Direct contact between a neuron soma and myocardial cells; an electronmicroscopic finding in the sinus venosus of the turtle heart.код для вставкиСкачать
Direct Contact between a Neuron Soma and Mvocardial Cells; An Electronmicroscopic Finding in the Sinus Venosus of the Turtle Heart 1 AKIO YAMAUCHI, YOKO FUJIMAKI AND KENICHI KUMAGAI Department of Anatomy, Iwate Medical University, School of Medicine, Morioka 020, J a p a n ABSTRACT Electron microscopy of the turtle sinus venosus shows an unusual neuron soma which is immediately surrounded by myocardial cells so as to form a somato-muscular complex. Studies of 36 sections cut through this neuron soma indicate that 27.4% of the somal surface studied is directly covered by cardiac muscle cells without any intervening basal lamina. Similarly, 18.8% of the somal surface studied is in contact with unmyelinated axons which at places make typical axosomatic synapses. A coupling of peripheral cisterns of the sarcoplasmic reticulum and the sarcolemma exists a t the somato-muscular contiguity as well as at the border between cardiac muscle cells. It is suggested that neural influences upon the pacemaking activity of the turtle heart sinus can well be exerted through the somato-muscular pathway. Additionally, an axon which is presynaptic to the neuron soma has been observed to be simultaneously in a synaptic relation with a cardiac muscle cell; a remarkable deviation from the classical picture of the efferent nervous outflow to the heart. The cardiac ganglion has been treated only briefly, if at all, in the previous ultrastructure studies of the innervation of the vertebrate heart (VirBgh and Porte, '61; Yamauchi and Burnstock, '68; Santer, '72; Yamauchi, '73). Recent studies which were more specifically devoted to the ganglionic synaptology have shed some light on the two different types of cardiac interneurons, one a granulated type occurring in the turtle heart (Chiba and Yamauchi, '73) and the other a non-granulated type in the fish heart (Yamauchi et al., '73). As regards the ordinary neurons in the cardiac ganglion, however, i t remains entirely uncertain whether they may be subdivided into classes according to the fine structure particularly of their synapses or whether they may represent a rather simple cell population in which all constituents are essentially alike. In the course of studies of ganglion cells in the sinus region of the turtle heart, we encountered a neuron soma immediately surrounded by cardiac muscle cells. An analysis of the fine structure of this pecuANAT. REC., 179: 491-4913, liar somato-muscular complex and its innervation will be presented in this paper. MATERIALS AND METHODS A soft-shelled turtle, Pseudemys scripta elegans, was decapitated and the heart quickly removed into a 2.5% solution of glutaraldehyde buffered in 1/15 M phosphate at pH 7.4. The sino-auricular region of the heart was then excised under a dissecting microscope, postosmicated, dehydrated in ethanol and embedded in Epon 812 (Luft, '61) through an infiltration medium, n-butyl glycidyl ether (Kushida, '63). Sections were cut at about 0.1 on a Porter-Blum microtome from the sinus wall, mounted on a series of grids, stained with uranyl acetate and lead citrate (Reynolds, '63), and viewed in the electron microscope. Recordings of one particular neuron soma in contact with cardiac muscle cells were made at 36 different sectional levels. Using a thin thread, lengths of the surface of that neuron were measured on the elecReceived Nov. 8, '73. Accepted Jan. 30, '74. 49 1 492 A. YAMAUCHI, Y. FUJIMAKI AND K. KUMAGAI tron micrographs at a magnification of 16,000 times obtained at each recorded level. OBSERVATIONS The wall of the sinus venosus of the heart of Pseudemys scripta elegans contains a well-developed cardiac muscle layer, which is attached to islets of smooth muscle cells located subendocardially. Nerve cell somata are for the most part encountered in the subepicardial connective tissue space, but a few of them are present within the muscle layer as well as in the subendocardial space in the sinus wall. The somato-muscular complex to be reported here as a single case in a single turtle heart consists of a neuron soma which is intimately surrounded by cardiac muscle cells at the border of myocardial layer and the subendocardial connective tissue space in the sinus wall (fig. 1). This neuron soma, like many other nerve cell bodies in the turtle heart, is rather small in size (about 10 X 15 p ) but is identifiable as such because, first of all, it receives the axon terminal synapsing upon its surface (fig. 2). Second, the soma1 nucleus is large and contains the electron-lucent karyoplasm with a very conspicuous nucleolus (fig. l ) , which is typical of the neuron in general. Third, the perikaryal cytoplasm of the soma contains at places a considerable