Z. Zellforseh. 108, 309--323 (1970) 9 by Springer-Verlag 1970

Uhrastructural Studies of Vagal Paraganglia in Syrian Hamsters* I-LI CHwN ** a n d R. D. YATES *** Department of Anatomy, College of Medicine, National Taiwan University Taipei, Taiwan, Republic of China and Department of Anatomy, The University of Texas Medical Branch Galveston, Texas, U.S.A. Received May 5, 1970

Summary. Typical vagal paraganglia of Syrian hamsters are encapsulated in connective tissue and consist of groups of epithelial cells. Ganglion cells, a few fenestrated capillaries, and bundles of unmyelinated nerve fibers are intermingled among the parenchymal cells. The parenchymal cells are of two types: chief or paraganglion and sustentacular or supporting cells. The processes of the supporting cells partly or completely surround the paraganglion cells. In addition to the nucleus, Golgi complex, mitochondria, parallel-arrayed granular endoplasmic reticulum, and lipofuscin pigment, the chief cells are characterized by the presence of numerous membrane-bound, electron opaque granules. After an injection of aH-dopa, labelings were concentrated over the chief cells and were associated predominantly with the granules. Following glutaraldehyde-diehromate treatment the granules gave a positive reaction for unsubstituted amines. These results suggest that the chief cells contain catecholamines in the electron opaque granules. Key-Words: Vagal Paraganglia - - Catecholamines - - Ultrastructure - - Radioautography - - Cytochemistry. Introduction The t e r m " p a r a g a n g l i o n " was first i n t r o d u c e d b y K o h n (1903) to describe s t r u c t u r e s c o m p o s e d of groups of epithelial cells which gave a chromaffin r e a c t i o n a n d were closely a s s o c i a t e d with s y m p a t h e t i c ganglia. Clusters of cells f o u n d in connection w i t h t h e p a r a s y m p a t h e t i c n e r v o u s s y s t e m do n o t give a conspicuous chromaffin reaction, a n d are classified b y W a t z k a (1934, 1943) as a c h r o m a f f i n in n a t u r e . A l t h o u g h t h e p a r a g a n g l i a in c e r t a i n species regress r a p i d l y d u r i n g p o s t n a t a l life (Coupland, 1956, 1960), G o o r m a g h t i g h (1936) f o u n d some discrete bodies along or w i t h i n t h e a b d o m i n a l b r a n c h e s of t h e vagus nerve in a d u l t mice, which he d e s i g n a t e d as v a g a l p a r a g a n g l i a a n d suggested t h a t t h e y were of t h e a c h r o m a f f i n t y p e p r o b a b l y e l a b o r a t i n g acetylcholine. W e h a v e localized p a r a g a n g l i a along t h e a b d o m i n a l b r a n c h e s of t h e vagus n e r v e in y o u n g a n d a d u l t S y r i a n h a m s t e r s , a n d our p r e l i m i n a r y electron microscopic studies h a v e r e v e a l e d n u m e r o u s m e m b r a n e b o u n d , electron o p a q u e granules similar in a p p e a r a n c e to t h e c a t e c h o l a m i n e storing ones of a d r e n a l m e d u l l a r y (Hagen a n d B a r r n e t t , 1960; Yates, 1963, 1964), s y m p a t h e t i c p a r a g a n g l i o n (Brundin, 1966; Virs a n d Both, 1967) a n d c a r o t i d b o d y glomus cells (Ross, 1959 ; L e v e r et al., 1959; D u n c a n a n d Yates, 1967 ; Chen * Research supported by USPHS Grants NS 05665, 00690 and HE 12751. A preliminary report of this research was presented before the American Society for Cell Biology, 1969. ** Sponsored by National Council on Science Development, Republic of China. *** Recipient of Career Research Development Award 1K3 GM 28064.

