Extraneuronal effects of 6-hydroxydopamine

Cell Tiss. Res. 174, 83 -97 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

Extraneuronal Effects of 6-Hydroxydopamine

Tissue Culture Studies on Adrenocortical Ceils of Rats*

K. Unsicker, J.H. Chamley** and J. McLean Department of Zoology, University of Melbourne, Parkville, Australia

Summary. The effects of various concentrations of 6-hydroxydopamine (6- OHDA) on rat adrenocortical cells in tissue culture were studied with phase contrast and electron microscopy. With 40 mg/l of 6-OHDA the first signs of alteration as revealed by microcinematography appeared in isolated cortical cells as early as 15 min after addition of the drug. There was a cessation of movement of cell organelles and an immobilisation of membrane undulations followed by the development of dark inclusion bodies. The cells underwent increasing shrinkage and collapsed by 11/2 h. Chromaffin cells were not affected until 45 min after exposure to the drug and neurons were the most resistant population. However 61/2 h after application of the drug most cells in the culture were dead. 6-OHDA applied in different doses and to adrenal explants did not alter the sequence of events. Ultrastructurally cortex cells underwent damage along two lines: they either showed lytic changes or developed various types of dense bodies before reaching the lyric stage.

Treatment of cortical cells with 40 mg/1 5-or 6-OHDA followed by exposure to buffered 2% glyoxylic acid and heat did not produce a fluorescence within the cells. Microspectrofluorimetry on amine models with noradrenaline, 5- and 6-OHDA revealed that neither 5-nor 6-OHDA are capable to form a fluorophore with glyoxylic acid.

Key-words: 6-hydroxydopamine - Extraneuronal effects - Rat - Adrenal - Tissue culture - Electron microscopy - Microspectrofluorimetry.

Send offprint requests to: Prof. Klaus Unsicker, Department of Anatomy, University of Kiel, 23 Kiel, West Germany Dedicated to Professor Berta Scharrer in honor of her 70th birthday

* Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellow- ship of the University of Melbourne to K.U., and a Research Fellowship and Grant-in-Aid from the Life Insurance Medical Research Fund of Australia and New Zealand to J.H.C. ** Present address: Department of Anatomy and Embryology, University College, London

84 K. Unsicker et al.

Introduction

The h ighly cy to toxic drug, 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) , is general ly though t to be selectively taken up and concen t ra t ed in ca techolaminerg ic neurons , espe- cial ly in their te rminals (cf. Sachs and Jonsson, 1975). However , in a previous s tudy it was shown to cause severe d a m a g e in ad renocor t i ca l ( interrenal) cells o f l izards and less severe changes in cells o f the ra t adrena l cor tex (Uns icker et al., 1976). The essential u l t r a s t rnc tu ra l features o f the d rug ' s effects on cor t ica l cells w e r e - (1) p o l y m o r p h i s m of m i t o c h o n d r i a ; (2) degenera t ive changes o f m i t o c h o n d r i a ; and (3) f o r m a t i o n o f dense bodies with myel in- l ike a n d / o r c rys ta l lo id in ternal pat tern . S imi la r changes have been r epor t ed to occur in s teroid p roduc ing inters t i t ia l cells o f the a m p h i b i a n testis af ter admin i s t r a t i on o f 6 - O H D A (Unsicker , 1975).

W h e n the capac i ty o f ra t ad renoco r t i ca l cells to take up norad rena l ine (NA) was tes ted in-vivo and in-vi t ro up take occur red at a concen t ra t ion o f 10-4 gm/ml in-vi t ro , ind ica t ing that up t ake o f 6 - O H D A is l ikely (Uns icker et al., 1976).

