Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.     

The Fine Structure of Degenerating Nerve Fibers, their Sheaths, and their Terminations in the Central Nerve Cord of the Cockroach (Periplaneta americana)

The connectives above and below the second thoracic ganglion and nerves to and from the mesothoracic leg were severed in Periplaneta americana. Isolated ganglia and severed nerve cord were examined in the electron microscope. In the connectives, sheaths of degenerating fibers remain continuous but become thicker and more dense. There is increase in number and more haphazard disposition of the neuroglial processes which ensheath the axons. The cytoplasm contains vacuoles. Dense droplets normally intercalated between the layers of neuroglial processes ensheathing the axons are strikingly increased in number. The axoplasm with its organelles forms dense clumps. Mitochondria in axons are enlarged, the intramitochondrial matrix is more dense, and the internal folds are disorganized. In ganglia, mitochondrial changes in terminal parts of the axons appear similar to those described in the parent axons in the connective. The synaptic portions of nerve fibers appear very dense. Alterations of the sheath are minimal. Synaptic particles in the degenerating axoplasmic coagulum undergo only slight morphological changes and are still present up to 6 days after severance of their nerve fibers. It is difficult to assess whether there are any alterations in the total number of synaptic particles during degeneration.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

Pervasive synaptic branch removal in the mammalian neuromuscular system at birth

Using light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate ～10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS. VIDEO ABSTRACT:     

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.     

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.     

Autonomic nerves terminating on smooth muscle cells of vessels in the pineal organ of various mammals

The significance of autonomic nerves reaching the pincal organ was already investigated in connection to the innervation of pinealocytes and mediating light information from the retina for periodic melatonin secretion. In earlier works we found that some autonomic nerve fibers are not secretomotor but terminate on arteriolar smooth muscle cells in the pineal organ of the mink (Mustela vison). Studying in serial sections the pineal organ of the mink and 15 other mammalian species in the present work, we investigated whether similar axons of vasomotor-type are generally present in the wall of pineal vessels, further, whether they reach the organ via the conarian nerves or via periarterial plexuses. In all species investigated, axons of perivasal nerve bundles were found to form terminal enlargements on the smooth muscle layer of pineal arterioles. The neuromuscular endings contain several synaptic and some granular vesicles. Axon terminals are also present around pineal veins. In serial sections, we found that the so-called conarian autonomic nerves reach the pineal organ alongside pineal veins draining into the great internal cerebral vein. Similar nerves present near arteries of the arachnoid enter the pineal meningeal capsule and septa by arterioles, both perivenous and periarterial nerves form terminals of vasomotor-type. The arteriomotor and venomotor regulation of the tone of the vessels of the pineal organ may serve the vascular support for circadian and circannual periodic changes in metabolic activity of the pineal tissue.     

Induced neuroma formation and target muscle perturbation in the giant fiber pathway of the Drosophila temperature-sensitive mutant shibire

Summary The temperature-sensitive mutation shibire (shi) in Drosophila melanogaster is thought to disrupt membrane recycling processes, including endocytotic vesicle pinch-off. This mutation can perturb the development of nerves and muscles of the adult escape response. After exposure to a heat pulse (6 h at 30° C) at 20 h of pupal development, adults have abnormal flight muscles. Wing depressor muscles (DLM) are reduced in number from the normal six to one or two fibers, and are composed of enlarged fibers that appear to represent fiber fusion; large spaces devoid of muscle fibers suggested fiber deletion. The normal five motor axons are present in the peripheral nerve PDMN near the ganglion. However, while some motor axons pass dorsally to the extant fibers, other motor axons lacking end targets pass into an abnormal posterior branch and terminate in a neuroma, i.e., a tangle of axons and glia without muscle target tissue. Hemisynapses are common in axons of the proximal PDMN and within the neuroma, but they are rarely seen in control (no heat pulse) shi or wild-type flies. All surviving muscle fibers are innervated; no muscle tissue exists without innervation. Fibrillar fine structure and neuromuscular synapses appear normal. Fused fibers have dual innervation, suggesting correct and specific matching of target tissue and motor axons. Motor axons lacking target fibers do not innervate erroneous targets but instead terminate in the neuroma. These results suggest developmental constraints and rules, which may contribute to the orderly, stereotyped development in the normal flight system. The nature of the anomalies inducible in the flight motor system in shi flies implies that membrane recycling events at about 20 h of pupal development are critical to the formation of the normal adult nerve-muscle pattern for DLM flight muscles.     