amount of the ribosomes which are set into small, polysomal rosetts, in a manner being recognized as characteris)tic of nerve cells (Peters et al., '70). And fourth, the thin sheet of satellite cell processes is present attached to a part of the surface membrane of the soma (fig. 1), At the contact zone between the soma and the muscle cell, the plasma membranes are apposed leaving a narrow gap about 200 A in width, where no basal lamina substance is detectable, Subsarcolemma1 cisternae containing a dense material are observed to occur not only at the somato-muscular, but also at the axo- muscular (fig. 2 ) and the myo-myal (fig. 1 ) junctions. When the surface lengths of the neuron soma with its initial processes are measured at 36 planes of thin sections, i t be- comes evident that 27.4% of the total surface is covered by cardiac muscle cells, 18.8% by axons. (table 1 ) . The vesiculated segment of axons forms an axosomatic synapse at two separate loci on the surface membrane of the neuron soma. Profiles of those axons synapsing to the neuron soma are large in size, and the axosomatic contact length measures up to 6 p. This finding suggests the occurrence of a large, probably calyciform axon terminal which covers a considerable area of a neuron soma within the turtle heart. Of particular interest is a case of the axosomatic synapse (fig. 2), where an axon terminal presynaptic to the neuron soma shows at the same time a broad contact with the cardiac muscle cell, which in turn abuts against the neuron soma. All the presynaptic boutons revealed in the present study show a predominance of agranular synaptic vesicles with a few, large granular ones: A vesicular content typical of cholinergic nerve terminals. DISCUSSION The sinus venosus of the turtle heart has been known, for a long time, to have a muscular wall where the dominating rhythm of the heart originates normally (Gaskell, 1883; Keith and Flack, '07; Meek and Eyster, 15-16). Much attention was also focused in these earlier studies on the nerve cells and fibers situated in the sinus wall, being in connection with the vagal and sympathetic nerve trunks. The somato-muscular complex disclosed presently in the sinus venosus of the turtle heart provides an unusual pathway of impulses from the postganglionic parasympathetic (see below) neuron to the effector, myocardial cells. Direct apposition between Fig. 1 A neuron soma-cardiac muscle complex i n the sinus venosus of a turtle heart. The neuron soma in this level of sections (Level 8 in table 1 ) contains a large nucleus ( N ) with a prominent nucleolus (Nu) and shows a small process (P). Two profiles of the cardiac muscle cell ( M l , M2) are in direct contiguity to the neuron soma. A n axon (A), which is i n contact with both a cardiac muscle cell ( M l ) and the neuron soma, forms synapses to those elements at the other sectional level shown i n figure 2. Arrows indicate subsarcolemmal cisternae occurring at the border between myocardial cells. S: Satellite cell sheath of the neuron soma. Calibration: 1 p . NEURON SOMA-CARDIAC MUSCLE CONTIGUITY 493 494 A. YAMAUCHI, Y. FUJIMAKI AND K. KUMAGAI Fig. 2 A typical synapse between the axon ( A ) and the neuron soma ( S ) is indicated by a double arrow. Level 10 i n table 1. Single arrows show the occurrence of subsarcolemmal cisternae at the junctions between the axon and a cardiac muscle cell ( M 1 ) and between the soma and cardiac muscle cells (M1 and M2). Calibration: 0.5 p. the neuron soma and the muscle cells would obviously enable fewer effectors to be brought under the more intense care of a postganglionic neuron than the ordinary connection between the postganglionic axon branches and the effector cells. In view of the presence of large, heavily vesiculated axon terminals presynaptic to the neuron soma described in the present study, there is little room for supposing that this particular neuron should represent the intracardial sensory neuron put forward by Skok ( ' 7 3 ) . On the other hand, a previous work (Chiba and Yamauchi, '73) has indicated an absence of adrenergic neuron soma in the heart of the turtle. The exception is the chromaffin cell type adrenergic interneuron whose ultrastructure was quite different from that of the ordinary neuron soma as shown in this study. Based on the above facts, i t is considered that the nature of the neuron soma presented here would certainly be postganglionic, cholinergic and most probably parasympathetic. As far as we know, a well-developed axosomatic synapse characterizes the parasympathetic (cardiac, ciliary and intestinal at least) ganglia of the vertebrates (cf. Szentagothai, '64; Taxi, '65; Gabella, '72; Yamauchi, ' 7 3 ) . Present observations of a preganglionic axon which directly terminates o n a myocardial cell provide evidence of a remarkable deviation from the classical concept of the arrangement of the parasympathetic outflow. The subsarcolemmal cisternae containing an amorphous material have been recognized in the myocardial cells of lower vertebrates (Sommer and Johnson, '70; Beringer and Hadek, ' 7 3 ) . An impression was obtained in this study that those cisternae in myocardial cells of the turtle sinus venosus are so indiscriminately distributed as to make their significance in the intercellular impulse transmission rather difficult to imagine. They might well bear a metabolic function, e.g., in transport or export of proteins that may be involved in the movement of calcium (Sommer and Johnson, ' 7 0 ) . 