Differentiation and transdifferentiation of adrenal chromaffin cells of the guinea-pig

Cell Tissue Res (1981) 215:341-367 Cell and Tissue Research �9 Springer-Verlag 1981

Differentiation and Transdifferentiation of Adrenal Chromaffin Cells of the Guinea-Pig I. Transplants to the Anterior Chamber of the Eye*

K. Unsicker, B. Tschechne, and D. Tschechne Department of Anatomy and Cell Biology, Philipp's University of Marburg, Marburg, Federal Republic of Germany

Summary. Autografts of adrenal chromaffin cells from adult guinea pigs to the anterior chamber of the eye were studied electron microscopically and both histo- and biochemically 4, 8 and 12 weeks after transplantation.

Transplanted chromaffin cells resembled in many respects their in-situ- counterparts: they stored varying amounts of granular vesicles, which were reduced in diameter, but displayed mostly cores of low or medium electron densities suggesting predominant storage of a secondary amine. Concomitant biochemical determinations of catecholamines (CA) and phenylethanolamine N-methyltransferase (PNMT) activity revealed a distinct reduction of total CA and PNMT activity, but no change in the proportion of adrenaline (A) to noradrenaline (NA) after 4 weeks. However, an increase of NA and almost equal amounts of A and NA were found after 8 weeks.

CA-histofluorescence and electron microscopy revealed that axon-like processes with varicosities extend from chromaffin cells and contain the large "chromaffin" vesicles (100-200 nm in diameter) in addition to small clear and dense-cored vesicles (40-80 nm in diameter). Processes of chromaffin cells grew in all directions over the host iris and were also found in close proximity to smooth muscle cells the sympathetic nerve supply of which had been cut by removing the superior cervical ganglion. Administration of 5- and 6-hydroxydopamine (OHDA) resulted in characteristic labeling and ultramorphological changes in axons, but caused alterations in chromaffin cell bodies only 8 weeks after transplantation. Transplanted chromaffin cells became reinnervated by nerve fibres that are considered to be cholinergic fibres derived from the ciliary ganglion. Transplanted chromaffin cells also exhibited synapse-like contacts with each other.

The present study shows that chromaffin cells from adult guinea pigs transplanted to the anterior chamber of the eye retain a large number of differentiated properties. Formation of axon-like processes by these cells

Send offprint requests to: Prof. K. Unsicker, Department of Anatomy and Cell Biology, Philipp's- University, Robert-Koch-Str. 6, D-3550 Marburg, Federal Republic of Germany

* Supported by grants from the Deutsche Forschungsgemeinschaft (Un 34/6; SFB 103)

0302-766X/81/0215/0341/$05.40

342 K. Unsicker et al.

indicates that the anterior chamber of the eye favours transdifferentiation, as does tissue culture (Unsicker et al. 1978a), eliciting a cell type that displays features of both a sympathetic neurone and a SIF-cell.

Key words: Adrenal chromaffin cells - Plasticity - Eye chamber transplants - Ultrastructure - Catecholamine biochemistry.

The adrenal medulla is a modified sympathetic ganglion, which has specialized in the synthesis, storage and release of the catecholamines (CA) adrenaline (A) and noradrenaline (NA). The endocrine chromaffin cells of the adrenal medulla share a variety of features with sympathetic adrenergic neurones: they originate from the neural crest (Weston 1970), receive preganglionic cholinergic synapses (Feldberg et al. 1934; Coupland 1965), produce almost identical carrier and enzyme proteins (von Euler 1972; Stj/irne 1972) and require Ca 2+ for the secretion of CA (Douglas 1968; Smith and Winkler 1972). On the other hand, there are also divergencies regarding their morphological, physiological and pharmacological behaviour: (i) the adrenal medulla synthesizes both NA and A, (ii) chromaffin cells possess no processes for transmitting impulses to other effector cells, and (iii) are not destroyed by 6-hydroxydopamine (6-OHDA), guanethidine and immunosympathectomy using an antiserum against a nerve growth factor (NGF) (Cheah et al. 1971; Burnstock et al. 1971; Levi-Montalcini and Angeletti 1966).

The differentiation of the sympathetic adrenergic neurone has been extensively studied. Orthograde trans-synaptic regulation through presynaptic cholinergic terminals, NGF, and non-neuronal cells have been found to be involved in the maturation of the adrenergic neurone and specification of its transmitter (see Black 1978, for review). In contrast, the genetic, humoral and other factors that govern and maintain differentiation of chromaffin cells and are responsible for the observed differences between these cells and sympathetic adrenergic neurones are largely unknown. The thesis that the adrenal cortex might contribute to the differentiation and maintenance of differentiation of the adrenal medulla by regulating the methylation of NA to A was originally suggested by H6fkelt (1952) and by Coupland (1953). This concept has been substantiated by developmental studies showing, for example, that in rat adrenals the rise in the methylating enzyme phenylethanolamine N-methyltransferase (PNMT) is preceded by a parallel increase in steroid synthesis by cortical cells (Roffi 1965; see also Pohorecky and Wurtman 1971; Weiner 1975, for reviews).

Studies of the chromaffin cells of the adult adrenal medulla have similarly shown the importance of corticosteroid hormones in inducing the methylation of NA (Wurtman and Axelrod 1966). Tyrosine hydroxylase (TH) and dopamine-/~- hydroxylase (DBH) also appear to be modulated by the secretions of the adrenal cortex. TH- (Wurtman and Axelrod 1966; Kvetuansky et al. 1970) and DBH-values (Weinshilboum and Axelrod 1970; Ciaranello et al. 1976) are significantly reduced in rat adrenals after hypophysectomy and can be restored by administration of ACTH (Mueller et al. 1970; Ciaranello et al. 1976), but not by administration of large doses of glucocorticoids, suggesting that factors other than gtucocorticoids, which originate from the adrenal cortex, may modify the activities of these enzymes. Investigations by Unsicker et al. (1978a), who failed to prevent the loss of A-storing

Adrenal Chromaffin Cells: Differentiation in Transplants 343

cells in cultures of dissociated adrenal medullary cells by administration of large doses of dexamethasone, might point in the same direction.

