: an excess substrate inhibition above 2 x 10(-3) M acetylcholine and Km of 1.6 x 10(-4) M, relative sensitivity to the specific inhibitors BW284C51 (I50 of 5 x 10(-8) M) and Iso-OMPA (I50 of 5 x 10(-4) M), and a half maximal thermal inactivation at 62.5 degrees C. These and additional results indicate that the 16S forms of AChE in both tissues are analogous molecules, which have a highly asymmetric conformation probably containing a collagen-like domain. The present findings are also consistent with the view that motor neurons provide at least a fraction of the 16S AChE present at the neuromuscular junction.

Spontaneous recovery from depolarizing drugs in rat diaphragm.

1. The end-plate region in surface fibres of rat diaphragm was located by the use of polarizing filters. 2. Carbachol (100 microM) produced depolarization at the end-plate to -55 mV, as shown by continuous recording, with some indication of spontaneous recovery in the presence of the drug. The miniature end-plate potentials disappeared and remained absent. 3. By repeated sampling it was found that the resting potential at the end-plate had largely recovered after 45 min in the presence of carbachol. Individual fibres showed much variation in the rate of recovery, and in some fibres the repolarization was rapid. 4. In the absence of K, carbachol produced depolarization at the end-plate without significant recovery, as shown by repeated sampling. 5. When muscles were exposed to ouabain (100 microM) in addition to carbachol the end-plate remained depolarized without recovery for 60 min. The effect of ouabain was reversible: withdrawal of ouabain (in the presence of carbachol) led to substantial recovery. 6. Suberyldicholine (100 microM) gave results which were similar to those produced by carbachol. 7. It was inferred that the spontaneous recovery of membrane potential in the presence of carbachol and of suberyldicholine is a process which is sensitive to K and to ouabain.

Biochemical stability of the AChE molecular forms after cytochemical staining: postnatal focalization of the 16S AChE in rat muscle.

A biochemical analysis of rat muscle acetylcholinesterase (AChE) is possible after a cytochemical staining at pH7 (Koelle and Friedenwald method). The solubilization properties, the physicochemical characteristics (sedimentation coefficients) of the multiple molecular forms of AChE are similar before and after the cytochemical procedure without fixation and without ammonium sulfide treatment. This is in contrast to the partial inactivation obtained after conventional pH 5 cytochemical staining, which selectively inactivates 16S AChE. We found that pH 7 cytochemical staining in these same conditions afford a satisfying morphological visualization of the motor end-plate AChE. The combined cytochemical-biochemical techniques allow a very precise dissection of motor end-plate containing (neural) and free (aneural) regions. In rat muscle (sternomastoid), high levels of 16S AChE are present in the aneural region in the first stages of postnatal development. At the end of the 1st month after birth, the 16S AChE becomes restricted to the neural (motor end-plate) region.

Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction

Exposure of sternomastoid muscles excised from 16-day embryonic rats to medium depleted of Ca 2+ or containing high concentrations of KCl leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca 2+ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.

Prolonged exposure to acetylcholine: noise analysis and channel inactivation in cat tenuissimus muscle.

1. Micro-electrodes were used to record membrane potential and associated noise at the end-plate region of cat tenuissimus muscle (37 degrees C), during applications of acetylcholine (ACh) in continuously flowing Krebs solution containing eserine and tetrodotoxin. 2. Densensitization was assessed from the frequency of channel opening calculated from the noise variance. 3. At higher concentrations of ACh (10-50 microM), desensitization occurred with an exponential fall to a plateau. 4. At low concentrations of ACh (1-2 microM) only slight desensitization occurred and at a much lower rate. Frequency of channel opening decreased at the rate of 0.045 +/- 0.024 min-1. Maximum frequency was (33 +/- 9) X 10(7)/sec while maximum depolarization was 20.5 +/- 1.4 mV (n = 11 cats). Depolarization was well maintained. 5. This slow rate of desensitization at low concentrations of ACh was confirmed in experiments where voltage clamped current, its associated noise, and miniature end-plate current amplitude were measured. 6. At low concentrations of ACh (1-2 microM) in the presence of eserine there was sustained block in neuromuscular transmission when twitch tension was measured. 8. It is concluded that the mechanism of neuromuscular block by ACh at around 1 microM concentration is by depolarization itself, not desensitization.

The effect of lanthanum ions on acetylcholine in frog muscle.

1. Frog sartorius muscles were treated with an irreversible cholinesterase inhibitor and then incubated in Ringer with 2 mM-LaCl3. The amounts of ACh in the tissue and medium were assayed by mass fragmentography, miniature end-plate potentials (min. e.p.p.s) were recorded and the end-plate was investigated by electron microscopy. 2. Addition of La3+ caused in normal, but not in denervated, muscles a discharge of both min. e.p.p.s and chemically detectable ACh. After 30 min both min. e.p.p.s and ACh release decreased. Between 4 and 5 hr after the addition of La3+ min. e.p.p.s had practically ceased and the rate of ACh release was almost back to that in the absence of La3+. 3. La3+ caused a 50% reduction in the ACh content of the tissue within the first 30 min; thereafter ACh gradually increased to 110% by 5 hr. At this time synaptic vesicles were practically absent in most terminals. The ACh was predominantly located in the end-plate regions of the muscles, before as well as after the incubation with La3+. ACh in end-plate free parts of the muscles was unchanged by La3+. 4. Hemicholinium-3 inhibited the synthesis of ACh in the muscles, but it had almost no influence on La3+-induced ACh release. 5. From these and other results, it is concluded that the ACh released by La3+ originates exclusively from the nerve terminals, that most likely this ACh is released via exocytosis from synaptic vesicles, and that the synthesis of ACh following the release of ACh takes place in the nerve terminals. The results further indicate that in freshly excised muscle the greater part (80-90%) of the ACh contained in the nerve terminals is located in the vesicles.

