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.     

Effect of temporary denervation on the development

Summary The influence of the motoneurone on the morphogenesis of the postsynaptic membrane of the myoneural junction was studied by denervation and subsequent re-innervation of the rat tibialis anterior muscles. This was effected by compressing the sciatic nerve at different stages of myoneural morphogenesis during the first month after birth. Changes in the postsynaptic structure were followed by histochemical demonstration of acetylcholinesterase (E.C. 3.1.1.7.) axtivity.Compression of the nerve brought development of the postsynaptic membrane to a standstill for about two to three weeks, the delay being greater in older rats. Subsequent structural development after re-innervation, although delayed for about two to three weeks, continued in the same way as in the controls.These observations indicate that the motoneurone exerts a long-lasting trophic stimulus on the development of the postsynaptic membrane of the muscle fibre and that this morphogenetic action continues throughout the structural maturation of the myoneural junction in the rat.     

Electron microscopic localization of cholinesterases in the rat myoneural junction

Summary The distribution of acetylcholinesterase (E.C. 3.1.1.7.) and other cholinesterases (E.C. 3.1.1.8.) in the fine structure of the myoneural junction of the rat diaphragma was studied, using acetylthiocholine and butyrylthiocholine as substrates. p]Acetylcholinesterase was located in the muscle sarcoplasm closely related to the postsynaptic membrane. Other cholinesterases are observed in the primary and secondary synaptic clefts and between the axon and the teloglial cells.     

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.     

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.     

Immunocytochemical identification of synaptotagmin 1, syntaxin 1, Ca2+ channel of the N-type, and nicotinic cholinoreceptor in motor neuromuscular junctions of somatic muscle of the earthworm Lumbricus terrestris

The earthworm somatic muscle contains myoneural synapses forming clusters of “synaptic buttons” in which the proteins syntaxin 1, synaptotagmin 1, and alpha 1B subunit of the Ca²⁺ channel of the N-type were identified. It is supposed that “synaptic buttons” contain a limited number of active zones, which is due to their small size (1–2 μm) and the pattern of distribution of proteins of the exoendocytotic cycle. The postsynaptic membrane of cholinergic synapses contains nicotinic acetylcholine receptors able to bind alpha-bungarotoxin. The area of the position of receptors on postsynaptic membrane is strongly restricted to the synaptic contact region.     

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.     

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.     

Effects of vinblastine and colchicine on the postsynaptic membrane at the frog neuromuscular junction

The possible effects of the alkaloids vinblastine and colchicine on the postsynaptic membrane of the frog neuromuscular junction were investigated using voltage-clamp techniques. Concentrations of vinblastine and colchicine which had been shown to exert no effect on the amplitude and duration of miniature endplate currents (MEPC) and the current-voltage relationship of low-quantal endplate currents (EPC) together with the coefficient of voltage-dependent EPC decay did produce a considerable rise in the amplitude of response to iontophoretically applied acetylcholine (ACh). In addition, vinblastine and colchicine accelerate MEPC and EPC during acetylcholine esterase inhibition while further depressing the amplitude of multi-quantal EPC succeeding at the rate of 10 Hz as well as response to regular (5–10 Hz) application of ACh from a micropipet. The dosage-frequency effects of vinblastine and colchicine on the postsynaptic membrane (as described) are presumed to be unconnected with the action of these agents on muscle fiber cytoskeleton but the results of accelerated desensitization of cholinoreceptors.S. V. Kurashov Medical Institute, Kazan. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 75–81, January–February, 1988.     

Electrophysiological and structural studies on the heart muscle of the lobster Homarus americanus

The electrophysiological properties and structure of heart muscle fibres of the lobster myocardium have been investigated. Discontinuities were observed in the expected decrement of electrotonic potentials in a study of the passive membrane properties of the muscle. Injection of Procion yellow revealed transverse barriers to dye movement, identified in electron micrographs as intercellular junctions. Unlike the intercalated discs of the vertebrate myocardium, which these regions superficially resemble, nexus regions of ;close' or ;gap' junctions are absent. The intercellular cleft is generally 400 A in width, locally reduced to 75-100 A The functional implications of these findings are discussed. Cardiac myoneural junctions, which include regularly repeating structures extending into the synaptic cleft from the postsynaptic membrane, are described.     