Tissue cul ture is a sui table tool to test and con t inuous ly m o n i t o r the degener- at ive effect o f drugs u p o n var ious k inds o f cells. Hil l et al. (1973) examined the ac t ion o f 6 - O H D A in tissue cul ture using sympathe t i c gangl ia of ne w born rats and chick embryos . In the fo l lowing repor t the effect of different concent ra - t ions o f 6 - O H D A on cor tex cells as revealed by phase con t ras t and e lec t ron mic ro scopy as well as m i c r o c i n e m a t o g r a p h y is descr ibed. A c ompa r i son is m a d e o f the ac t ions o f 6 - O H D A on medu l l a ry ' c h r o m a f f i n ' l cells, the axons, which they p roduce in tissue in tissue cul tures (cf. Uns icke r and Chamley , 1976, and in p repa ra t ion ) , medu l l a ry neurons and Schwann cells. In addi t ion , an a t t e mp t to d e m o n s t r a t e by h i s tochemica l means the up take o f 6 - O H D A into ad renocor t i - cal cells in t issue cul ture will be descr ibed.

Materials and Methods

Adrenal glands of 6 to 10 day old Sprague-Dawley rats were cultured in modified Rose chambers (cf. Chamley et al., 1972). Adrenals were quickly removed under sterile conditions and processed in two alternative ways;

(1) Explants of decapsulated cortices, either in combination or without adrenomedullary tissue were cultured at 37~ for 3-5 days in Medium 199 containing foetal calf serum 20%, and an additional 100 units/ml penicillin G.

(2) Isolated adrenal gland cells, either single or in small clusters were obtained by the following procedure: whole glands were cut into small pieces and incubated with collagenase for 1 h and then treated with 3-5 batches of trypsin. After each treatment single cells and small clumps were pipetted into foetal calf serum, centrifuged, resuspended in medium and injected into Rose chambers. The cells were grown for 2-3 days in medium identical to that used for explants.

6-OHDA hydrochloride (Labkemi AB, Sweden) was used at concentrations of 20, 40 and 80 mg/l (0.097, 0.195, 0.389 mM; cf. Hill et al., 1973) with the addition of 20 mg/1 of ascorbic acid. Since ascorbic acid at a concentration of 200 mg/1 has been reported to cause damage to nerve cell bodies and nerve fibres of cultured sympathetic chain ganglia (Hill et al., 1973), this drug was only applied in the lowest dose recommended by Hill and co-workers.

1 Nomenclature: The term 'chromaffin' was used to characterize adrenaline- and noradrenaline- storing cells, which can easily be distinguished from medullary neurons, both with the light and the electron microscope. No attempt was made to assess the affinity of these cells to chrome salts

In-vitro Effects of 6-OHDA on Adrenal Cortex 85

6-OHDA and ascorbic acid were dissolved in Hanks' Balanced Salt Solution and diluted with the culture medium. Several fields were selected in each chamber and photographed before adding the drugs. The changes occurring in the cultures were monitored with phase contrast optics from a few minutes after injecting the drugs to 14 h. A few cultures treated with 40 mg/l 6-OHDA were subjected to microcinematography time-lapse studies.

Electron Microscopy was carried out on cultures fixed after 2, 6 or 14 h for 1 h in phosphate buffered 2,5% glutaraldehyde (pH 7.4). The cultures were then washed in phosphate buffer, post- fixed in 2% aqueous OsO4, block-stained in a saturated aqueous solution of uranyl acetate, dehydrat- ed in a graded series of ethanol and embedded in Araldite. Sections were cut on a Huxley-LKB microtome, stained with uranyl acetate and lead citrate and viewed through a Joel 100B electron microscope.

Fluorescence Microscopy. A few cultures with trypsinised adrenal glands were treated with 40 mg/1 6-OHDA or 5-OHDA, with the addition of 20 mg/1 ascorbic acid for 1/2 h or 1 h. The cultures were then either washed for 1/2 h in fresh medium, or immediately underwent the following procedure: the chambers were opened and the tissue incubated with 2% glyoxylic acid in 0.1 M phosphate buffer (pH 7.0) for 1/2 h (Furness and Costa, 1975). The glyoxylic acid solution was then removed and the culture dried on the bench before being heated at 100~ for 5 or 15 min. The culture were mounted in paraffin oil and observed through a Leitz fluorescence microscope fitted with Ploem-optics.