Fig. 1. Light microscopic radioautograph taken from a thick Epon section, illustrating the general histological organization and distribution of labelings in a vagal paraganglion following 3H-dopa administration. The vagal paraganglion is attached to a branch of the vagus nerve (X). The perineurium of the vagus nerve is continuous (arrow) with the connective tissue capsule of the paraganglion. Four larger ganglion cells (Gg), numerous smaller paraganglion cells and a few thin-walled capillaries (C) are seen. The labelings which have been registered as a deposit of silver grains are associated predominantly with the paraganglion cells. • 570 Fig. 2. Photomicrograph taken from a thick Epon section. In this case paraganglion cells are aggregated within a branch of the vagus nerve and there is no indication of local enlargement of the nerve. In the adjacent thin sections these cells were identified with the electron microscope as paraganglion cells on the basis of the presence of membrane-bound electron opaque granules in their cytoplasm. • 570

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Fig. 3. A survey electron micrograph of the vagal paraganglion. A ganglion cell (Gg), a satellite cell (Sa) of the ganglion cell and several paraganglion cells (Pg) are seen in this picture. P a r t s of the cytoplasm of the satellite cell invest some of the paraganglion cells. Note specialized zones (arrows) along the apposing membranes of nerve terminals (Nt) a n d the paraganglion cells a n d a series of desmosome-like structures (D) along t h e two app0sing paraganglion cells, x 8,500

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a n d Yates, 1969; Chen etal., 1969). The present report is concerned with a description of the fine s t r u c t u r e of the vagal paraganglia i n S y r i a n h a m s t e r s a n d offers evidence for the presence of catecholamines i n the granules using electron microscopic, r a d i o a u t o g r a p h i c a n d cytochemical techniques.

Materials and Methods Nineteen Syrian hamsters weighing 30 to 100 g were used in these experiments. Six were injected intraperitoneally with a radioactive precursor of catecholamines, 3H-dihydroxyphenylalanine (specific activity, 0.680 C/raM, 29 f~C/g body weight) four hours before sacrifice. All animals were perfused with 3% glutaraldehyde in 0.1 M phosphate buffer (pH, 7.4). The vagal paraganglia corresponding to Goormaghtigh's paraganglion VII (1936) were excised together with the abdominal branch of the vagus nerve. The tissues were placed in fixatives of the same constituents as the perfusion fluid for two to four hours, washed in buffer containing 10% sucrose, postfixed in buffered 1% osmic acid for one hour, dehydrated and embedded in Epon 812. Thin sections showing light gold interference colors were cut on a PorterBlum Ultramicrotome, stained with lead citrate (Reynolds, 1963), and examined in an RCA-EMU3 G or Hitachi H U l l electron microscope. For radioautography thick and thin Epon sections were coated with Ilford L4 photographic emulsion, exposed for three to five weeks and subsequently developed in Microdol X. For electron microscopic detection of unsubstituted amines a slight modification of the potassium dichromate incubation method (Wood and Barrnett, 1964) was used. The detailed procedures for the radioautographic and cytochemical techniques have been described elsewhere (Chen and Yates, 1969).

Observations

Histological Organization The t y p i c a l vagal p a r a g a n g l i a of S y r i a n h a m s t e r s are small, node-like s t r u c t u r e s a t t a c h e d to a b d o m i n a l branches of the v a g u s nerve a n d s u r r o u n d e d b y a c o n n e c t i v e tissue capsule which is a n e x t e n s i o n of the p e r i n e u r i u m (Fig. 1). W i t h the electron microscope a t t e n u a t e d processes of the cells (probably of p i a - a r a c h n o i d origin) i n t e r d i g i t a t e b e t w e e n the collagen capsule a n d p a r e n c h y m a l cells. The pare n c h y m a l cells of the vagal paraganglia are of two t y p e s : chief or p a r a g a n g l i o n cells proper a n d s u s t e n t a c u l a r or supporting. T h e chief cells are m u c h more n u m e r o u s t h a n the s u p p o r t i n g ones a n d are f o u n d in groups or scattered singly w i t h i n t h e t r u n k or a b r a n c h of the vagus n e r v e (Fig. 2). Ganglion cells are f r e q u e n t l y observed w i t h i n the paraganglia. I n t e r m i n g l e d a m o n g the groups of p a r e n c h y m a l cells are a few thin-walled, fenestrated capillaries as well as u n m y e l i n a t e d n e r v e fibers. Some i n d i v i d u a l l y located, small, m y c l i n a t e d n e r v e fibers w i t h i n b u n d l e s of u n m y e l i n a t e d fibers are also noted.