Maintenance of function and adaptability of secretion of adrenal chromaffin cells by neuronal regulation of CA-synthesizing enzymes is a well-established fact (see Kirshner 1975 for review). Studies by Ciaranello and coworkers (1976) on the neuronal and hormonal regulation of DBH and PNMT indicate that both factors may interact at very complex levels. The extent to which preganglionic innervation contributes to the maturation of adrenal chromaffin cells during ontogeny has not been evaluated.

Finally, hereditary factors might well influence the development and maintenance of differentiation of the chromaffin cell. Using three inbred strains of mice, Ciaranello et al. (1972) have evaluated the effects of hypophysectomy, ACTH, dexamethasone and neural stimulation on PNMT levels and found that these strains exhibit interesting genetic differences regarding regulation of A synthesis.

Cell and tissue culture studies o f postnatal rat adrenal medulla have recently shown that chromaffin cells in the absence of an intact preganglionic innervation and cortical cells form processes spontaneously or in response to NGF treatment, adopt structural features similar to those seen in small granule-containing cells of sympathetic ganglia (Grillo 1966; Siegrist et al. 1968; Matthews and Raisman 1969; Williams and Palay 1969; Yokota 1973) or sympathetic neurones, and loose the ability to produce A (Unsicker and Chamley 1976; Unsicker et al. 1978a).

In an attempt to overcome the disadvantages of cell culture conditions due mainly to the rather artificial environment, we have investigated adrenal chromaffin cells in transplants to the anterior chamber of the eye and under the kidney capsule (Unsicker et al., in preparation). Both sites differ with respect to the innervation they provide for adrenal medullary grafts: chromaffin cells in the anterior chamber of the eye become reinnervated by cholinergic nerves, presumably from the ciliary ganglion (Unsicker et al. 1978b), while no reinnervation occurs with transplants under the kidney capsule (Carruba et al. 1974; Unsicker et al. 1977a). Thus, we aimed at obtaining information concerning the extent to which the distinctive relationship between the adrenal cortex and medulla and splanchnic nerves may define the expression of the phenotype of the adult chromaffin cell in terms of ultrastructure, CA content, and A and NA production. The guinea pig was chosen as an experimental animal, because its medulla contains a particularly high proportion of A-storing cells (Unsicker et at. i978c), which can be assumed to represent the most differentiated stage of the chromaffin cell.

Materials and Methods

Adult female albino guinea pigs (body weight 350-650 g) of an inbred strain were used in the present experiments.

Transplantation Technique

Animals were anaesthetized with Ketanest | (3ml/kg i.p.) and ether, and the abdominal cavity was opened on the left side under sterile conditions. The left adrenal was exposed and removed after ligature of the vessels. Medullary tissue was carefully freed from surrounding cortex in ice-cold Hanks' balanced salt solution under a binocular microscope and cut into pieces approximately 1 mm 3. The left eye was atropinized (one drop o f atropine, 10mg/ml) and under local anaesthesia with Pantocain | (3%


solution) two pieces of adrenal medullary tissue were inserted into the iridocorneal angle through a 2 mm wide slit made close to the corneoscleral junction.

Twenty-four animals had their left superior cervical ganglion including 5 mm of the preganglionic trunk and 5 mm of the postganglionic fibres removed 3-5 days prior to transplantation. These animals will be referred to as sympathectomized animals. Revascularization of anterior eye chamber transplants was studied by slit lamp examination. Four days after the transplantation, most eyes were clear and transplants were vascularized.

Electron Microscopy

Four, 8 or 12 weeks after transplantation the animals were perfused with 200 ml of 3.5 ~ glutaraldehyde in 0.1 M phosphate buffer (pH 7.4; 4--6 ~ C) via the descending aorta. Both eye balls were removed and both lenses and the posterior half of the bulbus were discarded. The anterior half of the bulbus was cut into 1-2 mm pieces, postfixed in phosphate-buffered glutaraldehyde for 2 h and rinsed for at least 2 h in phosphate buffer. After fixation in 2 ~ aqueous OsO4 for 2 h and dehydration in a graded series of ethanol, tissues were embedded in Araldite.

Adrenal glands from six guinea pigs served as controls and were processes for electron microscopy as described above.

Thin sections were cut on a Reichert-Sitte OmU 2 ultramicrotome, stained with a saturated solution of uranyl acetate in 70 ~ methanol and lead citrate (Reynolds 1963) 5 min each and viewed under a Zeiss EM 9A and a Siemens 101 electron microscope. 0.5-1 gm-thick sections were stained with 1 ~ toluidine blue or with azur II-methylene blue (Richardson et al. 1960).

Treatment with 5- and 6-Hydroxydopamine ( OHDA )

Two sympathectomized and two non-sympathectomized animals received three intraperitoneal injections of 300 mg/kg 5-OHDA-hydrochloride 30, 18 and 6 h prior to sacrifice. The drug was dissolved in 1 ml Hanks' balanced salt solution and supplemented with 0.1 mg ascorbic acid. Controls were injected with the vehicle only. Three sympathectomized and two non-sympathectomized animals were treated with 100mg/kg 6-OHDA-hydrochloride intraperitoneally 12h and immediately prior to per fusion.

Catecholamine Histochemistry

Iris stretch preparations from six animals carrying transplants and two irides from control animals were incubated with glyoxylic acid for demonstration of specific catecholamine (CA) histofluorescence (De la Torre and Surgeon 1976); 15 ~m-thick cryostat sections from adrenals of adult guinea pigs were treated in the same way. Preparations were examined with a Zeiss Universal fluorescence microscope fitted with epi-illuminescence. The argentaffin reaction was applied to semithin sections of Araldite-embedded postosmicated material (Gorgas and B6ck 1976) using the ammoniac silver nitrate solution described by Singh (1964).

Biochemical Determinations of Adrenaline ( A ) , Noradrenaline ( NA ) and Phenylethanolamine N-Methyltransferase (PNMT)

Eye balls with transplants from 17 sympathectomized and eyes from six control animals without transplants were used. In addition, A, NA and PNMT were determined in intact adrenal glands. Eye balls and adrenals were carefully freed from adhering tissue and homogenized in 6 ml of 5 mM tris-HC1 buffer (pH 7.0) containing 0.2~ Trition X-100 and 0.2~ bovine serum albumin (w/v both). The homogenate was divided into two parts for estimation of PNMT activity and catecholamines (CA).