For controls a few cultures were heated without having undergone the drug-treatment and/or the exposure to glyoxylic acid.

Amine Models. Noradrenaline (Levophed, Winthrop), 5-hydroxydopamine and 6-hydroxydopamme hydrocholorides (Labkemi AB, Sweden) with final concentrations of 10 -3 g]ml were prepared in 2% aqueous solution of glyoxylic acid (Fluka AG, Switzerland) which contained 1% human serum albumin (Commonwealth Serum Laboratories, Melbourne). The solutions were sprayed as microdro- plets on glass microscope slides which were allowed to dry at room temperature before incubation at 100 ~ C for 4 min to produce amine fluorescence.

MicrospectroJ7uorimetry. Emission spectra were obtained from uniform sized droplets with a Leitz- MPV microspectrofluorimeter equipped with an HBO 200 W Hg burner set up for Ploem epi- illumination. The excitation light, filtered through a 3 mm BG3 glass filter combined with an $405 (Type AL) interference band filter (which will transmit the 405 nm mercury line), was reflected into the 10 x objective with a TK 455 dichroic mirror which has 50% transmission and reflection at 355 nm. The fluorescent light was then filtered with a built-in K 460 suppression filter, and passed through a Schoeffel Quartz-prism monochromator (QPM 30) set at a slit width of 1 mm (half-band width 5 nm at 475 nm). The intensity of the light was measured with an EMI 9558 photomultiplier tube through a Schoeffel M600 photometer and recorded on a wavelength-time chart recorder as uncorrected instrument values.

Results

Controls

T r y p s i n - d i s p e r s e d a n d e x p l a n t e d c u l t u r e s o f r a t a d r e n a l 2 - 5 d a y s in c u l t u r e g a v e

r ise to c l e a r l y d i s t i n g u i s h a b l e cortical a n d medullary cel ls (F ig . 1). A f t e r 2 - 5 d a y s

m o s t o f t h e s e c o r t i c a l ce l ls w e r e r i c h in l ip id d r o p l e t s . T h e y w e r e p o l y g o n a l a n d

v a r i e d in size f r o m a b o u t 25 to 40 g d e p e n d i n g o n t he s t a t e o f f l a t t e n i n g .

N u c l e i w e r e r o u n d e d o r s l igh t ly o v a l w i t h o n e to t h r e e n u c l e o l i . In a c c o r d a n c e

w i t h A r m a t o a n d N u s s d o r f e r (1972) , we a l so o b s e r v e d a s m a l l n u m b e r ( less t h a n 1 0 % ) o f c o r t i c a l cel ls w h i c h h a d d a r k c y t o p l a s m a n d few, i f any , l ip id


Fig. 1. Trypsinised rat adrenal in tissue culture for 2 days. Control. Abstract from a time-lapse microcinematography film. The following types of cells are clearly discernible: Lipid-rich cortical cells (C), medullary chromaffin cells (M), medullary neurons (N), Schwann cells (S), processes of Schwann cells and axons (A). x 200

droplets. Both types of cells had a strong tendency to flatten out and to develop morphological features of fibroblasts from 4-6 days onwards, provided they were not exposed to A C T H or dbcAMP.

Ultrastructurally differentiated cortical cells (Fig. 3a) presented mitochondria with tubular cristae and a prominent smooth ER. In the absence of A C T H or dbcAMP these structures were gradually replaced by lamellated mitochondria and rough ER after 3 4 days in culture. For this reason experiments with 6 -OHDA were carried out on cells, which had been kept 2-3 days in culture.

