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.     

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.     

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.     

THE ULTRASTRUCTURE OF A REPTILIAN MYONEURAL JUNCTION

Myoneural junctions in Anolis are characterized by the formation of troughs in the surface of the muscle fibers in which small branches of the terminal axon lie. The muscle surface membrane lining the troughs is thrown into complex branching and anastomosing folds, which compose the subneural apparatus of Couteaux. A compound membrane 500 to 700 A thick separates axoplasm from sarcoplasm at the endings. This consists of five distinct layers and is described in detail. A thin layer of cytoplasm (probably Schwann) separates terminal axoplasm from extracellular space at the surfaces of the junctional troughs. Terminal axoplasm lacks axoplasmic filaments and contains numerous vesicular or tubular appearing structures about 300 to 500 A in diameter. Both terminal axoplasm and sarcoplasm contain numerous mitochondria.     

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.     

Ultrastructural features of neuromuscular junctions in the stapedius muscle of Gallus gallus

The ultrastructure of neuromuscular junctions in the twitch fibers of the stapedius muscle of Gallus gallus (domesticus) was investigated as part of a series of neurophysiological studies. Among the morphological features observed were elongated end-plates with numerous large and clear synaptic vesicles mixed with larger dense core vesicles and irregular or aperiodic “active sites” in the presynaptic membrane where synaptic vesicles were focused. The most remarkable features of these junctions were large synaptic clefts (50-80 nm) and the absence of junctional folds in the sarcolemmal surface. Unlike the large periodic junctional folds seen in the neuromuscular junctions of frogs and in the fast twitch fibers of the mammalian stapedius, the preparations studied only show small aperiodic invaginations (primitive folds) in the postsynaptic membranes. This morphological feature remains essentially constant from newly hatched to adult chickens. While these smooth junctions are consistent with earlier findings of inconspicuous junctional folds in the twitch fibers of the chicken posterior latissimus dorsi they are unlike those seen in the fast twitch fibers of the mammalian stapedius muscle, or other twitch fibers in general. The morphological findings of the present study may also suggest that the simple, unmodified neuromuscular junctions in the stapedius of Gallus may be a useful preparation for studies of synaptic membrane structures that employ the freeze-fracture technique.     

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.     

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.     

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat

Summary The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen -particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

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     

Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.     

Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.     

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.     

MEMBRANE SPECIALIZATION AT AN INSECT MYONEURAL JUNCTION

Myoneural junctions were examined in the asynchronous basalar flight muscle of the beetle Pachnoda ephippiata. The outer surface of the postjunctional membrane exhibits an array of prominent projections spaced at ~200 Å intervals which arise directly from the outer dense lamina of the plasma membrane and extend part way across the junctional cleft. The projections follow irregularities in the contour of the postjunctional membrane precisely and they end abruptly near the edge of the junctional region. No separation can be resolved between the projections and the underlying trilaminar plasma membrane after a variety of preparative methods, and the projections therefore appear to be a component part of the membrane. This specialization, which is distinctly different from that at desmosomes and hemidesmosomes, occurs nowhere else on the surface of the muscle and is interpreted as a mosaic of specialized membrane subunits which probably include the receptor sites for the transmitter.     

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.     

Regulation of turnover and number of acetylcholine receptors at neuromuscular junctions

The number and metabolic stability of acetylcholine receptors (AChRs) at neuromuscular junctions of rat tibialis anterior (TA) and soleus (SOL) muscles were examined after denervation, paralysis by continuous application of tetrodotoxin to the nerve, or denervation and direct stimulation of the muscle through implanted electrodes. After 18 days of denervation AChR half-life declined from about 10 days to 2.3 days (TA) or 3.6 days (SOL) and after 18 days of nerve conduction block to 3.1 days (TA). In contrast, the total number of AChRs per endplate was unaffected by these treatments. Denervation for 33 days had no further effect on AChR half-life but reduced the total number of AChRs to about 54% (SOL) or 38% (TA) of normal. Direct stimulation of the 33-day denervated SOL from day 18 restored normal AChR stability and counteracted muscle atrophy but had no effect on the decline in AChR number. The results indicate that motoneurons control the stability of junctional AChRs through evoked muscle activity and the number of junctional AChRs through trophic factors.     

Synaptic current between neuromuscular junction folds

Measurements of membrane infoldings of vertebrate subsynaptic membranes were taken to evaluate the possible electrophysiological implications. The shapes of standard interfolds of different neuromuscular junctions were established from micrographs available in the literature. Electrical properties were estimated using published fibre membrane and myoplasm electrical values. Models of synaptic current pathways were designed taking into account the small size of the postsynaptic patch activated by a transmitter quantum. This analysis reveals a resistance "in series" between the ACh-sensitive interfold crest and the remainder of the muscle fibre. The calculated cytoplasmic resistance of an interfold is between 0.2 and 3 Mohms which is in the same range as the fibre DC input resistance. The calculated interfold resistance appears to be dependent on the fibre type, the age and the pathology. Functional roles of junctional folds and dendritic spines are discussed.     

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.     

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.