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Cell Tiss. Res. 165, 477-508 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

Chromaff'm, Small Granule-Containing and Ganglion Cells in the Adrenal Gland of Reptiles A Comparative Ultrastructural Study*

K. Unsicker**

Department of Anatomy, University of Kiel, Germany

Summary. Chromaffin, small granule-containing (SGC)-cells, neurons and the innervation of these cells was studied in the adrenal gland of three species of reptiles (Testudo graeca, Lacerta dugesi, Natrix natrix).

1. After fixation with glutaraldehyde and osmium-tetroxide adrenaline (A)- and noradrenaline (NA)-storing cells can be distinguished by means of the different electron density of their granules: A-granules are moderately electron-dense, while NA-granules show a core of high electron density. The unusually high electron density of a few A-granules in Testudo occasionally required viewing of unstained sections which facilitated the discrimination of the two cell types in this species. In all species studied NA-granules display a remarkable polymorphism which is most pronounced in the tortoise. In this species A-granules are polymorphic, too. Both types of granules show wide variations in size, which are particularly great in the tortoise. This species also exhibits the largest average sizes for A-granules (285 nm), and NA-granules (354 nm). The corresponding parameters for Lacerta and Natrix, are 255 and 179 nm for A- and 323 and 304 nm for NA-granules, respectively. The rough ER in A-cells of the tortoise regularly occurs in the form of circular dilations ('ergastosomes', Kanerva and Hervonen, 1973).

Mitochondria sometimes contain longitudinal cristae with a crystalloid internal pattern. Large dense bodies which incorporate granules are abundant in NA-cells. Smaller dense bodies containing a few dense patches and membranes are present in both A- and NA-cells. Intermediate stages between dense bodies and what appear to be A- or NA-granules (if the latter have lost some of their amine-content) are frequently observed.

Send offprint requests to: Dr. Klaus Unsicker, Department of Anatomy, University of Kiel, 2300 Kiel, Federal Republic of Germany.

* Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3). This paper was presented in part at a Symposium on "Chromaffin, Enterochromaffin and

Related Cells" held at Gifu, Japan, 22-24 August, 1975. ** Thanks are due to Dr. Rogers, Department of Zoology, University of Melbourne, for his criticism of the manuscript.

Dedicated to Professor Dr. W. Bargmann in honor of his 70th birthday.

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2. Small granule containing (SGC)-cells are regularly found in the adrenal of reptiles. Their granules have an average size of 146 nm in Testudo (188 and 107 nm in Lacerta and Natrix, respectively). The cells exhibit a high nucleo-cytoplasmic ratio and an electron-lucent cytoplasm.

3. Various types of nerve cell are present in the reptilian adrenal. Small "light" neurons are similar to SGC-cells, but have only few granules, if any. Large "principal" neurons resemble ganglion cells in various autonomic ganglia. According to the morphology of their nuclei and the amounts and distribution of their ER, they may be divided into "light" and "dark" principal neurons.

4. Chromaffin, SGC- and nerve cells are innervated by cholinergic nerve terminals containing small, clear (500-600 A in diameter) and large, dense- cored vesicles measuring 730-1340 A (Testudo). Synapses are usually found on the cell bodies. A-cells in the tortoise which form long processes have their synapses mostly along these processes. A-cell processes are occasionally found in the media of arterioles. Pre- and postsynaptic membrane specialisations are extremely rare on SGC-cells.

5. Adrenergic nerve profiles, which are processes of neither chromaffin nor SGC-cells, may be identified after application of 5- and 6-hydroxydopamine in close proximity of chromaffin cells.

Key words: Adrenal gland - Reptiles - Chromaffin cells - Small granule- containing cells - Nerve cells - Innervation - 5- and 6-hydroxydopamine.

Introduction

The ultrastructure of adrenal and extra-adrenal chromaffin cells in mammals has been extensively dealt with in the past two decades. Detailed studies have been published on adrenal medullary cells of, e.g. rat (Lever, 1955; Er~ink6 and Hfinninen, 1960; Coupland, 1965b); hamster (Yates et al., 1962; Benedeczky and Smith, 1972), mouse (Wetzstein, 1957), hedgehog (Bargman and Lindner, 1964), rabbit (Coupland and Weakly, 1970), guinea-pig (Gorgas, 1968), cat (Wetzstein, 1957), and man (Benedeczky and Lapis, 1968; Yokoyama and Taka- yasu, 1969). Particular problems such as the development of mammalian chromaffin cells (Arnold and Hager, 1967; Hervonen, 1971), the differential fixation and staining characteristics of adrenaline (A)- 1 and noradrenaline (NA)-storing granules (Coupland et al., 1964; Tramezzani et al., 1964; Wood and Barrnett, 1964; Wood and Callas, 1966; Arnold and Hager, 1968), the formation of granules (Elfvin, 1967a; Holtzman etal., 1973), the discharge of the granule contents by exocytosis (Coupland, 1965a; Diner, 1967), the ultrastructure of the granular membrane (Agostini and Taugner, 1973), the ultra-cytochemical properties of various cell constituents as lysosomes, peroxisomes, Golgi apparatus and endoplasmic reticulum (ER) (Holtzman and Dominitz, 1967; Arnold and Holtzman, 1975), the morphological aspects of the cellular response to stimulation by acetylcholine (Butterworth and Man, 1958), reserpine (Yates, 1963; Elfvin, 1968), or insulin (Yates, 1964), and the innervation of adrenal

Adrenal Gland of Reptiles 479

chromaffin cells (Coupland, 1965 c; Grynszpan-Winograd, 1974; Prentice and Wood, 1975) have been considered as well.

Far less is known concerning the ultrastructural properties of adrenal chromaffin cells in non-mammalian vertebrates. There is a comparative study by Coupland (1971) covering a few species of elasmobranchs, amphibians, birds and mammals. An investigation on the fine structure and innervation of catecholamine-containing cells in the adrenal of 15 bird species (Unsicker, 1973 a-c) has shown a variety of species differences with regard to A- and NA-producing cells and their innervation. Furthermore, it has provided evidence for the exist- ence of three types of probably efferent nerve fibres to the gland (cholinergic, adrenergic and a third type, similar to purinergic nerves; c f Burnstock, 1972), small and large sympathetic nerve cells and a SGC-cell which bears some resem- blance to the SIF-cell found in various sympathetic ganglia (Er/ink6 and H/irk6nen, 1963, 1965; Grillo, 1964; Siegrist et al., 1968; Er/ink6 and Er/ink6, 1971 ; and others).

In terms of evolution reptiles are closely related to birds. With respect to their autonomic nervous system reptiles are the most primitive amniotic vertebrates using NA as an adrenergic transmitter in the peripheral autonomic nervous system (cf Burnstock, 1969), and producing mainly A in adrenal chromaffin cells (Holzbauer and Sharman, 1972). Despite their prominent phylogenetic position in respect to the evolution of the Amniota, information on the fine structure of their adrenal chromaffin cells is confined to a study of Xenodon (Wassermann and Tramezzani, 1963). The following report deals with the ultrastructure and innervation of chromaffin, SGC- and various kinds of ganglion cells present in the adrenal of three species of reptiles.

