Cell Tiss. Res. 178, 73-82 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
The Development of Innervation in the Rat Atrioventricular Node Ian M. Taylor Department of Anatomy, Faculty of Medicine, University of Toronto, Toronto, Canada
Summary. The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day a n d vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed. Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day. These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.
Key words: Atrioventricular node - Rat - Innervation - Ultrastructure.
Introduction Although the formation and conduction of impulses in the heart occurs within specialized myocardial cells, the function of these cells is profoundly influenced by the nerves of the heart. As Hoffman and Cranefield (1960) suggested, transmission of excitation from atrium to ventricle is delayed during passage Send offprint requests to: Dr. I.M. Taylor, Department of Anatomy, Medical Sciences Building University of Toronto, Toronto, Ontario M5S 1A8, Canada Acknowledgement. I wish to thank Mr. C.G.T. Watterson for his excellent technical assistance and Mrs. L. Wheeler for typing the manuscript. The experiments were supported by the Atkinson Charitable Foundation and the Medical Research Council of Canada.
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Fig. 1. Electron micrograph showing part of a large axon bundle lying in an intercellular space in the superior part of the node. The nerves are lying in a partial Schwann cell sheath, surrounded by a c o m m o n basal lamina. 2 Day R a t - - u n t r e a t e d x 17,100 Fig. 2. Part of a smaller axon bundle. The Schwann cell sheath is more complete than that shown in Figure 1. Vesicles may be seen in several axon profiles, vesicles with dense cores in axon A, vesicles with more palely stained cores in axon B and others that are agranular in axon C. 2 Day R a t - treated with 5-OHDA x 20,235
Innervation of Atrioventricular Node
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through the atrioventricular node and the duration of this delay is adjusted to changes in heart rate by activity of the vagus and sympathicus. Morphologically, the specialized tissue of the adult heart is associated with numerous nerves and ultrastructural evidence of axonal varicosities in close contact with the nodal cells has been presented to support the electrophysiological contention mentioned earlier (Thaemert, 1970, 1973). Many investigators agree that the atrioventricular nodal cells receive a dual nerve supply, although some species differences have been found. Initially, Torii (1962) identified two types of nerve terminations: axon varicosities containing dense cored vesicles that he attributed to the presence of adrenergic terminals, and varicosities containing only agranular vesicles which he felt were cholinergic endings. Subsequently, various workers using combined histochemical and ultrastructural techniques have amply confirmed his findings. Several authors, such as Navaratnam (1967), have speculated that nodal differentiation was induced by nerves and recent findings in both developing human and rat hearts have added weight to this suggestion (Taylor and Anderson, 1973; Finlay and Anderson, 1974; Taylor, 1976). The first nerves demonstrated histochemically at the light microscope level in relation to the developing atrioventricular node were adrenergic in type and they first appeared on the 4th postnatal day. Acetylcholinesterase containing nerves were not seen until the 13th day and the adult pattern of cholinesterase positive innervation was still not established by 31 days of age. Most recently it has been shown that the appearance of acetylcholinesterase in nodal cells is delayed in rats treated with an anti-nerve growth factor antiserum to inhibit the growth of adrenergic nerves. Although this enzyme normally begins to be histochemically evident on the fifth postnatal day, it could not be demonstrated before the 21st day in A N G F treated animals (Taylor, 1976). For these reasons, it is important to examine the innervation of the developing rat atrioventricular conducting tissue at the ultrastructural level in an effort to confirm the earlier findings made using light microscopical techniques and to clarify the relationships of those nerves to nodal cells.
Materials and Methods Eighty Wistar rats of varying maturity were used in the first part of this study. Counting the first 24 h after delivery as Day 1, eight animals were examined for each of the first seven days of age and for each of days twelve, seventeen and twenty-six. All the animals were decapitated and without preliminary washout perfused through the left ventricle using a solution of 4%
Fig. 3. Electron micrograph of a small bundle of five axons, partially covered by a Schwann process. 4 Day R a t - u n t r e a t e d x 17,100 Fig. 4. A group of naked axons lying between two nodal cells showing typically poor myofibrillar development. A further axon (arrowed) containing at least two vesicles with dense cores can be seen in intimate contact with the nodal cell N. No basal lamina intervenes between these two cells. 3 Day R a t - u n t r e a t e d • 17,100
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paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 at 4 ~ C. After removal from the chest, the tips of the ventricles were excised and the hearts immersed in cold fixative for up to two h. Each heart was then opened through the tricuspid ring and trimmed to give a block of tissue containing the base of the septal cusp of the tricuspid valve, the coronary sinus, and the atrioventricular node and bundle. Serial tissue slices 1 m m thick were prepared from this block by a n u m b e r of parallel cuts made at right angles to the base of the septal cusp of the tricuspid valve. These blocks were fixed for a further hour in ice-cold fixative, postosmicated, routinely dehydrated in alcohol and embedded in Epon. Ultrathin sections were stained with lead acetate and uranyl acetate and investigated with a Philips 300 electron microscope. In a further series of neonatal rats, some animals were fixed in 2% potassium permanganate at pH 7.0 (Richardson, 1966) and others by the glutaraldehyde-dichromate technique introduced by Bloom and Barrnett (1966). However, the results indicated very considerate damage to the specialized tissues and these methods designed for optimal preservation of granular vesicles dropped in favour of conventional glutaraldehyde/formaldehyde fixation following administration of 5-hydroxydopamine (Tranzer and Thoenen, 1967). Four litters of rats aged 1, 2, 3 and 6 days postnatal were used in the second study. Each litter was reduced to the 8 largest animals and of them, four from each age group were given a single intraperitoneal injection of 5 mg of 5-hydroxydopamine (5-OHDA) dissolved in water. The remaining four animals of each group were given an injection of water. Twenty-four hours later, all the animals were sacrificed and processed in an identical fashion to the first series of r~.ts.
