Comparative Morphology, Cytochemistry and Innervation of Chromaffin Tissue in Vertebrates

Home , Chromaffin cell

J. Anat. (1993) 183, pp. 327-342, with 14 figures Printed in Great Britain 327 Comparative morphology, cytochemistry and innervation of chromaffin tissue in vertebrates

DIETRICH W. SCHEUERMANN Laboratory of Cell Biology and Histology, University of Antwerp, Belgium

(Accepted I March 1993)

ABSTRACT Chromaffin cells were observed singly or in clusters in the heart and sympathetic cord of 2 genera of dipnoan fish, Protopterus and Lepidosiren. They were invariably found in close association with the autonomic sympathetic nervous system and at sites where chromaffin cells or their precursors are situated in mammals during ontogenetic development. X-ray microanalysis demonstrated that they contained a primary catecholamine which was identified microspectrofluorometrically as dopamine. The chromaffin cells were innervated by efferent axons with typical preganglionic sympathetic terminals which were acetylcholinesterase-positive. Although the general morphology and cytochemistry agree with those of developing intra-adrenal chromaffin cells in mammals, the morphological characteristics implicate them as active secretory gland cells. The dopamine transmitter phenotype seems to be determined by the maintenance throughout life of the separate and distant location of steroidogenic interrenal tissue from suprarenal elements.

Chromaffin cells originate from the neural crest INTRODUCTION lineage (for review, see Le Douarin, 1982) and share For over a century it has been known that cells of the many morphological and physiological characteristics adrenal medulla become brown when treated with with peripheral sympathetic neurons (Hervonen, chrome salts (Henle, 1865). Kohn (1898) coined the 1971; Fenwick et al. 1978; Coupland, 1980; Car- term 'chromaffin', confirming Koelliker's conception michael & Winkler, 1985), which is not true for that these cells belong to the sympathetic nervous enterochromaffin and mast cells (Andrew, 1963, 1974; system (Kohn, 1903). More recently, it has been Fontaine & Le Douarin, 1977). Coupland (1965a, shown that in the adrenal medulla a correlation exists 1972) therefore defined chromaffin cells as 'elements between the amount of catecholamines and the developed from neuroectoderm, innervated by pre- intensity of the chromaffin reaction (e.g. Eranko & ganglionic sympathetic fibres, capable of synthesising, Raisanen, 1961). Quite similar to the cells of the secreting and storing catecholamines'. The cells that adrenal medulla are extra-adrenal chromaffin cells do not conform to this definition should be called found in the retroperitoneal tissues of all mammalian 'chrome-reacting' cells. species (see e.g. Coupland, 1952). They are often more In the adrenal chromaffin cells of almost all prominent in lower vertebrates. mammals, 2 basic types of granules with different Cells containing indolethylamine derivatives, such electron densities can be recognised, mostly of the as enterochromaffin cells and mast cells, may yield a low-density type (Coupland et al. 1964; Wood & positive chromaffin reaction due to their 5-hydroxy- Barrnett, 1964; Coupland, 1971, 1972; Carmichael & tryptamine content (Hopwood, 1971; Coupland, Blair, 1973). Although the ultrastructure ofabdominal 1972; B6ck, 1982). Thus chromaffinity as such is not extra-adrenal chromaffin tissue resembles in many sufficiently specific to conform to Kohn's concept of respects that of the adrenal medulla, it contains 'chromaffin tissue' (Coupland, 1965a; Jacobowitz, mainly highly dense granules (Brundin, 1966). Ad- 1967; B6ck, 1982). ditional types of chromaffin cells harbour dense-cored

Correspondence to Professor D. W. Scheuermann, Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium.

23-2 328 D. W. Scheuermann vesicles markedly smaller than those found in intra- 1966; Coupland & Weakly 1968, 1970; Hervonen, and extra-adrenal chromaffin cells; they are therefore 1971), thus not always meeting the requirements to commonly referred to as small granule-containing qualify as true chromaffin cells. (SGC) cells, first discovered in sympathetic ganglia Experimental approaches to quail-chick chimera and more recently in the adrenal medulla of mammals (for review, see Le Douarin, 1982) corroborate Kohn's (Coupland et al. 1977; Kobayashi & Coupland, 1977; observations (1903) on embryos of the cat, rabbit and Kajihara et al. 1978; Unsicker et al. 1978), birds man, indicating that primitive sympathetic cells start (Unsicker, 1973) and reptiles (Unsicker, 1976). An- differentiating after a migratory period and that local other classification has been made on the basis ofboth environmental factors define their differentiation into the size of the granules and their relative electron chromaffin cells and their final destination. As in density, showing different intermediate types in the numerous instances of anatomy, the sequence seen in adrenal medulla ofthe mouse (Gorgas & B6ck, 1976), the process of individual development of higher the significance of which remains controversial. vertebrates might be paralleled in comparative As varied as the cytology of the different kinds of anatomy in passing from lower to higher forms. In the chromaffin cells are the biogenic amines they contain. early development of mammals, chromaffin cells are The predominant catecholamine secreted by the localised at sites where they are finally located in adult adrenal chromaffin cells of adult mammals is adren- primitive vertebrates. We will therefore refer to aline (A) (Coupland, 1965 a; Weiner, 1975). Nora- phylogenetic considerations on chromaffin tissue in drenaline (NA) is also secreted to some extent, some lower vertebrate classes. depending on the species investigated (Hagen & The 2 components which make up the adrenal Barrnett, 1960; Coupland et al. 1964; Winkler & gland in mammals remain separated in lower verte- Smith 1975). In extra-adrenal chromaffin cells, almost brates to varying extents. In cyclostomes and elasmo- only NA has been found (Shepherd & West, 1952; branchs both tissues are anatomically separate Brundin, 1966; Battaglia, 1969; Coupland & Weakly, throughout life, the interrenal tissue being the hom- 1970). Dopamine (DA) has been identified in SGC ologue of the cortex and more dispersed chromaffin cells in sympathetic ganglia (Norberg et al. 1966; cell groups of the medulla. Chromaffin cells appear in Bjorklund et al. 1970; Erank6 & Eranko, 1971; Libet the abdominal wall in close relationship to sym- & Owman, 1974; Chiba & Williams, 1975; Gorgas & pathetic neurons, along the vertebral column, the B6ck, 1976; Chiba et al. 1977; Hervonen et al. 1979; great vessels and in the heart (Balfour, 1878; Chevrel, Neffet al. 1983; Scheuermann et al. 1984; Forsgren et 1889; Grynfeltt, 1903; Giacomini, 1906; J. F. Gaskell, al. 1990). There seem to exist subtypes of SGC cells 1912; W. H. Gaskell, 1920; Krause, 1923; Lutz & with regard to catecholamine content, most of them Wyman, 1927; Goodrich, 1930; Young, 1933; Berkel- containing DA, others containing NA (e.g. rat, bach van der Sprenkel, 1934; Nicol, 1952; Augustins- Erank6 & Erank6, 1971; Heym et al. 1980; guinea son et al. 1956; Ostlund et al. 1960; Bloom et al. 1961; pig, Elfvin et al. 1975) and A (man, Schr6der et al. von Euler & Fange 1961; Coupland 1965 a; Gannon 1982). In SGC cells of the adrenal medulla of the et al. 1972). guinea pig NA and A (Unsicker et al. 1978) and in The organisation of the autonomic nervous system human thoracic sympathetic ganglia, NA, A and DA from cyclostomes to extant elasmobranchs shows a have been found (Schr6der et al. 1981). basic plan from which the more complex systems have Innervation of chromaffin cells by preganglionic evolved, leading on the one hand to actinopterygian sympathetic nerve fibres constitutes a major element fish, to which teleosts are believed to belong, and on in Coupland's definition. Most of the axons derive the other to Dipnoi and tetrapods. The autonomic from neurons, the perikarya of which lie in the nervous system in dipnoan fish is less differentiated intermediolateral column of the thoracolumbar cord, than in teleosts, with an organisation progressively and course in the splanchnic nerves through the adapted for terrestrial existence. Nicol (1952) and chromaffin cells (Coupland, 1965 c; Kesse et al. 1988; Holmes & Moorhouse (1956) emphasised that for an Parker et al. 1988, 1990). The morphological evidence appraisal of the chromaffin system of vertebrates, of the cholinergic nature of these nerves concurs with Dipnoi are better suited for its examination than the the finding that, in vivo, adrenal medullary chromaffin more divergent teleosts. cells are activated by the release of acetylcholine from It should be remembered that in dipnoan fish the the splanchnic nerves (Feldberg et al. 1934). Prior to chromaffin component of the adrenal gland is repre- birth, extra-adrenal chromaffin cells in some species sented by cells originating from the neural crest. These seem to be sparsely innervated, if at aft (Brundin, migrate through retroperitoneal mesenchyme to dif- Comparative cytology of chromaffin tissue 329