1. Abbreviations used: A = adrenaline; NA = noradrenaline; DA = dopamine; DBH=dopamine /~-hydroxylase; PNMT=phenylethanolamine-N-meth- yltransferase; CA---catecholamines; 5- and 6-OHDA=5- and 6-hydroxydopamine; SGC-cell=Small Granule Containing Cell; SIF-cell=Small Intensely Fluorescent Cell; ER = endoplasmic reticulum.

Materials and Methods

Six adult male specimens of the species Testudo graeca, Lacerta dugesi and Natrix natrix were used for the present study. The animals were anaesthetised with Nembutal ~ and perfused via the heart (tortoises and lizards) or aorta (grass snakes), respectively, with the following solutions:

1. Rinsing the vascular bed with Macrodex~ buffered with veronal acetate buffer to pH 7.4, 15-30 sec.

2. Fixation with phosphate buffered (0.1 m) 2.5% glutaraldehyde, pH 7.6, 15 30 min. 3. Perfusion with phosphate buffer (0.1 m), pH 7.6, 10 min.

In a few cases, the tissue was immersed into buffered glutaraldehyde subsequent to perfusion fixation for 1 hr and washed in phosphate buffer 1-2 hr. After removing the adrenal glands, slices approximately 1 m m thick were post-fixed in 2% aqueous OsO4, dehydrated in graded series of ethanol and embedded in Araldite.

Three tortoises and three lizards were treated with 5- or 6-OHDA, respectively, according to the following schedule: 5 -OHDA: three injections (i.p.) of 250 mg/kg at 12-hour intervals; 6- O H D A : three injections (i.p.) of 100 150 mg/kg at 24-hour intervals. The animals were killed

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within 6 hr after the last injection. For orientation thick sections (1 ~tm) were stained according to Richardson et al. (1960). Thin sections were stained with uranyl acetate in 70% methanol and lead citrate and observed through a Zeiss EM9A or a Siemens 101 electron microscope, respectively.

For measuring the sizes of A- and NA-granules in the three species and the granules of SGC-cells of Testudo, prints with a magnification of 18,000 were used, and the maximum diameter of the dense core of each granule was measured. On the basis of the measurement of at least 1,250 granules of each cell type (1,000 in SGC-cells) the size distribution was calculated according to Coupland (1968, 1971). The thickness of the sections was assumed to be 50 nm, and a correction factor of 20 nm was used for the min imum thickness allowing identification. Granule profiles up to the size of 556 A (group I) were not converted from profile to granule number since granules of this size can only be identified if they are entirely present in the section.

Results

Nomenclature

The term "chromaff in" as used in this study refers to A- and NA-producing cells whose granules can be distinguished by means of their different reactivity with glutaraldehyde (Coupland and Hopwood, 1966). It does not include a cell type with granular vesicles which are significantly smaller than granules in A- and NA-cells (SGC-cells) and whose staining properties in terms of "chromaff ini ty" remain to be assessed.

Testudo

A-Cells (Figs. 2, 3, 5, 7, 8) represent the majority of CA-cells. Low power micrographs show polygonal or elongated cells, or processes of these cells, containing granules with a wide range of electron densities and of varying sizes. The granules are packed in a more orderly arrangement in A- than in NA-cells (Figs. 2, 3). A characteristic feature of A-cells in Testudo is the occurrence of two classes of granules. Type 1 granules (Fig. 2) are more numerous and exhibit a smooth membrane; type 2 obviously represents a dilation of sacks of rough ER filled with a content of low to medium electron density (Figs..2, 5). Type 1 granules are normally round or oval in shape; however, irregular forms such as dumbbell and comma-shaped ones occur as well and may be more frequently found in some cells than in others. Type 1 granules display a high electron density on an average. A substructure of the granule is not constantly to be observed, and in a few instances the differentiation from a NA-granule may be difficult, particularly, when the dense core lies in an excentric position within the vesicle indicating a heavy reaction between granule contents and glutaraldehyde, which is frequently found with NA-granules. However, unstained sections always allow a clear differentiation of A- and NA-storing granules (Fig. 3). The light halo interposed between the dense core of an A- granule and the 6~75 A vesicle membrane is unusually narrow and often hard to detect. Occasionally the following forms of type 1 granules are observed: double and triple granules, i.e. two or three dense cores within one vesicle membrane, the dense core being dissolved into multiple dense spots, a highly electron dense core being situated within a core of less electron density. The size distribution of type 1 granules in A-cells of Testudo is given in Fig. 1. Their average size is 285 nm.


NA

i -'i i [

2 3 4 5 6 7 8 9 1 2 3 z, 5 6 ? 8 9 1 0

SGC

7-

5-

4-

3-

2-

1-

1 2 3 4

Testudo

X

Z

10-

9

8-

, ,

2 3 4 5 6

3-

2-

l -

i i i l l 1 2 3 A 5 6 7

Lacerta

6-

5

3

2-

i J i

2 3 4 5

3-

2-

1-

1 2 3 4 5 6 7

Size of granules in nm X 100

Natrix

Fig. 1. Size distribution of granules in chromaffin and SGC-cells in the adrenals of three reptilian species

Type 2 granules differ from previously described type 1 granules in several respects: they show less variation in size, they occupy a position close to the nucleus and Golgi area (Fig. 5) and never show the more or less even distribution of type 1 granules, they are connected with the non-dilated parts of the granular ER by narrow openings (Fig. 5a) and have a floccular content of constant medium electron density. Ribosomes cover only parts of the limiting membrane. Despite the remarkably close relationship between these ER sacks and the Golgi area we have not been able to trace the ER membranes to the Golgi cisternae and saccules. The Golgi areas of A-cells (Fig. 5 b) are prominent and consists of tightly packed cisternae, with dilated regions, running straight or in slight curves. In addition, there are smooth and coated vesicles. Dense cores occur within terminal dilations of cisternae, as well as within smooth vesicles and vesicles which show remnants of a fuzzy coat. Dense-cored vesicles (granules) are smaller in the Golgi region than in other parts of the cell.