PNMT activity was measured by the method of Axelrod (1962) with slight modifications. The homogenate was centrifuged for 30 min at 20,000 rpm in a Sorvall Superspeed RC2-B centrifuge; 20 gl of the supernatant were incubated at 37 ~ C for 5 min in a total volume of 200 gl containing 80 gl 5 mM tris- HC1 buffer (pH 7.0), 50 gl of 1 N tris-HC1 buffer (pH 8.6), 1.8 mM phenylethanolamine (dissolved in 25 ~tl 0.001 N HC1), 20 gl 0.001 N HCL and 5 lal S-adenosylmethionine solution (total 0.7 nmol, 17 14C-labelled, specific activity 56.7 mCi nmol-1). Throughout the incubation period the initial rate of enzyme activity was constant. The reaction was stopped by addition of 500 gl 0.5 M borate buffer (pH 10.0). Blank values were prepared by reversed addition of S-adenosylmethionine and borate buffer. The 14C-labelled N-methylated reaction product was extracted in 6ml 97 ~ toluene-3 ~ isoamyl alcohol


(v/v), which was added to the reaction mixture. The tube was shaken for 5 min and centrifuged. A 4 ml aliquot of the organic solvent was used for liquid scintillation counting (Packard model 3380) after addition of 10 ml scintillation fluid. NA and A were extracted from 2 ml of the homogenate by addition of 2 ml 10 % trichloroacetic acid for 20 min at 4 ~ C. After centrifugation at 5000 rpm for 10 rain at 4 ~ C, the sediment was reextracted with 6ml 5 % trichloroacetic acid and centrifuged as described above. Supernatants were pooled and the CA of an aliquot (2 ml) were obsorbed on and eluted from alumina. NA and A were measured by differential fluorometry (Unsicker et al. 1977b).

Statistics

For histograms (Fig. 18) comparing the size of storage vesicles in chromaffin cells and their processes in transplants 4, 8 and 12 weeks after surgery and in control adrenal glands, the diameters of the cores were measured at a magnification of 30,000 using a Zeiss particle-size determination device (Zeiss TGZ).

At least 2,000 granules were measured in each group. For biochemical data the Studenfs t-test was used to establish the significance of differences between means (Snedeeor and Cochran 1967). P<0.05 was chosen as the level of significance. The measurement of variation used in this study was the standard error of the mean (S.E.M.).

Drugs

Phenylethanolamine (ICN), S-adenosylmethionine (Boehringer, Mannheim), 14C-S-adenosyl- methionionine (NEN, Dreieichenhain), and 5- and 6-OHDA-hydrochloride (Labkemi, Sweden) were used in the present study.

Results

A. Adrenal Medulla, Controls

t . U t t r a s t ruc tu re

The u l t ras t ruc tura l appea rance o f the adrena l medu l l a o f the n o r m a l guinea pig was descr ibed by Uns icker et al. (1978c). There is a high p r e d o m i n a n c e (85-90 %) o f cells tha t con ta in mos t ly g ranu la r vesicles (average d iamete r 170 nm) with cores o f low or m e d i u m elect ron densit ies and, hence are cons idered to s tore ma in ly adrena l ine (A; Fig. 5) (see H o p w o o d 1971). These cells will be refer red to as chromaf f in A cells. Smal l g ranu le -con ta in ing (SGC) cells were a typica l fea ture o f the guinea pig adrena l medul la . A large ma jo r i t y o f S G C cells had g ranu la r vesicles with h ighly e lec t ron-dense cores measur ing 20 to 140nm (average 8 0 n m ) in d iameter , which were indicat ive o f s tor ing a p r imary amine (Fig. 6). F r o m b iochemica l analyses (Fig. 16) i t was conc luded tha t this p r imary amine was mos t l ikely no rad rena l ine (NA) ra the r than d o p a m i n e (DA). A mino r i t y o f S G C cells con ta ined s torage vesicles o f low e lec t ron densities.

S G C cells fo rmed processes, which ran in the vicinit ies o f medu l l a ry and cor t ica l b l o o d vessels and were a lso found ad jacen t to s t e ro id -p roduc ing endocr ine cells o f the ad rena l cor tex and to dendr i t ic profi les o f medu l l a ry sympa the t i c neurones. Chromaf f in A cells tha t fo rmed processes were no t observed. Sympa the t i c neurones were numerous in the guinea pig ad rena l medul la .

Processes o f n e u r o n e s and S G C cells con ta ined small ( abou t 50 n m in d iamete r ) clear and dense-cored vesicles, which were main ly loca ted wi thin var icose regions. Processes o f S G C cells con ta ined also the large g ranu la r vesicles charac te r i s t ic for this cell type.

A p p l i c a t i o n o f 5- a n d 6 - h y d r o x y d o p a m i n e ( O H D A ) d id no t cause any changes in the u l t r a m o r p h o l o g y o f c h r o m a f f i n A cells and o f S G C and neurona l cell bodies .


6-OHDA produced degeneration of neuronal, but not SGC cell processes. 5- OHDA was readily taken up into the small vesicle population of neuronal processes, but was rarely observed to label small vesicles in SGC cell processes.

2. Catecholamine Histochemistry

Fresh cryostat sections, which had been treated with glyoxylic acid for the visualization of catecholamine (CA) stores, exhibitied an intense yellow-green fluorescence of medullary cells, NA- and A-storing cells could not be distinguished according to their fluorescence colours.

3. Biochemistry

Results of the quantitative determinations of CA and phenylethanolamine N- methyltransferase (PNMT) are shown in Table/ and Fig. 16. A was the predominant CA in guinea-pig adrenal glands (approximately 90 ~o of the total CA); PNMT activity amounted to 900 pMol per min per gland of N-methylated reaction product.