Results
On the second postnatal day, two large bundles of nerve fibers are seen approaching the atrioventricular node from above and below the coronary sinus. The first descends through the interatrial septum and enters the superior part of the node while the second passes anteriorly from the posterior atrial wall, where ganglion cells can be seen, below the coronary sinus to enter the inferior and posterior part of the node. These nerve bundles contain mostly unmyelinated nerves but a few myelinated axons are evident in some specimens. Having entered the node, the nerves more or less ensheathed in Schwann cells lie in the intercellular spaces surrounded by bundles of collagen fibers and fibrocytes (Figs. 1, 2). These large bundles divide to form smaller fascicles of vesiculated axons that permeate the entire node by the fourth postnatal day. These smaller bundles of nerve fibers are still associated with Schwann cells which either fully or incompletely invest them (Fig. 3) but finally this covering is completely lost and naked axons are seen in proximity to nodal cells (Fig. 4). By day 2, an occasional naked axon or varicosity is seen enclosed within a cleft in a nodal cell or more frequently in the intercellular space between two neighbouring specialized cells (Figs. 5, 6). However, by day 6 practically every nodal cell appears to be near a nerve process somewhere along its length. Intimate contact between nodal cells, axons and terminal varicosities can be found as early as day 2 (Figs. 5, 6). Such contacts are defined as those where the space between the nerve process and the surface of the nodal cell is diminished to the point where it is devoid of a basal lamina. The nerve processes which participate in the formation of neuromuscular contiguities whether as individuals or as part of a bundle while doing so, possess varicosities and constricted intervaricose segments. For the most part, intervaricose segment are devoid of vesicles and mitochondria.
Fig. 5. A varicosity containing both dense cored vesicles (arrowed) and agranular vesicles situated deep within a cleft in a nodal cell. 2 Day R a t - u n t r e a t e d x 17,100 Fig. 6. A naked axon in intimate contact with two neighbouring nodal cells A and B. The absence of a basal lamina between these cells should be noted. 2 Day R a t - u n t r e a t e d x 17,100 Fig. 7. Several axon profiles are seen in relation to this nodal cell. Different types of vesicles may be picked out including some with dense cores (arrowed). This is the typical appearance of sections through the atrioventricular node on the sixth postnatal day. 6 Day Rat--untreated x 17,100
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Fig. g. Electron micrograph of three axons A, B and C forming an intimate relationship with a nodal cell N. Axon A contains many agranular vesicles and is in contact not only with the specialized cell N but also with its fellow axons. 2 Day Rat-untreated x 17,100 Fig. 9. Electron micrograph of vesiculated axon profiles located in an intercellular space of the atrioventricular node of a 5-OHDA treated six day old rat. Although it is sometimes difficult to distinguish an overlapping glycogen particle from the granular core of a vesicle, a proportion of the vesicles in axon A may safely be identified as granulated, x 31,350 Fig. 10. Electron micrograph of another axon bundle from the same animal. No intensely dense cored vesicles can be seen although a large vesicle with a moderately dense granule (arrow) may be observed. 6 Day Rat-treated with 5-OHDA x 31,350
V a r i c o s e v e s i c u l a t e d a x o n s are seen singly a n d in s m a l l b u n d l e s lying b o t h in r e l a t i o n to n o d a l cells (Figs. 7, 8) a n d in the c o n n e c t i v e tissue o f the a t r i o v e n t r i c u l a r n o d e (Figs. 9, 10). In the u n t r e a t e d n o r m a l a n d w a t e r - i n j e c t e d c o n t r o l a n i m a l s , the m a j o r i t y o f a x o n s c o n t a i n m a n y s m a l l a g r a n u l a r vesicles (Figs. 8, 10), 3 0 - 5 0 n m in d i a m e t e r a n d o c c a s i o n a l l y , a few large g r a n u l a r vesicles in a d d i t i o n (Figs. 5, 10). In t h e s e g r o u p s o f a n i m a l s , the g r a n u l e s , if present, t e n d to be faint. H o w e v e r , a f t e r t r e a t m e n t w i t h 5 - O H D A , a m i n o r i t y o f the
Innervation of Atrioventricular Node
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axon profiles contain both small and large vesicles with an unusually osmiophilic interior (Figs. 2, 9). Other axon profiles appear unchanged, containing small empty vesicles and a few large vesicles with a content of minimal to moderate electron opacity (Figs. 2, 9). Both kinds of vesiculated axons may occur in axolemmal contact with each other (Fig. 9) and they may have a common Schwann cell investment (Fig. 2). A count of 200 vesicles containing axon profiles seen in sections from 5-OHDA treated six day old animals shows that 167 contain small vesicles of the empty type exclusively, and 33 contain either small or large vesicles with a dense interior. No axons have been seen in untreated material which conform to descriptions of presumptive cardiac sensory nerve terminals. Nor have any large axon varicosities containing numerous mitochondria and dense bodies been observed which appear similar to Chiba's (1973) description of sensory neurons after 5-OHDA treatment.