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Fig. 2. Adrenaline (A)-, noradrenaline (NA)- and small granule containing (SGC)-cells in the adrenal of Testudo. A-cells display "normal" granules and "ergastosomes" (E), whereas NA-cells contain numerous large dense bodies (D) and electron translucent areas (e), which are devoid of cell organelles. Note the remarkable polymorphisln of A- and NA-granules and the relatively high electron density of a few A-granules. The SCG-cell shows an electron-lucent cytoplasm and granules which are considerably smaller than chromaffin granules. Both chromaffin and SCG-cells are surrounded by satellite cells (S). x 5,400


Fig. 3. Testudo. Unstained section with NA (left) and A-cells (right), which may be readily distinguished on the basis of the different electron density of their granules. Note differences in the distribution of granules in A- and NA-cells and the complex dense bodies (D) in NA-cells. x 10,000

Profiles o f rough ER are scarce and inconspicuous except in those areas where they f o r m the above men t ioned dilations. Free r ibosomes are found to be ub iqu i tous ly present. Sections th rough mi tochond r i a (Fig. 5) appear as round,

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Fig. 4. N A (above) and A-cells (below) in the adrenal of a snake (Natrix). Differentiation of the two cell types is easier than the tortoise. Axon terminals (S) in synaptic contact with an A-cell body. x 5,400

oval or slender profiles, which occasionally branch, They show a moderate number of transversely arranged cristae packed into a matrix of medium electron density which may display a few irregularly contoured dense particles. Distribu- tion of mitochondria within A-cells seems to be uniform. Particular granular- mitochondria-relationships were not observed. A special type of mitochondrion (Fig. 5 a inset) is characterised by two parallel cristae which run in a longitudinal

Fig. 5a and b. Testudo. Sacs of rough ER ("ergastosomes", E). (a) "Ergastosomes", intermingled with undilated cisternae of rough ER (R), chromaffin granules (G) and a large dense body (D) which is composed of materials of various electron densities. "Ergas tosomes" show a few protrusions (arrows) which could be connections with the undilated ER. Mitochondria with electron-dense particles (M). (b) "Ergastosomes" in the paranuclear region showing a close association with the Golgi area (GO). x 18,000. Inset: Mitochondrion with longitudinal cristae showing a crystalloid pattern, x 36,000

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direction and exhibit a crystalloid internal pattern. These mitochondria are similar to those seen in chromaffin cells of the rock and house sparrow (Unsicker, 1973a). Longitudinal cristae of the above type may be found along with more "normal" transverse ones within the same mitochondrion. Dense bodies (Fig. 5 a) are always surrounded by a limiting membrane and with regard to their number and internal structure differ from those present in NA-cells (see below). They are not regularly present in sections through A-cells, as they are in NA-cells. Most of them contain a lipid component and patches of high electron density which are interspersed among granular moderately dense areas. A continuous line can be drawn from larger A-granules displaying a homogenuous granularity of the dense core over granules containing irregular dense patches within their matrix towards dense bodies (Fig. 6c). Multivesicular bodies are distributed throughout the cytoplasm.

Glycogen particles occur in A-cells as single particles or in patches. Lipid droplets of various sizes are occasionally found. Cilia are commonly associated with A-cells extending into tunnels, which represent a deep invagination of the cell surface, or into the extracellular space. As a rule they have a 9+2 fibril pattern. Microtubules and filaments are widely distributed throughout the cell body without showing any preferential sites (with the exception of cell processes) or relations to granules and the cell surface, respectively. The cell membrane is a typical unit membrane. Profiles indicating exocytosis of granules have not been observed. Nuclei of A-cells may be ovoid, circular or with slight indentations.

NA-Cells (Figs. 2, 3) in comparison with A-cells, have a heterogeneous ground cytoplasm, with large electron light areas which contain scattered glycogen particles. The average number of NA-granules per cell profile, as judged from randomly taken pictures, varies considerably, in contrast to the packing of A-granules in A-cells. The size distribution of NA-granules is given in Fig. 1. The average profile diameter is 354 nm and granules extend in size to more than 1,100 nm. Most granules in NA-cells are highly electron dense. A few granules, which exhibit minor degrees of internal disorganisation or partial loss of amine from the granule (as far as it can be assessed by electron density), show an internal substructure in the form of globules (cf. Fig. 6a). Only in a very few NA-granules is the electron-dense core asymmetrically placed within a dilated vesicle membrane. Frequently granules are situated within membrane-bound dense bodies (Figs. 6a, b), which are very prominent in NA-cells of the tortoise and extend in size to several microns. Granules, which are found in dense bodies, are mostly devoid of a vesicle membrane, smaller than the average NA-granule, and often from irregularly shaped aggregates. Lipid

Fig. 6a-c . Testudo. Various forms of dense bodies in NA (6a and b) and A-cells (6c). (a) Dense bodies with aggregates of granules which lack a membrane and display various grades of internal disorganisation. This dense body also contains some filamentous material (F). x 30,000. (b) Myelin figures (M) and remnants of chromaffin granules (G) in a dense body. For details see text. x 120,000 (c) Unsta ined section showing a "normal" A-granule (A) and another one containing electron-dense patches (D). The latter form may be considered to represent a transitional stage towards a dense body. x 80,000


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Fig. 7a and b. Testudo. (a) Axon terminal (at) and process of an A-cell in a parallel course. The axon is filled with numerous small clear (cv) and large dense-cored vesicles (dr), tubules (t) and mitochondria (M). Site of synaptic contact (b). (b) Axon terminal embedded into the cell body of an NA-cell. Synaptic membrane specialisation (b). • t8,000


inclusions are rare in dense bodies. A typical feature of dense bodies in NA-cells are myelin figures (Fig. 6b) which, if cut in an orthograde direction, display alternating layers of high and low electron density. The dense lines are about 20-25 A wide, the light interspaces of medium electron density which separate them about 25-35 A. High power micrographs show that each dense line is in fact composed of two, with a 10 A wide light gap in between. Separation of the alternating layers, which is frequently observed, always occurs in the broader electron-lucent gap. Dense bodies with myelin figures are absent or scarce in NA-cells with minor numbers of granules. Mitochondria in NA-cells are identical in structure with those seen in A-cells. However, special cristae displaying a crystalloid internal pattern were not observed. Profiles of the rough ER are irregularly scattered and seldom arranged in stacks. Circumscribed dilations of rough ER like those described for A-cells do not occur. Free ribosomes are often seen singly or in clusters.

Granules are scarcer in the Golgi areas of NA- than A-cells. This makes Golgi fields in NA-cells more prominent and easily identifiable. Granules in the Golgi region do not show the same high electron density as granules located in other parts of the cell.

Other cell organelles such as cilia, tubules or filaments show no structural differences in comparison with A-cells.