B. Transplants of Adrenal Medullary Tissue to the Anterior Chamber of the Eye

1. Light Microscopy

Toluidine blue or azur II-methylene blue-stained 1 Bm-thick sections of Araldite- embedded material showed well vascularized transplants that were free of necrotic tissue (Fig. 1). Chomaffin tissue was arranged in cords or spherical groups. Single medullary cells were scattered in the periphery of the transplants. One transplant out of 12 contained a few adrenocortical cells. The light microscopic appearance of the grafted tissue was virtually identical in all transplants, irrespective of the time they had remained in the anterior eye chamber (4, 8 or 12 weeks).

2. Transplants after 4 Weeks

Cell Bodies. Grafted adrenomedullary cells showed excellent ultrastructural preservation. Cells lying singly or in groups of 3 to 10 cells were separated from blood vessels and connective tissue by a basal lamina. Most of the cells resembled the chromaffin A cells of controls. They contained the specific CA storage organelles, unconspicuous Golgi areas, few lysosomes, normal amounts of mitochondria of the cristae type and rough ER. The number of storage vesicles per unit area varied considerable between the cells (Fig. 4). Many chromaffin cells could not be classified in terms of A- or NA-cells, since storage vesicles with highly electron-dense and translucent cores were found within the same cells. However, well-defined A-cells still prevailed, while NA-cells could neither be identified by the argentaffin reaction (Fig. 2) nor in electron micrographs. Cores of storage vesicles were smaller in diameter than in controls (Figs. 4, 5). In a few transplants nerve cells resembled those found in the normal adrenal medulla (cf. Unsicker et al. 1978c). Only one transplant contained a group of adrenocortical cells.


Figs. 1-3. 1 Normal appearing adrenal chromaffin cells (A) embedded in the well-vascularized stroma of the iris 4 weeks after transplantation. Semithin section stained with azur II-methylene blue. x 125. 2 Transplanted chromaffin cells (A) stained for the argentaffin reaction to demonstrate primary monoamines such as NA or DA. Only mast cells (arrows) display positive argentaffin reaction product. x 125.3 Catecholamine histofluorescence (glyoxylic acid method) in a stretch preparation of the iris for

the visualization of transplanted chromaffin cells. Note the intense fluorescence, reaction in cell bodies and varicose processes emerging from the explant, x 150

Cell bodies of chromaffin and nerve cells did not display any ultrastructural changes after administration of 5- or 6-OHDA.

CellProcesses. Comparing the shapes of chromaffin cells in situ and in transplants, cells in the latter were more elongated, in particular when the cells were located at the edge of the transplants (Fig. 7). In many instances, the tapering ends of the cell bodies were seen to continue into processes, which could rarely be followed for more than 50 ~tm. However, in glyoxylic acid-treated whole mounts of irides that carried transplants, intensely fluorescent processes (Fig. 3) could be traced back to highly fluorescent cells, indicating that chromaffin cells most likely had the capacity to form processes of considerable length in eye chamber transplants. In electron micrographs taken from all parts of the iris including the regions of the sphincter and dilator muscles, bundles of axon-like processes were regularly found. Three different types of processes could be distinguished according to the populations of vesicles they contained: (1) Processes containing mostly small clear vesicles varying in diameter from 40 to 55 nm that did not take up 5-OHDA (Figs. 8, 10), and few large dense-cored vesicles (90-120nm in diameter). This type of process was frequently observed among smooth muscle cells of the sphincter muscle. This pattern of distribution and the negative results of the 5-OHDA uptake studies

Figs. 4-6. Adrenal chromaffin cells in a transplant to the anterior chamber of the eye 4 weeks after surgery (Fig. 4). The area depicted shows cell bodies and processes (p) of chromaffin cells. The specific storage vesicles (arrows) contain cores of low or medium electron densities. Storage vesicles are unevenly distributed among different cells and are clearly smaller than in controls (Fig. 5); they have approximately the size of storage vesicles characteristic of the small granule-containing cells (SGC) of the guinea-pig adrenal medulla in situ (Fig. 6). x 18,000


Fig. 7. Adrenal chromaffin cells grafted the anterior chamber of the eye for 4 weeks. Many cells (marked with black ink) are elongated in shape and have processes that emerge from the cell bodies, x 12,000


Fig. 8. Two "neuronal" cell processes in the stroma of the iris 4 weeks after sympathectomy and transplantation of the adrenal medullary tissue to the anterior chamber of the eye. Prior to sacrifice the animal received 5-OHDA, which was taken up into one of the processes (a) producing a highly electron- dense label in virtually all vesicles. The size of the vesicles ranges from 40 to 130 nm making it impossible to identify this process as belonging to a transplanted chromaffin or nerve cell. Another process (c) contains small clear vesicles, which have remained unlabelled and, hence, are not considered to store monoamines. Cytoplasm of a Schwann cell (SC). x 36,000

Fig. 9. Axonal profiles invested by Schwann cell cytoplasm in the stroma of the iris (8 weeks after transplantation and sympathectomy; 5-OHDA-treated). Four profiles contain small and very large dense-cored vesicles. The latter, which measure up to 180 nm (arrows), are chromaffin storage vesicles. x 21,500

sugges t ed tha t these a x o n s were cho l ine rg i c and h a d the i r cell bod ies in the ci l iary gang l ion . (2) Processes tha t cou ld be iden t i f ied as b e l o n g i n g to t r a n s p l a n t e d

c h r o m a f f i n cells, s ince they c o n t a i n e d s o m e rare typical " c h r o m a f f i n " s to rage vesicles ( d i ame te r : 100-200 nm) in a d d i t i o n to a m a j o r i t y o f smal l c lear a n d dense- c o r e d vesicles (40-80 n m in d i ame te r ) (Fig. 9). F r o m long i tud ina l sec t ions t h r o u g h


Fig. 10. Cholinergic (c) and adrenergic (a) terminal axons adjacent to smooth muscle cells of the m. sphincter pupillae 8 weeks after grafting adrenal medullary tissue to a host iris (sympathectomized, 5- OHDA-treated). Diameters of the large granular vesicles in adrenergic terminals measure up to 150 nm. x 14,000

these processes it became apparent that large port ions contained the small vesicle popula t ion only (see below). In mos t instances bo th"chromaf f in" and small vesicles were altered after the adminis t rat ion o f 5 - O H D A : electron densities o f the cores increased, whilst the electron-lucent halos between dense cores and vesicles membranes narrowed. (3) Processes that took up 5 - O H D A and conta ined vesicles f rom 40 up to approximately 120 nm could not unequivocally be identified as extending f rom transplanted chromaffin or nerve cells.

