Cholinergic, adrenergic, and purinergic neuromuscular transmission.

A general model of the autonomic neuromuscular junction is proposed which emphasizes muscle effector bundles with gap junctions (or 'nexuses') forming the low resistance pathways allowing electrotonic coupling between neighboring cells, and extensive terminal varicose nerve fibers with 'en passage' release of transmitter. Some variations in autonomic neuromuscular geometry are discussed. Junctional clefts vary from 15nm in densely-innervated tissues such as vas deferens and iris to 2,000 nm in some large elastic arteries. Postjunctional specializations take the form of subsynaptic cysternae (in vas deferens and iris) and aggregations of plasmalemmal vesicles (in circular intestinal muscle). Current views of the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized.     

The finestructure of nerve branches and neuromuscular junctions in the coxal adductor of the cockroach, Gromphadorhina portentosa

The neuromuscular junctions of a fast coxal adductor of Gromphadorhina portentosa show great variability in both axon terminal diameter and extent of post-junctional sarcoplasmic specializaton. Finestructural equivalents of both cone and brush type nerve endings are present. The large motor axons innervating this muscle are surrounded by a pervasive lemnoblast sheath, leaving the axon surface exposed only in the area of synaptic contact. Connective tissue covers the nerve and fills the spaces between sheath cell processes in the nerve trunk, but is lost after it enters the muscle. The role of sheath cells in nerve function is discussed in the light of these findings.     

Properties of sympathetic neuromuscular transmission and smooth muscle cell membranes in vascular beds.

In vascular smooth muscle tissues, the cycle of contraction-relaxation is mainly regulated by the cytosolic Ca, and many other factors, such as substances released from endothelial cells and perivascular nerve terminals (mainly sympathetic nerves). In this article, we introduce regional differences in specific features of ionic channels in vascular smooth muscle membranes (mainly on features of Ca, Na and K channels) in relation to mobilization of the cytosolic Ca. In many vascular tissues, neurotransmitters released from sympathetic nerve terminals activate post-junctional receptors, and subsequently modify ion channels (receptor-activated cation channel and voltage-dependent Ca channel), whereas in some tissues, ionic channels are not modified by receptor activations (pharmaco-mechanical coupling). However, activation of receptors, with or without modulation of ionic channels, regulates the cytosolic Ca through synthesis of second messengers. In addition, receptors distributed on prejunctional nerve terminals positively or negatively regulate the release of transmitters. Roles of neurotransmitters (mainly ATP and noradrenaline) are also discussed in relation to the generation of excitatory junction potentials.     

The epitheliomuscular cell of hydra: Its fine structure, three-dimensional architecture and relation to morphogenesis

Ectodermal epitheliomuscular cells of Hydra attenuata were studied by transmission and scanning electron microscopy, and a three-dimensional model was constructed. These cells are cuboidal to columnar, and each cell has one muscle process arising from the basal portion of the oral-facing surface and one from the aboralsurface. Adjacent epitheliomuscular cells are joined apicolaterally by septate junctions. Numerous gap junctions occur between adjacent epitheliomuscular cells and are irregularly distributed along the lateral and basal aspects. Finger-like interdigitations and specialized folds (couplers) also occur along the basal and lateral aspects and interlock adjacent epitheliomuscular cells. In the basal portion of these cells, myofilaments are aggregated into myonemes which are oriented in the oral-aboral axis of the polyp. Myonemes dominate the cytoplasm of muscle processes. Myofilaments are also aggregated in the basal cytoplasm of the cell body when the cell body is in contact with the mesoglea but are sparse or absent when the cell body rests upon other muscle processes. Epitheliomuscular cells and associated muscle processes rest upon other processes and the mesoglea and show variations in these relations. A muscle process and associated cell may rest upon another process; the process may then extend under the preceding process and cell body. This configuration, and variations, present a woven or braided network of muscle processes which collectively form a sheet of muscle on the mesoglea. The interdigitations, couplers and gap junctions between epitheliomuscular cells and the woven network of muscle processes present a cytological basis for the observations that the ectoderm in hydra behaves as a coherent sheet along the body column.     