SGC-Cells (Fig. 2): The tortoise adrenal contains cells, which can be distinguished both from A- and NA-cells and typical ganglion cells by the presence of small granules and an electron-lucent cytoplasm. There is a wide range of SGC-cells from cells with a few granules and a high nucleo-cytoplasmic ratio to cells filled with many granules and larger amounts of cytoplasm. The average size of granules is 146 nm, the size distribution is shown in Fig. 1. Most granular profiles are circular, but ovoid and dumbbell forms may be observed. The core which is highly electron-dense is normally found in a central position within the vesicle. Only rarely are the granules distributed throughout the entire cell body; usually they are situated peripherally along the cell membrane. Golgi areas are small and rarely contain granules. Free ribosomes are abundant, whereas short profiles of rough ER are only seldom encountered, particularly in cells with a low amount of cytoplasm. Dense bodies similar to those seen in NA-cells are common in larger SGC-cells, but do not incorporate granules or lipid. Mitochondrial profiles show no usual features. Microtubules measuring about 200 A in diameter occur in most cells. The nuclei are rounded or oval, sometimes with a few minor indentations, the chromatin underlying the nuclear membrane. Nucleoli are not particularly prominent and mitotic figures were not observed. While cells forming a morphological link between SGC- and A- or NA-cells were not observed, cells with features of both SGC-cells and what is to be described as small light neurons were sometimes seen.

Small "Light" Neurons: This type of cell is structurally similar to SGC-cells, especially to those containing very few granules. Small, "light" neurons are more common in the periphery of the adrenal gland than within the chromaffin cell cords and islets in the interior of the gland. Their round or oval nuclei are surrounded by a narrow rim of electron-lucent cytoplasm displaying sparsely distributed profiles of rough ER, an abundance of free ribosomes, singly or as polyribosomes, and round or oval mitochondria. The Golgi apparatuses

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Fig. 8a and b. Testudo. Processes of A-cells (A) in the vicinity of an A-cell (a) and adjacent to the media (M) of an arteriole (b). (a) A-cell processes exhibit typical features of axons, such as neurotubules and filaments, x 15,000 (b) Large process is completely covered by a satellite cell (SC) which also invests smaller axons (ax). x 7,200


Fig. 9a and b. Testudo. (a) Two types of vesicle-containing axons (al and a2) in a bundle of unmyelinated nerve fibres, a I displays the same kinds of vesicles found in cholinergic nerve terminals on chromaffin cells, a2 has small clear and large dense-cored vesicles typical of A-cell processes. (b) Adrenergic axon filled with an abundance of dense-cored vesicles of various sizes after the application of 5-OHDA. Chromaffin (CC) and SGC-cells are not affected by the drug. x 18,000

are about the size of those in SGC-cells. Granules are extremely rare ranging in size from about 600 to 1,250 nm.

Principal Neurons (Fig. 10) : Like the small "light" neurons, principal neurons are mostly found in and underneath the adrenal capsule, often associated with

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Fig. 10a and b. Light (a) and Dark (b) principal neuron in the adrenal of tortoise. For details see text. (a) x 15,000. (b) x 6,000


large nerve trunks. On the basis of electron opacity of the cytoplasmic matrix principal neurons may be divided into "light" and "dark" types. Both share a variety of structural features typical of sympathetic neurons (el Forssmann, 1964; Grillo, 1966; Unsicker, 1967; H6kfelt, 1968).

There is a fairly well developed Nissl substance consisting of free ribosomes, polysomes and irregularly arranged cisternae of rough ER which tend to be more sparse in the juxtanuclear and Golgi regions. Free ribosomes appear more numerous in "light" principal neurons; for the ER-cisternae the situation seems to be vice-versa. Principal neurons may contain dilations of ER-cisternae identical with those described above for A-cells which sometimes seen to cover an entire cell profile. Golgi apparatuses are composed of stacks of parallel cisternae and more or less dilated vesicles. Dense-cored vesicles (DCV), 700-1,100 A in diameter, are occasionally found dispersed throughout the cytoplasm. The many small mitochondria present in "l ight" and "da rk" neurons in equal amounts usually have transverse cristae. Microtubules are less frequently seen in "l ight" than in "da rk" neurons where they form tracks separating groups of cell organelles from each other. Dense bodies are membrane-limited and mostly consist of highly electron-dense components which may segregate forming myelin patterns. A few principal neurons display areas which may correspond to axon hillocks: they are rich in free ribosomes but poor in common cell organelles. One cell body may exhibit up to three of these regions.

Nuclei of "light" and "dark" principal neurons are somewhat different, the chromatin being denser and arranged in larger patches in nuclei of "dark" cells. On the other hand, nuclei of "light" cells appear more regularly rounded and nucleoli more prominent than those of "dark" neurons.

Satellite Cells and Cell-Cell-Interrelations: All of the cell types described above may come into close association, with an intercellular cleft, 150-200 A wide, interposed. However, more frequently they are enriched by satellite cells. There are no structural differences with regard to satellite cells surrounding different types of CA-storing or nerve cells, respectively, or investing nerve fibres. The central area of the satellite cell contains the nucleus, which is irregularly ovoid in shape, often with deep indentations, and stretches with its long axis parallel to the cell encircled. A fairly well developed Golgi field, centro- somes, dense bodies and lipid droplets are found in a juxtanuclear position. Processes, sometimes as thin as 100 nm, extend from the central region, often leaving parts of the cell surfaces of A-, NA-, SGC-cells and small, "light" neurons uncovered. Principal neurons which often occurred as individual cells were always observed to be encircled by a complete satellite cell sheath. The broader ones of these processes contain elongated mitochondrial profiles with hardly visible cristae, single stacks of rough ER, free ribosomes, microtubules and filaments.

Nerve Fibres and Synapses. Bundles of myelinated and unmyelinated nerve fibres are regularly encountered in and underneath the capsule of the adrenal gland, and in the interior of the organ, running alone or associated with chromaffin and nerve cells. Bundles may comprise up to more than 300 individual axons, numbers being higher at the surface than within the gland. Axons are invested by satellite cells in the usual manner. Larger bundles of nerve fibres

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are engulfed by slender, elongated cells with tapering processes. These cells show a rich membrane vesiculation, but only a sparse population of cell organelles such as rough ER, Golgi apparatuses, mitochondria or dense bodies. The nucleus exhibits dense patches of peripherally located chromatin and sometimes deep indentations. These cells which probably constitute the diffusion barrier separating endoneural and interstitial space (cf Shantaveerappa and Bourne, 1962; Klemm, 1970) alternate with layers of collagen fibres and are also found to invade large nerve trunks. However, they are separated from satellite cells and axons by a continuous basal lamina. The number of layers of perineural cells varies according to the thickness of the corresponding nerve bundles. Bundles having less than about 100 axons usually have a discontinuous perineural sheath or lack it completely. The size of individual axons varies between 0.2 and 5 la for non-myelinated and 1 to 4 g for myelinated ones, respectively. Myelinated axons have from 2 to approximately 20 mesaxon loops. Axons contain neurotubules often exhibiting a centrally situated filament. Neurofilaments are present, too. Mitochondria have transverse cristae embedded into a highly electron-dense matrix. Dense bodies are occasionally seen. According to the types of vesicles they contain two different types of nerve fibres can be distinguished (Fig. 9a). Type 1 displays DCV measuring 730-1,340 A with an average of 1,152 A, and small, clear vesicles ranging from 500-600 • in diameter. DCV are more frequently encountered than clear vesicles. If both are filling an axon profile, DCV tend to be more peripherally distributed. Both types of vesicles are found as well in synaptic endings on chromaffin cells and neurons. Axons of type 2 are larger than those of type 1 and account for the large-sized non-myelinated axons (up to 5 g) mentioned above. DCV present in these axons resemble A-granules with regard to size, shape and electron-density. Besides DCV axons of this type occasionally display small, clear vesicles scattered or in groups. They have approximately the same size as clear vesicles in type 1 axons, but are somewhat more polymorphic (Fig. 9 a).