Medullary cells (Fig. 1) occurred less frequently than cortical cells and were small (18-35 ~t) with sparse dark cytoplasm. Their most outstanding feature was their ability to migrate and develop long varicose processes. After exposure to buffered glyoxylic acid (Furness and Costa, 1975) cell bodies and processes exhibited a strong yellow fluorescence indicating the presence of considerable amounts of biogenic amines. It was concluded that these cells arose from the chromaffin moiety of adrenomedullary cells. Ganglion cells (Fig. 1) were found in a few instances. They had larger cell bodies (20 x 30 ~t to 30 x 45 ~t) which gave rise to 2 to 5 processes. Ultrastructurally, "ch romaf f in" cells resembled their in-vivo counterparts (Fig. 3 b). Adrenaline - and noradrenaline - producing cells could be distinguished on the basis of the different electron densities of the granules. For a detailed account on rat adrenal medulla in culture see Unsicker and Chamley (1976, and in preparation).

Fig. 2a-d . Trypsinised rat adrenals in tissue culture for 2 days treated with 40 mg/1 6 -OHDA and 20 mg/1 ascorbic acid. Abstract f rom a time-lapse microcinematography film. a Dark inclusion bodies have developed in cortical cells (arrows). b Cortical cells are shrunken and large bubbles (arrows) extend from their surfaces. Medullary cells are still normal, e After 3 h 37 rain cortical cells are collapsed. Medullary cells exhibit bizarre shapes. Schwann cells have densified nuclei, d All cells in the picture appear severaly damaged. For a detailed description see text. • 200


6-OHDA-Treated Cultures

Since most experiments were carried out with 40 mg/1 6-OHDA and 20 mg/1 ascorbic acid, the following description mainly refers to these experimental conditions.

In cultures with trypsinised cells the first alterations could be noted in time- lapse studies as early as 15 min after addition of the drugs. There was a cessation of movement of cell organelles in cortex cells and, shortly afterwards, immobilisation of membrane undulations. A few minutes later the pulsatory movements characteristic of Schwann cells ceased. Between 15 and 45 min after addition of the drugs dark bodies up to several microns in diameter were seen to develop in the cytoplasm of cortex cells (Fig. 2a). Medullary "chromaff in" and nerve cells were still entirely normal. The movement of organelles within these cells usually did not cease until 45 min after exposure to the drug, cell membrane undulations becoming immobilised at the same time.

Between 45 min and 11/2 h cortex cells underwent increasing shrinkage, until the formerly flattened cells finally collapsed into tight balls (Fig. 2 b). The col- lapse and shrinkage was accompanied by the formation of large bubbles extend- ing from the cells presumably indicating a rupture of the cell membrane (Fig. 2b and c). Nuclei of such cells showed a pronounced densification. After about 2 h, processes of Schwann cells and non-terminal nerve fibres orginating from medullary cells began to show irregular varicosities and spines, whereas frag- mentation of peripheral fibres was noted after only 1 h.

Between 2 and 11/z h, alterations in medullary "chromaff in" cells became obvious (Fig. 2c). They consisted of bizarre shapes and enlarged intercellular spaces due to the shrinkage of cells. It was not until 3 h that nuclei of these cells exhibited densified membranes or signs of pycnosis.

Neurons were the most resistant population in our cultures. After 4 h their Nissl substance appeared slightly clumped and after 41/2 they had clearly dimi- nished in size with nuclei shrinken and densified.

61/2 after application of the drugs most cells in the culture were dead. With 80 mg/1 6-OHDA, this stage was reached by 3 h, while with 20 mg/1 there was little effect on medullary cells and non-terminal nerve fibres, but cortical cells were clearly affected after 14 h.

The application of 6-OHDA to adrenal explants and their outgrowths caused the same sequence of events, but over a longer time course. Thus, with 40 mg/ml, effects on cortex cells were not observed until 3 h when the cell shape became bizarre and some cells formed irregular processes. At the same time medullary cells did not appear to be affected. With 20 mg/1 all types of cells looked healthy up to 6 h, while with 80 rag/1 most of the cortical and medullary cells were destroyed at this time.

In general, it was those cells which had grown out from the explant the earliest and furthest that were affected. No differences could be noted between the action of 6-OHDA on cultures of cortical cells grown alone and in combination with medullary tissue.