Insect Neuromuscular Synapses

Miniature end-plate potentials were used in studying severalaspects of the neuromuscular systems in the cockroach femur.The similar sizes and time courses of miniatures associatedwith fast and slow type excitatory axons suggest that they employthe same transmitter. There is other evidence also indicatingthat the essential difference between these two excitatory systemsis in the number of packets of transmitter released per nerveimpulse rather than different transmitter substances. From theshapes of miniatures it was suspected that typical muscle fibersmight have a branching structure. This was confirmed by histologicalexamination, intracellular stimulation, and intracellular dyeinjection. That inhibitory transmission is quantal is indicatedby hyperpolarizing miniatures which occur at random time intervals.Inhibitory transmission can be made to fail and recover in astepwise manner by manipulating the Ca/Mg ratio. In studiesof toxins which affect transmitter release at vertebrate motorend-plates, botulinal toxin was found to be without effect ateither excitatory or inhibitory junctions in cockroach muscle.However, black widow spider venom acted as it does in vertebrates,promoting massive release of transmitters and then permanentinactivation of the junctions.     

Talin at myotendinous junctions

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.     

Muscular force transmission: a unified, dual or multiple system? A review and some explorative experimental results

Structures contributing to force transmission in muscle are reviewed combining some historical and relatively recently published experimental data. Also, effects of aponeurotomy and tenotomy are reviewed shortly as well as some new experimental results regarding these interventions that reinforce the concept of myofascial force transmission. The review is also illustrated by some new images of single muscle fibres from Xenopus Laevis indicative of such transmission and some data about locations of insertion of human gluteus maximus muscle. From this review and the new material, emerges a line of thought indicating that mechanical connections between muscle fibres and intramuscular connective tissue play an important role in force transmission. New experimental observations are presented for non-spanning muscle (i.c., rat biceps femoris muscle), regarding the great variety of types of intramuscular connections that exist i n addition to myo-tendinous junctions at the perimuscular ends of muscle fibres. Such connections are classified as (1) tapered end connections, (2) Myo-myonal junctions, (3) myo-epimysial junctions and (3) Myo-endomysial junctions. This line of thought is followed up by consideration of a possible role of connections of intra- and extramuscular connective tissue in force transmission out of the muscle. Experimental results of an explorative nature, regarding the interactions of extensor digitorum longus (EDL), tibialis anterior (TA) and hallucis longus (HAL) muscles within a relatively intact dorsal flexor compartment of the rat hind leg, indicate that: (1) length force properties of EDL are influenced by TA activity in a length dependent fashion. Depending on TA length, force exerted by EDL, kept at constant origin insertion distance, is variable and the effect is influenced by EDL length itself as well; (2) Force is transmitted from muscle to extramuscular connective tissue and vice versa. As a consequence force exerted at proximal and distal tendons of a muscle are not always equal. The difference being transmitted by extramuscular connective tissue and may appear at the tendons of other muscles or may be transmitted via connective tissue directly to bone. It is concluded that the system of force transmission from skeletal muscle should be considered as a multiple system.     

Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca(2+) transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers.     