Bundles of axons approach and enter groups of chromaffin and SGC-cells. They penetrate the basal lamina surrounding these cells and become invested by somatic satellite cells. A clear distinction between the satellite cells investing axons or synaptic endings on various effector cells, respectively, is not possible.

There is a basic difference between synaptic endings at A- and NA-cells, respectively. In Testudo A-cells have a pronounced tendency for forming processes (Fig. 8 a) and synapses most frequently occur at these processes. Side-to- side contacts are most common (Fig. 7 a), with often more than one synaptic area along one cell process. Synaptic areas are usually smooth and invaginations of the terminal axons into the effector cell are rarely seen. Synaptic areas contain numerous small agranular vesicles 500-600 A in diameter, together with DCV of the same size as those seen in type 1 axons (see above) on the presynaptic side, whereas the postsynaptic side displays typical A-cell features, sometimes together with large amounts of neurotubules. In addition to vesicles the presynaptic areas exhibit mitochondria with transversely oriented lamellar cristae, scattered or aggregated glycogen particles, microtubules, and cisternae of smooth membranes. While clear "synaptic" vesicles occupy a position close to the presynaptic membrane, the other organelles including DCV appear to be distributed randomly. Pre- and postsynaptic membranes do not exhibit any unusual features.


In contrast to A-cells axon terminals at NA-cells contact the cell body itself (Fig. 7b). Very often synaptic endings are seen close to the centre of a group of chromaffin cells, where they are deeply embedded into NA-cells. As a rule the synaptic membranes are curved. Short spines of the NA-cell may penetrate into the terminal with the synaptic membranes located either beside the spine or on the spine itself. In terms of qualitative ultrastructure the contents of terminals is the same as in those of A-cells.

After administration of 5-OHDA a few axon profiles in close proximity to A- and NA-cells are filled with closely packed DCV measuring between 500 and 1,300 ,~ (average 760 ~ ) (Fig. 9 b). These vesicles have a highly electron-dense core. The light halo between core and vesicle membrane is very narrow or not discernible at all. A-, NA- and SGC-cells as well as the cell bodies of principal neurons and synapses at these cells are not labelled.

Synapses with SGC-cells are hard to find. Axon terminals similar to those ending on NA-cells make contact with the somata of SGC-cells. However, synaptic membrane specialisations are extremely rare. The same holds true for synapses with the small "l ight" neurons.

Principal neurons receive axo-somatic nerve terminals which exhibit predomi- nantly clear "synaptic" vesicles and a few DCV, 700-1,300/~ in diameter. Axo- dendritic contacts were not observed. Processes of what appears to be A-cells commonly join bundles of nerve fibres which innervate vessels in the adrenal gland (Fig. 8 b).

Lacerta and Natrix

The results obtained from Lacerta and Natrix, respectively, are to be dealt with only in so far as they differ from those described above.

A general impression when looking at A- and NA-cells of lizards and snakes concerns the minor polymorphism of the specific granules in these species compared to tortoises (Fig. 4). The size distributions of A- and NA-granules, respectively, for Lacerta and Natrix are shown in Fig. 1. The average sizes of A- granules in Lacerta and Natrix are 255 and 179 nm. The corresponding parameters for NA-granules are 323 and 304 nm. Moreover, groups of chromaffin cells look more compact in lizards and snakes than in tortoises. A-cells are almost exclusively found in the interior of the gland intermingled with internal cords. NA-cells on the other hand prevail at the surface, particularly at the dorsal side of the organ. They are rare in the proximity of interrenal and A-cells. Neither in Lacerta nor in Natrix do they form conspicuous processes. Hence, preganglionic axon terminals synapse with the perikaryon (Fig. 4) and axons of type 2 (see page 494) were never observed in the large nerve trunks. A-cells often contain a prominent rough ER arranged in parallel stacks. Round profiles of dilated ER-cisternae do not occur. NA-cells in both species show lower amounts of dense bodies, which rarely incorporate granules. SGC-cells as well as small, "l ight" neurons are more rarely encountered than in Testudo. In Lacerta granules vary in diameter from 600-1,250 ,~ with a mean diameter of 875 .&. The corresponding data for Natrix are 580 to 1,540 ,~, and 1,070 ,~. Synapses on SGC-cells are as rare as in the tortoise.

496 K. Unsicker

Administration of 6-OHDA reveals the response of a few destroyed axons in Lacerta. However, the fibres affected were never seen in direct contact with chromaffin or nerve cells. In addition, 6-OHDA causes profound changes in the ultrastructure of the steroid producing cells, which are reported elsewhere (Unsicker et al., 1975). Chromaffin, SGC- and nerve cells are not affected by 6-OHDA.

Discussion

Chromaffin Cells

The present paper confirms earlier reports on the occurrence of A- and NA- storing cells in the reptilian adrenal. Thus, the use of chromaffin and iodate reactions led Wright and Jones (1955) to assume that NA-producing cells mainly occupy the dorsal aspect of the adrenal gland in Lacerta viridis, while cells storing probably A were intermingled with the interrenal tissue. Similar results concerning the differential storage of both catecholamines and the distribution of A- and NA-cells were obtained by Gabe and Martoja (1961) for the Squamata and by Houssay et al. (1962) for the snakes, Xenodon merremii, Bothrops alternata and Bothrops neuwiedi, the lizard Teius teyon, the yacare Caiman latirostris, and the iguanas Tupinambis tegnixin and Tupinambis refescens. The latter authors as well as Wassermann and Tramezzani (1963) corroborated their findings, based primarily on histochemical techniques particularly that of Hillarp and H6kfelt (1955), by using chemical assays and chromatography of extracts. In addition, Wassermann and Tramezzani (1963) made electron microscope studies which showed clear differences between A- and NA-storing cells, especially with respect to their granules. They also noted a considerable polymorphism, a wide variation in size and a difficulty in preserving the membrane for NA- granules of Xenodon merremii, but a round shape of A-granules which exhibited a well-preserved membrane. The problems in preserving the membrane of NA- granules, which we did not have, are remarkable in so far as Wassermann and Tramezzani (1963) also performed a perfusion fixation. Polymorphism and a wide variation in size of chromaffin granules also occur in our material. Polymorphism of both NA- and A-granules is most pronounced in tortoises, whereas in lizards and snakes A-granules are round and profiles of NA-granules display minor deviations from the circular form. Although the polymorphism of chromaffin granules in reptiles is conspicuous, we must say that a moderate polymorphism of chromaffin granules is certainly not a typical feature of reptiles or other lower vertebrates. Employing high voltage electron microscopy, which allows three-dimensional analysis of cell organelles in relatively thick sections, Carmichael and Smith (1974) found that 14.9% of the granular vesicles in the cat adrenal medulla appeared tubular. They considered it likely that ovoid comma- and dumbbell-shaped forms result from tangential sectioning through these tubular forms.