Mediator liberation at the myoneural junctions of the frog sartorius muscle when axoplasmic transport has been disrupted with colchicine]

Colchicine application to the nerve innervating the frog sartorius muscle leads to reduction of the differences in functional condition of the myoneural synapses of this muscle by the signs of quantum content of the end plate potential (EPP) and by the changes of the EPP amplitudes in high frequency stimulation. Along with this there occurs a rarefaction of the frequency of miniature potential of the end plate, and reduction of its amplitude. Apparently, the latter is caused by reduction of effective resistance of the postsynaptic membrane. Changes of the induced and spontaneous secretion of the mediator are connected with disturbances of the axoplasmic transport just in the somatic nerve fibers.     

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.     

Effects of c-AMP, caffeine, theophylline, and vinblastine on spontaneous transmitter release at locust nerve-muscle junctions

The effects of c-AMP, phosphodiesterase inhibitors (caffeine and theophylline) and vinblastine on spontaneous transmitter release was investigated at locust neuromuscular junctions. c-AMP, theophylline, caffeine, and vinblastine caused facilitation of transmitter release. None of these drugs had any effect on the amplitude of miniature excitatory postsynaptic potentials (min. E.P.S.P.'s), or on the resting membrane potential, but vinblastine increased the proportion of 'large' min. E.P.S.P.'s. The effect of theophylline (but not c-AMP and caffeine) on min. E.P.S.P. frequency was found to be calcium dependent. The effects of these drugs on the locust glutamatergic synapse are compared with their actions at other synapses.     

Colchicine-binding activity and neurotubule integrity in a rat sympathetic ganglion

The colchicine-binding activity of rat superior cervical ganglia was examined. Ganglia were cooled and re-warmed in the presence of either Cu²⁺ or of metabolic inhibitors. Electronmicroscopy showed that these treatments depolymerized the neurotubules. This depolymerization of neurotubules did not affect the colchicine-binding activity of ganglion homogenates but caused a two-fold increase in colchicine-binding by whole ganglia. This suggests that colchicine binds only to depolymerized neurotubule subunits and that colchicine-binding by whole ganglia can be used as a measure of polymerization of the neurotubule protein.The major part of the colchicine-binding activity of ganglion homogenates was found in the soluble fraction and was unstable. In the absence of divalent cations, 10⁻⁴ M vinblastine stabilised the soluble colchicine-binding activity.     

Localization of the ruthenium red-positive substance in the myoneural junction of rat.

Ruthenium red staining of myoneural junctions was examined in the flexor digitorum brevis muscle of the rat. Ruthenium red-positive electron dense substance was observed to emerge from the outer layer of the presynaptic axolemma and post-synaptic sarcolemma towards the synaptic cleft. Also the cleft substance was intensely stained, usually consisting of a medium dense layer between pre- and postsynaptic membranes. The probable function of acid mucopolysaccharides in the neuromuscular transmission is discussed.     

Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notochord cells

The ascidian notochord follows a morphogenetic program that includes convergent extension (C/E), followed by anterior-posterior (A/P) elongation [1-4]. As described here, developing notochord cells show polarity first in the mediolateral (M/L) axis during C/E, and subsequently in the A/P axis during elongation. Previous embryological studies [3] have shown that contact with neighboring tissues is essential for directing M/L polarity of ascidian notochord cells. During C/E, the planar cell polarity (PCP) gene products prickle (pk) and dishevelled (dsh) show M/L polarization. pk and dsh colocalize at the notochord cell membranes, with the exception of those in contact with neighboring muscle cells. In the mutant aimless (aim), which carries a deletion in pk, notochord morphogenesis is disrupted, and cell polarization is lost. After C/E, there is a dynamic relocalization of PCP proteins in the notochord cells with dsh localized to the lateral edges of the membrane, and pk and strabismus (stbm) at the anterior edges. An A/P polarity is present in the extending notochord cells and is evident by the position of the nuclei, which in normal embryos are invariably found at the posterior edge of each cell. In the aim mutant, all appearances of A/P polarity in the notochord are lost.     