All kinds o f processes were usually invested by typical Schwann cells. Schwann cell-free varicosities were only found in close proximity to sphincter and dilator smooth muscle ceils (Fig. 10).

Innervation of the Sphincter and Dilator Muscle. In controls, unmyel inated axons, which had the capacity to take up 5 -OHDA, and axons, which were no t labelled with 5 -OHDA, approached smooth muscle cells at distances up to approximate ly 20 nm.


In accordance with previous findings by Nishida et al. (1969), the ratio of 5- OHDA-labelled, i.e., adrenergic axons and non-adrenergic, presumably cholinergic axons, was about 15: 85. In contrast, almost each axon varicosity in the region of the dilator muscle showed positive labelling after 5-OHDA treatment. The smallest distance observed between these axons and smooth muscle effector cells was 80 nm. In a few instances we observed non-adrenergic axons in close proximity do the dilator muscle, confirming earlier reports by Csillik and Koelle (1965) and Er~inko and R/iisfinen (1966). Removal of the ipsilateral superior cervical ganglion caused degeneration of adrenergic axons in the sphincter and dilator muscle regions. Four weeks after transplantation of adrenal medullary tissue to the anterior chamber of the eye, 5-OHDA-positive terminal axons were located in close proximity to smooth muscle cells of the sphincter and dilator muscle (Fig. 10).

Reinnervation of Transplanted Chromaffin Cells. In animals that had undergone sympathectomy prior to receiving an adrenomedullary transplant, synaptic axon terminals were found in contact with transplanted chromaffin cells (Figs. 1 l, 12), which resembled cholinergic synapses found at chromaffin cells in the normal adrenal medulla (Coupland 1965, 1972; Grynszpan-Winograd 1975). Synaptic endings were 3-5 gm in diameter and often embedded deeply into chromaffin cells. Small (40-60 mm in diameter) clear vesicles were located along the presynaptic membrane; the postsynaptic site exhibited typical postsynaptic membrane specilizations. The width of the synaptic cleft was approximately 25 nm. Most cells carrying this type of synapse were located in the periphery of transplants in close proximity to axons approaching the sphincter muscle. No synaptic endings containing 5-OHDA label could be observed contacting transplanted chromaffin cells.

Cell SurJace Specializations Between Transplanted Chromaffin Cells. As in the normal adrenal medulla, desmosomes were frequently encountered between adjacent chromaffin cells. A special type of contact resembling "somatic efferent" synapses of small granule-containing cells in sympathetic ganglia (cf. Siegrist et al. 1968; Matthews and Raisman 1969; Williams and Palay 1969; Yokota et al. 1973), which are seldom found in the normal adrenal medulla (Matthews, personal communication), was regularly observed between transplanted chromaffin cells. Identical types of contacts have also been described in transplants of chromaffin cells under the kidney capsule (Unsicker et al. 1977a). Asymmetrical membrane specializations associated with a string of chromaffin vesicles along the "presynaptic side" were typical features of these contacts (Fig. 13). "Somatic efferent" synapses were never observed on chromaffin cells, which received cholinergic type nerve endings.

3. Transplants after 8 Weeks

The vast majority of chromaffin cells did not exhibit any ultramorphological alterations compared to the 4-week stage. Storage vesicles in some cells showed increased electron densities after application of 5-OHDA. Administration of 6- OHDA caused the formation of swollen mitochondria and lamellar inclusion


Figs. 11 and 12. Non-adrenergic (probably cholinergic) terminal axons (s) making synaptic contacts with transplanted chromaffin cells (sympathectomized, 5-OHDA-treated). Synapses are characterized by small clear and large dense-cored (small arrows) vesicles, synaptic clefts (approximately 20 nm in width) and postsynaptic membrane specializations (large arrows), x 18,000; x 36,000


Fig. 13. "Somatic efferent" synapses (encircled areas) between three transplanted chromaffin cells. This special type of contact is characterized by a polarity in the arrangement of"chromaffin" storage vesicles, which are aligned (arrows) opposite to a postsynaptic membrane thickening. Desmosome (d), rough- surfaced endoplasmic reticulum (E), Golgi area (G). x 24,000

bodies in a few chromaff in cells and their processes. The results o f the quant i ta t ive de te rmina t ions o f CA and P N M T indica ted tha t figures for A and P N M T were unchanged , whilst N A levels were elevated c o m p a r e d to the 4-week stage. The A / N A - r a t i o was app rox ima te ly 1 : i (Table 1 and Fig. 16).

Transplants after 12 Weeks. C o m p a r e d to the 4- and 8-week stages there was a large number o f chromaff in cells with high nuc lea r -cy top lasmic rat ios. Diamete r s o f the cores o f ch romaf f in s torage vesicles were sl ightly smaller. A few chromaff in cells con ta ined vesicles with cores o f high, mode ra t e and low elect ron densities, ind ica t ing the s imul taneous s torage o f p r imary and secondary amines within the


Table 1. Quantitative determinations of adrenaline (A), noradrenaline (NA) and PNMT-activity in adrenal glands of normal adult guinea pig and in adrenomedullary grafts to the anterior chamber of the eye

Experimental NA A PN MT groups (in ng/gland or eye, (activity, expressed

respectively) in pMol min-1/gland or eye, respectively)

I Controls (n = 5) 840+ 280 12,000+ 2,000 900 + 100 (left adrenals)

II Controls (n = 6) 77+ 15 beyond limit 6.3+ 0.2 (right eyes) of detection

III Controls (n = 5) 26+ 13 beyond limit 4.3+ 0.4 (left sympathec- of detection tomized eyes)

IV Eyes containing 37+ 25 228+ 45 5.9_+ 1.1 transplants after 4 weeks (sympathectomized)

V Eyes containing 125+ 20 178+ 57 6.0+ 0.5 transplants after 8 weeks (sympathectomized)

same cell. Administration of 5-OHDA caused increased electron densities of granular vesicles in approximately one third of the chromaffin cells (Figs. 14, 15). Reinnervated chromaffin cells were frequently encountered at this stage.