Innervation of the carotid body. An experimental quantitative study

The innervation of the carotid body in the cat was studied by means of light- and electron-microscopic techniques. Sinus nerve resection, glossopharyngeal resection, bilateral cervical sympathectomy, excisions of two nerves, and injection of 6-hydroxydopamine (6-OH-DA) were performed in different groups of animals. It was found that resection of the sinus nerve produces a rapid phase of degeneration of intralobular fibers and synaptic boutons, followed by a reinnervation with a progressive reappearance of these elements. This reinnervation is retarded by sympathectomy and prevented by 6-OH-DA. It is therefore concluded that reinnervation is due to collateral regeneration of nearby sympathetic fibers. Resection of the sinus nerve produces an increase in the number of argentaffin cells and dense-cored vesicles in the cytoplasm of principal cells. These findings suggest the existence of efferent synaptic contacts between this nerve and principal cells. Part of the intralobular fibers and synaptic boutons degenerate after bilateral sympathectomy demonstrating that sympathetic axons connect synaptically to the principal cells. Sympathetic fibers reach the carotid body, not only from branches of the cervical plexuses but also from fibers running in the adventitia of the common carotid artery, and via glossopharyngeal and sinus nerves. The vagus nerve contributes a few fibers to the parenchymal lobules of the carotid body.     

Distribution and the fine structure of serotonin-containing fibers in the rat small intestine

Serotonin immunoreactive fibers were observed under the electron microscopy in all layers of the small intestine, with greatest abundance in the mucosa. Submucosal blood vessels were often surrounded by immuno positive nerves. In the inner circular muscle layer the immunoreactive serotonin positive fibers were closely associated with the smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labelled fibers surrounded the immunonegative neural cell bodies, but rarely formed conventional synaptic junctions. It is concluded that the serotoninergic system of the small intestine may influence the activity of associated structures in a diffuse non-synaptic manner.     

Innervation of the limb accessory flexor muscle in several decapod crustaceans. I. Anatomy

The innervation of the accessory flexor muscle of the limbs of several decapod crustaceans was studied by means of vital staining, with methylene blue and electron microscopy. Three patterns of innervation were found. In the first pattern, the distal (DAFM) and proximal (PAFM) heads of the accessory flexor muscle were supplied by two axons (a thick and a thin) which travel in a private nerve along the length of the merus. This pattern was found in the crab (Cancer) and the lobster (Homarus), and conforms to the classical pattern established in the literature. In the second pattern, the nerve to the DAFM is made up of conjoined branches of the flexor and accessory flexor nerves. Consequently, the DAFM receives at least five axons in the portunid crabs, Carcinus, Callinectes, and Ovalipes, and occasionally six axons in Ovalipes. The PAFM in those portunids receives the usual two axons. In the third pattern, based on preliminary observations on the grapsid crab, Pachygrapsus, “super-innervation” of the accessory flexor muscle appears to include not only five axons to the DAFM but also at least three to the PAFM. In all species, methylene blue staining of the axon terminations revealed a regular pattern of blebs which are thought to correspond to synaptic terminals as revealed by electron microscopy.     

Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.     

