Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Effect of oculomotor and trigeminal nerve section on the ultrastructure of different myoneural junctions in the rat extraocular muscles

Summary The intramuscular nerves and myoneural junctions in the rat rectus superior, medialis and inferior muscles from 10 hours to about 10 days after section of the trigeminal and oculomotor nerves were studied with the electron microscope. Two different kinds of myoneural junctions are to be observed; one type derives from myelinated nerves and is similar to the ordinary myoneural junctions (motor end plates) of other striated skeletal muscles, while the other type derives from unmyelinated nerves, is smaller in size and has many myoneural synapses distributed along a single extrafusal muscle fibre.Section of the trigeminal nerve caused no changes in the myoneural synapses. After section of the oculomotor nerve degenerative changes occur in both the myelinated and unmyelinated nerves and in both types of myoneural junctions. In the axon terminals of both the myelinated and unmyelinated nerves the earliest changes are to be observed 10 to 15 hours after section of the nerve. First, swelling of the axoplasm, fragmentation of microtubules and microfilaments and swelling of mitochondria takes place, somewhat later agglutination of the axonal vesicles and mitochondria. The axon terminals are separated from the postsynaptic muscle membrane by hypertrophied teloglial cells about 24 hours after section of the nerve. The debris of the axon terminals is usually digested by the teloglial cells within 42 to 48 hours in both types of myoneural junction.Changes in the postsynaptic membrane are observed in the myoneural junctions of the unmyelinated nerves as disappearance of the already earlier irregular infoldings, whereas no changes take place in the infoldings of the motor end plates. The postsynaptic sarcoplasm and its ribosomal content increase somewhat.The earliest changes occur along unmyelinated axons 10 to 15 hours and along myelinated axons 15 to 24 hours after nerve section. The unmyelinated axons are usually totally digested within 48 hours, whereas the myelinated axons took between 48 hours and 4 days to disappear. The degeneration, fragmentation and digestion of the myelin sheath begin between 24 and 42 hours and still continues 10 days after the operation.The results demonstrate that in the three muscles studied structures underlying the physiologically well known double innervation of the extraoccular muscles are all part of the oculomotor system.We are grateful to Professor Antti Telkkä, M. D. Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

Effects of colchicine and vinblastine on neurotubules of the sciatic nerve and cholinesterases in the developing myoneural junction of the rat

Summary Colchicine (0.1 M) or vinblastine (0.01 M) was locally applied on the sciatic nerves of newborn rats. Both colchicine and vinblastine caused reversible disappearance of axonal neurotubules and appearance of increased amounts of neurofilaments at the site of application. Subsequent morphogenesis of myoneural junctions in the tibialis anterior muscle was studied after histochemical demonstration of acetylcholinesterase (AChE; E.C. 3.1.1.7) and non-specific cholinesterase (Ns. ChE; E.C. 3.1.1.8) activity in the myoneural area.Development of the postsynaptic muscle plasma membrane of the myoneural junction was arrested in the ipsilateral, but not in the contralateral control side, for a period of about three weeks following treatment with the test substances. After this delay the myoneural morphogenesis continued normally and neurotubules were seen in the axoplasm.Since disruption of neurotubules is likely to cause blockage of the intratubular axoplasmic transport system, it seems possible that the neurotrophic influence responsible for the development of the postsynaptic muscle membrane is mediated through a secretory product transported along axons intratubularly to the nerve endings.     

Formation of myoneural and myotendinous junctions in the chick embryo

Summary The mode of formation of the myoneural and myotendinous junctions was investigated in the thigh muscles of the chick embryo. Myotendinous junctions first appeared on day 11 of incubation, whereas myoneural junctions developed on day 12. Intracellular AChE activity in the muscles increased by the 12th day of incubation, and decreased rapidly after the formation of the myoneural junctions. Light and electron microscopically, AChE activity was demonstrated in the nuclear envelope, sarcoplasmic reticulum, Golgi complex, and in large granules which appeared to be derived from the Golgi complex. Large granules showing an intense AChE activity accumulated in the sarcoplasm at the poles of the muscle fiber before the formation of myotendinous junctions. After the translocation of this intracellular enzyme onto the sarcolemma, most likely the result of an exocytosis of the granules, the myotendinous junctions were formed. The AChE-rich granules present in the middle of myotubes developed into spindle- or comma-shaped cisternae which were located in the sarcoplasm just below the presumptive motor endplates. The present results suggest that the transport of AChE-rich granules to the sarcolemma is the first step in the formation of myoneural and myotendinous junctions.This work was carried out under grant 38848 from the Ministry of Education of Japan     

An improved combined cholinesterase stain and silver impregnation method for quantitative analysis of innervation patterns in frog muscle

The original method combining Karnovsky's cholinesterase stain and Bodian silver impregnation has been modified to stain both myelinated and unmyelinated axons and to reduce background staining. The improvements were obtained by adding nitric acid to a paraformaldehyde-acetone fixative and by carrying out the silver impregnation of axons in an alcoholic solution. The method is especially suitable for quantitative estimation of the different kinds of nerve sprouting as well as for study of the remodeling of neuromuscular junctions in normal and experimental frog muscles.     