Discussion
The existence of nerves in the prenatal rat heart has been established by Hall (1951) and Muir (1955) using silver impregnation techniques. However, light microscope histochemical studies of this organ have yielded varied results. Owman et al. (1971) demonstrated nerves in various parts of the heart before birth using the fluorescence technique of Falck and Hillarp (1962). They did not describe any fluorescing nerves in relation to the atrioventricular conducting tissue but more recent study (Taylor, 1976) has revealed the presence of such nerves in the interatrial septum and the atrioventricular node by the fifth postnatal day. In contrast, Finlay and Anderson (1974) were unable to visualize any nerves in the rat heart using the cholinesterase method of Gomori (1952) until the twelfth postnatal day when they saw a nerve bundle in the interatrial septum. Three possible explanations for these results may be made. Firstly, that the cholinesterase method is not sensitive enough to demonstrate nerves which are present and do contain acetylcholinesterase, secondly that the cholinergic nerves which may be present are immature and do not possess acetylcholinesterase activity, and thirdly that the growth of acetylcholinesterase containing nerves into the heart occurs separately from and after the ingrowth of adrenergic nerves. These suggestions should now be considered in the light of the ultrastructural evidence. This study indicates that nerves are certainly present in the atrioventricular conducting tissue by the second postnatal day, that there is a profuse innervation by the fourth day and that by the sixth day nerves are seen in relation to nearly every specialized cell. The electron-microscopical method for identifying adrenergic and cholinergic neurons using 5-OHDA as a tracer (Tranzer and Thoenen, 1967; Tranzer et al., 1969) has been widely applied to the study of peripheral autonomic innervation. Many workers have shown that nearly all the small and large vesicles within
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the adrenergic terminals of treated animals possess an intensely osmiophilic interior. Using this criterion for differentiating between adrenergic and cholinergic terminals, it may be concluded that the node has a dual innervation. Both adrenergic and cholinergic nerves are seen for the first time in relation to the atrioventricular node on the same day. Assuming that cholinergic neurons in the heart contain acetylcholinesterase, the hypothesis that acetylcholinesterase containing nerves may grow into the node at a later date than the adrenergic innervation cannot therefore be supported. Both adrenergic neurons with intensely granulated vesicles and cholinergic neurons with unchanged vesicles can be seen within the node of 5 - O H D A treated animals by day 2. This implies that neither the Falck-Hillarp nor the G o m o r i technique is able to demonstrate those nerves which can be visualized at the ultrastructural level in the early postnatal period. It is possible that the Falck-Hillarp technique is not quite sensitive enough to detect adrenergic nerves between days 2 and 4 when they may contain reduced amounts of nor-adrenaline. However, while this technique is demonstrating adrenergic terminals directly, the G o m o r i method is being used as a possible indicator of cholinergic nerves by staining acetylcholinesterase rather than by visualizing acetylcholine directly. The ultrastructural presence of small agranular vesicles in apparently cholinergic nerves does not necessarily mean that acetylcholinesterase will be present (Israel and Gautron, 1969). The inability to detect such nerves in the developing atrioventricular node may therefore reflect either a lack of enzymic activity or inadequate sensitivity of the method used or some combination of these two. Further study will be required to elucidate this problem. Thaemert (1970) noted that axon varicosities in the tail of the mature mouse atrioventricular node contained primarily agranular vesicles which suggested a preponderance of cholinergic innervations. His material was osmium fixed and the more certain results which might follow from 5 - O H D A treatment are not so far available. However, a similar finding has been made in the present study although it is possible that the adult pattern of innervation is different to that seen in the immature animals examined here. In addition, Anderson (1972a, b) has emphasized the considerable species variation in the innervation of the conducting tissue and Thaemert (1973) has noted regional differences in density of innervation of the atrioventricular node. Many reports of the innervation of the atrioventricular conducting system have been made and most authors agree that the node is supplied with unmyelinated nerves. In contrast it has been found that while the axon bundles approaching the developing rat atrioventricular node contain mostly unmyelinated axons, about 5% are myelinated although no nerve seen within the node has possessed a myelin sheath. A curious feature of many but not all reports of nodal innervation is the lack of intimate contact between nodal cells and axon terminals. However, Thaemert (1970) and Hayashi (1971) have described the existence of many neuromuscular contacts within the nodes of mouse and dog respectively. Thaemert (1973) subsequently found that the frequency of such contacts varied from one area of the node to another. The posterior lateral portion of the
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tail o f the m u r i n e n o d e e x h i b i t e d i n t i m a t e n e u r o m u s c u l a r contact c o m m o n l y - w h e r e a s they were r a t h e r i n f r e q u e n t in the m e d i a l a n d a n t e r i o r p a r t s o f the node. T h u s choice o f s a m p l i n g a r e a m a y help to explain the lack o f n e u r o m u s c u l a r contiguities r e c o r d e d in m a n y reports. T h a e m e r t ( 1 9 7 0 ) c a t e g o r i z e d the v a r i o u s types o f n e u r o m u s c u l a r r e l a t i o n s h i p s f o u n d in the inferior p o r t i o n o f the m o u s e node. In s u m m a r y , he f o u n d t h a t nerves m a y dwell in shallow o r d e e p g r o o v e s o n the surfaces o f n o d a l cells, in s a r c o l e m m a - l i n e d tunnels, o r in cul-de-sacs inside n o d a l cells. In the a b s e n c e o f serial sections in the p r e s e n t study, the types o f n e u r o m u s c u l a r r e l a t i o n s h i p s present in the n e o n a t a l rat n o d e c a n n o t be s t a t e d b u t diligent search has failed to reveal a n y profiles o f s a r c o l e m m a - l i n e d tunnels. U l t r a s t r u c t u r a l l y , there seems n o r e a s o n to d o u b t t h a t the nerves d e m o n s t r a t e d in this s t u d y o f the p o s t n a t a l rat a t r i o v e n t r i c u l a r n o d e are f u n c t i o n i n g despite the failure o f light m i c r o s c o p e h i s t o c h e m i c a l techniques to visualize them. V a r i o u s a u t h o r s such as N a v a r a t n a m (1967) have suggested t h a t n o d a l d i f f e r e n t i a t i o n is i n d u c e d b y n e u r a l function. A l t h o u g h the effects o f the d e v e l o p i n g i n n e r v a t i o n on the u l t r a s t r u c t u r e o f the d i f f e r e n t i a t i n g n o d e have yet to be studied, it has been f o u n d (Taylor, 1976) t h a t n e o n a t a l s y m p a t h e c t o m y by the a d m i n i s t r a t i o n o f an anti nerve g r o w t h f a c t o r a n t i s e r u m ( A N G F ) causes c o n s i d e r a b l e d e l a y in the a p p e a r a n c e o f a c e t y l c h o l i n e s t e r a s e in the n o d a l a n d b u n d l e cells o f the rat a t r i o v e n t r i c u l a r node. A s the e n z y m e n o r m a l l y a p p e a r s b y the 5th d a y as c o m p a r e d to 21 days in A N G F t r e a t e d a n i m a l s , it seems r e a s o n a b l e to c o n c l u d e t h a t the a d r e n e r g i c i n n e r v a t i o n at a n y rate is f u n c t i o n a l in the a t r i o v e n t r i c u l a r n o d e o f the n e o n a t a l rat.