Synaptic differentiation between two phasic motoneurons to a crayfish fast muscle

Synaptic differentiation among crustacean phasic motoneurons was investigated by characterizing the synaptic output and nerve terminal morphology of the two axons to the adductor exopodite muscle in the crayfish uropod. The muscle is of the fast type with short sarcomeres (2–3 μm) and a low thin to thick filament number (6:1). On single muscle fibers, excitatory postsynaptic potentials generated by the large-diameter axon are significantly larger than those by the small-diameter axon suggesting a presynaptic origin for these differences. Nerve terminals arising from these two axons have typical phasic features, filiform shape and a low (6–8%) mitochondrial density. Synaptic contacts are similar in size between the two axons as is the length and number of active zone dense bars at these synapses. The large-diameter axon, however, exhibits a twofold larger area of nerve terminal than the small-diameter axon resulting in a higher density of synapses per muscle fiber. Hence, differences in synaptic density may in part account for differences in synaptic output between these paired phasic axons. Electronic Publication     

An Attempt to Account for the Diversity of Crustacean Muscles

Crustacean muscles are known to contain muscle fibers of variableproperties and to be innervated by phasic and/or tonic motoneuronswhich may possess synapses of diverse physiological properties.Frequently, phasic motor axons innervate short-sarcomere phasicmuscle fibers and tonic motor axons innervate long-sarcomeretonic muscle fibers, but some muscles receiving a single (tonic)motor axon contain both phasic and tonic muscle fibers. Althoughit is not known whether neural trophic influences are involvedin muscle differentiation, some neural trophic effects havebeen found in crustaceans, and it is reasonable to assume thatsuch influences may be involved in establishing the definitiveproperties of the muscle. Several other postulates must be made:(1) Phasic and tonic motor axons differ in their trophic effectiveness:(2) muscle fibers innervated relatively early in developmentby a tonic motor axon acquire the properties of tonic musclefibers, while those innervated later become intermediate orphasic muscle fibers; (3) the developmental stage of a growingor regenerating axon terminal plays a role in determinationof synaptic properties. Studies on regenerating limb buds supportthe hypothesis, which can account for the genesis of all observedtypes of crustacean neuromuscular system. Further experimentalwork is necessary to test the hypothesis.     

Nerve ultrastructure of the arteries of the brain stem]

Ultrastructure of the nerve apparatus in the arteries of the brain base has been studied in cats. The structure of peri- and adventitial nerves has been investigated electron microscopically. Three types of efferent axons and four types of synaptic vesicles (small agranular and granular, large granular, large electron opaque vesicles) have been revealed. Vesicle-containing axons in the brain arteries approach the external smooth muscle cells of about 80 nm. Terminal axonal dilatations possessing direct and mediated connections with muscular cells of the middle tunica have been revealed.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Regeneration of the projection and synaptic connections of tympanic receptor fibers of Locusta migratoria (Orthoptera) after axotomy

The tergite nerve N6 of the first abdominal segment of the locust Locusta migratoria contains receptor fibers, from the tympanic organ, and hair sensilla as well as motoric axons. The nerve was axotomized in nymphal instars or adults, and the regeneration of nerve fibers was studied. The sensory fibers regrow and regenerate their projection pattern within the central nervous system. They recognize their specific neuropile areas even after entering the ganglion through different pathways. The receptor fibers of the tympanic organ reestablish synaptic connections to auditory interneurons, even though the physiological characteristics of the interneurons are not fully restored. This regenerative capability contrasts with the lack of regeneration of peripheral structures in locusts, but supports the described plasticity in the auditory system of monaural locusts (Lakes, Kalmring, and Engelhard, 1990). The motor fibers do not regenerate nerves innervating muscles of the body wall.     

Localization of catecholamines and acetylcholinesterase in the terminal nerve fibres of the rabbit myometrium

Summary The autonomic nerves of the myometrium of the rabbit were studied in order to demonstrate simultaneously the adrenergic nature of an axon and the localization of acetylcholinesterase (AChE) in the same axons. The synaptic vesicles of the adrenergic axons and nerve terminals remained partially filled with the electron dense material typical for them after formaldehyde fixation and short incubation time for AChE. AChE stain was localized regularly on the axons which contained agranular synaptic vesicles and also on axons which contained dense cored synaptic vesicles beeing probably adrenergic. The role of AChE on the adrenergic axons is discussed.     

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.