*E::s9^XS9n;fii*RX

2F 330 D. W. Scheuermann ferentiate into chromaffin cells along different lines, (pH 7.1) for 5 h (GA-dichromate method). The tissues yet do not form in conjunction with the interrenal were dehydrated through acetone and embedded in organ a glandula suprarenalis (Holmes, 1950). Steroi- Durcupan. Semithin sections were stained with tol- dogenic tissue has been found in the wall of the renal uidine blue/basic fuchsin. Thin sections were viewed veins in 2 genera of Dipnoi: Protopterus and Neo- both unstained and counterstained with uranyl acetate ceratodus (Janssens et al. 1965; Call & Janssens, and lead citrate. Granules of thin sections of tissues 1975), but nowhere in association with chromaffin treated with the simultaneous and the subsequent cells. A preliminary electron microscope investigation, GA-dichromate technique were analysed with a Kevex which identified intracellular biogenic amines using energy-dispersive X-ray microanalyser system in a X-ray microanalysis and microspectrofluorometry, Siemens Elmiskop 101 in TEM and STEM to localise has provided evidence that in Protopterus aethiopicus the incorporation of chromium at the site of the chromaffin cells are located in the wall of the auricle reaction product (Wood, 1974). and sinus venosus of the heart. The present study on chromaffin tissue of Dipnoi has been expanded for the heart of the species Protopterus annectens and the Histochemistry genus Lepidosiren and for both with regard to For the chromaffin cells in the sympathetic cord close to the demonstration of cholinergic nerves electron intercostal branches of the dorsal aorta. microscopically the Karnovsky & Roots (1964) technique for acetylcholinesterase (AChE) activity was used, followed by amplification of the Hatchett's brown by polymer generation with 3,3'-diamino- MATERIALS AND METHODS benzidine tetrahydrochloride (DAB) according to the method of Adult specimens of Protopterus aethiopicus, Protop- Hanker et al. (1973), with acetylthiocholine as terus annectens and Lepidosiren paradoxa were ex- substrate. amined (2 of each). These African and South American lungfish were kept in a low-water aquarium Fluorescence at 24 °C under near-physiological conditions and microscopy apparently displayed normal behaviour. The heart This was carried out on unfixed small pieces of the was removed after anaesthesia with Hypnorm same tissues from the remaining fish of each species; (J. Philips-Duphar). Segments of the sympathetic they were freeze-dried for 5 d at -45 °C and at a cords, close to the origin of the intercostal branches pressure of 10-2 Torr in a Leybold-Heraeus GTOO1 of the dorsal aorta, were dissected out. apparatus with phosphorus pentoxide as a water trap. The temperature of the dried tissues was slowly raised to 25 °C, the vacuum broken and the tissues exposed Electron microscopy for 2 h at 80 °C to paraformaldehyde vapour generated from paraformaldehyde powder equilib- Small tissue samples of the sympathetic chain, the rated at 60% relative humidity (Hamberger et al. sinus venosus and the auricle of the heart from 1 1965). The tissues were embedded in filtered paraffin animal from each species were fixed by immersion under vacuum, sectioned at 10 gm and mounted in either in 4% glutaraldehyde (GA) buffered at pH 7.2 fluorescence-free paraffin oil (Bayol F, Serva Feinbio- by 0.15 M sodium cacodylate for 8 h, or a mixture chemica, Heidelberg, Germany). The fluorescence containing 3 % GA and 2.5 % potassium dichromate. specificity was examined by means of the Corrodi test Part of the GA-fixed tissues was postfixed in a 1 % (Corrodi et al. 1964) performed with a solution of aqueous OSO4 solution for 2 h. Another part was 0.03 % sodium borohydride in 90% isopropanol further treated with a solution of 4% GA containing (2 min), followed by a second exposure to form- 2.5 % potassium dichromate in a 0.2 M acetate buffer aldehyde vapour for 2 h at 80 °C for fluorescence

Fig. 1. Chromaffin cells in the right wall of the auricle of Lepidosiren, filled with abundant highly dense granules on fixation with GA and Os04 Unmyelinated nerve fibres (NJ) with associated Schwann cell cytoplasm (S) and their basement membrane (arrows) as observed in the inset. The space separating the 2 basement membranes contains collagen fibrils (arrowhead). Bars, 0.5 gtm (main figure), 0.2 gm (inset). Fig. 2. Chromaffin cells in the right wall of the Protopterus annectens auricle with homogeneously dense granules. Unmyelinated nerve fibres (NJ) with associated Schwann cell cytoplasm (S) run amongst chromaffin cells; to the right are cross sections through bare invaginated nerve endings. Bar, 1 gm. Comparative cytology of chromaffin tissue 331

-t4, 4< .'-

Fig. 3. Process of a chromaffin cell partly covered with Schwann cell cytoplasm (S) in the right wall of the auricle of Protopterus annectens. Membrane-bound granules appear to fuse with the plasma membrane (arrows). Bar, 0.2 gm. Fig. 4. A chromaffin cell with an expanded Golgi complex (G) in the right wall of the auricle ofLepidosiren. Golgi vesicles and cisterns contain immature granules (arrows). Bar, 0.2 gim. regeneration. For recordings of excitation spectra and models were prepared from DA HCl and NA HCl fading curves, sections were mounted on quartz according to the method of Reinhold & Hartwig microscope slides (Suprasil Heraeus Quartzschmelze, (1982). Fading curves were registered and spectra Hanau, Germany). Microspectrofluorometric analysis corrected following the procedure previously outlined in the extended excitation range from 240-460 nm, (Stilman & Scheuermann, 1987). particularly to differentiate NA from DA, was carried Measurements of the vesicle diameters were carried out with an automated microspectrofluorometer de- out on micrographs of tissue fixed in GA, postfixed veloped in this laboratory as a modification of the with OSO4, counterstained on grid with uranyl acetate MPV II (Leitz, Wetzlar, Germany) with the instru- and lead citrate, and examined at a final magnification mental configuration as previously reported (Stilman of x 55000. A test grid of 2160 lines/mm was used as & Scheuermann, 1987). a control. The largest diameter was measured with a To obtain formaldehyde-induced fluorophores at Kontron MOP/AMO2 image analysis system. neutral pH, sections were deparaffinized with xylene We have been unable to achieve specific immuno- and exposed to ammonia gas. To ensure an accurate cytochemical visualisation of either DA or dopamine- differentiation of formaldehyde-induced fluorescence ,B-hydroxylase because of the unavailability of ap- (FIF), acidification of samples is required (Bj6rklund propriate species-specific antisera. et al. 1972; Reinhold & Hartwig, 1982; Stilman & Scheuermann, 1987). Deparaffinised sections were therefore treated for 10 min with hydrochloric acid (HCl) vapour. Fluorescence spectra from droplet 332 D. W. Scheuermann