References Anderson, R.H.: The disposition, morphology and innervation of the cardiac specialized tissue in the guinea pig. J. Anat. (Lond.) 111,453~168 (1972a) Anderson, R.H. : Histologic and histochemical evidence concerning the presence of morphologically and distinct cellular zones within the rabbit atrioventricular node. Anat. Rec. 173, 7-24 (1972b) Bloom, F.E., Barrnett, R.J.: Fine structural localization of noradrenaline in vesicles of autonomic nerve endings. Nature (Lond.) 210, 599 601 (1966) Chiba, T.: Electron microscopic and histochemical studies on the synaptic vesicles in mouse vas deferens and atrium after 5-hydroxydopamine administration. Anat. Rec. 176, 3548 (1973) Ehinger, B., Falck, B., Sporrong, B.: Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals. Z. Zellforsch. 107, 508-521 (1970) Falck, B., Hillarp, N.A., Thieme, G., Torp, A.: Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354 (1962) Finlay, M., Anderson, R.H.: The development of cholinesterase activity in the rat heart. J. Anat. (Lond.) 117, 239 248 (1974) Gomori, G. : Microscopic histochemistry-principles and practice. Chicago: University of Chicago Press 1952 Hall, E.K.: Contractility in the embryonic heart. Anat. Rec. 111, 381-399 (1951) Hayashi, S.: Electron microscopy of the heart conduction system of the dog. Arch. Histol. Japon. 33, 67-86 (1971) Hoffman, B.F., Cranefield, P.F.: Electrophysiology of the heart. New York: McGraw-Hill Book Co. 1960 Israel, M., Gautron, J.: Cellular and subcellular localization of acetylcholine in electric organs. In: Cellular dynamics of the neuron (S.H. Barondes, ed.). New York-London: Academic Press 1959
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Muir, A.R.: The sinuatrial node of the rat heart. Quart. J. exp. Physiol. 40, 378-386 (1955) Navaratnam, V.: The ontogenesis of cholinesterase activity within the heart and cardiac ganglia in man, rat, rabbit and guinea pig. J. Anat. (Lond.) 99, 459-467 (1967) Owman, C.H., Sj6berg, N.-O., Swedin, G. : Histochemical and chemical studies on pre- and postnatal development of the different systems of " s h o r t " and "'long" adrenergic neurons in peripheral organs of the rat. Z. Zellforsch. 116, 319-341 (1971) Richardson, K.C. : Electron microscopic identification of autonomic nerve endings. Nature (Lond.) 210, 756 (1966) Taylor, I.M.: The relationship between innervation and cholinesterases of the rat atrioventricular node. J. Histochem. Cytochem., in the Press (1976) Taylor, I.M., Anderson, R.H.: Cholinesterase and the atrioventricular node and bundle in the human fetus up to midterm. J. Histochem. Cytochem. 21,464-468 (1973) Thaemert, J.C.: Atrioventricular node innervation in ultrastructural three dimensions. Amer. J. Anat. 128, 239 264 (1970) Thaemert, J.C.: Fine structure of the atrioventricular node as viewed in serial sections. Amer. J. Anat. 136, 4 3 4 6 (1973) Torii, H.: Electron microscope observation of the S-A and A-V nodes and Purkinje fibers of the rabbit. Jap. Circ. J. 26, 39-77 (1962) Tranzer, J.P., Thoenen, H.: Electron microscopic localization of 5-hydroxydopamine (3,4,5-trihydroxy-phenyl-ethylamine), a new "false" sympathetic transmitter. Experientia (Basel) 23, 743 744 (1967) Tranzer, J.P., Thoenen, H.: Ultramorphologische Ver~inderungen der sympathischen Nervenendigungen der Katze nach Vorbehandlung mit 5- und 6-Hydroxy-Dopamin. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 25% 343-344 (1967) Tranzer, J.P., Thoenen, H., Snipes, R.L., Richards, J.G. : Recent developments on the ultrastructural aspect of adrenergic nerve endings in various experimental conditions. Progr. Brain Res. 31, 33-46 (1969) Yamauchi, A., Chiba, T.: Adrenergic and cholinergic innervation of the turtle heart ventricle. Z. Zellforsch. 143, 485-493 (1973)
Accepted October 5, 1976
Cell and Tissue Research 9 by Springer-Verlag 1977
The Development of Innervation in the Rat Atrioventricular Node Ian M. Taylor Department of Anatomy, Faculty of Medicine, University of Toronto, Toronto, Canada
Summary. The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day a n d vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed. Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day. These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.