Electron Microscopy carried out on cultures which had been treated with various concentrations of 6-OHDA for 2, 6 or 14 h confirmed and extended the light

In-vitro Effects of 6 -OHDA on Adrenal Cortex 89

Fig. 3a and b. Cortical a and medullary " c h r o m a f f i n " b cells 2 days in culture. Note well developed tubular mitochondria (M), profiles of smooth ER (R) and lipid droplets (L) in cortical cells. The medullary cell in Figure 3b shows electron-dense granules of the noradrenaline type. a x 15.000, b x 18.000


Fig. 4. Medullary "chromaffin" cell (lower half) and cortical cell (upper half) after exposure to 40 mg/t 6-OHDA and 20 mg/l ascorbic acid for 2 h. The cortical cell has undergone almost complete lysis, while the medullary cell appears largely unchanged, x 12.000

mic roscop ic f indings. F igure 4 shows a medu l l a ry " c h r o m a f f i n " cell side by side wi th the r emnan t s of a cor tex cell which has unde rgone a lmos t comple te lysis after 2 h exposure to 40 rag/1 6 - O H D A . The cell m e m b r a n e is broken. The fine s t ructure o f the m e d u l l a r y cell appea r s largely unchanged. However , there seems to be some shr inkage as indica ted by the re t rac ted cell membrane .

Two dif ferent k inds of u l t r a s t ruc tu ra l d a m a g e could be observed with cor t ica l


Fig. 5. Dense inclusion bodies seen in cortical cells after 2 h treatment with 40 mg/l 6-OHDA and 20 mg/1 ascorbic acid. Some of the dense bodies clearly show a limiting membrane and a finger print-like internal pattern, x 40.000

cells: they e i ther showed lytic changes such as swell ing o f m i t o c h o n d r i a and g r o u n d cy top l a sm a n d rup tu re o f the cell m e m b r a n e , or they deve loped var ious types o f dense bodies before reaching the lytic stage. A few examples o f such dense bodies are shown in F igures 5 and 6.

One type exhib i ted a f inger pr in t - l ike in terna l pa t t e rn (Fig. 5), e m b e d d e d into an e lec t ron-dense matr ix . The para l le l lines a r r anged in concent r ic a r r ay


Fig. 6. Various types of dense bodies found after exposure of cortical cells to 40 mg/l 6-OHDA and 20 mg/1 ascorbic acid for 2 hrs. x 30.000

did not show the periodicity of the lamellar type of inclusion body seen under in-vivo conditions (Unsicker et al., 1976). Pairs of closely applied membranes, 40 50 A thick, alternated with electron-lucent areas, which appeared to be part of the matrix. Cross-sections of the alternating layers revealed a stippled pattern. Inclusion bodies with a true myelin pattern were only rarely found. Another group of dense bodies was characterized by the heterogenety of its contents (Fig. 6). They contained patches of granular materials of various electron den-


Fig. 78 and b. Cortical (C) and medullary (M) ceils after exposure to 40 mg/1 6-OHDA and 20 mg/l ascorbic acid for 1/2 h and subsequent incubation with 2% glyoxylic acid b. No specific fluorescence can be detected in cortical cells, compared with controls a

sities and membranes partly arranged in whorls. All types of dense bodies observed were membrane-delimited.

Fluorescence Microscopy showed fluorescent adrenal medullary ceils with fibres of varying fluorescent intensities originating from them. Cortical cells were clearly identifiable by their larger size, nuclei and lipid inclusions. Treatment with 5- or 6 -OHDA did not produce a fluorescence in cortical cells (Fig. 7). In order to clarify whether 5- or 6 -OHDA was capable of forming a fluorophore with glyoxylic acid under these conditions, models with noradrenaline or 5- or 6 -OHDA in albumin droplets were studied microspectrofluorometrically (Fig. 8). While noradrenaline exhibited an intense peak of emission at 475 nm which is characteristic for the fluorophore formed on condensation with glyoxylic acid, both 5- and 6-OHDA showed only a wak peak at 445 nm, identical to that given by the albumin of the control. This result is in accordance with