(lysosome-like) bodies were distributed in the cytoplasm (Fig. 9). Schwann cells with their nuclei exhibiting concentrated chromatin partly covered the surface of granule-containing cells and sympathetic neurons and enclosed nerve fibres (Figs 1 (inset), 2, 3, 6). Granule-containing cells were the most numerous. Their nuclei bore dense collections ofheterochromatin next to the nuclear membranes (Figs 1, 2). They were polygonal or fusiform, amply digitated with processes of varying length, and often found apposed to the basal lamina of the endocardium (Fig. 5) or of capillaries in the epicardium and even among con- tractile myocardial cells. Their most distinctive feature was the presence of a large number of dense-cored vesicles, in the 40-400 nm diameter range (measured Jk to the surrounding membrane), although cells with larger and others with smaller dense-cored vesicles were encountered (Fig. 6). In many instances a light halo occurred between the limiting membrane and the dense core (Figs 7, 8). In larger vesicles, the dense core lay centrally or adhered eccentrically to the membrane. After initial fixation in GA and postosmication, all types of chromaffin cells of the dipnoan species investigated were found to contain exclusively the homogeneously highly dense granular type, with a high degree of polymorphism, resembling the NA- Fig. 5. Relationship between the endocardium (E) of the auricular containing cells described in the extra-adrenal chro- chamber (C) of Protopterus annectens, showing chromaffin cells Some immediately adjacent to the flattened endothelium. Chromaffin cells maffin cells of higher vertebrates. chromaffin can also be found apposed to the epicardium (Ep). The chromaffin cells contained much smaller dense granules, in the cells contain numerous lysosome-like (autophagic) bodies (arrow- 40-60 nm diameter range, and belonged to SGC cells heads). Fixation by the GA-dichromate method. Bar, 2 gm. (Fig. 10). In sections of tissue treated with the 2 GA- dichromate techniques, X-ray microanalytical re- RESULTS cordings of the granules showed characteristic chro- In the dipnoan fish investigated, the sympathetic mium peaks when compared to nongranular areas, chain, without any obvious ganglionic swellings, suggesting that the granules could contain NA (Fig. appeared as a small cord lying on either side of the 13a, b). aorta, confirming the findings of Giacomini (1906), Some of the granular vesicles appeared to fuse with Jenkin (1928) and Holmes (1950). Stained semithin the plasma membrane (Fig. 3). Exocytotic figures sections examined by light microscopy showed single were observed with coated vesicles, indicating dis- or grouped ganglion cells and chromaffin cells close to charge of the contents of the granule for cate- the origin of segmental arteries which leave the dorsal cholamine secretion, followed by recycling of the aorta. In the auricle and sinus venosus of the heart, granule membrane. A well-developed Golgi field was chromaffin cells were seen to occur in clusters. On situated near the nucleus (Figs 4, 8). Some cisterns of electron microscopy, at least 3 cell types could be the Golgi apparatus contained granular material that distinguished: ganglion cells, Schwann cells and seemed to represent an early stage in the formation of granule-containing cells. Ganglion cells accounted for granules (Fig. 4). The rough endoplasmic reticulum only a very small proportion of the cell population. was frequently arranged in stacks of parallel arrays These cells, which were 20-35 jim in diameter, (Fig. 7). Mitochondrial profiles, glycogen particles, have evenly dispersed chromatin and a prominent free ribosomes, microtubules and microfilaments were nucleolus. Short arrays of rough endoplasmic re- common. Autophagic bodies in which chromium was ticulum associated with free ribosomes and dense detected by X-ray microanalysis were particularly Comparative cytology of chromaffin tissue 333

M-1 ~-

1L --- A Q L

Fig. 6. Cluster of chromaffin cells with highly dense granules lying adjacent to the intercostal branches of the dorsal aorta in Lepidosiren. A Schwann cell (S), the nucleus of which exhibits concentrated chromatin, accompanies nerve fibres (NJ. The cytoplasm contains large lysosomes and microfilaments. Bar, 1 gm. numerous, suggesting that undischarged secretory fluorescence (Fig. 14a). A positive Corrodi test material is stored in these organelles, thereby dem- demonstrated the specificity of the histochemical onstrating the mechanism of crinophagy (Figs 5, 11). reaction for monoamines. When excited at 415 nm, Chromaffin cells of dipnoan fish, inclusive of SGC the fluorescence emission maximum was found at cells, were richly innervated by numerous unmye- 480 nm, indicative ofcatecholamine(s) (Fig. 14b). The linated axon profiles apposed to the cell surface excitation spectrum, at neutral pH, showed a maxi- membrane with a flattened or club-shaped terminal mum peak at about 410 nm and additional low peaks (Fig. 11). They contained a few slender mitochondria, at 330 and 260 nm (Fig. 14d). The mean peak ratio numerous small (40-60 nm) clear-cored and a few values 410/260, always exceeding 1, are indicative of larger (120-200 nm) vesicles with moderately electron- a primary catecholamine. The maximum excitation dense cores. Well-developed afferent synaptic struc- peak at 410 nm, obtained at neutral pH, revealed, on tures with obvious membrane densities on both the treatment with HCI, a shift to 380 nm, with additional axolemma and chromaffin cell membrane were en- peaks at 320 and 260 nm, yielding mean peak countered (Fig. 11). The dark deposit of the AChE ratio values 380/320 nm > 0.8 (Fig. 14e). Prolonged reaction was most prominently localised on the outer acidification, even up to 20 min, did not induce further surface of the plasma membrane of the chromaffin changes. The fading rate showed a decrease of less cells and between the membrane ofthe nerve fibre and than 20% compared with the original fluorescence the chromaffin cell (Fig. 12). Nerve endings with intensity after 5 min of illumination with the most vesicular profiles with moderately electron-dense effective excitation wavelength, both before and after cores, typical of neuropeptide content, were also HC1 treatment (Fig. 14c)., observed. Sections of tissue segments of the auricular wall of the heart and of the sympathetic cord prepared for fluorescence microscopy revealed a bright blue-white 334 D. W. Scheuermann

.2v~ ..... _ z Atiaw..~ ~ ~ ~ 1~~A

i.111~ AN

Fig. 7. Section through a chromaffin cell in the wall of the auricle of Protopterus annectens showing a stack of parallel cisterns of rough endoplasmic reticulum near the nucleus. Bar, 0.1 gm. Fig. 8. Expanded Golgi complex near the nucleus of a chromaffin cell in the auricular wall of Lepidosiren. Bar, 0.1Ilm.

bound to the dense core of vesicles, reveals that DISCUSSION chromaffin cells are the storage site of a primary In a previous electron microscopic study (Scheuer- catecholamine. Indeed, it has been shown exper- mann, 1980) it was demonstrated that the ultra- imentally that NA and DA-containing cells react in a structural characteristics of chromaffin cells localised similar way with GA, forming a precipitate as a water- in the heart of Protopterus aethiopicus are basically insoluble amine-GA complex (Cannata et al. 1968; similar to those already described in the adrenal Hopwood, 1971; Mastrolia et al. 1981), thereby medulla of higher vertebrates (for reviews, see Coup- enabling the reaction with chromium salts. Adren- land 1965b, 1989). In addition, microspectrofluoro- aline, however, is lost in the fixing GA solution metric investigation has shown that these chromaffin (Coupland et al. 1964, 1976; Tramezzani et al. 1964; cells contain a primary catecholamine, namely DA Coupland & Hopwood, 1966; Cannata et al. 1968; (Scheuermann et al. 1981). Hopwood, 1971). Marked granular electron density and poly- Three different catecholamines, A, NA and DA, morphism among granules in the cells concerned do may be synthesised in chromaffin cells of vertebrates not permit the recognition of the existence of 2 clearly as hormones in a 4-step process (see Blaschko, 1959; distinct types of granules. These findings are in Kirshner, 1975) to manufacture A. For their direct in keeping with the observations on chromaffin tissue in situ identification, advanced microspectrofluorometry some lower vertebrates such as primitive urodeles (e.g. is a fairly sensitive and specific histochemical method Accordi, 1991). (Bjorklund et al. 1968). Indolethylamine derivatives The chromaffin reaction in the cytoplasmic granules can be differentiated from catecholamines by record- after initial fixation with GA at pH 7.2 and X-ray ing the fluorescence emission spectra (Bjorklund et al. microanalysis, indicating that chromium is specifically 1975; Stilman & Scheuermann 1987; and others). Comparative cytology of chromaffin tissue 335