Membrane systems in skeletal muscle of the lizard Anolis carolinensis

The iliotibialis muscle of the iguanid lizard Anolis carolinensis is composed of fibers that display highly developed membrane systems. One of these systems is composed of the so-called “transverse” tubules, which may branch in both the horizontal and vertical planes and which usually are connected to the surface sarcolemma via intermediary passages composed of caveolar vesicles. The remaining membrane system comprises the various elements of the sarcoplasmic reticulum (SR). Three reasonably distinct yet confluent conformations of SR can be found: (1) highly developed “conventional” SR which surrounds each myofibril and is segmented into specialized regions according to the divisions of the sarcomeres, such as junctional SR at the A–I levels, “network” SR massed over the I/Z/I band complexes, and fenestrated collars at the M lines; (2) subsarcolemmal SR whose junctional SR cisternae form peripheral couplings with the inner surface of the external cell membrane; and (3) extensive masses of proliferated SR that form roughly cylindrical bodies oriented longitudinally within intermyofibrillar spaces. These last (“columnar SR”) are found at all depths of the fibers and may be collected into less discrete aggregates that lie just underneath the sarcolemma, including the soleplate regions of motor end plates. The degree of development of the Anolis skeletal muscle membrane systems may reflect the requirements for rapid movement of the fibers.

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture.

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 microliters nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 microliters: 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p < 0.001) larger amount of endplate 16S AChE activity (140--145%) than the corresponding controls (100%). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.

Effects of drug-induced paralysis and depolarisation on acetylcholine receptor and cyclic nucleotide levels of chick muscle cultures

Neuromuscular interactions play an important role in the development and maintenance of several myofiber properties including the regulation of its chemosensitivity. In adult innervated skeletal muscle, receptors for the neuromuscular transmitter, acetylcholine, are confined to the endplate region of the myofiber membrane. By contrast, non-innervated embryonic myotubes or denervated adult muscle fibers bear acetylcholine receptors (AChR) on their entire ‘extrasynaptic’ cell surface (reviewed [ 1,2]). Different investigators showed that the developmental localisation of AChR to the postsynaptic part of the sarcolemma involves: (i) The aggregation of receptors under the nerve terminal [3,4]; (ii) The repression of the synthesis of extrasynaptic AChR [S-7]. Several lines of evidence suggest the latter process to be mediated by neurally-induced muscle activity: (1) Electrical stimulation of adult denervated muscle prevents appearance of extrasynaptic AChd [8] and blocks the incorporation of radiolabelled amino acids into the receptor protein [9]; (2) Chronic paralysis of the motor endplate by cholinergic antagonists increases the synthesis of extrasynaptic AChR in adult and embryonic myofibers [6,10,11]; (3) In muscle cell cultures, the rate of AChR synthesis is decreased by electrical stimulation or depolarising agents [ 12,131; under the same conditions, paralysis of the spontaneously contracting myotubes by tetrodoxin leads to an increase of newly synthesized receptor molecules. At present, the molecular events underlying the regulation of extrasynaptic AChR by muscle activity are unknown. In experiments with chick myotubes in tissue culture, dibutyryl cGMP added to the medium was found to reduce the rate of AChR synthesis [ 131, whereas CAMP derivatives increased the latter [ 13,141. Moreover, both cholinergic and electrical stimulation of adult muscle are known to induce transient increases in intracellular cGMP which depend on the presence of extracellular Ca*+ [ 15-171. It, therefore, was postulated that Ca*+ and cGMP may constitute the intracellular messengers of the activity-mediated control of AChR [13]. In this letter, it is shown that there is no obvious correlation between the AChR and the cyclic nucleotide levels of chick muscle cell cultures under conditions of drug-induced paralysis and depolarisation.

Endogenous electric field around muscle fibres depends on the Na+-K+ pump.

We describe here experiments which reveal a new physiological specialization in the endplate (synaptic) region of skeletal muscle fibres. Using a vibrating microelectrode which can detect small currents flowing in extracellular fluid, we have found that the membrane in the endplate region behaves as though a steady positive current is generated in this location. Current re-enters the fibre in the extrajunctional region. Further experiments show that this current is dependent on the activity of the sodium pump. The electric field created by this current may be important for long-term interactions between muscle and nerve.

The fate of foreign endplates in cross-innervated rat soleus muscle.

After transplantation of the superficial fibular and the medial plantar nerve to neighbouring sites in the proximal region of adult rat soleus muscles many muscle fibres were initially innervated by axons in both foreign nerves after resection of the original soleus nerve. The foreign endplates were formed at ectopic sites and were often separately located on individual muscle fibres. After 3-4 weeks many endplates had been eliminated and most muscle fibres were innervated by only a single foreign axon. Many muscle fibres still had multiple esterase-staining endplate sites in the region innervated by the foreign nerve. On examination by electronmicroscopy, some of these sites were seen to have lost their presynaptic terminal while the postsynaptic structure of the endplate remained intact. Other sites were only partially occupied by motor axon terminals. On each muscle fibre there was always at least one fully occupied endplate region. In some instances separate endplate sites on the same muscle fibre were innervated by branches of the same motor axon. We conclude that the elimination of endplates is due to a competitive interaction between motor axons innervating the same muscle fibre. Morphologically, the elimination of functional endplates is caused by a retraction of nerve terminals from the postsynaptic site.