The figures for the distribution of sizes of A- and NA-granules demonstrate that there are species dependent variations in size. The largest average size


and individual granules occur in Testudo. This species seems to have the largest chromaffin granules reported so far for vertebrates. However this could be due to the fact that greater amounts of the larger granules are perhaps lysosomes which cannot be recognised because of the relatively high electron density of (A) granules in Testudo.

Species differences in relative size of A- and NA-storing granules in various forms of vertebrates (dogfish, frog, domestic fowl, rat, cat, man) have also been reported by Coupland (1971, 1972).

It is widely accepted that glutaraldehyde fixation followed by osmium tetroxide impregnation forms a reliable basis for the identification of NA- and A- granules with the electron microscope. Glutaraldehyde denaturates the soluble proteins of the chromaffin granule including chromogranin A and precipitates NA, while A, on the other hand, is lost from its storage sites and leaves the proteins in its granule relatively unmasked (cf. Coupland et al., 1964; Tramezzani et al., 1964; Coupland and Hopwood, 1967). Accordingly, one should expect NA-granules to be highly electron-dense and A-granules to appear finely granular. As a whole this is what can be seen in chromaffin cells of reptiles used for the present study as well as in other vertebrates (Coupland, 1971). However, in the tortoise, some A-granules have a high electron density which may reach that of NA-granules and render the discrimination difficult. This is of particular importance with regard to the fact that mixtures of A- and NA-granules have been reported to occur in mammalian chromaffin cells in foetal and early postnatal life (Elfvin, 1967a; Coupland and Weakly, 1968, 1970) and under in vitro conditions in NA-cells after inducing the NA into A converting enzyme PNMT with a corticosterone-treatment (Coupland and MacDougall, 1966). However, leaving the sections unstained always allowed a clear differentiation of A- and NA-granules, and mixtures of the two types were never observed.

Irregularities of preservation and[or staining of the contents of chromaffin granules, which sometimes occur, may allow an evaluation of their internal structure. Thus, NA-granules may exhibit a globular substructure which might reflect the macro-molecular organisation of the protein components in the granule.

As has been pointed out by Bloom (1972) there is surprisingly little rough ER compared to the amount of protein liberated in adrenomedullary cells. Adrenal chromaffin cells of reptiles are no exception in this respect. The quantity and pattern of rough ER in A-cells of the tortoise, however, are different from those seen in the same type of cells in other species. There are round dilations of rough ER containing a floccular material of moderately electron opacity, identical to what has been described by Kanerva and Hervonen (1973) in autonomic neurons of the rat paracervical ganglion as "ergastosomes". Kanerva and Ter~iv~iinen (1972) had noted similar inclusions in the vacuolated nerve cells of the same ganglion. Kanerva and Hervonen (1973) have offered several speculations as to the functional significance of the contents of "ergastosomes", among them:

1. they could store a protein with endocrine activities, or a binding protein for some component of the metabolic l~roduction of the neurons;

2. they could be an indication of peripheral neurosecretion.

498 K. Unsicker

We think that amongst others, the possibility of an overproduction of enzymes or proteins required for the synthesis or binding of catecholamines should be considered as well for the following reasons: NA-cells in the tortoise possess a conspicuous number of dense bodies apparently involved in the destruction of surplus granules (see below), NA-cells in lizards and snakes have these dense bodies, too, however in much smaller amounts. If environmental or behav- ioural conditions require a reduced rate of secretion of both NA and A, and if A-cells, as is obvious from their ultrastructure, do not have a granule destruction device as effective as that in NA-cells, reduced secretion could be achieved by means of a retention of storage of proteins normally associated with the synthesis and binding of catecholamines (e.g. tyrosine hydroxylase, DOPA decarboxylase, DBH, PNMT, chromogranin A). This speculation, however, does not reflect the current concepts of inhibition mechanisms of catecholamine synthesis as, for example, reduced firing rates of the pre-ganglionic neurons of tyrosine hydroxylase end product inhibition (Nagatsu et al., 1964).

Another possibility for the interpretation of the "ergastosomes" in tortoise A-cells, which should be considered, is the direct formation of chromaffin granules from ER, as has been suggested by Ratzenhofer and Mtiller (1967). A conclusive answer concerning the role of the "ergastosomes" could perhaps be obtained by experiments using reserpine, which has been reported to increase the amounts of ER in adrenal medullary cells of the hamster (Yates, 1964). Preliminary experiments with 5 mg/kg Serpasil| failed to show significant changes in the amount and pattern of ER in CA-cells of the tortoise (Unsicker, unpublished results).

Dense bodies are prominent cell organelles in NA-producing cells of all reptiles studied. They are particularly numerous in the tortoise where they can be seen to engulf granules of various numbers and sizes. In accordance with Coupland (1971, 1972), who has observed identical cytosomes in adrenomedullary cells (NA-cells) of the dogfish and the domestic fowl, we would suggest that these dense bodies are involved in the intracellular destruction of chromaffin granules. This view is supported by the finding that NA-cells with only a few granules usually lack dense bodies. A similar concept has been brought forward by Smith and Farquhar (1966) for the mammotrophs in the anterior pituitary. The myelin figures usually present in dense bodies of reptilianNA-Cells might represent alternating layers of catechol-polymers and lipid moleculaes coming from the high lipid content (22% dry weight, Hillarp, 1959) of the granules. Dense bodies are far less numerous in A- than in NA-cells and do not show myelin-like inclusions. There is a good deal of evidence that lysosomes of chromaffin cells arise in the Golgi region (Coupland et al., 1968) or in the GERL, where Golgi saccules and ER are closely related (Holtzman and Dominitz, 1968). Positive acid phosphase reaction product can be localised in that area (Coupland, 1968) as well as in dense bodies and autophagic vacuoles (Holtzman and Dominitz, 1968). Structures containing ER and mitochondria fragments, ribosomes and granules have been referred to by Holtzman and Dominitz as autophagic vacuoles in A-cells of the rat. These structures are more or less identical with what we call dense bodies. The latter term has been reserved by these authors for acid phosphatase-positive, membrane-delimited bodies 0.5


1 ~ in diameter, containing patches of grains, membranes and vacuolated areas. These bodies may correspond to some larger granules in our material, which, most clearly in unstained sections, contain irregular dense patches of high electron density and which form transitional stages towards dense bodies, as far as it can be judged from their ultrastructure. If this is right, a considerable number of what appear as secretory granules in our findings could be lysosomal in nature.

