Subneural Apparatus Research Articles

A rapid method for demonstrating skeletal muscle motor innervation in frozen sections.

Longitudinal 50-100 mum-thick frozen sections of muscle are picked up on slides coated with 3% EDTA and after drying are incubated to demonstrate acetylcholinesterase. Subsequent incubation in 0.5% K3Fe(CN)6 is followed by fixation for 30 minutes in formol-calcium or formol-saline. After washing, the slides are incubated in 20% aqueous AgNO3 containing 0.1% CuSO4 for 2-30 minutes at 37 C. Following development in a 1% solution of quinol (w/v) 5% with respect to NaSO3 (w/v), axons and subneural apparatus stain dark brown to black in contrast to the less well stained muscle fibers and nuclei. This procedure permits study of the pattern of neuromuscular innervation in skeletal muscle 3 1/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.

Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle.

A local anesthetic, methyl-bupivacaine was injected into the planta of adult mice, and the ultrastruct of motor end-plates was studied during the degenerative and regenerative cycle induced in lumbrical muscles. Muscle degeneration took place during the first day after drug administration. The postsynaptic part of the neuromuscular junction completely degenerated as did the whole injured muscle fibre. Nerve terminals, however, remained unaffected. By the second day after muscle injury, axon terminals were enclosed within Schwann cell cytoplasm and thus became separated from the residual sarcolemmal tube. One to three days later, when myotubes were formed by fusion of the surviving myoblasts, the layer of Schwann cell cytoplasm on nerve terminals was discontinuous. Subsequently nerve terminals approached the regenerating muscle cell and the subneural apparatus began to differentiate. Slight depressions and furrows appeared on the myotube surface below the nerve ending and the myotube membrane, covered with basement membrane, became undercoated by dense material in this region. Where the distance between nerve ending and myotube was reduced to that found in the normal neuromuscular junction, i.e. to about 500 A, junctional folds were formed. Fourteen days after drug administration, newly formed end-plates were indistinguishable from those in normal control lumbrical muscles.

Effect of scorpion (Buthus minax, L. Koch) venom on succinic dehydrogenase and cholinesterase activity of mouse tissue.

The histochemical activities of succinic dehydrogenase and cholinesterase in mouse tissues after administration of scorpion ( Buthus minax ) venom have been studied. There were obvious changes in the enzyme activities, of the chronically treated animals, in the kidney, heart and liver. The decreases in cholinesterase activity were greatest in the white matter of the brain, in the portal tract and in some liver cells, while the spleen showed increased activity. A decrease in succinic dehydrogenase activity was observed in the kidney, heart, and liver of the chronically treated mice, while the spleen showed no change in the activity. The striated muscles of the acutely treated mice were fragmented and vacuolated with a loss of their striation. The intramuscular nerve axons became beaded and the subneural apparatus and their terminal expansions were smaller.

Initial changes in the neuromuscular synapses of denervated rat diaphragm

The onset of fine structural changes in the axonal terminals of the neuromuscular junction was investigated in the diaphragm of the rat over periods of 3–48 h after phrenicotomy. Changes in the axonal terminals begin as early as 3 h after the operation and are demonstrated by swelling and destruction of mitochondria, reduction in the number of synaptic vesicles and appearance of membrane whorls which envelope the degenerated mitochondria, the dense granules or synaptic vesicles. Fragmentation of the nerve terminals is noted within 6 h after the operation. Up to 12 h, the glial processes have not yet penetrated the synaptic cleft between the subneural apparatus and nerve terminal. In the subsequent 10–h, the intra-axonal changes are intensified and the axonal fragments are completely involved in and phagocytosed by the Schwann cells. At the end of the first 24 h, the Schwann cells replace the destroyed nerve terminals. Under identical experimental conditions, an exceptional variety and great differences in the degree of degenerative changes are observed. Degenerative nerve endings are established with filamentous reaction.

Complete staining of neuromuscular innervation with bromoindigo and silver.

Frozen sections of musde fixed in buffered formaldehyde (pH 7.3) are first incubated for localization of esterase using 5-bromoindoxyl acetate as substrate. The sections are then mounted on slides and stained by a urea-silver nitrate method for axons. Result: subneural apparatus—blue; axons—black; other tissue components in various shades of grey.