To differentiate between DA, NA and A, differences in the corrected excitation spectra of their formaldehyde-induced fluorophores, recorded with a microspectrofluorometer extended to a wavelength range of 240-460 nm at neutral pH, permitted the exclusion of the presence of secondary amines. Hence the recorded excitation spectra with peak ratio values 410/260, always exceeding 1, are indicative ofprimary catecholamines (Reinhold & Hartwig, 1982; Stilman & Scheuermann, 1987) and thus support the results of X-ray microanalysis, confirming the exclusion of A. Microspectrofluorometrically, NA and DA can be distinguished by the differing behaviour of their fluorophores after conversion into their nonquinoidal form upon exposure to HCl vapour. The maximum excitation peak of the acidified DA fluorophore shifts to 380-370 nm, with a lower excitation peak at 320 nm, and is not influenced by prolonged HCl treatment, whereas the acidified NA fluorophore only transiently exhibits a maximum excitation peak at this wavelength (Bjorklund et al. 1968). The predominant occurrence of DA in the cells concerned is clearly apparent from the stable peak ratio values 380/ Fig. 9. Section through the sympathetic cord of Lepidosiren with 320 nm, even on prolonged HCl treatment, always part of a nerve cell body (N) covered by Schwann cell cytoplasm (S). Note the clear nucleus (Nu), Nissl bodies (Ni) and dense bodies (Db) exceeding 0.8. The gradual change of the maximum in the neuronal cytoplasm. At the top, a chromaffin cell process can excitation peak of NA from 380-370 to 320 nm gives be seen adjacent to the neuron. Bar, 0.5 rm. rise to peak ratios below 0.7 (Bjorklund et al. 1972, 1975; Hartwig et al. 1979; Reinhold & Hartwig, 1982; Stilman & Scheuermann, 1987). The low and slow photodecomposition rate of the fluorophores in acid state on exposure to UV light is also specific to DA, because the NA fluorophore shows a markedly higher and faster photodecomposition rate. The recorded data accord with those obtained from DA-containing droplet models (Stilman & Scheuermann, 1987). It cannot be excluded, however, that the chromaffin cells in the species studied store a small fraction of NA besides DA, since their simultaneous in situ occurrence cannot be evaluated with the available methods. Abrahamsson et al. (1979a) found that NA and A were present in chromaffin tissue of the heart of Protopterus aethiopicus after a nonspecific fluor- ~I escence microscopy investigation followed by the biochemical method of Haggendal 44 quantification (1963). Moreover, the presence of the enzyme dopamine-,8-hydroxylase (DBH) and of phenyl-etha- nolamine N-methyltransferase (PNMT) in the auricle was studied in tissue extracts (Abrahamsson et al. Fig. 10. High magnification of a small granule-containing cell with 1979b). The large amount of nonchromaffin tissues small sized electron-dense granules in the auricular wall of homogenised, as the authors themselves admitted, Protopterus annectens. Note the dense core and the limiting together with the small amount of chromaffin tissue membrane with an intervening clear halo. Bar, 0.1Igm. proper, does not make for an easy interpretation. The morphological characteristics of SGC cells of 336 D. W. Scheuermann

Fig. 11. A bulbous presynaptic axonal ending containing small clear centred vesicles and a few larger dense-cored vesicles (small arrows) to a synaptic connection with a chromaffin cell (ChC) in the right auricular wall of Lepidosiren. The arrow indicates the direction of the polarity of the synapse. Large autophagic bodies (arrowheads). Bar, 0.1 im. Fig. 12. AChE reaction product (arrows) is shown between an axonal dilatation and a chromaffin cell in the auricular wall of Protopterus annectens. Small clear vesicles are aggregated near the presynaptic axonal membrane (arrowhead). AChE reaction product is localised in the synaptic cleft. Bar, 0.2 gm.

Dipnoi appear to be identical to those found in (a) different autonomic ganglia ofa variety ofvertebrates. Immunocytochemical techniques have shown that most of them contain DA and some NA or A (for Cr review, see Eranko et al. 1986). Since biochemical analysis provided evidence that the adrenal medulla contains small amounts of DA (Shepherd & West, RP Cr 1953; Eade, 1958), it has been suggested that adrenal SGC cells store DA (Grynszpan-Winograd, 1975). 0 20 keV The anatomical proximity of the adrenal cortex to the medulla, and hence of locally high concentrations (b) of glucocorticoid hormones to chromaffin cells, has been proved to activate the enzymes DBH (Gewirtz et al. 1971) and PNMT (Wurtman & Axelrod, 1966), catalysing in the steps in the formation of NA from DA and of A from NA. This is substantiated by observations that during fetal life, the catecholamine content of the adrenal chromaffin cells in mammals is

CI Cr Fig. 13. X-ray microanalytical spectrum, recorded from a dense K Cr granule after treatment by the GA-dichromate technique, showing Fe a large amount of chromium (a), as compared with nondense 0 20 keV cytoplasmic areas (b). Comparative cytology of chromaffin tissue 337 Q'I QA max (b) 100% (C) 1 ?

0 1. . 05 + v~~~~~~~~~~~~ ta 50%*

F A AAA AA~~~~~~~~I.

n0/ OT|'~~~~~~ 0 1 2 3 4 5 min 400 500 600 700 nm Q2A QAi (e) QA max (d) QA max 1 1

I+ +

+ + ++ ++ + ,.,, ~+ + ++ + + I

+ N ~+ +

+ + 0-5 + ~ 0*5-

. . 0", ., , I .Is

200 300 400 500 nm 200 300 400 500 nm

Fig. 14. (a) Section through the auricular wall of Lepidosiren showing cells with a bright FIF. (b, c, d, e) Spectral recordings of the FIF cells of (a) (b) Characteristic emission spectrum of catecholamines. (c) Fading curve typical of dopamine. (d) Excitation spectrum obtained at neutral pH with peak ratio values 420-260 nm > 1, characteristic of primary catecholamines. (e) Excitation spectrum obtained at acid pH, with prolonged HCl treatment, demonstrating the characteristic peaks of the dopamine fluorophore. exclusively NA, A appearing later in correlation with Innervation of the chromaffin cells of Dipnoi seems the cortical development (Shepherd & West, 1951; in most aspects to correspond to that of the intra- West et al. 1951; H6kfelt, 1952; Coupland, 1953, adrenal chromaffin cells in higher vertebrates. Afferent 1956; Niemineva & Pekkarinen, 1953; Coupland & synaptic contacts to granule-containing cells and clear Weakly, 1968). Comparative investigations have synaptic vesicles, resembling the typical preganglionic shown that an increasing proportion of A in the sympathetic terminals described by Richardson adrenal medulla occurs during evolution, indicating (1964), appear to be morphologically mature. Ultra- that subsequent enzymatic activities catalysing bio- structural histochemistry for AChE indicates that synthesis of catecholamines are parallel events in these nerve fibres may indeed be considered to be ontogenetic and phylogenetic development, which are cholinergic in nature. Acetylcholine may be involved both subject to the biochemical influence of local in the release of amines from catecholamine-con- levels of cortical steroids (Coupland, 1953, 1968; taining tissues (for review, see Lewis, 1975). Although West, 1955; Wright & Jones, 1955; Ghosh, 1962; sympathetic preganglionic (cholinergic) nerve fibres Lempinen, 1964; Coupland & MacDougall, 1966; make up the majority of efferent fibres to chromaffin Eranko et al. 1966; Eranko & Erank6, 1972; Lewis, cells in dipnoan fish, the morphological features of 1975; Accordi, 1991). some dense-cored vesicles in nerve terminals resemble In chromaffin cells of dipnoan fish, catecholamine those of peptide-secreting neurons. Recently, in some synthesis ends mainly with DA, just as it does in some mammalian species both a postganglionic adrenergic nuclei of the brain and in type I cells of the carotid and vagal efferent and afferent innervation, as well as body (for review, see Verna, 1979). The DA trans- a spinal afferent innervation, have been found (Kesse mitter phenotype seems to be determined by the et al. 1988; Coupland et al. 1989; Parker et al. 1990). maintenance throughout life of the separate and The neural circuits, involved in the homeostatic distant location of steroidogenic interrenal tissue from mechanisms regulated by chromaffin cells, are as yet suprarenal elements. unknown in lower vertebrates. 338 D. W. Scheuermann