Fig. 4. This micrograph illustrates a part of the cell body and a large cytoplasmic process of paraganglion cells. Both the cell body and the process are surrounded by attenuated processes of supporting cells. In the cell body the components of the granular endoplasmic reticuhim (Rer) are organized into a system of parallel-arrayed cisternae. A few microtubules and profiles of smooth-surfaced endoplasmic reticulum (Ser) are present in the cell process. • 19,000 Fig. 5. A part of a paraganglion cell. In addition to the Golgi complex (G) there are some profiles of branching, tubular, smooth membranes (Ser) in the cytoplasm. The basal part of a cilium (Ci) of a supporting cell is cut obliquely. Note that the limiting membrane of some electron opaque granules appear to be in direct contact with the plasma membrane. • 39,000 22 z. Zellforsch.,Bd. 108

Fig. 6. A part of a paraganglion cell showing numerous membrane-bound electron opaque granules a n d an aggregation of fine filaments (F) in the cytoplasm. X 39,000 Fig. 7. Portions of two paraganglion cells. The tissue had been subjected to potassium dichromate incubation. Note t h a t most of the granules in the paraganglion cells (Pg) show a positive reaction for unsubstituted amines. Su, supporting cell. X 22,000

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The Fine Structure o/the Parenchymal Cells The chief cells arc oval or irregular in shape with two or more cytoplasmic processes some of which extend into the interstitial connective tissue spaces. The nucleus, oval or irregular, is eccentrically located and well developed Golgi complexes are usually observed in a juxtanuclear position. The most prominent structures of the chief cells are membrane-bound, electron opaque granules 500 to 2,000 A in diameter which are mostly spherical, but oval and rod-shaped ones are noted. There is a narrow, lucid space between the limiting membrane and the dense core of the granules (Figs. 3, 6, 8, 10). The appearance of the granules is, therefore, somewhat different from the norepinephrine-storing ones in adrenal medullary cells (Coupland and Hopwood, 1966) in which the core of the granules is frequently eccentrically located with a large lucid space immediately inside the limiting membrane. The granules are distributed throughout the cytoplasm but they tend to accumulate largely at the periphery and in the cytoplasmic processes of the chief cells. The limiting membrane of some of the granules appears to be in contact with the plasma membrane (Figs. 5, 6, 8) but profiles suggesting exteriorization of the granules into the extracellular spaces are very rarely encountered. Pigment granules exhibiting the characteristics of lipofuscin in neurons are a frequent occurrence (Fig. 9). The granular endoplasmic reticulum is not well developed but in m a n y cases its components form a discrete system of parallel-arrayed cisternae (Fig. 4) as described in the glomus cells of the carotid body (Garner and Duncan, 1958; Ross, 1959; Biscoe and Stehbens, 1966; Hess, 1968). Occasionally an aggregation of fine filaments is encountered in the cytoplasm of the chief cells (Fig. 6) and solitary cilia with a " 9 - ~ 0 " pattern are found extending from the cells. Whenever two chief cells appose one another there is a series of desmosomes along adjacent plasma membranes (Fig. 3). Light and dark chief cells can be distinguished on the basis of electron opacity of the cytoplasmic matrix (Fig. 3). The dark cells are fewer in number t h a n the light ones, exhibit more free ribosomes and mitoehondria but no appreciable differences in the number of granules are noted. Following glutaraldehyde-dichromate t r e a t m e n t without further staining, the core of the granules in the chief cells becomes electron opaque and readily distinguishable from other organelles in the cytoplasm (Fig. 7). The supporting cells are irregular in shape with a few attenuated cytoplasmic processes which invest the chief cells. The nucleus is irregular and a deep indentation is often observed. The components of the granular endoplasmic retieulum are arranged in an irregular fashion and cilia with a " 9 + 0 " pattern are observed occasionally (Figs. 5, 8). The fine structure of both the chief and supporting cells is essentially the same as t h a t of glomus and supporting cells of the carotid body (Chen and Yates, 1969; Chen et al., 1969). Nerve Terminals The nerve terminals which appose the chief cells are usually club-shaped. I n the nerve terminals there are numerous vesicles 300 to 500 A in diameter and a few small mitoehondria with a matrix denser than t h a t seen in the chief cells (Figs. 3, 9, 10). Other vesicles with an electron opaque core (Figs. 9, 10) and a few profiles of anastomosing tubules of smooth membranes (Fig. 9) are seen on occasion in the 22*