A STUDY OF THE INNERVATION OF THE TAENIA COLI

An electrophysiological and anatomical study of the guinea pig taenia coli is reported. Changing the membrane potential of single cells cannot modulate the rate of firing action potentials but does reveal electrical coupling between the cells during propagation. The amplitude of the junction potentials which occur during transmission from inhibitory nerves is unaffected in many cells during alteration of the membrane potential, indicating electrical coupling during transmission. The taenia coli is shown to consist of smooth muscle bundles which anastomose. There are tight junctions between the cells in the bundles, and these probably provide the pathway for the electrical coupling. The smooth muscle cells towards the serosal surface of the taenia coli are shown electrophysiologically to have an extensive intramural inhibitory innervation, but a sparse sympathetic inhibitory and cholinergic excitatory innervation. These results are in accordance with the distribution of these nerves as determined histochemically. As single axons are only rarely observed in the taenia coli, it is suggested that the only muscle cells which undergo permeability changes during transmission are those adjacent to varicosities in the nerve bundles. The remaining muscle cells then undergo potential changes during transmission because of electrical coupling through the tight junctions.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E2 (PGE2) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE2 (10(-10)-10(-9)M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10(-5) M) and FPL55712 (10(-6) M). In doses over 10(-8)M, PGE2 reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10(-5) or 5 x 10(-5) M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE2 (10(-9) M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 x 10(-7) M). However, indomethacin (10(-5) M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE2 in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.     

The relationship between nerves and smooth muscle cells in the rat iris

Summary The dilatator muscle cells form short projections into the stroma of the iris. Close to these projections run several nerve bundles. The unmyelinated axons often show enlargements (varicosities) containing mitochondria and vesicles. Several of the varicosities are partly denuded of the Schwann cell and are covered only by a basement membrane. The varicosities are then separated from the muscle cells only by basement membranes and a 0.1–1 stromal space. The ultrastructure of the iris dilatator muscle thus also fits the view that the autonomic ground plexus with its varicosities forms the real innervation apparatus.The smallest space between axon and muscle has a width of 700–900 Å and is cemented with basement membrane material. It is suggested that the main function of these contact sites is not to transmit a nerve impulse but to anchor the nerves to their effector organ.This study has been supported by grants from the Swedish State Research Council (U 267) and the United States Public Health Service (N B 2854-04).     

Neurobiology of Polyorchis. II. Structure of effector systems

The gross and fine morphology of the major effector systems in the anthomedusan, Polyorchis penicillatus, is described and discussed in relation to the known physiological and behavioral properties of these systems. Swimming is controlled by an anastomosing network of giant neurons within the inner nerve ring and radial nerves. Although these neurons may be coupled by gap junctions it is likely that they form a syncytium. The photosensitivity of the “giants” is attributed to reflexive membranes within the cytoplasm. Giant neurons act as both the pre- and postsynaptic cell when forming synapses with other neurons of the inner nerve ring. Neuromuscular synapses between “giants” and the striated swimming muscle are found around the margin and along the radii. Swimming muscle cells are connected laterally by gap junctions and end-to-end by desmosomes which are sometimes elaborated with extra-thick filaments. Unstriated sphincter and radial muscles, the major muscles associated with crumpling, are both greatly folded over mesogloeal ridges and have processes that cross the mesogloea to contact the ring and radial canals, respectively. Synapses or other sites that might be responsible for exciting these muscles during crumpling have not been found. The ability of the endodermal lamella and canals to propagate action potentials can be accounted for by the numerous gap junctions that are seen in these tissues. The precise location where excitation is transferred to the nervous system to initiate crumpling is not known but epithelial bridges crossing the mesogloea are likely routes. Synapses between neurons originating in the outer nerve ring and tentacle longitudinal muscle can account for the control of tentacle length. Neurons of the outer nerve ring also synapse onto velar, radial fibers and the sphincter muscle. The inner and outer nerve rings have nervous connections. The organisation of the outer nerve ring and the arrangement of nerves within the endodermal plexus is described. A diagram showing the major connections and interactions of components of the effector systems is presented.     

John Newport Langley and His Construction of the Autonomic (Vegetative) Nervous System

The paper describes the scientific career of Professor John Newport Langley, an outstanding English physiologist and histologist, member of the London Royal Society and its Vice-President. The main scientific achievements of Langley are dealing with anatomy and physiology of the autonomic (vegetative) nervous system that he considered as an entirely efferent system. The autonomic nervous system was divided by Langley into three parts: sympathetic, parasympathetic, and enteral (intestinal) systems. He has also established functional differences of the autonomic nervous system from the somatic one. He suggested the presence of synaptic contacts on muscle and secretory cells, as well as the existence in these synaptic structures of a peculiar receptor substance providing interaction of nerve fibers with postganglionic neurons and effector cells of visceral organs. The examples of development of several Langley's concepts at present are given.     