Key words: Atrioventricular node - Rat - Innervation - Ultrastructure.
Introduction Although the formation and conduction of impulses in the heart occurs within specialized myocardial cells, the function of these cells is profoundly influenced by the nerves of the heart. As Hoffman and Cranefield (1960) suggested, transmission of excitation from atrium to ventricle is delayed during passage Send offprint requests to: Dr. I.M. Taylor, Department of Anatomy, Medical Sciences Building University of Toronto, Toronto, Ontario M5S 1A8, Canada Acknowledgement. I wish to thank Mr. C.G.T. Watterson for his excellent technical assistance and Mrs. L. Wheeler for typing the manuscript. The experiments were supported by the Atkinson Charitable Foundation and the Medical Research Council of Canada.
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Fig. 1. Electron micrograph showing part of a large axon bundle lying in an intercellular space in the superior part of the node. The nerves are lying in a partial Schwann cell sheath, surrounded by a c o m m o n basal lamina. 2 Day R a t - - u n t r e a t e d x 17,100 Fig. 2. Part of a smaller axon bundle. The Schwann cell sheath is more complete than that shown in Figure 1. Vesicles may be seen in several axon profiles, vesicles with dense cores in axon A, vesicles with more palely stained cores in axon B and others that are agranular in axon C. 2 Day R a t - treated with 5-OHDA x 20,235
Innervation of Atrioventricular Node
75
through the atrioventricular node and the duration of this delay is adjusted to changes in heart rate by activity of the vagus and sympathicus. Morphologically, the specialized tissue of the adult heart is associated with numerous nerves and ultrastructural evidence of axonal varicosities in close contact with the nodal cells has been presented to support the electrophysiological contention mentioned earlier (Thaemert, 1970, 1973). Many investigators agree that the atrioventricular nodal cells receive a dual nerve supply, although some species differences have been found. Initially, Torii (1962) identified two types of nerve terminations: axon varicosities containing dense cored vesicles that he attributed to the presence of adrenergic terminals, and varicosities containing only agranular vesicles which he felt were cholinergic endings. Subsequently, various workers using combined histochemical and ultrastructural techniques have amply confirmed his findings. Several authors, such as Navaratnam (1967), have speculated that nodal differentiation was induced by nerves and recent findings in both developing human and rat hearts have added weight to this suggestion (Taylor and Anderson, 1973; Finlay and Anderson, 1974; Taylor, 1976). The first nerves demonstrated histochemically at the light microscope level in relation to the developing atrioventricular node were adrenergic in type and they first appeared on the 4th postnatal day. Acetylcholinesterase containing nerves were not seen until the 13th day and the adult pattern of cholinesterase positive innervation was still not established by 31 days of age. Most recently it has been shown that the appearance of acetylcholinesterase in nodal cells is delayed in rats treated with an anti-nerve growth factor antiserum to inhibit the growth of adrenergic nerves. Although this enzyme normally begins to be histochemically evident on the fifth postnatal day, it could not be demonstrated before the 21st day in A N G F treated animals (Taylor, 1976). For these reasons, it is important to examine the innervation of the developing rat atrioventricular conducting tissue at the ultrastructural level in an effort to confirm the earlier findings made using light microscopical techniques and to clarify the relationships of those nerves to nodal cells.
Materials and Methods Eighty Wistar rats of varying maturity were used in the first part of this study. Counting the first 24 h after delivery as Day 1, eight animals were examined for each of the first seven days of age and for each of days twelve, seventeen and twenty-six. All the animals were decapitated and without preliminary washout perfused through the left ventricle using a solution of 4%
Fig. 3. Electron micrograph of a small bundle of five axons, partially covered by a Schwann process. 4 Day R a t - u n t r e a t e d x 17,100 Fig. 4. A group of naked axons lying between two nodal cells showing typically poor myofibrillar development. A further axon (arrowed) containing at least two vesicles with dense cores can be seen in intimate contact with the nodal cell N. No basal lamina intervenes between these two cells. 3 Day R a t - u n t r e a t e d • 17,100
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paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 at 4 ~ C. After removal from the chest, the tips of the ventricles were excised and the hearts immersed in cold fixative for up to two h. Each heart was then opened through the tricuspid ring and trimmed to give a block of tissue containing the base of the septal cusp of the tricuspid valve, the coronary sinus, and the atrioventricular node and bundle. Serial tissue slices 1 m m thick were prepared from this block by a n u m b e r of parallel cuts made at right angles to the base of the septal cusp of the tricuspid valve. These blocks were fixed for a further hour in ice-cold fixative, postosmicated, routinely dehydrated in alcohol and embedded in Epon. Ultrathin sections were stained with lead acetate and uranyl acetate and investigated with a Philips 300 electron microscope. In a further series of neonatal rats, some animals were fixed in 2% potassium permanganate at pH 7.0 (Richardson, 1966) and others by the glutaraldehyde-dichromate technique introduced by Bloom and Barrnett (1966). However, the results indicated very considerate damage to the specialized tissues and these methods designed for optimal preservation of granular vesicles dropped in favour of conventional glutaraldehyde/formaldehyde fixation following administration of 5-hydroxydopamine (Tranzer and Thoenen, 1967). Four litters of rats aged 1, 2, 3 and 6 days postnatal were used in the second study. Each litter was reduced to the 8 largest animals and of them, four from each age group were given a single intraperitoneal injection of 5 mg of 5-hydroxydopamine (5-OHDA) dissolved in water. The remaining four animals of each group were given an injection of water. Twenty-four hours later, all the animals were sacrificed and processed in an identical fashion to the first series of r~.ts.