Discussion

Transplants of adrenal medullary tissue to the anterior chamber of the eye have earlier been employed as a tool for studying the prerequisites for the maintenance of differentiation of chromaffin cells. Coupland (1959), Piezzi and Cavicchia (1973), Piezzi et al. (1975), and Pohorecky and collaborators (1970) have investigated cell types and CA production of rat adrenal medulla in eye-chamber transplants using morphological and biochemical techniques; they obtained contradictory results. Whilst Coupland (1959) was able to demonstrate both NA- and numerous A- storing cells up to 2 weeks after transplantation using the iodate-rnethod, Piezzi et al. (1975) found only very few A-, but mostly NA-storing cells electron microscopically after 21 days. The latter result correlates with quantitative determinations of A and P N M T by Pohorecky et al. (1970) showing that both the activity of the enzyme and the amount of A were strongly reduced. Olson and Malmfors (1970) reported strong CA-specific histofluorescence in transplanted chromaffin cell bodies and processes.

In accordance with previous reports, we confirm that adrenal medullary tissue from adult guinea pigs survives well for at least 12 weeks in transplants to the anterior chamber of the eye.

356 K. Unsicker et al,

Figs. 14 and 15. Chromaffin cells 12 weeks after transplantation. After application of 5-OHDA many storage vesicles appear to be severely altered (arrows), exhibiting segregation of their highly electron- dense cores. A synaptic terminal (s) contains clear vesicles, which have not incorporated 5-OHDA. x 12,000; x 24,000

The following aspects of the present investigation will be discussed in detail: (1) The problem of maintained differentiation versus progressive dedifferentiation of transplanted chromaffin cells as revealed by electron microscopy, histochemistry and quantitative determinations of A, NA and PNMT; (2) transdifferentiation of transplanted chromaffin cells leading to the formation of neurone-like cells and the ingrowth of axons into the regions of the denervated dilator and sphincter muscles; (3) the similarities of transplanted chromaffin cells and small granule-containing cells in sympathetic ganglia; (4) the formation of storage vesicles and the possibility of simultaneous storage of A and NA within the same axon; and (5) the reinnervation of chromaffin cells by non-adrenergic, probably cholinergic axons.

1. Differentiation versus Dedifferentiation of Transplanted Chromaffin Cells

The functional topographical anatomy of mammalian adrenal chromaffin cells is characterized by an intimate relationship with the adrenal cortex and with


CA in ng

12.000-

5,000.

5oo; 300-

200-

100-

~NA [C3A

in situ 4 weeks trpL (whole left (whole eye) adrenal)

8 weeks trp[, (whole eye)

PNMTin )mol.min -1 /gland or eye 1.000-

500-

100- I0-~ ii

I 5- ~

'ii i

in ;itu (whole left ad renal )

i i 4 weeks trpL 8 weeks trpl. (whole eye) (whole eye)

Fig. 16. Catecholamines and PNMT in normal and transplanted guinea-pig adrenal medulla (ocular transplants)

preganglionic cholinergic nerve fibres from the splanchnic nerve. The adrenal cortex, by secreting high doses of glucocorticoid hormones (Jones et al. 1977), is commonly held to influence CA production via activating PNMT (Wurtman and Axelrod 1966; Pohorecky and Wurtman 1971), whilst cholinergic nerve terminals stimulate secretion and regulate the biosynthesis of CA (Feldberg et al. 1934; Douglas 1975; Kirshner 1975).

The present study reveals that adrenal chromaffin cells that have been deprived of their normal supply, of excessive doses of glucocorticoid hormones and preganglionic nerves retain the basic ultramorphological features of their in situ- counterparts.

Their specific large-granular storage vesicles allow distinction of transplanted chromaffin from nerve cells. In contrast to controls, transplanted chromaffin cells possessed storage vesicles that (1) were more unevenly distributed between different cells, (2) had smaller central cores, and (3) tended to display with time features typical of the storage of a primary amine (cf. Hopwood 1971). The finding that cores of storage vesicles were smaller than in controls is in accordance with reports that vesicles of cultured chromaffin cells (Unsicker and Chamley 1977) also contained smaller cores. This reduction of core-diameters under transplant and culture conditions is difficult to explain. It might be argued that these conditions favour the survival of small granule-containing cells (see below) present in the mammalian adrenal medulla including that of the guinea pig (Unsicker et al. 1978c).

However, most of the small granule-containing cells in the guinea-pig adrenal medulla have typical NA storage vesicles and constitute a 5-10 % minority of the


total chromaffin cell population; hence, these cells can hardly be identical with transplanted chromaffin cells containing small storage vesicles.

Although vesicles displaying the ultramorphological features typical of primary amine storage were more frequently encountered with progressive age of the transplants, the majority of chromaffin cells exhibited A-typical storage vesicles even after 12 weeks. Concomitant biochemical determinations revealed a relative increase of the NA content of the transplants between 8 and 12 weeks with no significant changes of A during the same period. In contrast, Piezzi and Cavicchia (1973), Piezzi et al. (1975), and Unsicker and Chamley (1977) using rat chromaffin cells reported a massive disappearance of A-storing cells within two weeks in culture and in transplants to the anterior eye chamber. Divergent results obtained with rat and guinea-pig adrenal medullary cells might be explained by age-dependent or genetically controlled differences in PNMT-activities. Ciaranello and Axelrod (1973) have shown that adrenal PNMT-activities may differ even between two strains of inbred mice probably due to mutation of a gene that controls PNMT degradation.