The role of nerve lysosomal enzymes in the pathogenesis of denervation atrophy. Electromyographic and histochemical study in rats

Section of sciatic nerves of rats produced fibrillations within 3 days. Foci of hyalination leading to necrosis corresponded to segments of muscles containing end plates. The electrolyte content, mainly Ca, was increased, NADH2-TR activity was decreased and membrane ATP-ase was increased. The known increase in hydrolytic enzyme activities in denervated muscles was due to spilling of lysosomal enzymes from degenerating axons at the myoneural junction. This explains the discrepancy between morphological studies indicating paucity of lysosomes in normal muscles and the high hydrolytic enzyme activities in denervation. We propose that denervation changes are at least partly due to the effect of lysosomal spillage from degenerating axons.     

Structural development of myoneural junctions in the human embryo

Summary The structure of myoneural junctions in the tibialis anterior and intercostal muscles was studied after histochemical reaction for myoneural acetylcholinesterase (E.C. 3.1.1.7) in human embryos.Myoneural junctions of a primitive form were first seen in the intercostal muscle at the age of 8.6 weeks (crown-rump length of 3.2 cm), and in the tibialis anterior muscle at the age of 10 weeks (4.3 cm). The postsynaptic membrane was devoid of any junctional folds typical of adult synapses up to the age of about 19 weeks. At first small, the junctional folds gradually became deeper and more prominent during the following weeks, and some ramification of the previously coherent postsynaptic area took place. Myoneural morphogenesis was not completed at birth, although well developed postsynaptic Moldings were present.     

The role of nerve lysosomal enzymes in the pathogenesis of denervation atrophy

Summary Section of sciatic nerves of rats produced fibrillations within 3 days. Foci of hyalination leading to necrosis corresponded to segments of muscles containing end plates. The electrolyte content, mainly Ca, was increased, NADH₂-TR activity was decreased and membrane ATP-ase was increased.The known increase in hydrolytic enzyme activities in denervated muscles was due to spilling of lysosomal enzymes from degenerating axons at the myoneural junction. This explains the discrepancy between morphological studies indicating paucity of lysosomes in normal muscles and the high hydrolytic enzyme activities in denervation. We propose that denervation changes are at least partly due to the effect of lysosomal spillage from degenerating axons.     

Axonal protrusions in the small multiple endings in the extraocular muscles of the rat

Summary A special type of myoneural junction has been observed in the extraocular muscles of the rat with electron microscopy. These axon terminals are derived from unmyelinated nerves and contain synaptic vesicles and mitochondria. The terminals are invested by teloglia cells and separated by a synaptic cleft of about 500 Å from a slow-type muscle fibre. From the nerve ending a pseudopod-like evagination projects into the muscle cell. The membranes of this evagination and the muscle cells are only separated by a narrow cleft of about 100 Å, which is devoid of the basement membrane-like material typical of ordinary myoneural junctions. The evagination contains fewer axonal vesicles than other regions of the terminal axoplasm and the postsynaptic part of the muscle plasma membrane in this special region does not exhibit the postsynaptic thickening characteristic of ordinary myoneural junctions.The author thanks ProfessorAntti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

Localization of acetylcholinesterase in the rat myoneural junction

Summary The distribution of myoneural acetylcholinesterase (AChE; EC. 3.1.1.7) was studied electron microscopically with the copper ferricyanide method at pH 6.0, using acetylthiocholine iodide as substrate and iso-OMPA to exclude other cholinesterase activity.It was observed that the results obtained with this method are affected by changes in the reaction temperature, inhibitor concentration and fixation time. Changes which retard the rate of hydrolysis of AChE were observed to transfer the localization of the reaction endproduct in intact junctions from the extracellular side of the postsynaptic membrane to the intracellular side of the postsynaptic membrane. From the results it is concluded that the site of most intense AChE corresponds to the region of increased subsarcolemmal electron density of the postsynaptic membrane.     