Results
On the second postnatal day, two large bundles of nerve fibers are seen approaching the atrioventricular node from above and below the coronary sinus. The first descends through the interatrial septum and enters the superior part of the node while the second passes anteriorly from the posterior atrial wall, where ganglion cells can be seen, below the coronary sinus to enter the inferior and posterior part of the node. These nerve bundles contain mostly unmyelinated nerves but a few myelinated axons are evident in some specimens. Having entered the node, the nerves more or less ensheathed in Schwann cells lie in the intercellular spaces surrounded by bundles of collagen fibers and fibrocytes (Figs. 1, 2). These large bundles divide to form smaller fascicles of vesiculated axons that permeate the entire node by the fourth postnatal day. These smaller bundles of nerve fibers are still associated with Schwann cells which either fully or incompletely invest them (Fig. 3) but finally this covering is completely lost and naked axons are seen in proximity to nodal cells (Fig. 4). By day 2, an occasional naked axon or varicosity is seen enclosed within a cleft in a nodal cell or more frequently in the intercellular space between two neighbouring specialized cells (Figs. 5, 6). However, by day 6 practically every nodal cell appears to be near a nerve process somewhere along its length. Intimate contact between nodal cells, axons and terminal varicosities can be found as early as day 2 (Figs. 5, 6). Such contacts are defined as those where the space between the nerve process and the surface of the nodal cell is diminished to the point where it is devoid of a basal lamina. The nerve processes which participate in the formation of neuromuscular contiguities whether as individuals or as part of a bundle while doing so, possess varicosities and constricted intervaricose segments. For the most part, intervaricose segment are devoid of vesicles and mitochondria.
Fig. 5. A varicosity containing both dense cored vesicles (arrowed) and agranular vesicles situated deep within a cleft in a nodal cell. 2 Day R a t - u n t r e a t e d x 17,100 Fig. 6. A naked axon in intimate contact with two neighbouring nodal cells A and B. The absence of a basal lamina between these cells should be noted. 2 Day R a t - u n t r e a t e d x 17,100 Fig. 7. Several axon profiles are seen in relation to this nodal cell. Different types of vesicles may be picked out including some with dense cores (arrowed). This is the typical appearance of sections through the atrioventricular node on the sixth postnatal day. 6 Day Rat--untreated x 17,100
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Fig. g. Electron micrograph of three axons A, B and C forming an intimate relationship with a nodal cell N. Axon A contains many agranular vesicles and is in contact not only with the specialized cell N but also with its fellow axons. 2 Day Rat-untreated x 17,100 Fig. 9. Electron micrograph of vesiculated axon profiles located in an intercellular space of the atrioventricular node of a 5-OHDA treated six day old rat. Although it is sometimes difficult to distinguish an overlapping glycogen particle from the granular core of a vesicle, a proportion of the vesicles in axon A may safely be identified as granulated, x 31,350 Fig. 10. Electron micrograph of another axon bundle from the same animal. No intensely dense cored vesicles can be seen although a large vesicle with a moderately dense granule (arrow) may be observed. 6 Day Rat-treated with 5-OHDA x 31,350
V a r i c o s e v e s i c u l a t e d a x o n s are seen singly a n d in s m a l l b u n d l e s lying b o t h in r e l a t i o n to n o d a l cells (Figs. 7, 8) a n d in the c o n n e c t i v e tissue o f the a t r i o v e n t r i c u l a r n o d e (Figs. 9, 10). In the u n t r e a t e d n o r m a l a n d w a t e r - i n j e c t e d c o n t r o l a n i m a l s , the m a j o r i t y o f a x o n s c o n t a i n m a n y s m a l l a g r a n u l a r vesicles (Figs. 8, 10), 3 0 - 5 0 n m in d i a m e t e r a n d o c c a s i o n a l l y , a few large g r a n u l a r vesicles in a d d i t i o n (Figs. 5, 10). In t h e s e g r o u p s o f a n i m a l s , the g r a n u l e s , if present, t e n d to be faint. H o w e v e r , a f t e r t r e a t m e n t w i t h 5 - O H D A , a m i n o r i t y o f the
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axon profiles contain both small and large vesicles with an unusually osmiophilic interior (Figs. 2, 9). Other axon profiles appear unchanged, containing small empty vesicles and a few large vesicles with a content of minimal to moderate electron opacity (Figs. 2, 9). Both kinds of vesiculated axons may occur in axolemmal contact with each other (Fig. 9) and they may have a common Schwann cell investment (Fig. 2). A count of 200 vesicles containing axon profiles seen in sections from 5-OHDA treated six day old animals shows that 167 contain small vesicles of the empty type exclusively, and 33 contain either small or large vesicles with a dense interior. No axons have been seen in untreated material which conform to descriptions of presumptive cardiac sensory nerve terminals. Nor have any large axon varicosities containing numerous mitochondria and dense bodies been observed which appear similar to Chiba's (1973) description of sensory neurons after 5-OHDA treatment.
