The Fine Structure of a Multiterminal Innervation of an Insect Muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

The fine structure of a multiterminal innervation of an insect muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

Electron Microscopy of Peripheral Nerves and Neuromuscular Junctions in the Wasp Leg

The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast. The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs. The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles. The physiologic roles of the specialized structures are considered.     

The fine structure of a rectifying electrotonic synapse

The synapses between the lateral giant axon and the giant motor axon found in the abdominal ganglia of the ventral nerve cord of the crayfish Procambarus clarkii are electronic. The junctional membrane rectifies, favoring impulse transmission from lateral giant fiber to giant motor fiber. This rectifying electronic junction consists of closely apposed membranes indistinguishable from ordinary arthropod gap junctions. The apposed membranes contain intramembrane particles that are approximately 12.5 nm in width. These particles have a central depression and are arranged in a loosely ordered array with a center-to- center spacing of about 20 nm. The only obvious morphological evidence of asymmetry is the presence of vesicles (about 80 nm in diameter) in the cytoplasm adjacent to the junctional region of the presynaptic lateral giant fiber. Vesicles are not present in the adjacent cytoplasm of the postsynaptic giant motor fiber; however, mitochondria and smooth tubular endoplasmic reticulum are more frequent in the cytoplasm of the giant motor fiber.     

Morphological differences between excitatory and inhibitory nerve terminals in cockroach coxal muscles

Two morphologically distinct types of neuromuscular junction on the coxal leg muscles of the cockroach, Periplaneta americana, which have been physiologically described as innervated by fast, slow and inhibitory nerve fibers, have been found. In one type of neuromuscular junction the axon terminal contains many round clear synaptic vesicles and contacts several sarcoplasmic extensions from the muscle fiber. The muscle processes adhere to the axon terminal for a short distance (short contact or SC type). The axon terminal of the other type of neuromuscular junction directly contacts the muscle fiber and no extensions of the muscle fiber are formed. The contact region is comparatively long (long contact or LC type). The nerve terminal contains many polymorphic synaptic vesicles. From a correlation of the present morphological findings and the previous physiological results, it may be suggested that the SC type of nerve terminal represents both fast and slow nerve terminals and the inhibitory terminal is of the LC type.     

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.     

The fine structure of nerve endings on rat thyroid follicular cells

Summary Axon profiles in thyroid glands obtained from adult male Wistar rats were studied electron-microscopically, using common and serial thin sections.Bouton profiles of nerve fibers, resembling the terminal or en passant type, often appeared closely associated with vascular smooth muscle cells via basement membranes. These structures are probably adrenergic, since they contained mainly small-core vesicles (mean diameter: 41.2 nm), in addition to a few large-core (mean diameter: 88.4 nm) and flattened vesicles.Nerve fibers containing microtubules and sometimes mitochondria and vesicles were seen lying between basement membranes and follicular cells. The incidence of nerve fiber contacts on profiles of follicular cells was 0.0177±0.0092 (S.D.). Using serial sections, follicles were seen to have up to two nerve endings, separated from the plasma membranes of the follicular cells by a gap of 22 nm. They contained mainly flattened vesicles and several large-core vesicles (mean diameter: 95.1 nm). Small-core vesicles were rarely seen in these nerve endings. Furthermore, subsurface cistern-like rough endoplasmic reticulum was found immediately under the plasma membranes of follicular cells facing membranes of nerve endings. These results suggest that the nerve fibers in contact with follicular cells are different from the adrenergic type.     

THE ORGANIZATION OF FLIGHT MUSCLE IN AN APHID, MEGOURA VICIAE (HOMOPTERA) : With a Discussion on the Structure of Synchronous and Asynchronous Striated Muscle Fibers

The organization of the indirect flight muscle of an aphid (Hemiptera-Homoptera) is described. The fibers of this muscle contain an extensive though irregularly disposed complement of T system tubules, derived as open invaginations from the cell surface and from the plasma membrane sheaths accompanying the tracheoles within the fiber. The sarcoplasmic reticulum is reduced to small vesicles applied to the T system surfaces, the intermembrane gap being traversed by blocks of electron-opaque material resembling that of septate desmosomes. The form and distribution of the T system and sarcoplasmic reticulum membranes in flight muscles of representatives of the major insect orders is described, and the extreme reduction of the reticulum cisternae in all asynchronous fibers (to which group the aphid flight muscle probably belongs), and the high degree of their development in synchronous fibers is documented and discussed in terms of the contraction physiology of these muscle cells.     