Ultrastructure of heart synapses]

The nerve endings in the intramural ganglia of the rat's heart are connected by synaptic and non-synaptic junctions with the dendrites and neuron bodies. Peri-membrane indurations of synaptic complexes may look relatively symmetrical or asymmetrical depending on the orientation of the section in relation to the elements of the Akert's presynaptic lattice. In non-synaptic junctions the indurations are symmetrical, but the presynaptic one may be more complicated in its structure. The synaptic complexes are disposed in the field of synaptic dilatations of axons and in the sites of interlacing thin "preterminal" parts of axons with dendrites. They connect preganglionic fibres with dendrites, neuron bodies and with the filamentous and fungiform thorns.     

Ultracytochemical demonstration of cholinesterase activity in the atrium of the guinea-pig heart

Summary The ultrastructural distribution of cholinesterase (ChE) activity was examined in the atrium of the guinea-pig heart using Karnovsky's method.The reaction products showing the ChE activity were found mainly in the neural elements in the atrium. There were axons showing no reaction products intermingled with axons showing the positive enzymatic activity. In the enzymatically positive axons, the reaction products were observed in the axon-Schwann interspace, the interspace between neighboring axons and the corresponding plasma membranes in nerve fiber bundles as well as preterminal axons. It was of interest to find that not only axons containing ordinary agranular vesicles, but also axons with larger granular vesicles showed the ChE activity.In terminals the enzymatic activity was found also in the axon-Schwann interspace, the interspace between the axolemma and the sarcolemma and the corresponding plasma membranes of the enzymatically positive areas. In the neuron the enzymatic activity was positive in the endoplasmic reticulum and the interspace between the neuronal plasma membrane and the corresponding plasma membrane of the satellite cell. Plasma membranes facing to these enzymatically positive spaces were also positive for the enzymatic activity.This investigation has been supported by a research fund from Takeda Chemical Industries, Ltd., Osaka, Japan.A part of this work has been presented at the 8th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Kyoto on Nov. 30–Dec. 1, 1967, and also at the 24th Annual Scientific Meeting of the Japanese Society of Electron Microscopy held in Kofu on May 11–12, 1968.     

Development of the myoneural junction in the rat

Summary The structure of the myoneural junction in the striated muscle of rat embryos and postnatal rats was studied by electron microscopy in order to assess at ultrastructural level the roles of neuronal and muscular elements and the sequence of events resulting in the formation of a functionally mature synaptic organization.From the observations it is concluded that the axon terminals enveloped by Schwann cells contain vesicles prior to apposition of the prospective synaptic membranes. Subsequently, subsarcolemmal thickening of the postsynaptic membrane takes place after the synaptic gap has been formed by disappearance of the teloglial cell from between the synaptic membranes but before the primary synaptic cleft in the strict sense is formed. Secondary synaptic clefts are formed later, when the primary synaptic cleft is regular in width, by local finger-like invaginations of the postsynaptic membrane, which thereafter expand basally, in a plane transverse to the axis of the axon terminal, to resemble flattened flasks. The junction is formed between multinucleated muscle cells and multiple axons, which at first lie side by side and later, when formation of adult-type secondary synaptic clefts is in progress, become separated by folds of the sarcoplasm and the teloglia. In extraocular muscles of adult rats the sarcoplasmic reticulum is closely associated with the postjunctional sarcoplasm.In the light of earlier observations on the development of contractibility after nerve stimulation, cholinesterase histochemistry and muscle fibre physiology, these observations are interpreted to indicate that functional differentiation of the myoneural synapse results from induction by the motor axon and that the association of the sarcoplasmic reticulum with the postjunctional sarcoplasm in adult extraocular muscles is related to modified fibre physiology.The author wishes to thank Prof. Antti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for placing the electron microscopic facilities at his disposal.     

Effect of nerve compression on the morphogenesis of the cholinesterase containing structures of the rat myoneural junction

Summary The sciatic nerve of the newborn rat was unilaterally compressed to cause temporary denervation and subsequent re-innervation of immature myoneural junctions prior to the development of postsynaptic infoldings in the rat tibialis anterior muscle. Subsequent changes in the postsynaptic structure were followed by histochemical demonstration of acetylcholinesterase activity.It was found that postsynaptic infoldings into the myoneural junctions appeared between 16–21 days of age on the temporarily denervated side but already at 5 days of age on the contralateral unoperated side. Also further development on the operated side was delayed by 10–15 days as compared to the control side.The observations indicate that the nerve-ending exerts a stimulating influence on the development of the postsynaptic structures and, in addition, that this action is long-lasting, as compared to the more transitory induction of myoneural acetylcholinesterase activity shown earlier to take place in the rat embryo at the 18-day stage.     

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.     