Discussion
The existence of nerves in the prenatal rat heart has been established by Hall (1951) and Muir (1955) using silver impregnation techniques. However, light microscope histochemical studies of this organ have yielded varied results. Owman et al. (1971) demonstrated nerves in various parts of the heart before birth using the fluorescence technique of Falck and Hillarp (1962). They did not describe any fluorescing nerves in relation to the atrioventricular conducting tissue but more recent study (Taylor, 1976) has revealed the presence of such nerves in the interatrial septum and the atrioventricular node by the fifth postnatal day. In contrast, Finlay and Anderson (1974) were unable to visualize any nerves in the rat heart using the cholinesterase method of Gomori (1952) until the twelfth postnatal day when they saw a nerve bundle in the interatrial septum. Three possible explanations for these results may be made. Firstly, that the cholinesterase method is not sensitive enough to demonstrate nerves which are present and do contain acetylcholinesterase, secondly that the cholinergic nerves which may be present are immature and do not possess acetylcholinesterase activity, and thirdly that the growth of acetylcholinesterase containing nerves into the heart occurs separately from and after the ingrowth of adrenergic nerves. These suggestions should now be considered in the light of the ultrastructural evidence. This study indicates that nerves are certainly present in the atrioventricular conducting tissue by the second postnatal day, that there is a profuse innervation by the fourth day and that by the sixth day nerves are seen in relation to nearly every specialized cell. The electron-microscopical method for identifying adrenergic and cholinergic neurons using 5-OHDA as a tracer (Tranzer and Thoenen, 1967; Tranzer et al., 1969) has been widely applied to the study of peripheral autonomic innervation. Many workers have shown that nearly all the small and large vesicles within
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the adrenergic terminals of treated animals possess an intensely osmiophilic interior. Using this criterion for differentiating between adrenergic and cholinergic terminals, it may be concluded that the node has a dual innervation. Both adrenergic and cholinergic nerves are seen for the first time in relation to the atrioventricular node on the same day. Assuming that cholinergic neurons in the heart contain acetylcholinesterase, the hypothesis that acetylcholinesterase containing nerves may grow into the node at a later date than the adrenergic innervation cannot therefore be supported. Both adrenergic neurons with intensely granulated vesicles and cholinergic neurons with unchanged vesicles can be seen within the node of 5 - O H D A treated animals by day 2. This implies that neither the Falck-Hillarp nor the G o m o r i technique is able to demonstrate those nerves which can be visualized at the ultrastructural level in the early postnatal period. It is possible that the Falck-Hillarp technique is not quite sensitive enough to detect adrenergic nerves between days 2 and 4 when they may contain reduced amounts of nor-adrenaline. However, while this technique is demonstrating adrenergic terminals directly, the G o m o r i method is being used as a possible indicator of cholinergic nerves by staining acetylcholinesterase rather than by visualizing acetylcholine directly. The ultrastructural presence of small agranular vesicles in apparently cholinergic nerves does not necessarily mean that acetylcholinesterase will be present (Israel and Gautron, 1969). The inability to detect such nerves in the developing atrioventricular node may therefore reflect either a lack of enzymic activity or inadequate sensitivity of the method used or some combination of these two. Further study will be required to elucidate this problem. Thaemert (1970) noted that axon varicosities in the tail of the mature mouse atrioventricular node contained primarily agranular vesicles which suggested a preponderance of cholinergic innervations. His material was osmium fixed and the more certain results which might follow from 5 - O H D A treatment are not so far available. However, a similar finding has been made in the present study although it is possible that the adult pattern of innervation is different to that seen in the immature animals examined here. In addition, Anderson (1972a, b) has emphasized the considerable species variation in the innervation of the conducting tissue and Thaemert (1973) has noted regional differences in density of innervation of the atrioventricular node. Many reports of the innervation of the atrioventricular conducting system have been made and most authors agree that the node is supplied with unmyelinated nerves. In contrast it has been found that while the axon bundles approaching the developing rat atrioventricular node contain mostly unmyelinated axons, about 5% are myelinated although no nerve seen within the node has possessed a myelin sheath. A curious feature of many but not all reports of nodal innervation is the lack of intimate contact between nodal cells and axon terminals. However, Thaemert (1970) and Hayashi (1971) have described the existence of many neuromuscular contacts within the nodes of mouse and dog respectively. Thaemert (1973) subsequently found that the frequency of such contacts varied from one area of the node to another. The posterior lateral portion of the
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tail o f the m u r i n e n o d e e x h i b i t e d i n t i m a t e n e u r o m u s c u l a r contact c o m m o n l y - w h e r e a s they were r a t h e r i n f r e q u e n t in the m e d i a l a n d a n t e r i o r p a r t s o f the node. T h u s choice o f s a m p l i n g a r e a m a y help to explain the lack o f n e u r o m u s c u l a r contiguities r e c o r d e d in m a n y reports. T h a e m e r t ( 1 9 7 0 ) c a t e g o r i z e d the v a r i o u s types o f n e u r o m u s c u l a r r e l a t i o n s h i p s f o u n d in the inferior p o r t i o n o f the m o u s e node. In s u m m a r y , he f o u n d t h a t nerves m a y dwell in shallow o r d e e p g r o o v e s o n the surfaces o f n o d a l cells, in s a r c o l e m m a - l i n e d tunnels, o r in cul-de-sacs inside n o d a l cells. In the a b s e n c e o f serial sections in the p r e s e n t study, the types o f n e u r o m u s c u l a r r e l a t i o n s h i p s present in the n e o n a t a l rat n o d e c a n n o t be s t a t e d b u t diligent search has failed to reveal a n y profiles o f s a r c o l e m m a - l i n e d tunnels. U l t r a s t r u c t u r a l l y , there seems n o r e a s o n to d o u b t t h a t the nerves d e m o n s t r a t e d in this s t u d y o f the p o s t n a t a l rat a t r i o v e n t r i c u l a r n o d e are f u n c t i o n i n g despite the failure o f light m i c r o s c o p e h i s t o c h e m i c a l techniques to visualize them. V a r i o u s a u t h o r s such as N a v a r a t n a m (1967) have suggested t h a t n o d a l d i f f e r e n t i a t i o n is i n d u c e d b y n e u r a l function. A l t h o u g h the effects o f the d e v e l o p i n g i n n e r v a t i o n on the u l t r a s t r u c t u r e o f the d i f f e r e n t i a t i n g n o d e have yet to be studied, it has been f o u n d (Taylor, 1976) t h a t n e o n a t a l s y m p a t h e c t o m y by the a d m i n i s t r a t i o n o f an anti nerve g r o w t h f a c t o r a n t i s e r u m ( A N G F ) causes c o n s i d e r a b l e d e l a y in the a p p e a r a n c e o f a c e t y l c h o l i n e s t e r a s e in the n o d a l a n d b u n d l e cells o f the rat a t r i o v e n t r i c u l a r node. A s the e n z y m e n o r m a l l y a p p e a r s b y the 5th d a y as c o m p a r e d to 21 days in A N G F t r e a t e d a n i m a l s , it seems r e a s o n a b l e to c o n c l u d e t h a t the a d r e n e r g i c i n n e r v a t i o n at a n y rate is f u n c t i o n a l in the a t r i o v e n t r i c u l a r n o d e o f the n e o n a t a l rat.