Structure and staining characteristics of myofiber external lamina.

A variety of cytochemical methods were used to investigate the composition of mouse sarcolemma. We found that the sarcolemma is a complex composed of the myofiber cell membrane, a thin zone external to the cell membrane that contains groups that bind colloidal iron and thorium at low pH and a thicker amorphous layer that fails to stain with colloidal iron and thorium at low pH. The entire complex is periodic acid-Schiff-positive and stains with ruthenium red and strongly acid solutions of phosphotungstic acid. Although the specificity of these cytochemical stains is controversial, data obtained with them and from preliminary analyses of myofiber external lamina (EL) indicate that the myofiber cell coat is chiefly composed of glycoprotein containing a large number of carboxyl groups. The EL within the subneural apparatus of the neuromuscular junction differs from noninnervated areas in the fusion of Schwann cell and myofiber EL, the absence of collagen microfibrils, the more intense binding of divalent cations and the less intense stain with phosphotungstic acid in strongly acid solutions.

Direct connections between the T system and the subneural apparatus in mouse neuromuscular junctions demonstrated by lanthanum.

Direct connections between the T system and the subneural apparatus in mouse neuromuscular junctions demonstrated by lanthanum.

The neuromyopathy of vincristine in the guinea pig: An electrophysiological and pathological study

This report deals with the toxic effects of vincristine on guinea pigs. The brain, spinal cord, dorsal root ganglia, peripheral nerve, neuromuscular junction and skeletal muscle were studied with the light microscope, and the latter 3 tissues were also examined with the electron microscope. In an acute experiment in which adult guinea pigs received a single intraperitoneal injection of vincristine (1 mg/kg body weight) and were studied at intervals thereafter, the changes of early axonal degeneration occurred with the rapidity of Wallerian degeneration in the sciatic, posterior tibial, digital and intramuscular nerves, and in the terminal axonal expansions of the motor end-plates. Some of the dorsal root ganglia and anterior horn neurons had an increased number of nucleoli, but, like the remainder of the central nervous system, were otherwise normal by light microscopy. There were only minor changes in the subneural apparatus of the motor end-plates. Skeletal muscle underwent rapid degeneration with changes in the longitudinal sarcoplasmic reticulum and the development of complex “spheromembranous bodies”. The latter may be derived from the longitudinal sarcoplasmic reticulum. Proximal muscles were more severely involved than distal. In a chronic experiment, adult guinea pigs received twice-weekly intraperitoneal injections of vincristine (0.03 mg/kg body weight) for up to 14 weeks. The sciatic, posterior tibial and digital nerves contained fibres at all stages of axonal degeneration. All fibre sizes and all levels of nerve were affected. Relatively few fibres were involved. The dorsal root ganglia and central nervous system were normal by light microscopy. There was increased branching of terminal intramuscular nerves, an increased number of motor end-plates per muscle fibre, and ultrastructural indications of denervation of many motor end-plates. The skeletal muscle showed no grouped atrophy of denervation. It appears that denervated fibres became rapidly reinnervated by peripheral regeneration of nerves. The skeletal muscle at the end of the chronic experiment was largely normal, though in animals moribund early in the chronic experiment there was gross necrosis and phagocytosis of muscle. Either the muscle became resistant to vincristine or particularly sensitive animals died early. Fatty degeneration of the liver, and dilatation of the bowel and bladder were seen in animals receiving vincristine. The maximum motor nerve conduction velocity in the sciatic nerve fell significantly to 81% of normal after an average of 8 weeks of the chronic experiment, the change occurring in the first few weeks in serial studies. The mechanism of vincristine neurotoxicity is discussed in terms of current concepts of axoplasmic transport and the function of neurotubules and neurofilaments. The biochemical effect of vincristine on skeletal muscle requires further study. In the guinea pig, unlike man, the myopathic effect of vincristine exceeds the neurotoxic.