495 NEURON SOMA-CARDIAC MUSCLE CONTIGUITY TABLE 1 Measurements of the surface of a neuron soma in the turtle heart sinus venosus, made at 36 levels sampled from 60 serial sections mounted on 20 successive grids. The interval from one level to the next is not more accurately known than to be less than about 0.8 p. The whole circumference o f the neuron soma with its initial segment of processes was estimated at all levels except for level 7, where about a quarter of a neuronal profile was intersected by a grid bar. Level Total length of neuron surface (cm at 16,000 magnification) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 91.7 86.2 85.0 77.5 81.2 82.0 68.8 79.2 60.1 73.3 52.7 61.7 60.9 63.3 64.3 64.7 71.0 69.9 69.3 54.4 53.5 54.1 51.0 50.6 53.5 47.8 46.7 45.7 53.5 48.3 50.2 43.5 39.7 42.2 52.4 42.9 S u m total The neuron surface in contact with vesiculated and non-vesiculated axons 2192.8 ACKNOWLEDGMENTS We are grateful to Miss R. Yokota for advice and Miss K. Tsushida for her expert assistance in typing the manuscript. LITERATURE CITED Beringer, T., and R. Hadek 1973 Ultrastructure of sinus venosus innervation in Petromyzon marinus. J. Ultrastruct. Res., 42: 312323. Chiba, T., and A. Yamauchi 1973 Fluorescence and electron microscopy of the monoaminecontaining cells in the turtle heart. Z. Zellforsch., 140: 25-37. The neuron surface in contact with myocardial cells 22.5 21.7 20.6 10.2 15.5 15.4 14.1 17.2 8.1 14.5 8.1 7.5 7.8 9.0 7.4 6.7 9.8 7.6 10.6 17.6 16.0 15.8 22.5 21.5 24.7 10.5 6.2 5.0 5.2 5.5 4.2 4.0 4.7 5.0 4.6 4.2 17.0 15.9 15.7 16.3 24.7 25.5 23.7 23.O 18.2 23.8 17.8 17.5 20.8 22.0 21.0 22.5 24.0 24.7 23.7 17.8 15.5 14.7 8.0 8.6 8.1 9.1 10.5 10.7 13.3 14.0 17.0 11.0 9.5 10.5 13.6 11.0 411.5 (18.8%) 600.7 (27.4%) Appearance of the nucleus of the neuron ++ ++ ++ ++ + ++ ++ ++ ++ ++ +-I++ ++ ++ + - - Gabella, G. 1972 Fine structure of the myenterjc plexus i n the guinea-pig illeum. J. Anat., 11I : 69-97. Gaskell, W. H. 1883 On the innervation of the heart, with especial reference to the heart of the tortoise. J. Physiol., 4 : 43-127. Keith, A,, and M. Flack 1907 The form and nature of the muscular connections between the primary divisions of the vertebrate heart. J. Anat., 41: 172-189. Kushida, H. 1963 An improved epoxy resin “Epok 533,” and polyethylene glycol 200 as a dehydrating agent. J. Electron Microscopy (Tokyo), 12: 167-174. 496 A. YAMAUCHI, Y. FUJIMAKI AND K. KUMAGAI Luft, J. H. 1961 Improvements i n epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-411. Meek, W. J., and J. A. E. Eyster 1915-16 The origin of the cardiac impulse in the turtle's heart. Am. J. Physiol., 39: 291-296. Peters, A,, S. L. Palay and H. de F. Webster 1970 The Fine Structure of the Nervous System. Harper and Row, New York, pp. 7-14. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Santer, R. M. 1972 Ultrastructural and histochemical studies on the innervation of the heart of a teleost, Pleuronectes platessa L. Z. Zellforsch., 131: 519-528. Skok, V. I. 1973 Physiology of Autonomic Ganglia. Igaku Shoin Ltd., Tokyo, pp. 49-53. Sommer, J. R., and E. A. Johnson 1970 Comparative ultrastructure of cardiac cell membrane specializations. A review. Am J. Cardiol., 25: 164-194. Szentagothai, J. 1964 The structure of the au- tonomic interneuronal synapse. Acta neuroveg., 26: 336-359. Taxi, J. 1965 Contribution a 1'Ctude des connexions des neurons moteurs du systhme nerveux autonome. Ann, Sci. Natur. Zool., 7: 4 13-674. Viragh, S., and A. Porte 1961 Bl6ments nerveux intracardiaques et innervation du myocarde. Btude au microscope electronique dans le coeur de rat. 2. Zellforsch., 55: 282-296. Yamauchi, A. 1973 Ultrastructure of the innervation of the mammalian heart. In: Ultrastructure of the Mammalian Heart. C. E. Challice and S. VirAgh, eds. Academic Press, New York, pp. 127-178. Yamauchi, A., and G . Burnstock 1968 An electron microscopic study on the innervation of the trout heart. J. Comp. Neurol., 132: 567-586. Yamauchi, A., Y. Fujimaki and R. Yokota 1973 Fine structural studies of the sino-auricular nodal tissue in the heart of a teleost fish, Misgurnus, with particular reference to the cardiac internuncial cell. Am. J. Anat., 138: 407-430.