Golgi areas in chromaffin cells of reptiles resemble those in various other vertebrates (c f Coupland, 1971). In both types of cells, Golgi cisternae and vesicles contain a granular material of medium electron density, perhaps pre- lysosomes or binding proteins for catecholamines. As in the rat, the Golgi region of NA-cells is almost devoid of chromaffin granules.

Since De Robertis' and Vas Ferreira's (1957) early work on the mechanisms of discharge of the granule contents, several morphological observations have supported the concept of exocytosis (Coupland, 1965a, b; Diner, 1967) which is in accord with biochemical evidence (see Smith and Winkler, 1972, for review). Profiles indicating exocytosis of chromaffin granules were not observed in this study. This is not surprising, since it becomes apparent from a review of literature that some species are more suitable for the demonstration of exocytosis than others.

The arrangements of microtubules in chromaffin cells and their relation to granules deserves a special study for two reasons:

1. microtubules have been suggested to participate in the transport of secretory granules in endocrine cells of the pancreas (Lacy et al., 1968); and

2. colchicine and vinblastine inhibit the secretion of catecholamines evoked by carbamylcholine from the perfused adrenal gland (A.D. Smith, unpublished observation, quoted after Smith and Winkler, 1972). An evaluation of the second observation, however, has to take into account the high concentrations of the drugs required, which could speak in favour of a direct, unspecific inhibitory action on secretion.

Vacuoles filled with a colloid-like substance and situated within chromaffin cells or surrounded by them (cf. Unsicker, 1969; A1-Lami, 1970: hamster; Un- sicker, 1973a: house sparrow and chaffinch) do not exist in the reptilian species studied.

SGC-Cells

A substantial literature on SGC-cells has accumulated in the past decade. These cells have been reported to occur in the superior cervical ganglion (Er~ink6 and H~irkonen, 1965; Siegrist et al., 1966; H6kfelt, 1969; Matthews and Rais- man, 1969; Van Orden etal . , 1970; Yokata, 1973), in the inferior mesenteric ganglion (Elfvin, 1968), in the hypogastric ganglion (Watanabe, 1971), in peri- bronchial paraganglia (B6ck, 1970), in the turtle heart (Chiba and Yamauchi, 1973; Yamauchi et al., 1975), in the paracervical ganglion (Kanerva, 1971, 1972; Kanerva and Ter~v~iinen, 1972), in the adrenal medulla of rat (Diner, 1965) and man (Hervonen, 1971) during development, and in the adrenal gland of

500 K. Unsicker

adult birds (Unsicker, 1973a). Many, if not all, of these SGC-cells seem to be identical with SIF-cells, first described by Ergnk6 and H/irk6nen (1963) in the rat superior cervical ganglion. In sympathetic chain ganglia of pig, cat and rat, these cells have been shown to contain dopamine (Bj6rklund et al., 1970). On the other hand, Er/ink6 and Er/ink6 (1971) have demonstrated NA in SIF-cells of the rat superior cervical ganglion. As for the function of these cells, four major lines of hypothesis are discussed:

1. SGC-cells may be capable of acting as interneurons within the ganglia. Support for this hypothesis comes from the demonstration of efferent synapses which may contact dendrites, cell bodies, or even axon collaterals of principal sympathetic neurons Matthews and Raisman, 1969; Yokota, 1973).

2. An endocrine, catecholamine-secretory function has to be considered like- wise, since parts of these cells may be exposed to the intercellular space in close proximity to blood capillaries (Matthews and Raisman, 1968; Siegrist et al., 1968; Yokota, 1973). On the other hand, Van Orden et al. (1970) have found that the catecholamine stores of SIF-cells are very stable and not depleted under physiological circumstances.

3. Chun and Yates (1970) and Erfink6 and ErS.nk6 (1971) have proposed a chemoreceptor function; and finally

4. It is possible that SGC-cells constitute a non-functional relic of a primitive system (Norberg and Sj6qvist, 1966; Hervonen, 1971).

As far as the SGC-cells in the adrenal gland of reptiles are concerned, we would not adopt hypothesis 1., 3. or 4. An interneuron capacity is unlikely because of the lack of efferent synapses, and to be regarded as non-functional these cells are probably too numerous. The presence of highly electron-dense granules suggests the storage of a primary amine, as NA or DA. Determinations of catecholamines in the adrenals of reptiles (cf. Holzbauer and Sharman, 1972) have not revealed the presence of DA so far. However, Anton and Sayre (1964) have found DA in the heart, brain, kidney and liver of the turtle. A particularly high concentration of DA (44.7 lag/g tissue) was recorded in the spleen of this animal. Nothing can be said with regard to the storage sites of DA in this organ (mast cells-nerve cell bodies-nerve fibres), hence, a combined chemical and microspectrofluorimetric approach is suggested for settling the questions where DA is stored in the spleen and whether it is more than a precursor of NA in the adrenal of reptiles. Most mammalian adrenal glands contain only small amounts of DA probably as a precursor of NA (see Holzbauer and Sharman, 1972, for references). However, in sheep adrenal glands, Dengler (1957) found 2% of DA and Lishajko (1968, 1969) suggested that it is stored in separate particles in sheep adrenals.

Principal Neurons

Nerve cells are consistently found in the adrenal medulla of mammals (Hillarp, 1947; Unsicker, 1967; Lewis and Shute, 1969; Hervonen, 1971) and birds (Un- sicker, 1973a). The fine structure of what we termed principal neurons in the


adrenal of reptiles is very similar to that of ganglion cells in autonomic ganglia (Forssmann, 1964; Grillo, 1966; Kudo, 1971 ; Kanerva and Ter/iv/iinen, 1972; and others) and in the adrenal gland of mammals and birds (Unsicker, 1967, 1973 a).