Fine Structure Observations of Denervation and Reinnervation of Neuromuscular Junctions in Mouse Foot Muscle

The fine structure changes which occurred during denervation and reinnervation of neuromuscular junctions for a period of thirty weeks after nerve section were studied in muscles of the mouse foot. Axons were absent by the end of the first week after nerve section and there was retraction of Schwann cells and perineural epithelial cells. The primary synaptic cleft became shallow, the secondary clefts, shorter and wider, but the basement lamina filling the subneural apparatus was unaltered. The characteristic thickening of the muscle surface membrane in the primary synaptic cleft persisted, and there were increased numbers of clustered ribosomes. A loss of coated vesicles was observed in the sole-plate sarcoplasm. The earliest returning axon sprouts were first observed at three weeks and became numerous by four weeks. These originally contained few and variably sized synaptic vesicles but no coated vesicles. In the succeeding weeks the primary clefts deepened, the secondary clefts became elongated and narrower, and, by fifteen to thirty weeks, all neuromuscular junctions studied had normal fine structure. No fine-structure evidence of de novo formation of neuromuscular junctions was obtained by electron microscopy, although occasional small neuromuscular junctions formed by escaped fibers were observed in dual-stained sections studied in the light microscope. Persistence of tenuous processes of both Schwann and perineural epithelial cells in the immediate vicinity of denervated neuromuscular junctions appeared to play a major role in guiding returning axons to old sites of innervation.

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. II. The distribution of DFP-reactive sties at motor endplates of a vertebrate twitch muscle.

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-(3)H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per micro(3) of cleft tissue or 12,000 sites per micro(2) of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)-a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.

Fine structure of neuromuscular junctions after nerve section and implantation of nerve in denervated muscle

The first evidence of spontaneous reinnervation of neuromuscular junctions occurred 4 weeks after nerve section. In these experiments, the regenerating axons returned to old subneural apparatuses. There was little tendency to form new junctions as judged by light and electron microscopic studies. When the peroneal nerve was cut and reimplanted in the m. tibialis anterior at a new site distant from the original innervation band, a new innervation band developed near the point of entry of the transplanted nerve 6–8 weeks after operation. Electron microscopic study revealed atrophic subneural apparatuses with characteristic features of previous denervation and other junctions with fine structure resembling that observed in the embryonic morphogenesis of neuromuscular junctions. Similar patterns of neuromuscular junction reinnervation were observed in experiments in which after denervation, a foreign nerve was implanted into the muscle. Under these circumstances, an additional observation was the formation of abnormal tubular structures probably related to the regeneration of the transverse tubular system in atrophic muscle.

Morphological and Cytochemical Studies on the Synapses in the Autonomic Nervous System

Publisher Summary This chapter presents morphological and cytochemical studies on the synapses in the autonomic nervous system. At the ultrastructural level, the interneuronal synapses exhibit morphological differentiations both in the nerve endings, such as synaptic vesicles, and in the effector, such as the thickening of the postsynaptic membrane in synaptic complexes or active zones. The motor endplate is characterized by a special differentiation of the muscular membrane, which is the subneural apparatus of Couteaux. In the tissues innervated by the autonomic nervous system, it is not possible to distinguish, with the techniques available, the limited areas morphologically differentiated both on the nerve side and on the effector side that might be clearly recognized as synaptic contacts. Because of the lack of morphological evidence and precise physiological data on a true synaptic transmission, it is inadequate to call synapses the neuroeffector connections of the autonomic nervous system, but because the peripheral part of the autonomic nerve fibers can release a transmitter, this can be called the “efficient part” of the sympathetic or parasympathetic fibers, and the study of its properties can take place with that of the presynaptic fibers sensu stricto.

THE EFFECTS OF BOTULINUM TOXIN ON THE PATTERN OF INNERVATION OF SKELETAL MUSCLE IN THE MOUSE

Sublethal quantities of botulinum toxin were injected into the gastrocnemius and hamstring muscles of one leg in mice. Local paralysis and atrophy occurred and persisted for a few weeks. Histological examination showed no degeneration of nerve fibres or of motor nerve endings. Silver impregnations showed that during the period of muscle paralysis there was growth of motor nerve fibres from the terminal arborizations. Nerve fibre overgrowth was accompanied by elongation and abnormal configuration of sites of cholinesterase activity. As muscle function recovered the nerve fibre overgrowth receded and well‐formed though still abnormally shaped subneural apparatuses were seen. Nine months after the injection of the toxin, the pattern of innervation of the muscle was still abnormal.The growth of motor nerve fibres after the injection of botulinum toxin appears to be a response to the failure of neuromuscular transmission and is not analogous to regeneration of nerve fibres after trauma.