Studies on the juxtaglomerular apparatus

The juxtaglomerular apparatus of the rat was studied after freeze-fracturing with special respect to intercellular junctions. It was found that juxtaglomerular granulated cells of the vas afferens are interconnected by gap junctions to adjacent cells (granulated cells, possibly also smooth muscle cells). Gap junctions have also been found on the surface of lacis cells and mesangial cells. It is therefore concluded that these cells of the juxtaglomerular apparatus and the glomerulus--granulated cells (possibly also smooth muscle cells) of the vas afferens, lacis cells and mesangium cells--form a functional system reacting in a coordinated manner to physiological stimuli.     

Ultrastructural distribution of AChE in Catenula leptocephala (Nuttycombe, 1956)

Summary Acetylcholinesterase activity (AChE, E.C. 3.1.1.7) was examined in different tissues of Catenula leptocephala (Nuttycombe, 1956). Eserine and iso-OMPA were used to distinguish AChE from non-specific cholinesterases (ChE, E.C. 3.1.1.8). The enzyme was located mainly in the brain neuropil, the peripheral nervous system, neuromuscular junctions, on the membrane of muscle cells and of cells with rhabdites. The distribution of the enzyme suggests that cholinergic transmission occurs in Catenula leptocephala, while simultaneously the presence of AChE on the membranes of muscle cells points to the receipt of cholinergic stimulation. The role of AChE in differentiation and maturation of cells with rhabdites is also discussed in this paper.     

Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duodenum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce perinuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular contacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium showing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-Kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.     

A cytologic analysis of the bag cell control of egg laying in Aplysia

A fine structural analysis of the ovotestis in Aplysia was undertaken in order to analyze the site of action of the bag cell hormone. Five stages of oocyte development are described. Of particular interest is the fact that the yolk seems to be synthesized primarily by the granular endoplasmic reticulum. In addition, small muscle cells whose long, thin processes surround the follicle of the ovotestis have been pointed out. This paper suggests that bag cell extract has a direct action on these small muscle cells causing them to contract and thus expel oocytes from the ovotestis. The evidence for this suggestion is that (1) these muscle cells are the most obvious effector cells in the ovotestis, (2) there are no signs of neural innervation of these muscles, (3) the time course for the liberation of the oocytes is so short that any other method of oocyte release is unlikely, (4) there is no cytologic evidence for any other expulsion process except muscular contraction, and (5) the ripe oocytes are attached to other cells of the wall of the ovotestis only by very small, simple junctions, thus making them the most likely cells to be expelled by muscular contraction.     

Gap junctions synchronize action potentials and Ca2+ transients in Caenorhabditis elegans body wall muscle

The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.     

Inter- and intramyotomal gap junctions in the axolotl embryo.

In early tailbud embryos of the axolotl (Ambystoma mexicanum), cells of the anterior myotomes begin to elongate and align along the longitudinal axis of the animal. Soon thereafter, gap junctions appear between the differentiating myotubes. These junctions occur between adjacent cells within a myotome (intramyotomal) and between the cells of adjacent myotomes which are separated from one another by narrow connective tissue septa (intermyotomal). The latter are found at the ends of the elongating cells where muscle-tendon insertion will occur and nerve-muscle synapses will form. The gap junctions are transient: They appear with the onset of myofibrillar formation at the time that nerve fibers enter the intermyotomal septa. The junctions last until the cells have differentiated into mature striated muscle cells and neuromuscular synapses are fully developed.These gap junctions may provide a means for the direct intercellular spread of electrical excitation between the differentiating muscle cells and so account for the observed myogenic contraction of myotomes. We also suggest that these junctions may form a means for cellular communication and interaction during the development of the axial musculature.