Dense-cored vesicles have been seen to fuse with the differentiate into mature chromaffin cells, in a similar surface membrane of chromaffin cells. The latter are way as they would in the adrenal medulla of adult localised near the endothelium of the blood stream higher vertebrates. This concurs with the finding that with only an intervening basement membrane and a chromaffin cells in developing sympathetic ganglia small amount of connective tissue. These structures and those in the adrenal medulla ofmammals respond suggest that the granule content is released from the basically in a similar way to cortical hormones cell by exocytosis (Diner, 1967; Smith, 1971; Bene- (Erank6 et al. 1982). deczky & Smith, 1972; Grynszpan-Winograd, 1975; Nagasawa, 1977; Kobayashi & Serizawa, 1979; Gronblad et al. 1980). The enlarged Golgi apparatus REFERENCES and the presence and amount of rough endoplasmic ABRAHAMSSON T, HOLMGREN S, NILSSON S, PETTERSSON K (1979 a) reticulum may represent additional morphological On the chromaffin system of the African lungfish, Protopterus parameters that identify the chromaffin cells of the aethiopicus. Acta Physiologica Scandinavica 107, 135-139. ABRAHAMSSON T, JONSSON A-C, NILSSON S (1979b) Catecholamine Dipnoi investigated as mature, highly active secretory synthesis in the chromaffin tissue of the African lungfish, cells. Protopterus aethiopicus. Acta Physiologica Scandinavica 107, It seems conceivable that sympathetic preganglionic 149-151. cholinergic nerves on in fish ACCoRDI F (1991) The chromaffin cells of urodele amphibians. chromaffin cells dipnoan Journal of Anatomy 179, 1-8. regulate primarily the secretion of DA. The pro- ANDREW A (1963) A study of the developmental relationship gression of the biosynthetic pathway of catechola- between enterochromaffin cells and the neural crest. Journal of mines in chromaffin tissues, leading to DA, NA and A Embryology and Experimental Morphology 11, 307-324. ANDREW A (1974) Further evidence that enterochromaffin cells are as the end product, may be modulated by some other not derived from the neural crest. Journal of Embryology and mechanisms. Experimental Morphology 31, 589-598. Chromaffin cells in Protopterus and Lepidosiren, as ANGELAKOS ET, KING MP, MILLARD RW (1969) Regional reported in the present investigation, are almost distribution of catecholamines in the hearts of various species. Annals of the New York Academy of Sciences 156, 219-240. invariably found in close association with the auto- AUGUSTINSSON K-B, FANGE R, JOHNELS A, OSTLUND E (1956) nomic sympathetic nervous system and at sites where Histological, physiological and biochemical studies on the heart chromaffin cells or their precursors are situated in of two cyclostomes, hagfish (Myxine) and lamprey (Lampetra). Journal of Physiology 131, 257-276. mammals during ontogenetic development (Kohn, BALFOUR F-M (1878) A Monograph on the Development of 1903; Iwanow, 1932; Coupland, 1952, 1980; Elasmobranch Fishes. London. Hervonen, 1971). In a variety of mammalian species, BATTAGLIA G (1969) Ultrastructural observations on the biogenic chromaffin cells occur in or near the fetal heart amines in the carotid and aortic-abdominal bodies of the human during fetus. Zeitschriftfir Zellforschung 99, 529-537. successive developmental stages (Kohn, 1903; Dail et BENEDECZKY I, SMITH AD (1972) Ultrastructural studies on the al. 1973; Papka, 1976). Their occurrence in the adult adrenal medulla of golden hamster: origin and fate of secretory state has also been reported (Jacobowitz, 1967; granules. Zeitschriftifur Zellforschung 124, 367-386. BERKELBACH VAN DER SPRENKEL H (1934) Nebenniere. In Handbuch Ehinger et al. 1968; Angelakos et al. 1969; Ellison & der vergleichenden Anatomie der Wirbeltiere, Bd. 2, I. Halfte (ed. Hibbs, 1974). It is noteworthy that chromaffin cells of L. Bolk, E. G6ppert, E. Kallius & W. Lubosch). Berlin, Vienna: the heart are considered to belong to SGC cells Urban and Schwarzenberg. et al. BJORKLUND A, EHINGER B, FALCK B (1968) A method for (Ehinger 1968; Ellison & Hibbs, 1974; Papka, differentiating dopamine from noradrenaline in tissue sections by 1974). Although the significance of the latter cells is microspectrofluorometry. Journal of Histochemistry and Cyto- still debated, it has been suggested that they occupy an chemistry 16, 263-270. intermediate position between mature chromaffin and BJORKLUND A, CEGRELL L, FALCK B, RITZEN M, ROSENGREN E (1970) Dopamine-containing cells in sympathetic ganglia. Acta primitive sympathetic cells (Siegrist et al. 1968; Physiologica Scandinavica 78, 334-338. Kobayashi & Coupland, 1977; Unsicker et al. 1978). BJORKLUND A, EHINGER B, FALCK B (1972) Analysis offluorescence The findings reported make it plausible that the excitation peak ratios for the cellular identification of noradrenaline, dopamine or their mixtures. Journal ofHistochemistry occurrence of catecholamine-containing cells in the and Cytochemistry 20, 56-64. heart of mammals is reminiscent of the process of BJORKLUND A, FALCK B, LINDVALL 0 (1975) Microspectrofluoro- migration of primitive sympathetic cells during phy- metric analysis of cellular monoamines after formaldehyde or logeny. Once they have reached their final destination glyoxylic acid condensation. In Methods in Brain Research (ed. J. Wiley), pp. 249-294. london: P. B. Bradley. in the microenvironment of the adrenal medulla, they BLASCHKO H (1959) The development of current concepts of acquire the common morphological pattern and catecholamine formation. Pharmacological Reviews 11, 307-316. innervation. In primitive vertebrates, precursors of BLOOM G, OSTLUND E, EULER US VON, LISHAJKO F, RITZEN M. et al. (1961) Studies on catecholamine-containing granules of chromaffin cells may stop their migration to ac- specific cells in cyclostome hearts. Acta Physiologica Scandinavica cumulate in the heart and great vessels, where they 53 (Suppl. 185), 1-34. Comparative cytology of chromaffin tissue 339