MOTOR END PLATE REACTIVITY TO DIVALENT METAL IONS HISTOCHEMICAL STUDIES

Motor end plates in the tibialis anterior muscle of the rat were demonstrated by metal sulfide deposits following injection of aqueous solutions of lead, stannous, cadmium, zinc or cupric ions into the muscle in vivo or in vitro. The appearance of the end plates was similar to the structure demonstrated by cholinesterase staining, with visualization of the subneural apparatus. Neither metal binding nor cholinesterase activity was affected 4 weeks after dissection of the sciatic nerve, indicating that the metal binding site is postsynaptic. Freezing or formalin fixation of muscle prevented binding of all metal ions to the end plate without greatly affecting cholinesterase activity, indicating that these two activities of the end plate are distinct. Prior administration of acetylcholine, d-tubocurarine, neostigmine or diisopropyl fluorophosphate inhibited binding to the end plate of cadmium and zinc ions but did not alter binding of lead and stannous ions. By formation of a lake with alizarin red S previously injected in vivo intramuscularly, the release of calcium ions at the motor end plate following stimulation of the muscle through the nerve or administration of neostigmine was demonstrated. These results suggest a close relationship of the site of binding of divalent metal ions in the motor end plate to the site of calcium release, and a close but not identical relationship to the site of cholinesterase activity and the acetylcholine receptor.

Structure and Organization of the Myoneural Junction

This chapter focuses on the the myoneural junction obtained by histochemistry of cholinesterase and electron microscopy. One of the most stimulating findings is the reduction or the absence of secondary synaptic clefts in the motor endings of some somitic primitive muscles, of some “slow” muscles, and of intrafusal muscle fibers. This feature suggests that one of the morphological factors of differentiation of the myoneural synapse should be the degree of folding of the sarcolemma. It may be assumed that one of the results of this enlargement of the postsynaptic surface is to increase its richness in cholinesterase. This hypothesis is supported by the fact that the cholinesterase activity of the myoneural junction of the tonic fibers of the frog is weaker than the activity of the end bushes supplying the fast fibers. Thus, the structure of the subneural apparatus should represent one of the factors conditioning the electrical and mechanical characteristics of the muscle fibers. Some features of comparative morphology of the motor synapse are also mentioned in the chapter. The question of the double innervations of striated muscles both by the α- and γ-motor system and the functional differentiation of the motor endings related to extrafusal and intrafusal muscle fibers are discussed in the light of modern morphological and physiological concepts.

Fine structure of nerve endings in extraocular muscle.

Introduction Recent electron microscopic studies have demonstrated the presence of two distinct types of muscle fiber, Fibrillenstruktur and Felderstruktur, 1 in the extraocular muscles of guinea pig, 2 cat, 3 and man. 4 In the Fibrillenstruktur fibers, the myofibrils have a punctate appearance, and the sarcoplasmic reticulum surrounds clearly the individual fibrils. The Felderstruktur fibers, on the other hand, have larger myofibrils which are not regularly separated from each other and appear to be fused together. Physiologically, the Fibrillenstruktur fibers appear to be associated with relatively rapid twitch responses, whereas the Felderstruktur fibers respond to stimulation with prolonged contracture. 3 There are, in addition, two kinds of nerve endings in the extraocular muscles which can be histochemically differentiated by cholinesterase staining; these are the en plaque and en grappe endings. 2-7 The en plaque endings stain intensely and show the same configuration as the subneural apparatus of motor end-plates in

Cholinesterases and motor end-plates in developing duck skeletal muscle.