The fine structural localization of acetylcholinesterase in the muscle spindle of the rat

Summary Muscle spindles from lumbricalis muscles of the rat were incubated for acetylcholinesterase with a modified thiocholine-method of Lewis and Shute and examined by light and electron microscopy.All types of motor nerve ending showed heavy deposits of reaction product in the synaptic cleft. The underlying sarcoplasmic reticulum, transverse tubular system, and, when present, the envelope of sole plate nuclei were also stained.In the sensory region, the reaction was negative in the interface between the plasma membranes of the primary sensory terminal and muscle. One of two secondary sensory endings identified showed distinct reaction product in the cleft; the other secondary sensory ending showed no such reaction.Precipitates were present on the sarcolemma of the intrafusal muscle fibers in the polar and adjacent myotube regions, but not at the spindle equator. Extrafusal and intrafusal myelinated -nerve fibers and preterminal motor axons showed staining of the axolemma. Fibers with thick myelin sheaths and preterminal sensory axons were free of acetylcholinesterase activity, as were the unmyelinated nerve fibers.We wish to thank Mrs. D. Schilling and Mrs. Ch. Beyer for technical assistanc     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

MYONEURAL JUNCTIONS OF TWO ULTRASTRUCTURALLY DISTINCT TYPES IN EARTHWORM BODY WALL MUSCLE

The longitudinal muscle of the earthworm body wall is innervated by nerve bundles containing axons of two types which form two corresponding types of myoneural junction with the muscle fibers Type I junctions resemble cholinergic neuromuscular junctions of vertebrate skeletal muscle and are characterized by three features: (a) The nerve terminals contain large numbers of spherical, clear, ~500 A vesicles plus a small number of larger dense-cored vesicles (b) The junctional gap is relatively wide (~900 A), and it contains a basement membrane-like material, (c) The postjunctional membrane, although not folded, displays prominent specializations on both its external and internal surfaces The cytoplasmic surface is covered by a dense matrix ~200 A thick which appears to be the site of insertion of fine obliquely oriented cytoplasmic filaments The external surface exhibits rows of projections ~200 A long whose bases consist of hexagonally arrayed granules seated in the outer dense layer of the plasma membrane The concentration of these hexagonally disposed elements corresponds to the estimated concentration of both receptor sites and acetylcholinesterase sites at cholinergic junctions elsewhere. Type II junctions resemble the adrenergic junctions in vertebrate smooth muscle and exhibit the following structural characteristics: (a) The nerve fibers contain predominantly dense-cored vesicles ~1000 A in diameter (b) The junctional gap is relatively narrow (~150 A) and contains no basement membrane-like material, (c) Postjunctional membrane specialization is minimal. It is proposed that the structural differences between the two types of myoneural junction reflect differences in the respective transmitters and corresponding differences in the mechanisms of transmitter action and/or inactivation.     

Two types of neuromuscular junctions in the pharyngeal retractor muscle ofHelix lucorum

The pharyngeal retractor muscle of the snailHelix lucorum is innervated by a pair of nerves containing axons of two types, for which there are two corresponding types of myoneural junctions with the muscle cells. The junctions of type I correspond to the thick axons. The terminals of these axons, which contain numerous spherical transparent vesicles (41±5 nm) and fewer vesicles of the dense-core type (67±3 nm), make contact mainly with noncontracting sarcoplasmic projections of the muscle cells. Junctions of type II correspond to thin axons, containing many granules. The terminals of these axons make contact with contractile parts of the muscle cells and they contain a heterogeneous population of vesicles: small spherical clear vesicles (44±2 nm), granules with fine-grained contents (135±5 nm), and a few spherical dense-core vesicles. The distance between the muscle cells is usually great — over 50 nm, but in the region of the sarcoplasmic processes the surface membranes come together to form a gap which in some areas does not exceed 10 nm.N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 5, pp. 539–542, September–October, 1977.     

Formation of Axon to Myocyte Contacts in Drosophila Cell Cultures

Cultures of Drosophila embryonic cells offer new opportunitiesfor studying myoneural junctions. In culture, neuroblasts andmyoblasts differentiate and yield neurons and myocytes. Someaxons grow across the surface of the culture vessel and attachto myocytes, forming functional myoneural junctions. Therefore,all stages in junction formation may be examined in vitro underconditions where pharmacological, electrophysiological, andother commonplace approaches are facilitated. This method offersan additional, most powerful approach for studying the junctions,that of genetic analysis. Drosophila mutations may be soughtwhich affect junction formation and function. Altered cellsand junctions from mutants may then be compared to those fromwild-type animals in order to dissect the gene-directed stepsunderlying junction phenomena.