BOCK P (1982) The Paraganglia. Berlin, Heidelberg, New York: COUPLAND RE, PYPER AS, HOPWOOD D (1964) A method for Springer-Verlag. differentiating between noradrenaline- and adrenaline-storing BRUNDIN T (1966) Studies on the preaortal paraganglia of newborn cells in the light and electron microscope. Nature 201, 1240-1242. rabbits. Acta Physiologica Scandinavica 70 (Suppl. 290), 1-54. COUPLAND RE, HOPWOOD D (1966) The mechanism of the CALL RN, JANSSENS PA (1975) Histochemical studies of the differential staining reaction for adrenaline- and noradrenaline- adrenocortical homologue in the kidney of the Australian storing granules in tissues fixed in glutaraldehyde. Journal of lungfish, Neoceratodus forsteri. Cell and Tissue Research 156, Anatomy 100, 227-243. 533-538. COUPLAND RE, MACDOUGALL JDB (1966) Adrenaline formation in CANNATA MA, CHIoccHIo SR, TRAMEZZANI JH (1968) Specificity noradrenaline-storing chromaffin cells in vivo induced by of the glutaraldehyde silver technique for catecholamines and corticosterone. Journal of Endocrinology 36, 317-324. related compounds. Histochemie 12, 253-264. COUPLAND RE, WEAKLEY BS (1968) Developing chromaffin tissue CARMICHAEL SW, BLAIR BC (1973) Normal ultrastructure of the in the rabbit: an electron microscopic study. Journal ofAnatomy day old dog adrenal medulla. Journal ofAnatomy 115, 113-118. 102, 425-455. CARMICHAEL SW, WINKLER H (1985) The adrenal chromaffin cell. COUPLAND RE, WEAKLEY BS (1970) Electron microscopic ob- Scientific American 253, 4-49. servation on the adrenal medulla and extra-adrenal chromaffin CHEVREL R (1889) Sur I'anatomie du systeme nerveux grand tissue of the postnatal rabbit. Journal ofAnatomy 106, 213-23 1. sympathique des Elasmobranches et des poissons osseux. Arc- COUPLAND RE, KOBAYASHI S, CROWE J (1976) On the fixation of hives de Zoologie Expe'rimentale et Gene'rale 2me Ser. 5, 1-196. catecholamines including adrenaline in tissue sections. Journal of CHIBA T, WILLLAMS TH (1975) Histofluorescence characteristics Anatomy 122, 403-413. and quantification of small intensely fluorescent (SIF) cells in COUPLAND RE, KOBAYASHI S, TOMLINSON A (1977) On the presence sympathetic ganglia of several species. Cell and Tissue Research of small granule chromaffin (SGC) cells in the rodent adrenal 162, 331-341. medulla. Journal of Anatomy 124, 488-489. CHIBA T, BLACK JR AC, WILLLAMS TH (1977) Evidence for COUPLAND RE, PARKER TL, KESSE WK, MOHAMED AA (1989) The dopamine-storing interneurons and paraneurons in rhesus innervation of the adrenal gland. III. Vagal innervation. Journal monkey sympathetic ganglia. Journal of Neurocytology 6, of Anatomy 163, 173-181. 441-453. DML WG, PALMER GC, KOPLIK L (1973) Ontogeny of the CORRODI H, HILLARP NA, JONssON G (1964) Fluorescence methods autonomic nervous system in the human fetal heart. Anatomical for the histochemical demonstration of monoamines. 3. Sodium Record 175, 301. borohydride reduction of the fluorescent compounds as a DINER 0 (1967) L'expulsion des granules de la medullo-surrenale specificity test. Journal of Histochemistry and Cytochemistry 12, chez le hamster. Comptes Rendus Hebdomadaires des S&ances de 582-586. `Academie des Sciences, serie D, 265, 616-619. COUPLAND RE (1952) The prenatal development of the abdominal EADE NR (1958) The distribution of the catecholamines in para-aortic bodies in man. Journal of Anatomy 86, 357-372. homogenates of the bovine adrenal medulla. Journal of Physi- COUPLAND RE (1953) On the morphology and adrenaline- ology 141, 183-192. noradrenaline content of chromaffin tissue. Journal of Endo- EHINGER FALCK H, SPORRONO B (1968) Adrenergic 194-203. B, B, PERSSON crinology 9, and cholinesterase-containing neurons of the heart. Histochemie COUPLAND RE (1956) The development and fate of the abdominal chromaffin tissue in the rabbit. Journal ofAnatomy 90, 527-537. 16, 197-205. ELFVIN HOKFELT GOLDSTEIN M Fluorescence COUPLAND RE (1965 a) The Natural History ofthe Chromaffin Cell. L-G, T, (1975) London: Longmans. microscopical, immunohistochemical and ultrastructural studies COUPLAND RE (1965b) Electron microscopic observations on the on sympathetic ganglia ofthe guinea pig, with special reference to structure of the rat adrenal medulla. I. The ultrastructure and the SIF cells and their catecholamine content. Journal of organization of chromaffin cells in the normal adrenal medulla. Ultrastructure Research 51, 377-396. Journal ofAnatomy 99, 231-254. ELLISON JP, HIBBs RG (1974) Catecholamine-containing cells ofthe COUPLAND RE (1965c) Electron microscopic observations on the guinea pig heart: an ultrastructural study. Journal of Molecular structure of the rat adrenal medulla. III. Normal innervation. and Cellular Cardiology 6, 17-26. Journal of Anatomy 99, 255-272. ERANK6 L, ERANKO 0 (1972) Effect of hydrocortisone on COUPLAND RE (1968) Corticosterone and methylation of nora- histochemically demonstrable catecholamines in the sympathetic drenaline by extra-adrenal chromaffin tissue. Journal of Endo- ganglia and extra-adrenal chromaffin tissue of the rat. Acta crinology 41, 487-490. Physiologica Scandinavica 84, 125-133. COUPLAND RE (1971) Observations on the form and size dis- ERANKO L, PAIVARINTA H, SoiNILA S, HAPPOLA 0 (1986) Trans- tribution ofchromaffin granules and on the identity ofadrenaline- mitters and modulators in the superior cervical ganglion of the and noradrenaline-storing chromaffin cells in vertebrates and rat. Medical Biology 64, 75-83. man. In Memoirs of the Society for Endocrinology No. 19, ERANKO 0, Riis&NN L (1961) Cytophotometric study on the Subcellular Organization and Function in Endocrine Tissues (ed. chromaffin reaction, the iodate reaction and formalin-induced H. Heller & K. Lederis), pp. 611-635. Cambridge: Cambridge fluorescence as indicators of adrenaline and noradrenaline University Press. concentrations in the adrenal medulla. Journal ofHistochemistry COUPLAND RE (1972) The chromaffin system. In Handbook of and Cytochemistry 9, 54-58. Experimental Pharmacology XXXIII: Catecholamines (ed. ERANKO 0, LEMPINEN M, RAIsANEN L (1966) Adrenaline and H. Blaschko & E. Muscholl), pp. 16-45. Berlin, Heidelberg, New noradrenaline in the organ of Zuckerkandl and adrenals of York: Springer. newborn rats treated with hydrocortisone. Acta Physiologica COUPLAND RE (1980) The development and fate of catecholamine Scandinavica 66, 253-254. secreting endocrine cells. In Biogenic Amines in Development (ed. ERANKO 0, ERANKO L (1971) Small, intensely fluorescent granule- H. Parvez & S. Parvez), pp. 3-29. Amsterdam, New York, containing cells in the sympathetic ganglion of the rat. In Oxford: Elsevier/North-Holland Biomedical Press. Histochemistry of Nervous Transmission, vol. 34. Progress in COUPLAND RE (1989) The natural history of the chromaffin cell- Brain Research (ed. 0. Erinko), pp. 39-51. Amsterdam: Elsevier. twenty-five years on: in the beginning. Archives ofHistology and ERANKO 0, PICKEL VM, HARKONEN M, ERANKO L, JOH TH, REIS Cytology 52 (Suppl.), 331-341. DJ (1982) Effect of hydrocortisone on catecholamines and the 340 D. W. Scheuermann