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nerve endings. Profiles suggesting exteriorization of the content of the 300---500 A vesicles into the synaptic cleft (Fig. 10) and of budding of the vesicles from the tubular smooth membranes are observed (Chen, 1968). Specialized zones, appearing as condensations of cytoplasm, are observed at the synaptie sites both on the nerve terminal and chief cell sides (Figs. 3, 9, 10). These specialized zones are similar to those seen between glomus cells and nerve terminals in the carotid body (Biscoe and Stehbens, 1966 ; Chen, 1968 ; Kobayashi, 1968) and at synapses in the central nervous system (Gray and Guillery, 1966).

Radioautography Fig. 1 is a light microscopic radioautograph showing the distribution of reduced silver grains over a paraganglion after an injection of aH-dihydroxyphenylalanine. The silver grains are predominantly concentrated over the chief cells with only a very few grains over nerve cells, nerve fibers or blood vessels. The electron microscope shows that the labelings are concentrated not only over the chief cells but also are closely associated with membrane-bound electron opaque granules (Fig. 11).

Discussion The paraganglia are conventionally classified into chromaffin or sympathetic and achromaffin or parasympathetic types (Nomina Histologica, 1965). Goormaghtigh (1936) further divided the aehromaffin type into sensory paraganglia connected with afferent fibers, and vagal paraganglia connected with efferent fibers of the vagus nerve. The achromaffin paraganglia were considered to elaborate acetylcholine (Goormaghtigh, 1936) but not catecholamines (Seto, 1949). The chromaffin reaction at the light microscopic level is not a very reliable method for the evaluation of the cellular catecholamine content when there is only a small amount of amine in the cell. Lever et al. (1959) noted that catecholamines disappeared very rapidly from the cells during fixation and that no chromaffin reaction could be definitely detected in the glomus cells of the carotid body unless the fixative was perfused. Recent fluorescent microscopic (Rahn, 1961; Niemi and Ojala, 1964; Hamberger etal., 1966; Blfimcke et al., 1967; Dearnaley et al., 1968), cytochemical (Chiocchio et al., 1967 ; Chen et al., 1969) and radioautographic (Gershon and Ross, 1966; Chen and Yates, 1969) studies have revealed the presence of biogenic amines in the carotid body glomus cells. The present electron microscopic studies have demonstrated that the vagal chief cells also possess membrane-bound, electron opaque granules which are similar in Fig. 8. Portions of three adjacent paraganglion cells separated by cytoplasmic processes of a supporting cell (Su). The larger spaces between the paraganglion and the supporting cells are probably artifacts produced during tissue preparation. Note a few microtubules and the basal body of a cilium with rootlets (Rt) in the supporting cell. • Fig. 9. This micrograph shows two profiles of nerve terminals which directly appose a paraganglion cell. Several specialized zones (Sz) are seen at the synaptic sites. The nerve terminals are filled with numerous synaptic vesicles and some larger vesicles containing electron opaque materials, the density of the latter substances is less than that of the granules in the paraganglion cell. A few profiles of tubular, smooth membranes (arrows) are also discernible in the nerve terminals. P, pigment granule, x 30,000