5- and 6-OHDA, which differ from DA with respect to a third phenolic hydroxyl group, but are transported into adrenergic nerves by their specific amine uptake mechanism (for review, see Thoenen 1972), were applied in this study to determine whether transplanted chromaffin cells possess an amine uptake system. In contrast to chromaffin cells in the normal guinea pig adrenal medulla, which do not show any ultrastructural changes after application of 5- or 6-OHDA (Unsicker et al. 1978c), many but not all transplanted chromaffin cells exhibited ultrastructural alterations after administration of both drugs. 5-OHDA caused an increase in the electron densities of cores in chromaffin storage vesicles after 8 and 12, but not after 4 weeks. Likewise, 6-OHDA did not affect transplanted chromaffin cells before the 8-week stage.

Morphological changes in chromaffin cells after treatment with 6-OHDA have previously been described for the newborn rat (Unsicker et al. 1976). Cell bodies of sympathetic adrenergic neurones, which are 6-OHDA resistant in the adult stage, are affected by this drug in newborn animals (Angeletti and Levi-Montalcini 1970; Erfink6 and Er/ink6 1972). Thus, 5- and 6-OHDA-specific alterations of transplanted chromaffin cells might suggest that these cells adopt an amine uptake mechanism, which resembles that of their immature precursors.

In summary, it can be stated that adrenal chromaffin cells from adult guinea pigs transplanted to the anterior chamber of eye remain largely differentiated regarding their ultrastructure. However, the reduction of the capacity to synthesize A and the development of an uptake mechanism for 5- and 6-OHDA may indicate a certain degree of functional dedifferentiation.

2. Transdifferentiation of Transplanted Chromaffin Cells

It has previously been shown that adrenal chromaffin cells from young and adult animals may adopt a neurone-like instead of an endocrine phenotype under a variety of experimental conditions. This phenotypical change will be referred to as "transdifferentiation". Olson and Malmfors (1979; see also Olson 1970) have


reported the formation of varicose catecholamine-containing axons, when adrenal chromaffin cells were transplanted to a formerly smypathetically denervated iris. The present study corroborates and extends this observation emphasizing that transdifferentiation of chromaffin cells (a) is not restricted to a certain mammalian species, and (b) is accompanied by typical ultramorphological and biochemical changes. Outgrowth of axon-like processes from chromaffin tissue has also been observed by Unsicker et al. (1977a) in kidney transplants. Furthermore, it is well established that extraadrenal chromaffin cells may emit processes in situ (Furness and Sobels 1976). In vitro studies using explants and enzyme-dissociated cells from adrenal medullae have largely extended our knowledge on the conditions that induce or inhibit axon outgrowth from chromaffin cells.

Chromaffin cells from young postnatal rats extend processes spontaneously after a few days in culture. Spontaneous axon outgrowth requires a factor produced by non-chromaffin cells that are present in those cultures. Formation of processes is markedly enhanced by NGF and can be inhibited by the administration of the glucocorticoid hormone dexamethasone, dibutyrylic cyclic AMP and cholera toxin (Unsicker and Chamley 1976, 1977; Unsicker et al. 1978a; Unsicker and Ziegler 1980; Ziegler and Unsicker 1980). In contrast, adrenal chromaffin cells from newborn guinea pigs (Unsicker et al., in preparation) and adult cattle (Unsicker et al. 1980) exhibit only scarce fibre outgrowth in culture. However, in transplants to the anterior eye chamber of Nunu-mice bovine chromaffin cells produce an abundance of long varicose axons within the iris (Unsicker et al. 1980). Thus, it appears possible that NGF or other nerve growth factors produced by the iris stroma are responsible for the induction of fibre outgrowth from transplanted chromaffin cells. This suggestion would be in line with results recently obtained by Ebendahl and co-workers (1980), who have presented evidence for NGF activity both in cultured rat irides and in irides after sensory and sympathetic denervation.

3. Similarities of Transplanted Chromaffin Cells and Small Granule-containing Cells in Sympathetic Ganglia

Adrenal chromaffin cells transplanted to the anterior chamber of the eye resemble in many respects the small granule-containing cells occurring in sympathetic ganglia (Grillo 1966; Siegrist et al. 1968; Matthews and Raisman 1969; Williams and Palay 1969; Yokota 1973) and in the adrenal medulla (Coupland et al. 1977; Unsicker et al. 1978c; compare Figs. 4 and 6), and are considered to be identical with the small intensely fluorescent (SIF) cells first described by Er/ink6 and H/irk6nen (1965; see also Jacobowitz 1970; Van Orden et al. 1970). Both transplanted chromaffin and SIF-cells contain storage vesicles, which are smaller than those of normal adrenal chromaffin cells; both have processes, which, in the case of SIF- cells, can make synaptic contacts with principal adrenergic neurones, or, as far as transplanted chromaffin cells are concerned, may contact smooth muscle. An ultrastructural analysis of the vesicle populations in processes formed by transplanted chromaffin cells and by SIF-cells also supports the concept of a close relationship between both cell types (Williams 1967; Kobayashi and Coupland 1977).


4. Storage Vesicles of Transplanted Chromaffin Cells and the Problem of Dual A mine Storage within the Same Axon

In addition to the large storage vesicles typical of adrenal chromaffin cells, transplanted chromaffin cells contain a second population of smaller (40-80 nm) agranular and granular vesicles, which were rarely encountered within the cell soma, but were frequently found in varicose and terminal parts of cell processes. The latter type of vesicles is commonly observed in axons of sympathetic neurones (Grillo 1966; for review, see Burnstock and Costa 1975). These small vesicles often contained an excentrically located core of high electron density, which would be compatible with their storage of a primary amine, presumably NA. The large increase of biochemically determined NA in transplants between weeks 4 and 8, i.e., during a period of extensive axon proliferation, would favour the assumption that the small vesicles contain NA. Their NA might be produced in the cell bodies, or alternatively might be taken up from the blood. Since a majority of the large chromaffin-storage vesicles contain A, as judged by ultramorphological criteria, a dual transmitter storage of A and NA in transplanted chromaffin cells is likely. From the differential distribution of large "chromaffin" and small vesicles, the first being the dominating type of vesicle in cell bodies, the other one being preferentially located in processes, a spatial dominance of A and NA might be concluded. The general validity of Dale's principle - one neurone, one transmitter - has been repeatedly questioned (cf. Burnstock 1976). The chromaffin cell appears to store both NA and A during certain periods of development (Coupland and Weakly 1968, 1970) and under in vitro (Coupland and MacDougall 1965; Unsicker and Chamley 1977) and transplant conditions. Whether both amines can be used as transmitters remains to be elucidated.

