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.     

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.     

Molluscan visceral muscle fine structure. General structure and sarcolemmal organization in the smooth muscle of the intestinal wall of Buccinum undatum L.

Fine structure of intestinal muscle in the gastropod Buccinum undatum is described. Myofibrillar organization is typical of non-pseudostriated molluscan muscles. The dense body system is poorly developed but there are extensive attachment plaques. The sarcolemma is elaborately modified. Deep infoldings of the membrane give the cells an irregular outline. Such infoldings enclose extracellular matrix and are associated with attachment plaques. Arising from these and from the general sarcolemma are numerous tubular membranous invaginations ending blindly at varying depth in the sarcoplasm. These structures have a helical coat of particles on the cytoplasmic face. Associated with both types of invagination are subsarcolemmal vesicles. The possibility that the tubular invaginations are analogues of vertebrate smooth muscle caveolae or striated muscle T-tubules and that the vesicles are the corresponding sarcoplasmic reticulum is discussed. The occurrence of such structures in molluscan muscle and elsewhere is reviewed.     

An Electron Microscopic Study of the Intestinal Villus : I. The Fasting Animal

The structure of the intestinal villus of the rat was studied in thin sections of tissue fixed in buffered osmium tetroxide and embedded in methacrylate. The simple columnar epithelium investing the villus is surmounted by a striated border consisting of slender projections of the cell surface. These microvilli are arranged in almost crystalline, hexagonal array, and increase the apical surface area of the cell by a factor of 24. The core of each microvillus is filled with fine fibrils which arise from the filamentous substance of the terminal web underlying the striated border. Each microvillus is covered by a tubular extension of the plasma membrane of the epithelial cell. Pinocytotic vesicles originating from the plasma membrane occur at the bases of the intermicrovillous spaces. The nucleus, mitochondria, and the endoplasmic reticulum of the epithelial cell display no unusual features. Small bits of ergastoplasm occur in the apical cytoplasm. A thin basement membrane separates the epithelium from the lamina propria which consists of vessels, nerves, and numerous lymphocytes, eosinophiles, mast cells, plasma cells, smooth muscle fibers, and macrophages suspended in a delicate stroma of fibroblasts and collagen fibers. Intercellular fat droplets often occur in this stroma, even in animals fasted for 40 hours. The blood capillaries are distinguished by their extremely attenuated, fenestrated endothelial cells. The lacteal has a thicker endothelium which, although not fenestrated, appears to have significant interruptions, especially at the margins between neighboring lining cells. Strands of smooth muscle always accompany the lacteal but do not form an integral part of its wall. Unmyelinated nerves, many of which are too small to be distinguished with the light microscope, course through the lamina propria in association with the vessels. The nerve fibers evidently do not cross the basement membrane into the epithelium. Neuromuscular junctions or other terminal apparatus were not found.     

An electron microscopic study of the intestinal villus. I. The fasting animal

The structure of the intestinal villus of the rat was studied in thin sections of tissue fixed in buffered osmium tetroxide and embedded in methacrylate. The simple columnar epithelium investing the villus is surmounted by a striated border consisting of slender projections of the cell surface. These microvilli are arranged in almost crystalline, hexagonal array, and increase the apical surface area of the cell by a factor of 24. The core of each microvillus is filled with fine fibrils which arise from the filamentous substance of the terminal web underlying the striated border. Each microvillus is covered by a tubular extension of the plasma membrane of the epithelial cell. Pinocytotic vesicles originating from the plasma membrane occur at the bases of the intermicrovillous spaces. The nucleus, mitochondria, and the endoplasmic reticulum of the epithelial cell display no unusual features. Small bits of ergastoplasm occur in the apical cytoplasm. A thin basement membrane separates the epithelium from the lamina propria which consists of vessels, nerves, and numerous lymphocytes, eosinophiles, mast cells, plasma cells, smooth muscle fibers, and macrophages suspended in a delicate stroma of fibroblasts and collagen fibers. Intercellular fat droplets often occur in this stroma, even in animals fasted for 40 hours. The blood capillaries are distinguished by their extremely attenuated, fenestrated endothelial cells. The lacteal has a thicker endothelium which, although not fenestrated, appears to have significant interruptions, especially at the margins between neighboring lining cells. Strands of smooth muscle always accompany the lacteal but do not form an integral part of its wall. Unmyelinated nerves, many of which are too small to be distinguished with the light microscope, course through the lamina propria in association with the vessels. The nerve fibers evidently do not cross the basement membrane into the epithelium. Neuromuscular junctions or other terminal apparatus were not found.     