enzymes synthesizing them in the developing sympathetic HANKER JS, THORNBURG LP, YATES PE, MooRE HG III (1973) The ganglion. Histochemical Journal 14, 461-478. demonstration of cholinesterases by the formation of osmium EULER US VON, FXNGE R (1961) Catecholamines in nerves and blacks at the sites of Hatchett's brown. Histochemie 37, 223-242. organs of Myxine glutinosa, Squalus acanthias and Gadus HARTWIG HG, REINHOLD CH, LYNCKER I (1979) Distribution callarias. General and Comparative Endocrinology 1, 191-194. patterns of formaldehyde-induced fluorescence of hypothalamic FELDBERG W, MINz B, TsuDzIMuRA H (1934) Mechanisms of monoamines. A computer-assisted analysis. Cell and Tissue nervous discharge of adrenaline. Journal of Physiology 81, Research 201, 499-502. 286-304. HENLE (1865) Uber das Gewebe der Nebenniere und der Hypo- FENWICK EM, FAJDIGA PB, HowE NBS, LIVETT BG (1978) physe. Zeitschriftfuir rationelle Medizin 3 Reihe, 24. Functional and morphological characterization of isolated HERVONEN A (1971) Development of catecholamine-storing cells in bovine adrenal medullary cells. Journal of Cellular Biochemistry human fetal paraganglia and adrenal medulla. A histochemical 76, 12-30. and electron microscopical study. Acta Physiologica Scandinavica FONTAINE J, LE DOuARIN NM (1977) Analysis of endoderm (Suppl.) 368, 1-94. formation in the avian blastoderm by the use of quail-chick HERvoNEN A, ALHO H, HELEN P, KANERVA L (1979) Small, chimaeras. The problem of the neuroectodermal origin of the intensely fluorescent cells of human sympathetic ganglia. Neuro- cells ofthe APUD series. Journal ofEmbryology andExperimental science Letters 12, 97-101. Morphology 41, 209-222. HEYM C, ADDICKS K, GEROLD N, SCHR6DER H, KONIG R et al. FORSGREN S, MoRAVEc M, MoRAVEc J (1990) Catecholamine- (1980) Catecholamines in paraganglionic cells of the rat superior synthesizing enzymes and neuropeptides in rat heart epicardial cervical ganglion: functional aspects. In Histochemistry and Cell ganglia; an immunohistochemical study. Histochemical Journal Biology of Autonomic Neurons, SIF Cells, and Paraneurons (ed. 22, 667-676. 0. Erank6, S. Soinila & H. Piiivarinta), pp. 87-94. New York: GANNON BJ, CAMPBELL GD, SATCHELL GH (1972) Monoamine Raven Press. storage in relation to cardiac regulation in the Port Jackson shark HOKFELT B (1952) Noradrenaline and adrenaline in mammalian Heterodontus portusjacksoni. Zeitschrift fur Zellforschung und tissues. Acta Physiologica Scandinavica (Suppl.) 92, 1-134. mikroskopische Anatomie 131, 437-450. HOLMES W (1950) The adrenal homologues in the lungfish GASKELL JF (1912) The distribution and physiological action of the Protopterus. Proceedings of the Royal Society of London B 137, suprarenal medullary tissue in Petromyzon fluviatilis. Journal of 549-562. Physiology 44, 59. HOLMES W, MooRHousE DE (1956) The peri-renal tissue of GASKELL WH (1920) The Voluntary Nervous System. New York: Protopterus. A contribution to the history of the adrenal. Longmans, Green. Quarterly Journal of Microscopic Science 123-154. GEWIRTZ G, KVETNANSKY 97, R, WEISE VK, KOPIN JJ (1971) Effect of HOPWOOD D The hypophysectomy and adrenal (1971) histochemistry and electron histochemistry dopamine beta-hydroxylase ac- of chromaffin tissue. tivity in the rat. Molecular Pharmacology 7, 163-168. Progress in Histochemistry and Cyto- GHosH A (1962) A chemistry 3, 1-66. comparative study of the histochemistry of the IWANOW avian adrenals. General and Comparative Endocrinology (Suppl.) G (1932) Das chromaffine und interneurale System des 1, 75-80. Menschen. Zeitschrift fur die gesamte Anatomie 29, 87-280. GiACOMINI E (1906) Sulle capsule surrenali e sul simpatico dei JACOBOWITZ D (1967) Histochemical studies of the relationship of Dipnoi. Ricerche in Protopterus annectens. Atti della Reale chromaffin cells and adrenergic nerve fibres to the cardiac ganglia Accademia dei Lincei 15, 394-398. of several species. Journal of Pharmacology and Experimental GOODRICH ES (1930) Studies on the Structure and Development of Therapeutics 158, 227-240. Vertebrates. London: Macmillan. JANSSENS PA, VINSON GP, CHESTER JONES I, MOSLEY W (1965) GORGAS K, BOCK P (1976) Morphology and histochemistry of the Amphibian characteristics of the adrenal cortex of the African adrenal medulla. I. Various types of primary catecholamine- lungfish (Protopterus sp.). Journal ofEndocrinology 32, 373-382. storing cells in the mouse adrenal medulla. Histochemistry 50, JENKIN PM (1928) Note on the sympathetic nervous system of 17-31. Lepidosiren paradoxa. Proceedings of the Royal Society of GRONBLAD M, AKERMAN KE, ERANK6 0 (1980) Ultrastructural Edinburgh 48, 55-69. evidence ofexocytosis from glomus cells after incubation ofadult KAJIHARA H, AKIMOTO T, IIJIMA S (1978) On the chromaffin cells in rat carotid bodies in potassium-rich calcium-containing media. dog adrenal medulla: with special reference to the small granule Brain Research 189, 576-581. chromaffin cells (SGC). Cell and Tissue Research 191, 1-14. GRYNFELrr E (1903) Theses presentees a la Faculte des Sciences de KARNOVSKY MJ, RooTS L (1964) A 'direct-coloring' thiocholine Paris. Recherches Anatomiques et Histologiques sur les Organes method for cholinesterase. Journal of Histochemistry and Cyto- Surrenaux des Plagiostomes, pp. 1-137. Lille: L. Danel. chemistry 12, 219-221. GRYNSZPAN-WINOGRAD 0 (1975) Ultrastructure of the chromaffin KESSE WK, PARKER TL, COUPLAND RE (1988) The innervation of cell. In Handbook of Physiology, vol. 6. Adrenal Gland (ed. the adrenal gland. I. The source of pre- and postganglionic nerve H. Blaschko, G. Sayers & A. D. Smith), pp. 295-308. Washing- fibres to the rat adrenal gland. Journal of Anatomy 157, 33-41. ton, D.C.: American Physiological Society. KIRSHNER N (1975) Biosynthesis of the catecholamines. In Hand- HAGEN P, BARRNErr RJ (1960) The storage of amines in the book of Physiology, vol. 6. Adrenal Gland (ed. H. Blaschko, chromaffin cells. In Adrenergic Mechanisms (ed. J. R. Vane, G. Sayers & A. D. Smith), pp. 341-355. Washington, D.C.: G. E. W. Wolstenholme & M. O'Connor), pp. 83-99. London: American Physiological Society. Churchill. KOBAYASHI S, COUPLAND RE (1977) Two populations of micro- HAGGENDAL J (1963) An improved method for fluorimetric vesicles in the SGC (Small Granule Chromaffin) cells of the determination of small amounts of adrenaline and noradrenaline mouse adrenal medulla. Archivum Histologicum Japonicum 40, in plasma and tissues. Acta Physiologica Scandinavica 59, 251-259. 242-254. KOBAYASHI S, SERIZAWA Y (1979) Stress-induced degranulation HAMBERGER B, MALMFORS T, SACHS C (1965) Standardization of accompanied by vesicle formation in the adrenal chromaffin cells paraformaldehyde and of certain procedures for the histo- of the mouse. Archivum Histologicum Japonicum 42, 375-388. chemical demonstration of catecholamines. Journal of Histo- KoHN A (1898) Uber die Nebenniere. Prager Medizinische chemistry and Cytochemistry 13, 147. Wochenschrift 23. Comparative cytology of chromaffin tissue 341