5. Reinnervation of Transplanted Chromaffin Cells

A central issue of the present study was a possible reinnervation of transplanted chromaffin cells. Adrenal chromaffin cells in situ are innervated by preganglionic cholinergic nerves (Coupland 1965, 1972; Grynszpan-Winograd 1975). True adrenergic synapses, which should not be confused with adrenergic axons adjacent to chromaffin cells, have seldom been documented (Mustonen and Teriiv/iinen 1971 ; Becker 1972; Furness and Sobels 1976). Carruba and co-workers (1974), who transplanted adrenal medullary tissue together with the superior cervical ganglion under the kidney capsule, were unable to detect a reinnervation of chromaffin cells by postganglionic adrenergic sympathetic neurones. Similar results were obtained with sympathetic ganglia in vivo: after interruption of normal preganglionic inputs a functional reinnervation could be reestablished with cholinergic parasympathetic and somatic nerves, but not with sympathetic adrenergic neurones (Hillarp 1946; Ceccarelli et al. 1971; McLachlan 1974). In contrast, in vitro studies have shown that chromaffin cells (Unsicker et al. 1978d) and sympathetic adrenergic neurones (Landis 1976; O'Lague et al. 1976) may be reinnervated by adrenergic neurones. No indications of such an "unspecific" adrenergic reinnervation have been found in the course of the present study. Synapses observed on transplanted chromaffin cells were formed even in sympathectomized animals, did not take up 5-OHDA, and displayed all ultrastructural features indicative of cholinergic terminals. We


@

Ac.

in situ in vitro (rat)

CH.G.

ACt C.[

in t ransp lan ts to the an ter io r chamber of the eye (gu inea pig)

Fig. 17. Innervation of adrenal chromaffin cells in situ, in vitro and in transplants to the anterior chamber of the eye. In situ chromaffin cells of the adrenal medulla (34) are innervated by preganglionic nerve fibres, which have their cell bodies located in the lateral column of the spinal cord (s) and release acetylcholine (ACH) at the synaptic site. Under in vitro conditions rat adrenal chromaffin cells may become reinnervated by cholinergic and adrenergic (NA) axons arising from explants of guinea-pig ciliary (C.G.) and sympathetic chain ganglia (Ch.G.) (Unsicker et al. 1978b). The third part of the figure demonstrates results obtained in this study. Transplanted chromaffin cells are reinnervated by cholinergic axons from the ciliary ganglion, but not by adrenergic neurones located in the superior cervical ganglion (S.C.G.)

therefore assume tha t these te rmina ls be long to chol inergic axons f rom the ci l iary gangl ion, which conta ins exclusively chol inergic neurones in the guinea pig ( W a t a n a b e 1972). Thus, a mixed p a r a s y m p a t h e t i c / s y m p a t h e t i c neu rona l chain consis t ing o f three neurones was fo rmed between the o c u l o m o t o r p a r a s y m p a t h e t i c b ra ins tem nucleus and the iris (Fig. 17). In our model , "correc t" synapses a p p e a r to be es tabl i shed on the basis o f the "correc t" t r ansmi t t e r ra ther than on the basis o f nerves o f "correc t" origin.

H o w m a y the differing results concerning re innerva t ion o f ch romaf f in cells in vi t ro and in v iva be in t e rp re ted? Cells s tud ied in cul tures (Uns icker et al. 1978d) were taken f rom young animals, whilst adul t animals were used in the present study, rais ing the poss ibi l i ty tha t m a t u r a t i o n o f cell membranes being involved in the recogni t ion process is an i m p o r t a n t fac tor for specific synapse fo rmat ion . Ana lyses o f g lycopro te ins o f axons and innerva ted cells a t different deve lopmenta l stages might con t r ibu te to solving this p rob lem. Env i ronmen ta l factors poss ib ly absent in cultures, but present in v iva to prevent unspecif ic r e innerva t ion should also be


n

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5~ 100 150 200 250 280 d in nm

rl 4 weeks non- sym pothectomized 450.

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Fig. lg. Size distribution of the cores of "chromaffin" storage vesicles in cell bodies and processes of adrenal chromaffin cells 4, 8 and 12 weeks after transplantation to the anterior chamber of the eye and in controls. The results document that (1) storage vesicles in transplanted cells are smaller than in controls, (2) storage vesicles in chromaffin cell processes are smaller than in the cell bodies, and (3) there are no obvious differences in the size distribution of "chromaffin" storage vesicles in sympathectomized and non-sympathectomized animals


n

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systematically studied in order to gain more insight into the mechanisms governing synapse formation.

6. Conclusions

The results presented above have clearly shown that transplantation to the anterior chamber of the eye is a valuable tool for studying differentiation ofchromaffin cells under conditions that resemble, in certain respects, those of tissue cultures, without implicating some of the major inherent disadvantages of cultures. Although adrenal chromaffin cells in transplants become reinnervated, they do not retain their full scope of differentiated properties, suggesting that humoral factors provided by the adrenal microenvironment play an important role in the maintenance of differentiation of the adrenal medulla. Formation of axon-like processes growing into the sphincter and dilator muscle regions by transplanted chromaffin cells indicates that the anterior chamber of the eye permits transdifferentiation of chromaffin cells eliciting a cell type that displays features of both a sympathetic adrenergic neurone and a SIF-cell.

Acknowledgements. The authors thank Professor K. L6ffelholz, Dr. R. Lindmar and Mrs. U. Wolf, Department of Pharmacology, University of Mainz, for conducting the biochemical determinations of catecholamines and PNMT activity. Skillful technical assistance was provided by U. K6nig, and R. Korff, Ch. Fiebiger, C. Riehl and H. Schneider helped with the photographic work and the preparation of the manuscript.

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Accepted October 25, 1980

Documents

Differentiation and transdifferentiation of adrenal chromaffin cells of the guinea-pig