Development of the myoneural junction in the rat

Summary The structure of the myoneural junction in the striated muscle of rat embryos and postnatal rats was studied by electron microscopy in order to assess at ultrastructural level the roles of neuronal and muscular elements and the sequence of events resulting in the formation of a functionally mature synaptic organization.From the observations it is concluded that the axon terminals enveloped by Schwann cells contain vesicles prior to apposition of the prospective synaptic membranes. Subsequently, subsarcolemmal thickening of the postsynaptic membrane takes place after the synaptic gap has been formed by disappearance of the teloglial cell from between the synaptic membranes but before the primary synaptic cleft in the strict sense is formed. Secondary synaptic clefts are formed later, when the primary synaptic cleft is regular in width, by local finger-like invaginations of the postsynaptic membrane, which thereafter expand basally, in a plane transverse to the axis of the axon terminal, to resemble flattened flasks. The junction is formed between multinucleated muscle cells and multiple axons, which at first lie side by side and later, when formation of adult-type secondary synaptic clefts is in progress, become separated by folds of the sarcoplasm and the teloglia. In extraocular muscles of adult rats the sarcoplasmic reticulum is closely associated with the postjunctional sarcoplasm.In the light of earlier observations on the development of contractibility after nerve stimulation, cholinesterase histochemistry and muscle fibre physiology, these observations are interpreted to indicate that functional differentiation of the myoneural synapse results from induction by the motor axon and that the association of the sarcoplasmic reticulum with the postjunctional sarcoplasm in adult extraocular muscles is related to modified fibre physiology.The author wishes to thank Prof. Antti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for placing the electron microscopic facilities at his disposal.     

THE FINE STRUCTURAL LOCALIZATION OF ACETYLCHOLINESTERASE AT THE MYONEURAL JUNCTION

A study of the cytochemical localization of acetylcholiriesterase activity, combining histochemistry with electron microscopy, showed that the final product of the reaction, which was deposited at or near enzyme sites, occurred at four places in the myoneural junction. These included: plasma membrane of the muscle covering the junctional folds, the primary and secondary synaptic clefts, parts of the plasma membrane covering the axon terminal, and vesicular structures in the terminal axoplasm. No reaction occurred in the presence of 10^-4 eserine or DFP, whereas 10^-5 DFP inhibited the reaction at all sites except in the vesicles of the terminal axon. These findings are discussed with reference to the histochemical method used and to the occurrence of esterolytic activity in the vesicles, as well as to some of the current hypotheses concerning the relationship of the site of acetylcholinesterase and synaptic transmission.     

Ultrastructural study on the morphogenesis of the neuromuscular junction in the skeletal muscle of the chick

Summary The morphogenesis of the neuromuscular junction was examined at the ultrastructural level in the skeletal muscle of the lower limb of the chick. The fine structure of the neuromuscular junction of the adult fowl was essentially the same as that in other vertebrates; the junction consists of the axon terminal, the Schwann cell, and the muscle fiber. The first visible sign of neuromuscular junction formation, in embryos of 13 days in ovo, was the membrane thickening of the sarcolemma which develops into the postsynaptic membrane. The axons approaching the muscle fibers were incompletely ensheathed by a Schwann cell and contained vesicles. The subsequent differentiation of the junctional sarcoplasm, the axoplasm, and the Schwann cell cytoplasm takes place from 13 to 18 days in ovo and the junction nearly reaches maturity at around 20 days in ovo. The formation of complicated anastomoses and branching of the junctional infoldings seems to occur after hatching. These ultrastructural observations are in good agreement with histochemical findings (cholinesterase method) in terms of the chronology of the morphogenesis of the junction.This investigation was supported in part by U. S. Public Health Service Grant MH 12269-01, administered by Dr. Kazuo Ogawa. It was initiated on the suggestion of Prof. J. Nakai, Department of Anatomy, Faculty of Medicine, Tokyo University, and a part of it was performed in his laboratory. The author is greatly indebted to Prof. K. Ogawa, Department of Anatomy, Kansai Medical School, for his guidance and encouragement, and to Dr. S. Igarashi, Department of Anatomy, Tokyo University, for some technical advice.     

Observations on the Fine Structure of the Turtle Atrium

The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.     

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.     

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.     

About the T-system in the myofibril-free sarcoplasm of the frog muscle fibre

Previous investigations of the T-system in skeletal muscle fibres described the inter-myofibrillar relationships between T-tubules and the sarcoplasmic reticulum. They disregarded the arrangement of the T-system in the myofibril-free sarcoplasm in the area of muscle fibre nuclei. In the present investigation, the T-system was filled by means of lanthanum incubation and the myofibril-free sarcoplasm was ultrastructural examined by means of thin (< or = 100 nm) as well as thick sections (> 300 nm-1 microm) with the electron microscope. The investigation of thick sections revealed that T-tubules meander through this myofibril-free sarcoplasm and tangle up at the poles of muscle fibre nuclei and in the area of fundamental nuclei of the motor end plate. They are, far from myofibrils, in proximity to these nuclei, the Golgi apparatus and mitochondria. On basis of this proximity and their openings at the muscle fibre surface, a contribution at the drainage of metabolic products and at the local calcium control is discussed.     