Figs. 10 and 11

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appearance to the granules in the carotid body glomus cells. Following the administration of a radioactive precursor of catecholamines the labelings are predominantly associated with the granules in the vagal paraganglion cells. With the cytochemical technique for the detection of unsubstituted amines the granules, gave a positive reaction, which indicated t h a t they contained catecholamines, probably norepinephrine or dopamine (Wood, 1966). Our findings are in agreement with those of Brundin (1966) who used fluorometrie techniques and N a k a t a (1964) who employed the chromaffin reaction at the light microscope level. Both these workers demonstrated norepinephrine in the preaortal paraganglia of rabbits, cats and dogs. The dense core of the norepinephrine storing granules in adrenal medullary cells fixed with glutaraldehyde and osmic acid is usually eccentrically located which has been attributed to a vigorous chemical reaction between the content of the granule and the fixative (Coupland and Hopwood, 1966). The dense-cored vesicles in the adrenergie nerve endings which are known to contain norepinephrine are similar in appearance to those in the chief cells of the vagal paraganglia. The amount of amines in the granules in both the nerve endings and the chief cells m a y be too small to produce disproportional shrinkage of the dense cores as seen in medullary cells. From our radioautographie and cytochemical studies we have found that all the chief cells of the vagal paraganglia contain unsubstituted catecholamines. The conventional classification of chromaffin and achromaffin paraganglia is a light microscopic distinction not supported by electron microscopic findings and should be abandoned. 57o direct evidence for the presence of acetylcholine in the paraganglion cells is available from our studies. Recent fluorescent microscopic studies have demonstrated a few small catecholamine-containing cells in the sympathetic ganglia (Jacobowitz, 1961; Er~nk5 and HiirkSnen, 1965; Norberg et al., 1966; Jacobowitz and Woodward, 1968; Winckler, 1969). Electron microscopic studies have shown t h a t these small cells contain numerous membrane-bound granules in the cytoplasm (Grillo, 1966; Siegrist etal., 1966, 1969; Williams, 1967a, b; Elf~fin, 1968; Matthews and Raisman, 1968, 1969; Yates and Mascorro, 1970). These cells exhibit m a n y similarities in fine structure to the chief cells reported in this study. Williams (1967a, b), Siegrist etal. (1968) and Matthews and Raisman (1969) described afferent and efferent synapses in respect to the granule containing cells in the sympathetic ganglia. They suggested t h a t the chief cells are interneurons between the presynaptic nerve fibers and post-ganglionic neurons and t h a t these interneurons m a y modify neuronal activity (inhibitory) either by means of synapses or b y functioning as endocrine structures. I n our studies of the vagal paraganglia we found no axons originating from the granule containing ceils. The nerve terminals which appose the chief cells of the paraganglia are provided with numerous small vesicles, the diameter of which is in the range of synaptic vesicles Fig. 10. Portion of a paraganglion cell and two sections of nerve terminals. Profiles suggesting exteriorization of the content of the vesicles in the nerve terminals into the synaptic gap are seen in at least three places (arrows). • 39,000 Fig. 11. Electron microscopic radioautograph. The distribution of silver grains roughly matches that of the membrane-bound electron opaque granules in paraganglion cells. • 13,000