References Anderson, R.H.: The disposition, morphology and innervation of the cardiac specialized tissue in the guinea pig. J. Anat. (Lond.) 111,453~168 (1972a) Anderson, R.H. : Histologic and histochemical evidence concerning the presence of morphologically and distinct cellular zones within the rabbit atrioventricular node. Anat. Rec. 173, 7-24 (1972b) Bloom, F.E., Barrnett, R.J.: Fine structural localization of noradrenaline in vesicles of autonomic nerve endings. Nature (Lond.) 210, 599 601 (1966) Chiba, T.: Electron microscopic and histochemical studies on the synaptic vesicles in mouse vas deferens and atrium after 5-hydroxydopamine administration. Anat. Rec. 176, 3548 (1973) Ehinger, B., Falck, B., Sporrong, B.: Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals. Z. Zellforsch. 107, 508-521 (1970) Falck, B., Hillarp, N.A., Thieme, G., Torp, A.: Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354 (1962) Finlay, M., Anderson, R.H.: The development of cholinesterase activity in the rat heart. J. Anat. (Lond.) 117, 239 248 (1974) Gomori, G. : Microscopic histochemistry-principles and practice. Chicago: University of Chicago Press 1952 Hall, E.K.: Contractility in the embryonic heart. Anat. Rec. 111, 381-399 (1951) Hayashi, S.: Electron microscopy of the heart conduction system of the dog. Arch. Histol. Japon. 33, 67-86 (1971) Hoffman, B.F., Cranefield, P.F.: Electrophysiology of the heart. New York: McGraw-Hill Book Co. 1960 Israel, M., Gautron, J.: Cellular and subcellular localization of acetylcholine in electric organs. In: Cellular dynamics of the neuron (S.H. Barondes, ed.). New York-London: Academic Press 1959
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Muir, A.R.: The sinuatrial node of the rat heart. Quart. J. exp. Physiol. 40, 378-386 (1955) Navaratnam, V.: The ontogenesis of cholinesterase activity within the heart and cardiac ganglia in man, rat, rabbit and guinea pig. J. Anat. (Lond.) 99, 459-467 (1967) Owman, C.H., Sj6berg, N.-O., Swedin, G. : Histochemical and chemical studies on pre- and postnatal development of the different systems of " s h o r t " and "'long" adrenergic neurons in peripheral organs of the rat. Z. Zellforsch. 116, 319-341 (1971) Richardson, K.C. : Electron microscopic identification of autonomic nerve endings. Nature (Lond.) 210, 756 (1966) Taylor, I.M.: The relationship between innervation and cholinesterases of the rat atrioventricular node. J. Histochem. Cytochem., in the Press (1976) Taylor, I.M., Anderson, R.H.: Cholinesterase and the atrioventricular node and bundle in the human fetus up to midterm. J. Histochem. Cytochem. 21,464-468 (1973) Thaemert, J.C.: Atrioventricular node innervation in ultrastructural three dimensions. Amer. J. Anat. 128, 239 264 (1970) Thaemert, J.C.: Fine structure of the atrioventricular node as viewed in serial sections. Amer. J. Anat. 136, 4 3 4 6 (1973) Torii, H.: Electron microscope observation of the S-A and A-V nodes and Purkinje fibers of the rabbit. Jap. Circ. J. 26, 39-77 (1962) Tranzer, J.P., Thoenen, H.: Electron microscopic localization of 5-hydroxydopamine (3,4,5-trihydroxy-phenyl-ethylamine), a new "false" sympathetic transmitter. Experientia (Basel) 23, 743 744 (1967) Tranzer, J.P., Thoenen, H.: Ultramorphologische Ver~inderungen der sympathischen Nervenendigungen der Katze nach Vorbehandlung mit 5- und 6-Hydroxy-Dopamin. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 25% 343-344 (1967) Tranzer, J.P., Thoenen, H., Snipes, R.L., Richards, J.G. : Recent developments on the ultrastructural aspect of adrenergic nerve endings in various experimental conditions. Progr. Brain Res. 31, 33-46 (1969) Yamauchi, A., Chiba, T.: Adrenergic and cholinergic innervation of the turtle heart ventricle. Z. Zellforsch. 143, 485-493 (1973)
Accepted October 5, 1976