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.     

OBSERVATIONS ON THE FINE STRUCTURE OF PHARYNGEAL MUSCLE IN THE PLANARIAN DUGESIA TIGRINA

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.     

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.     

Water Permeability of Isolated Muscle Fibers of a Marine Crab

This report deals with the diffusional and nondiffusional water fluxes of muscle fibers of the crab, Chionoecetes bairdi. Graphical analysis of the deuterium exchange indicates that two fiber compartments exist for water. The first, comprising about 60–70% of the fiber water, probably represents the sarcoplasm which is bounded externally by the plasma membrane. The second compartment might represent intracellular organelles. The ratio between the nondiffusional and diffusional fluxes is very much larger than that found earlier for erythrocytes and for the giant axon of the squid. A ratio of such size is unlikely to be caused by unstirred layers and more accurate determinations of the water flux must include study of the influence of the complex morphology of these muscle fibers.     

Early stages of flight muscle development in the blowfly Lucilia cuprina: A light and electron microscopic study

In Lucilia cuprina the dorsal longitudinal flight muscles of the adult develop from two sets of three larval muscles. About the end of the first day after puparium formation the three degenerating larval fibres on each side of the thorax cleave longitudinally into six. The numerous neighbouring myoblasts, which multiply by mitotic divisions, fuse gradually with the developing fibres, so the myoblast nuclei become incorporated into the sarcoplasm to form the muscle syncytium. The changes in length of the muscle fibres are not uniform. Between 30 to 56 hr after puparium formation the fibres shorten, but after this they rapidly increase in length. The structures of the tendon-like strands and of the anterior extremity of the growing fibres are described.     

Fine structure of the alary muscles of the American cockroach

The alary muscles of the cockroach, Periplaneta americana, are striated with an A-band of 3·0 to 3·5 μm long. Each muscle fibre was 10 to 12 μm in diameter and Z-lines appeared as small discrete units staggered throughout the sarcoplasm. Mitochondria were conspicuously located near the Z-line areas and were absent from the middle portion of the sarcomere. A transverse membrane system was present which formed dyad structures with a relatively sparse sarcoplasmic reticulum. Cockroach alary muscles were innervated by axons containing electron-dense granules of near 100 nm in diameter. These are thought to be typical of ‘neurosecretory’ axons based on their ultrastructural appearance.     

Ultrastructural study of neuromuscular junction in rectus femoris muscle of streptozotocin-diabetic rats.

The neuromuscular junctions (NMJ) from rectus femoris muscle in streptozotocin (STZ)-induced diabetic rats were examined by electron microscopy eight weeks after the STZ injection. When compared to controls and vehicle-injected groups, both the axon terminal and the junctional sarcoplasm showed serious alterations including mitochondrial degeneration, presence of myeloid bodies, breakdown of presynaptic membrane and changes in the form of the synaptic vesicles. The results suggest that NMJ can contribute to the pathogenesis of diabetic proximal myopathy.     

THE FINE STRUCTURE OF NEUROMUSCULAR JUNCTIONS AND THE SARCOPLASMIC RETICULUM OF EXTRINSIC EYE MUSCLES OF FUNDULUS HETEROCLITUS

The extrinsic eye muscles of the killifish (F. heteroclitus) were fixed in OSO₄ (pH 7.6) and subsequently dehydrated, embedded, and sectioned for electron microscopy. The fine structures of neuromuscular junctions and of sarcoplasmic reticulum were then observed. The neuromuscular junction consists of the apposition of axolemma (60 to 70 Å) and sarcolemma (90 to 100 Å), with an intervening cleft space of 200 to 300 Å, forming a synaptolemma 400 to 500 Å thick. The terminal axons contain synaptic vesicles, mitochondria, and agranular reticulum. The subsynaptic sarcolemma lacks the infolding arrangement characteristic of neuromuscular junctions from other vertebrate skeletal muscle, making them more nearly like that of insect neuromuscular junctions. A comparison between the folded and non-folded subsynaptic membrane types is made and discussed in terms of comparative rates of acetylcholine diffusion from the synaptic cleft and resistances of the clefts and subsynaptic membranes. The sarcoplasmic reticulum consists of segmentally arranged, membrane-limited vesicles and tubular and cisternal elements which surround individual myofibrils in a sleeve-like arrangement. Triadic differentiation occurs at or near the A-I junction. Unit sleeves span the A and I bands alternately and consist of closed terminal cisternae interconnected across the A and I bands by tubular cisternae. The thickness of the sarcoplasmic membranes increases from 30 to 40 Å in intertriadic regions to 50 to 70 Å at the triads. The location of the triads is compared with previously described striated muscle from Ambystoma larval myotomes, cardiac and sartorius muscles of the albino rat, mouse limb muscle, chameleon lizard muscle, and insect muscle, with reference to their possible role in intracellular impulse conduction.