End-plates in the thigh muscles of duck embryos were first recognized with myristoylcholine as substrate at the 19th day of incubation. Each appeared as a cholinesterase-positive dot surrounded by a small halo which rapidly increased in size during the 20th and 21st days. The endplates were usually oval in shape, averaging 33 µ x 25 µ with a subneural apparatus 5-12µ wide. The latter contained refringent lamellas arranged transversely in a palisade fashion. From the 21st day to the day of hatching (27-29 days) the number of end-plates progressively increased. After hatching, the myristoylcholine-reacting end-plates were difficult to locate. With acetylthiocholine as substrate, the embryonal end-plates were not demonstrable; however, the posthatched tissues showed numerous end-plates. The nerve trunks and nerve fibers gave a faintly positive myristoylcholine reactions in all stages after the 19th day of incubation. On the basis of the effects of eserine and diisopropyl fluorophosphate, the structures reacting with myristoylcholine and acetylthiocholine contained specific chohinesterase. The end-plates containing nonspecific cholinesterase also appeared on the 19th day of incubation and appeared to increase gradually in number until the 15th postembryonic day; thereafter they seemed to decrease.

Submicroscopic organization of the postsynaptic membrane in the myoneural junction; a polarization optical study.

Cross-striated muscles of frogs and rats were fixed in 3.3 per cent lead nitrate solution. Frozen sections 30 micra thick were mounted in different media and observed by polarization microscopy. The subneural apparatus of myoneural junctions exhibits a strong birefringence in these sections. Birefringence is exerted by a highly organized lipoprotein framework (postsynaptic material) which builds up the "organites" (junctional folds) of the postsynaptic membrane. Synaptic cholinesterase is closely associated with this material. Freezing and/or formalin fixation results in a destruction of the molecular organization of the postsynaptic material, but does not influence the synaptic enzyme activity. It is hypothesized from this study that the junctional folds (postsynaptic "organites") consist of regularly arranged, sheet-like lamellar micellae in the frog and of less regular, mainly radially arranged submicroscopic units in the rat. The micellar organization as revealed by polarization analysis is in good agreement with the electron microscopic findings reported in the literature. Intramicellar protein molecules of the resting postsynaptic membrane are arranged longitudinally, lipids transversely. Supramaximal stimulation or treatment with acetylcholine + eserine results in a disorganization of proteins and a rearrangement of lipids. Denervation results in a rearrangement of lipids without any significant alterations of proteins. All these functional stresses influence only the molecular and not the micellar structure of the membrane. The function of the organized lipoprotein framework as an acetylcholine receptor is suggested.

Observations on the fine structure and cytochemistry of mouse and human intercostal neuromuscular junctions.

The fine structure of the mouse and human intercostal muscle neuromuscular junction was studied after brief fixation in a new formol-sucrose fixative. This primary formalin fixation was followed by brief postosmication in buffered 1 per cent osmium tetroxide. Muscle blocks were embedded in methacrylate or Epon 812 epoxy resin. Marked similarities between mouse and human motor end-plates were observed. Neuromuscular junctions from both mouse and human intercostal muscle showed synaptic vesicles, primary and secondary synaptic clefts, and layered differentiation of the amorphous surface material (ASM) present on the surface of the Schwann cell plasma membrane and on the muscle surface membrane in the region of the neuromuscular junction. An attempt to stain the ASM with lead was unsuccessful. Observations on thick and thin plastic-embedded sections stained by PAS after diastase digestion showed that the ASM within the subneural apparatus is PAS positive. Alcian blue stained the endoneurium and perineurium of peripheral nerve bundles and portions of the end-plates. The similarity of the PAS-positive ASM to other basement membranes described in other sites is discussed and its possible physiologic significance within the subsynaptic apparatus is considered.

The histochemical localization of acetylcholinesterase in the fine structure of neuromuscular junctions of mouse and human intercostal muscle.

The localization of acetylcholinesterase activity in the fine structure of mouse and human neuromuscular junctions was studied by the method of Barrnett and Palade. A formolsucrose solution was used for primary fixation followed by osmification. It was found that deposits of lead sulphide, the product of the histochemical reaction, were found in axonal and sarcoplasmic mitochondria and most prominently in the primary and secondary synaptic clefts of the subneural apparatus. Rarely were reactive sites found near the M-band. Control experiments utilizing inhibitors and high concentrations of Pb(NO3)2 were performed. The possible significance of acetylcholinesterase activity in the areas described is discussed.