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in presynaptic nerve terminals (Gray and Guillery, 1966), and in some cases budding of vesicles from the tubular smooth membranes and profiles suggesting exteriorization of the content of the vesicles into the synaptic gaps were seen. I t has been assumed that chemical substances are involved in the transmission of nerve impulses at synaptic sites and that a transmitter must be released from the presynaptic side into the synaptic cleft. We believe that the profiles seen in the nerve terminals is evidence that transmitter substances are discharged into the synaptic cleft. Our findings indicate that the nerve terminals which apposc the chief cells of the paraganglia are probably efferent rather than afferent. Since de Castro's morphological investigations (1928) and Heymans and his co-worker's physiological studies (1930) the carotid body has been considered to be a chemoreceptor. Hollinshead (1941, 1946) suggested the possible existence of similar chemoreceptors in the abdominal region. The striking similarities in histological organization, fine structure, innervation of the hamster paraganglia and carotid body (Chen, 1968) and the presence of catecholamines in both organs implies that these structures may be homologous. If the carotid body is indeed a chemoreceptor then the vagal paraganglia may also have a chemoreceptive function. Unfortunately our morphological studies were not designed to investigate this question specifically but the presence of numerous thin-walled fenestrated capillaries in both the paraganglia and the carotid body suggests that these organs are in an advantageous position for monitoring minor changes in blood constituents. On the other hand the richness of blood vessels and of thin-walled fenestrated capillaries in the organs may simply play a role in the rapid transport of secretory material from the cells to the circulatory system. Siegrist et al. (1968) noted chromaffin cells in the superior cervical ganglion of the rat in close proximity to fenestrated capillaries and they suggested possible endocrine activity for these cells. Such a function was also postulated by Yates and Mascorro (1970). The amines which, according to some investigators (Dontas, 1957; Joels and White, 1967; Ishii, 1967) act as transmitters in the carotid body do not seem to be involved in respiratory chemoreflexes induced by hypoxia (Chen and Yates, 1968; AI-Lami and Murray, 1968; Chen, et al., 1969). In addition, the secretion of amines from the carotid body chief cells seems to be controlled by efferent fibers (Eyzaguirre and Uehizono, 1961; Biscoe and Sampson, 1967) in the sinus nerve (Chen and Yates, 1968). Our evidence indicates that the structures described in this paper as well as the carotid body are examples of paraganglia which are closely associated with cranial nerves and that both structures are effector rather than solely receptor organs. References

A1-Lami, F., Murray, R. : Fine structure of the carotid body of normal and anoxic cats. Anat. Rec. 160, 697--718 (1968). Biscoe, T. J., Sampson, S. R. : Spontaneous activity recorded from central cut end of the carotid sinus nerve of the cat. Nature (Lond.) 216, 294--295 (1967). -Stehbens, W. E. : Ultrastructure of the carotid body. J. Cell Biol. 30, 563--578 (I966). Bliimcke, S., Rode, J., Niedorf, H. R. : The carotid body after oxygen deficiency. Z. Zellforsch. 80, 52--77 (1967). Brundin, T. : Studies on the preaortal paraganglia of newborn rabbits. Acta physiol, scand. 70, Suppl. 290 (1966).