KoHN A (1903) Die Paraganglien. Archiv far mikroskopische ScHEuERMANN DW (1980) A study of chromaffin cells in the heart Anatomie 62, 263-365. of Protopterus. Folia Morphologica 28, 93-98. KRAUSE R (1923) Mikroskopische Anatomie der Wirbeltiere. Berlin, ScBEuERmANN DW, STILMAN C, REINHOLD Cii, DE GROODT- Leipzig: Walter de Gruyter. LASSEEL MHA (1981) Microspectrofluorometric study of mono- LE DouARIN N (1982) The Neural Crest. Cambridge: Cambridge amines in the auricle of the heart of Protopterus aethiopicus. Cell University Press. and Tissue Research 217, 443-449. LEMPINEN M (1964) Extra-adrenal chromaffin tissue of the rat and SCHEUERMANN DW, DE GROODT-LASSEEL MHA, STILMAN C (1984) the effect of cortical hormones on it. Acta Physiologica A light and fluorescence cytochemical and electron microscopic Scandinavica 62, (Suppl. 231), 1-91. study of granule-containing cells in the intrapulmonary ganglia LEwis GP (1975) Physiological mechanisms controlling secretory of Pseudemys scripta elegans. American Journal ofAnatomy 171, activity of adrenal medulla. In Handbook of Physiology, vol. 6. 377-399. Adrenal Gland (ed. H. Blaschko, G. Sayers & A. D. Smith), pp. SCHRODER H, GEROLD N, HEYM C (1981) Immunofluorescence of 309-319. Washington, D.C.: American Physiological Society. dopamine-beta-hydroxylase in small intensely fluorescent cells of LIBET B, OWMAN C (1974) Concomitant changes in formaldehyde- human sympathetic ganglia. Acta Anatomica 109, 47-50. induced fluorescence of dopamine interneurones and in slow SCHRODER H, LACKNER K, HEYm CH (1982) Simultaneous dem- inhibitory post-synaptic potentials of the rabbit superior cervical onstration of catecholamines and phenylethanolamine-N- ganglion, induced by stimulation ofthe preganglionic nerve or by methyltransferase in the same tissue section by means of a muscarinic agent. Journal of Physiology 237, 635-662. formaldehyde-induced fluorescence (FIF) and tetramethyl- LUTZ BR, WYMAN LC (1927) The chromaphil tissue and interrenal rodamine-isothiocyanate (MRITC) immunofluorescence. Journal bodies of elasmobranchs and the occurrence of adrenin. Journal of Neuroscience Methods 6, 281-286. ofExperimental Zoology 47, 295-307. SHEPHERD DM, WEST GB (1951) Noradrenaline and the suprarenal MASTROLIA L, GALLO VP, LA MARCA A (1981) Adrenal homologue medulla. British Journal of Pharmacology 6, 665-674. in Scardinius erythrophthalmus (Teleostei, Cyprinidae): light and SiuEPiRD DM, WEsT GB (1952) Noradrenaline and accessory electron microscopic observations. Bollettino di Zoologia 48, chromaffin tissue. Nature 170, 42-43. 127-138. SHEPHERD DM, WEST GB (1953) Hydroxytyramine in medulla of NAGASAWA J (1977) Exocytosis: the common release mechanism of sheep, ox and cow. Journal of Physiology 120, 15-19. secretory granules in glandular cells, neurosecretory cells, SIEGRIST G, DOLIVO M, DUNANT Y, FOROGLOU-KERAMEUS C, DE neurons and paraneurons. Archivum Histologicum Japonicum 40, RIBAUPIERRE FR et al. (1968) Ultrastructure and function of the 31-47. chromaffin cells in the superior cervical ganglion of the rat. NFrF NH, KAROUM F, HADJICoNSTANTINOU M (1983) Dopamine- Journal of Ultrastructure Research 25, 381-407. containing small intensely fluorescent cells and sympathetic SmTH AD (1971) Summing up: some implications of the neuron as ganglion function. Federation Proceedings 42, 3009-3011. a secreting cell. Philosophical Transactions ofthe Royal Society of NICOL JAC (1952) Autonomic nervous systems in lower chordates. London, Series B, Biological Sciences 261, 423-437. Biological Reviews of the Cambridge Philosophical Society 27, STILMAN C, SCHEUERMANN DW (1987) Computer-operated micro- 1-49. spectrofluorimetry to identify formaldehyde-induced fluoro- NEEMINEVA K, PEKKARiNEN A (1953) Determination of adrenalin phores of biogenic monoamines and precursor substances in and noradrenalin in the human foetal adrenals and aortic bodies. models and tissue sections. Histochemistry 86, 255-267. Nature 171, 436-437. TRAMEZZANI JH, CioccCHio S, WASSERMAN GF (1964) A technique NORBERG KA, RITZEN M, VOGERSThDT V (1966) Histochemical for light and electron microscopic identification of adrenaline- studies on a special catecholamine-containing cell type in and noradrenaline-storing cells. Journal of Histochemistry and sympathetic ganglia. Acta Physiologica Scandinavica 67, 260-270. Cytochemistry 12, 890-899. OSTLUND E, BLOOM G, ADAMS-RAY J, RITZEN M, SIEGMAN M et al. UNSICKER K (1973) Fine structure and innervation of the avian (1960) Storage and release of catecholamines and the occurrence adrenal gland. I. Fine structure of adrenal chromaffin cells and of a specific submicroscopic granulation in the hearts of ganglion cells. Zeitschrift fiir Zellforschung 145, 389-416. cyclostomes. Nature 188, 321-325. UNSICKER K (1976) Chromaffin, small granule-containing and PAPKA RE (1974) A study of catecholamine-containing cells in the ganglion cells in the adrenal gland of reptiles. A comparative hearts of fetal and postnatal rabbits by fluorescence and electron ultrastructural study. Cell and Tissue Research 165, 477-508. microscopy. Cell and Tissue Research 154, 471-484. UNSICKER K, HABURA-FLUH 0, ZWARG U (1978) Different types of PAPKA RE (1976) Studies of cardiac ganglia in pre- and postnatal small granule-containing cells and neurons in the guinea-pig rabbits. Cell and Tissue Research 175, 17-35. adrenal medulla. Cell and Tissue Research 189, 109-130. PARKER TL, KEssE WK, TOMLINSON A, COUPLAND RE (1988) VERNA A (1979) Ultrastructure ofthe carotid body in the mammals. Ontogenesis of preganglionic sympathetic innervation of rat International Review of Cytology 60, 271-330. adrenal chromaffin cells. In Progress in Catecholamine Research, WEINER N (1975) Control of the biosynthesis of adrenal cate- part A: Basic Aspects and Peripheral Mechanisms (ed. A. cholamines by the adrenal medulla. In Handbook of Physiology, Dahlstr6m, R. H. Belmaker & M. Sandler), pp. 227-232. New vol. 6. Adrenal Gland(ed. H. Blaschko, G. Sayers & A. D. Smith), York: Alan R. Liss. pp. 357-366. Washington, D.C.: American Physiological Society. PARKER TL, MOHAMED AA, COUPLAND RE (1990) The innervation WEST GB (1955) The comparative pharmacology of the suprarenal of the adrenal gland. IV. The source of pre- and postganglionic medulla Quarterly Review of Biology 30, 116-137. nerve fibres to the guinea-pig adrenal gland. Journal ofAnatomy WEST GB, SHEPHERD DM, HuNTER RB (1951) Adrenaline and 172, 17-24. noradrenaline concentrations in adrenal glands at different ages REINHOLD C, HARTWIG H-G (1982) Progress in microfluorometric and in some diseases. Lancet ii, 966-969. identification of monoamine fluorophores. Brain Research Bull- WINKLER H, SmITH AD (1975) The chromaffin granule and the etin 9, 97-105. storage ofcatecholamines. In Handbook ofPhysiology, Section 7, RICHARDSON KC (1964) The fine structure of the albino rabbit iris Endocrinology, vol. 6, Adrenal Gland (ed. H. Blaschko, G. Sayers with special reference to the identification of adrenergic and & A. D. Smith), pp. 321-339. Washington, D.C.: American cholinergic nerves and nerve endings in its intrinsic muscles. Physiological Society. American Journal of? Anatomy 114, 173-205. WOOD JG (1974) Positive identification of intracellular biogenic

24 ANA 183 342 D. W. Scheuermann

amine reaction product with electron microscopic X-ray analysis. WURTMAN RJ, AXELROD J (1966) Control of enzymatic synthesis of Journal of Histochemistry and Cytochemistry 22, 1060-1067. adrenaline in the adrenal medulla by adrenal cortical steroids. WooD JG, BARRNETT RJ (1964) Histochemical demonstration of Journal of Biological Chemistry 241, 2301-2305. norepinephrine at a fine structural level. Journal ofHistochemistry YOUNG JZ (1933) The autonomic nervous system of Selachians. and Cytochemistry 12, 197-209. Quarterly Journal of Microscopical Science 75, 571-624. WRIGHT A, JoNES IC (1955) Chromaffin tissue in the lizard adrenal gland. Nature 175, 1001-1002.