The presence of a few axons, which can be labelled with 5-OHDA or react with 6-OHDA, could suggest that at least some principal neurons in the reptilian adrenal are adrenergic, provided that these axons do not belong to SGC-cells or small "light" neurons. This would classify those principal ganglion cells as "shor t" adrenergic neurons (cf Owman and Sj6strand, 1965). This hypothesis could be tested since "short" adrenergic neurons differ from "long" ones in several functional respects. They react differently to immunosympathectomy (Iversen et al., 1966) and have a lower in vivo sensitivity to 6-OHDA (Malmfors and Sachs, 1968) and reserpine (Owman and Sj6berg, 1967; Sj6strand and Swedin, 1968). On the other hand, since sympathetic and parasympathetic nerve cells may show the same ultrastructure (Yoshida, 1968), it cannot be excluded that some principal neurons are cholinergic. Hervonen's (1971) observations on the developing human adrenal suggest that "the possibility of direct postganglionic innervation of a minor part of medullary cells should be considered".This is compatible with the finding that after sectioning the extrinsic nerves some fibres persist in the gland (Hollinshead, 1936; Swinyard, 1937; Robinson, 1975, personal communication). Evidence for persisting synapses on adrenomedullary cells in long-term tissue cultures would suggest the possibility of a postganglionic innervation of the medulla. Two kinds of principal neurons have been found in the adrenal gland of reptiles: the "light" and the "dark" ones. Similar observations on the occurrence of light and dark autonomic nerve cells have been made in the paracervical ganglion of the rat by Kanerva (1972), who also stated differences between the two types with regard to the structure of nuclei and the quantities of free ribosomes and rough ER. In a later study on the same ganglion using perfusion fixation, Kanerva (1972) was not able to confirm the former results and regarded the differences between "light" and "dark" neurons to be presumably an artifact. Since perfusion fixation did not abolish the differences between the two cell types in our study, we do not consider them as artifacts. Nerve cells in the adrenal of reptiles have also been observed by Braun (1882), Vincent (1896) and Ciaccio (1906). Braun and Ciccaio mention the occurrence of intermediate stages between sympathetic nerve cells and chromaffin cells. For the discussion of "ergastosomes", which also occur in principal neurons, see page 497.

Nerve Fibres and Synapses

There is a general agreement that adrenal medullary cells in mammals are innervated by preganglionic sympathetic cholinergic nerve fibres (Feldberg et al., 1934; Hillarp, 1947; Marley and Prout, 1968). Morphological evidence for the cholinergic nature of most of the nerve fibres and terminals in the adrenal gland of reptiles derives from the facts that, firstly, only very few fibres contain dense-cored vesicles after the application of 5-OHDA or show signs of degenera-

502 K. Unsicker

tion after treatment with 6-OHDA, respectively (see below), and secondly, the ultrastructural features of the terminals are those of cholinergic nerve endings, as have been described to occur in various autonomic ganglia (Grillo, 1966) the adrenal medulla of mammals (Coupland, 1965 c, 1972; Grynszpan-Winograd, 1974) and birds (Unsicker, 1973b), and in the CNS (cf De Robertis et al., 1963; Whittaker, 1965). These nerve endings are characterised by the presence of two different populations of vesicles: clear vesicles approximately 500-600 fk in diameter, and dense-cord vesicles measuring 730-1,340 A with a mean diameter of 1,152 A. The same types of vesicles are seen in nerve endings contacting SGC-cells and principal neurons suggesting that these cells receive a cholinergic nerve supply as well. At chromaffin cells and principal neurons typical thicken- ings of pre- and postsynaptic membranes are usually observed at the site of synaptic contacts. Similar membrane specialisations are very rare on SGC-cells, even in those cases when large accumulation of synaptic vesicles are seen. Since membranes specialisations in developing synapses on human fetal chromaffin cells appear simultaneously or after the accumulation of synaptic vesicles (Her- vonen, 1971), this might indicate that quite a few synapses on SGC-cells (and SGC-cells themselves?) are still under development.

In the adrenal gland of the tortoise there is a clear dimorphism in the innervation of A- and NA-cells. A-cells are contacted via their processes, which facilities side-to-side contacts, whereas synaptic terminals on NA-cells end at the perikaryon. The various morphological aspects of synaptic endings on NA- cells in rept i les- e.g. lying within deep invaginations of the cell surface, enlarging the area of contact by forming complex interdigitations with the effector ce l l - are very similar to those seen in birds (Unsicker, 1973b). Due to a less marked formation of chromaffin cell processes in lizards and snakes there is no evidently distinct innervation of A- and NA-cells in these species: quantitative studies, as have been carried out on the innervation of A- and NA-cells in the adrenal medulla of the hamster (Grynszpan-Winograd, 1974), would be required to reveal a different innervation of A- and NA-cells in Lacerta and Natrix, if it exists.

The way, in which nerve fibres reach their effector cells in the reptilian adrenal, is also very similar to what we have seen in birds (Unsicker, 1973b). Nerve fibres penetrate the basal lamina surrounding clusters of chromaffin cells, become engulfed by somatic satellite cells, and frequently terminate in or near the centre of a group of cells for the formation of synapses. Despite the frequent occurrence of this mode of arrangement of nerve fibres and terminals we have not observed clearly polarised chromaffin cells (cf Unsicker, 1973 b).

Cell processes, which according to their granule equipment, belong to A-cells, are a common feature of the tortoise adrenal. They join the preganglionic nerve plexus and may be even found in the vicinity of smooth muscle cells in the wall of some adrenal blood vessels. The formation of processes may be regarded as a primitive feature of chromaffin cells whose development from a sympathetic stem cell towards a highly specialised catecholamine producing cell has stopped at an intermediate stage resulting in a cell with the functional characteristics of a chromaffin cell but also with a few remaining morphological


characteristics of a sympathetic neuron. It should be mentioned in this context that extra-adrenal chromaffin cells tend to be fusiform (Coupland and Weakly, 1970), and that mammalian medullary cells transplanted to the anterior eye chamber (Olson and Malmfors, 1970) or grown in tissue culture (Unsicker and Chamley, in press) show a strong tendency for developing processes. The presence of these A-cell processes in the proximity of the media of arterioles suggests that. they cannot be regarded as mere relics in term of evolution. However, if they are to be regarded as functional processes this would raise the question of A as an adrenergic transmitter in reptiles.

By using 5- and 6-OHDA, which are known to mark selectively adrenergic nerve terminals (see Thoenen, 1972, for review) we have identified adrenergic nerves in the adrenal of reptiles. Including the first discovery of adrenergic nerves in the vicinity of chromaffin cells (Unsicker, 1973c; birds) and the reports by Coupland (1972) and Prentice and Wood (1975) on adrenergic nerves in the adrenal medulla of dogfish and cat, there is now consistent evidence for the presence of adrenergic nerve fibres in the adrenal medulla of amniotic and anamniotic vertebrates. A convincing interpretation concerning their significance can hardly be delivered. A local c~-adrenoreceptor mediated feed-back inhibition of catecholamine release from the adrenal medulla, similar to the mechanism working at adrenergic nerve endings (Starke, 1972), has recently been proposed by Starke et al. (1974). This concept has been supported by the finding of Gutman and Boonyaviroy (1974) that NA suppresses catecholamine secretion from the adrenal medulla. Adrenergic terminals could perhaps be responsible for establishing unusually high concentrations of N A exceeding those with result from catecholamines released from chromaffin cells.

Nerve fibres characterised by the presence of large DCV which exceed the size of the DCV in cholinergic nerves and which show only a thin light rim between the dense core and the vesicle membrane are constantly found in the adrenal gland of birds. This type of nerve fibre which resembles the fibres of the purinergic type (cf. Burnstock, 1972, for review) was not observed in the adrenal gland of the reptilian species studied.

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Received August 18, 1975