Motor nerve terminal loss from degenerating muscle fibers

The fate of nerve terminals following elimination of postsynaptic target cells was studied in living mouse muscle. Several days after muscle fiber damage, observations of previously identified neuromuscular junctions showed that motor nerve terminal branches had rapidly disappeared from degenerating muscle fibers. Following muscle fiber regeneration, loss of terminal branches ceased and nerve terminals regrew, reestablishing some of the original sites and adding new branches. The distribution of acetylcholine receptors reorganized in the regenerated muscle so that perfect alignment was reestablished with the newly configured nerve terminals. These results argue that the maintenance of the full complement of nerve terminal branches at a neuromuscular junction is dependent on the presence of a healthy muscle fiber. Similarly, regenerating muscle is dependent on the nerve terminal for the organization and maintenance of postsynaptic receptors.     

ARCHITECTURE AND NERVE SUPPLY OF MAMMALIAN SMOOTH MUSCLE TISSUE

Smooth muscle tissue from mouse urinary bladder, uterus, and gall bladder has been studied by means of the electron microscope. The smooth muscle cells are distinctly and completely separated from each other by a cytolemma comparable to the sarcolemma of striated muscle. The tissue is thus cellular and not syncytial. With this evidence, supported by electron microscopy of other tissues, we question the existence of true syncytia in animal tissues. Individual cell membranes necessary for the electrophysiologic events exist in smooth muscle, and its nerve and conduction in a tissue such as uterus or bladder can occur at the cellular level as well as at the tissue area level. The smooth muscle cell contains myofilaments, nucleus, endoplasmic reticulum, mitochondria, Golgi complex, centrosome, and pinocytotic vesicles. These structures are described in some detail, and their probable interrelations and functions are discussed. The autonomic nerves innervating smooth muscle cells are composed of axons and lemnoblasts. The axon is suspended by the mesaxon formed by the infolded plasma membrane of the lemnoblast. The respective plasma membranes separate axon and lemnoblast from each other and from surrounding muscle cells. The axons of autonomic nerves never penetrate the plasma membrane of the muscle cell, but pass or intrude into muscle cell pockets, forming a contact between axonal plasma membrane and smooth muscle plasma membrane. The lemnoblast shows well developed endoplasmic reticulum with Palade granules, mitochondria, and a long, elliptical nucleus. The axon contains neurofilaments, mitochondria, and synaptic vesicles; the quantity of the latter two being significantly greater in the periphery of lemnoblasts and near axon-muscle contact regions. We regard the contact regions as the synapses between the autonomic nerves and the smooth muscle cells.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : II. Cytological and Cytochemical Studies on the Neuromuscular Junction of Dedifferentiating Muscle in the Regenerating Limb of the Newt Triturus

Following amputation of the limb of the newt, Triturus viridescens, muscle fibers dedifferentiate giving rise to mesenchymal cells. The earliest changes detected in neuromuscular junctions of dedifferentiating muscle fibers are the appearance of a few vacuoles and decrease in density of the terminal axoplasm. Later, synaptic vesicles become tightly clustered in the axon termination, and their content appears denser than normal. Then, vesicles diminish in number until few are seen in the ending. While these changes are occurring, the area of contact of nerve with muscle becomes smaller. Junctional folds persist only where the nerve maintains contact with muscle, but these are shorter than normal and appear as slight ridges on the muscle surface. Subsequently, the nerve withdraws from the muscle cell and is completely invested by Schwann cell cytoplasm, and all traces of junctional folds are lost at the former region of contact. Cholinesterase activity was localized with the thiolacetic acid-lead nitrate method. Even before marked morphological changes occur in the junction, DFP- and physostigmine-sensitive activity in the cleft between nerve and muscle is decreased in intensity. Activity continues to decrease as the area of nerve-muscle contact diminishes and junctional folds disappear. When the nerve has withdrawn from the muscle surface, only a few small deposits of lead are left in the intervening region. These results show that as muscle becomes less specialized during dedifferentiation, the neuromuscular junction also loses the cytological and cytochemical specializations associated with synaptic function.     

Structural definition of the neuromuscular system in the swimming-paddle opener muscle of blue crabs

Blue crabs are excellent swimmers, using their highly modified last pereiopods as sculling paddles. Hence, the hypertrophied paddle opener muscle was examined for adaptations of its motor innervation by an excitor and a specific inhibitor axon. The muscle has a uniform composition of slow fibers with long (6-12 microm) sarcomere lengths. Individual fibers are richly innervated with approximately two-thirds excitatory and one-third inhibitory innervation. The profuse excitatory innervation reflects the high activity levels of this motoneuron in swimming. Adaptation to sustained activity associated with swimming is also reflected in the motor nerve terminals by a high concentration of energy source, which is equally divided between glycogen granules and mitochondria, the former providing a more rapid source of energy. The excitor axon makes predominantly neuromuscular synapses, but also a few synapses onto the inhibitor axon. The location of these excitatory axoaxonal synapses suggests regional modulation of the inhibitor axon. The specific inhibitor axon makes less than two-thirds of its synapses with the muscle fiber, regulating contraction via postsynaptic inhibition. The remaining inhibitory synapses are onto the excitor axon, signaling very strong presynaptic inhibition. Such presynaptic inhibition will effectively decouple the opener muscle from the stretcher muscle even though both are innervated by a single excitor axon.     