INNERVATION OF THE FIBRILLAR FLIGHT MUSCLE OF AN INSECT: TENEBRIO MOLITOR (COLEOPTERA)

The structure of peripheral nerves, and the organization of the myoneural junctions in flight muscle fibers of a beetle is described. The uniaxonal presynaptic nerve branches display the "tunicated" structure reported in the case of other insect nerves and the relationship between the axon and the lemnoblast folds is discussed. The synapsing nerve terminal shows many similarities with that of central and peripheral junctions of other insects and of vertebrates (e.g., the intra-axonal synaptic vesicles) but certain important differences have been noted between this region in Tenebrio flight muscle and in other insect muscles. Firstly, the axon discards the lemnoblast before the junction is established and the axon effects a circumferential synapse with the plasma membrane of the fiber, which alone shows the increased thickness often observed in both pre- and postsynaptic elements. Secondly, in addition to the synaptic vesicles within the axon are present, in the immediately adjacent sarcoplasm, great numbers of larger postsynaptic vesicles which, it is tentatively suggested, may represent the sites of storage of the enzymatic destroyer of the activating substance similarly quantized within the intra-axonal vesicles. The spatial relationship between the peripherally located junctions and the portion of the fiber plasma membrane internalized as circumtracheolar sheaths is considered, and the possible significance of this with respect to impulse conduction is discussed briefly.     

Carboxylic esterases in developing myoneural junctions of rat striated muscle

Summary The distribution of acetylcholinesterase (AChE; E.C. 3.1.1.7), other cholinesterases (ns.ChE; E.C. 3.1.1.8) and eserine-resistant carboxylic esterases (ns. E; E.C. 3.1.1.1) was studied in the developing myoneural junction of the rat tibialis anterior muscle from the 16th intrauterine day onwards. Acetyl-and butyrylthiocholine were used as substrates for AChE and ns.ChE, and -naphthyl acetate for ns. E.Acetylcholinesterase was first visible in 18-day rat embryos, ns.ChE in 21-day embryos and ns. E in 1-day-old postnatal rats and thereafter. Both AChE and ns.ChE activities were localized at the level of the plasma membrane in the middle of the muscle fibres. In the early stages this area of activity had the appearance of a plate-like structure, which deepened to form a depression in the surface of the muscle fibre by the 2nd to the 4th postnatal day. About 5 days later subneural lamellaes appeared in this structure, and ramification and segmentation took place, their extent increasing concomitantly with the increase of enzyme activity. The adult pattern was attained by the age of one month. Precise localization of ns. E was not possible in the immature stages, mainly owing to the granularity of the reaction end-product. After the age of about 10 days, however, the distribution of the reaction end-product suggested a mainly presynaptic location. Other cholinesterases and ns. E, but not AChE, were detected in the neurilemma cells along nerve fibres. This neurilemmal enzyme activity gradually diminished after birth and was lost at about the age of 3 weeks.These observations demonstrate that the formation of the junction between the nerve and the muscle fibre takes place just before the first appearance of AChE activity in a sharply delineated area of the plasma membrane. The structural changes made apparent by the distribution of the reaction end-products are assumed to be linked to the spatial rearrangement of the synaptic membranes, seen in earlier electron microscopic studies.     

Innervation and membrane specialization at neuromuscular junctions in submaxillaris muscle of the frog

The submaxillaris muscle of the frog after zinc iodide-osmium staining reveals the presence of polyneural innervation. Cholinesterase staining shows that the longer terminals have postsynaptic folds whereas the smaller terminals (up to 5 micron) lack them. Thin-section electron microscopy shows that muscle fibers with or without an M line have terminals with and without postsynaptic folds. The terminals with postsynaptic folds have presynaptic membrane outpocketings above folds. These outpocketings are rudimentary or absent in the terminals without postsynaptic folds. In longer junctions, the P face of the presynaptic membrane has double rows of paired particles on active zone ridges perpendicular to the axis of the muscle. In smaller junctions active zone ridges are rudimentary or absent and double rows of particles form various patterns. Postsynaptic active zones in longer junctions consist of clusters of particles leaving gaps in between, whereas in the smaller junctions they lack gaps. The polyneural innervation and different deployment of membrane particles at neuromuscluar junctions could be a factor responsible for different physiological properties of this muscle.     

Tbc1d15–17 regulates synaptic development at the Drosophila neuromuscular junction

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15–17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15–17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15–17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15–17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15–17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15–17 and its potential substrate Rab7 in regulating synaptic development.