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Castro, F. de: Sur la structure et l'innervation du sinus carotidien de l'homme et des mammif~res. Nouveaux faits sur l'innervation et la fonction du glomns caroticum. Trab. Lab. Invest. Biol. (Madr.) 2~, 331--380 (1928). Chen, I-li: Biogenic amines in t h e glomus cells of t h e hamster carotid body: An electron microscopic, radioautographic, a n d cytochemical study. Dissertation, University of Texas Medical Branch (1968). - - Yates, R. D. : A n electron microscopic s t u d y of the effects of hypoxia a n d reserpine on the glomus cells of the carotid body. Anat. Rec. 160, 330 (1968). - - - - The effects of nerve stimulation or transection on the glomus cells of the carotid body. J. Cell Biol. 89, 24a (1968). - - - - The electron microscopic radioautographic studies of the carotid body following injections of labeled biogenic amine precursors. J. Cell Biol. 42, 794--803 (1969). - - - - Duncan, D. : The effects of reserpine and hypoxia on the amine-storing granules of the hamster carotid body. J. Cell Biol. 42, 804--816 (1969). Chiocehio, S. R., Biscardi, A. M., Tramezzani, J. H. : 5-hydroxytryptamine in the carotid body of t h e cat. Science 158, 790--791 (1967). Coupland, R. E. : The development and fate of the abdominal chromaffin tissue in the rabbit. J. Anat. (Lond.) 90, 527--537 (1956). The post-natal distribution of the abdominal chromaffin tissue in the guinea pig, mouse a n d white rat. J. Anat. (Lond.) 94, 244--256 (1960). - - Hopwood, P. : The mechanism of the differential staining reaction for adrenalin and noradrenalin storing granules in tissue fixed in glutaraldehyde. J. Anat. (Lond.) 1D0, 2 2 7 - 243 (1966). Dearnaley, D. P., Fillenz, M., Woods, R. I. : The identification of dopamine in the r a b b i t ' s carotid body. Proc. roy. Soc. B 170, 195--203 (1968). Dontas, A. R. : Effects of reserpine a n d hydralazine on carotid a n d splanchnic nerve activity and blood pressure. J . Pharmacol. exp. Ther. 121, 1--7 (1957). Duncan, D., Yates, R. D. : Ultrastructure of the carotid body of the cat as revealed b y various fixatives and the use of reserpine. Anat. Rec. 157, 667--681 (1967). Elfvin, L. G. : A new granule-containing nerve cell in the inferior mesenteric ganglion of t h e rabbit. J. Ultrastruct. Res. 22, 37--44 (1968). Er~nk5, 0., H~rkSnen, M. : Monoamine-containing small cells in the superior cervical ganglion of the r a t a n d organ composed of them. Acta physiol, scand. 63, 511--512 (1965). Eyzaguirre, C., Uchizono, K. : Observations on the fiber content of nerves reaching the carotid body of the cat. J. Physiol. (Lond.) 159, 268--281 (1961). Garner, C.M., Duncan, D. : Observations on the fine structure of the carotid body. Anat. Rec. 139, 691--709 (1958). Gershon, M. D., Ross, L. L. : Location of sites of 5-hydroxytryptamine storage a n d metabolism b y radioautography. J. Physiol. (Lond.) 186, 477--492 (1966). Goormaghtigh, N. : On the existence of abdominal vagal paraganglia in the a d u l t mouse. J. Anat. {Lond.) 71, 77--90 {1936). Gray, E. G., Guillery, R. W. : Synaptic morphology in the normal a n d degenerating nervous system. Int. Rev. Cytol. 19, 111--182 {1966). Grillo, K. : Electron microscopy of sympathetic tissues. Pharmacol. Rev. 18, 387--399 (1966). Hagen, P., Barrnett, R. J . : The storage of amines in the chromaffin cell. I n : Adrenergie mechanisms (eds. J. R. Vane, G. E. W. Wolstenholme and M. O'Connor) p. 83. Boston: Little, Brown a n d Co. 1960. Hamberger, B., Ritzen, M., Wers~ll, J. : Demonstration of cateeholamines a n d 5-hydroxyt r y p t a m i n e in the h u m a n carotid body. J. Pharmacol. exp. Ther. 152, 197--201 (1966). Hess, A. : Electron microscopic observations of normal a n d experimental cat carotid bodies. I n : Arterial chemoreceptors (ed. R. W. Torrance), p. 51--56. Oxford-Edinburgh: Blackwell Scientific Publications 1968. Heymans, C., Bouckaert, J . J . , Dautrebande, L. : Sinus carotidien et r6flexes respiratoires. II. Influences respiratoires r6flexes de l'acidose, de l'alcalose, de l'anhydride carbonique, l'ionhydrog~ne et anoxemie. Sinus carotidiens et 6changes respiratoires clans les poumons et au-dels des poumons. Arch. int. Pharmaeodyn. 89, 400--447 (1930). -

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R. D. : An electron microscopic study of the effects of reserpine on adreno-medullary cells of the Syrian hamster. Anat. Ree. 146, 29--45 (1963). - - A light and electron microscopic study correlating the chromaffin reaction and granule ultrastructure in the adrenal medulla of the Syrian hamster. Anat. Rec. 149, 237--250 (1964). - - Mascorro,J. A. : Electron microscopic studies of sympathetic paraganglia. Anat. Rec. 166, 400 (1970). I-li Chen Department of Anatomy College of Medicine National Taiwan University No. 1, Section 1, Jen-ai Road Taipei, Taiwan Republic of China