Rattlesnake shaker muscle: II. Fine structure

The shaker muscle of the rattlesnake is able to sustain a rate of contraction around 50/sec for at least 3 hr. Most of the muscle is composed of a single, major fiber type which contains large numbers of small myofibrils, abundant sarcomplasmic reticulum, numerous mitochondria, and large deposits of glycogen. The neuromuscular junction of the major fiber type is extensive with several nerve terminal expansions and junctional folds that are both numerous and deep. The structure of the major fiber type suggests that it must be responsible both for the speed of contraction as well as for the indefatigability of the shaker muscle. The ultrastructure of the much less numerous minor fibers in the shaker muscle suggest that their role may be a tonic one.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : I. Cytological and Cytochemical Studies on the Neuromuscular Junction of Differentiating Muscle in the Regenerating Limb of the Newt Triturus

Development of the neuromuscular junction on differentiating muscle was investigated in the regenerating limb of the newt Triturus. Motor end-plate formation begins when vesicle-filled axon terminations approach differentiating muscle cells that have reached the stage of a multinucleate cell containing myofibrils. Slight ridges or elevations occur on the muscle surface, and there is an increase in density of the cytoplasm immediately beneath the plasma membrane of the elevation. The axon becomes more closely approximated to the muscle cell and comes to lie in a shallow depression or gutter on the surface of the muscle. The surface ridges increase in length and constrict at their bases to form junctional folds. In the axon terminal, focal accumulations of vesicles are found where the axon contour projects slightly opposite the secondary synaptic clefts. Cholinesterase activity in the developing junctions was demonstrated by the thiolacetic acid-lead nitrate method. Enzymatic activity is not found on intercellular nerve fibers or the muscle surface prior to close approximation of axon endings and muscle. Eserine- and DFP-sensitive activity appears concurrently with morphological differentiation. Activity occurs in membranous tubulovesicles in the sarcoplasm subjacent to the neuromuscular junction and in association with the sarcolemma. The largest reaction deposits occur at the tips of the emerging junctional folds. Smaller and less numerous localizations occur on the axon membrane and within the axoplasm. It is concluded from these studies that the nerve endings have an inductive effect on both the morphological and chemical specializations of the neuromuscular junction.     

Ultra rapid calcium events in electrically stimulated frog nerve terminals.

Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca(2+) within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed.     

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.     

THE ULTRASTRUCTURE OF THE NEUROMUSCULAR JUNCTIONS OF MAMMALIAN RED, WHITE, AND INTERMEDIATE SKELETAL MUSCLE FIBERS

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.     

Allotransplanted nerves regenerate inhibitory synapses on a crayfish muscle: Possible postsynaptic specification

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.     

Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures

Whole-mount stereo electron microscopy has been used to examine the cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor (AChR) clusters in cultures of Xenopus nerve and muscle cells. The cells were grown on Formvar-coated gold electron microscope (EM) finder grids. AChR clusters were identified in live cultures by fluorescence microscopy after labeling with tetramethylrhodamine-conjugated alpha-bungarotoxin. After chemical fixation and critical-point drying, the cytoplasmic specializations of identified cells were examined in whole mount under an electron microscope. In the presynaptic nerve terminal opposite to the AChR cluster, synaptic vesicles were clearly suspended in a lattice of 5-12- nm filaments. Stereo microscopy showed that these filaments directly contacted the vesicles. This lattice was also contiguous with the filament bundle that formed the core of the axon. At the AChR cluster, an increased cytoplasmic density differentiated this area from the rest of the cytoplasm. This density was composed of a meshwork of filaments with a mean diameter of 6 nm and irregularly shaped membrane cisternae 0.1-0.5 micron in width, which resembled the smooth endoplasmic reticulum. These membrane structures were interconnected via the filaments. Organelles that were characteristic of the bulk of the sarcoplasm such as the rough endoplasmic reticulum and the polysomes, were absent from the cytoplasm associated with the AChR cluster. These results indicate that the cytoskeleton may play an important role in the development and/or the maintenance of the neuromuscular synapse, including the release of transmitter in the nerve terminal and the clustering of AChRs in the postsynaptic membrane.