100"

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u. 40, | ._~

,o, a2_ ~oo 560 606 ~oo 5do 606 ~oo 56o 606 Joo 560 6oo

8 Fluorescence Wavelength (nm) Fig. 8. Emission spectra of amine models in dried albumin droplets treated with glyoxylic acid. A noradrenaline; B 6-hydroxydopamine; C 5-hydroxydopamine; and D Control. Uncorrected instrument values

former observations showing that 6-OHDA administered in organ bath did non produce any fluorescence in adrenals, heart and spleen of rats (Unsicker, Allan and Newgreen, unpublished). Contrary to our results with 5-OHDA, Ehinger and Falck (1969) have shown in model experiments that this substance is capable of forming highly fluorescent products with formaldehyde. This is amazing since glyocylic acid is considered to be more efficient than formaldehyde in the fluorophore forming reactions (Bj6rklund et al., 1972).

D i s c u s s i o n

The present results underline the general toxicity of 6-OHDA. If administered in sufficiently high doses it eventually destroys both neurons and non-neuronal cells.

Moreover, the results of our tissue culture experiments indicate that some types of cells are more susceptible to 6-OHDA or one of the agents formed durings its auto-oxidation, than others. Adrenocortical cells were affected earlier than the medullary tissue components including the nerve fibres arising from medullary chromaffin cells. Different doses of 6-OHDA only had an effect


upon the rapidity of the sequence of changes occurring in the cells, but did not alter the sequence itself. As a whole, trypsinised cells were affected earlier than explants and cells further away from explants earlier than those in close proximity to explants. Hill et al., (1973) showed that neurons which migrated into the outgrowth of sympathetic chain explants were earlier and more severly affected by 6-OHDA and guanethidine than cells remaining in the explant which are probably less exposed to the drugs. With trypsinised cells it is likely that they are slightly pre-damaged by the separation procedure thus becoming more readily attacked by the drug.

The first morphological sign of impairment in all cell types studied was the cessation of cell organelle movement followed by the immobilisation of the cell membrane. This was not observed earlier than 15 min after exposure to 40 mg/1. Under tissue culture conditions which permit free access of a drug to all types of cells, this may be regarded to be a relatively long period. If 6-OHDA acted only on the cell membrane, the first morphological signs of impairment should be expected to occur earlier than 15 min after starting the experiment. Hence, it might be argued that uptake of 6-OHDA into non-nervous cells is a prerequisite for establishing its actions, as it is in neuronal tissues (Malmfors and Sachs, 1968). In the previous study it has been shown that rat adrenocortical cells may exhibit formaldehyde-induced fluorescence after exposure to 10-4 g/ml noradrenaline in-vitro. This result could likewise suggest that 6-OHDA may enter these cells, although the concentration required remains to be established.

In the series of events occurring in cortical cells after exposure to 6-OHDA the formation of dark bodies was the next step in some of the cells while others underwent immediate shrinkage and released some of their contents. Ultrastructurally cortex cells were observed to undergo damage along two different lines. Both pathways ended up in a necrotic stage, but in one of them cortex cells developed various types of dense bodies in an intermediate stage. These dense bodies differed ultrastructurally from those seen after applying 6-OHDA in-vivo. One type exhibited concentrically arranged pairs of membranes resembling lamellar bodies seen in hepatic cells after treatement with weakly amphiphilic drugs (Ltillmann-Rauch, 1974; Lfillmann-Rauch and Scheid, 1975). Another type was characterized by an abundance of heterogeneous components comprising mainly membranes in various arrangements. Cell organelles could not be identified within these bodies. Whether autophagic or beterophagic processes are involved in the formation of these lysosome-like bodies is as yet unknown. In contrast to the 6-OHDA-effects observed in-vivo inclusion bodies with a myelin-like internal pattern (Unsicker et al., 1976) were seldom found and crystalloid inclusion bodies were never encountered. The failure to demonstrate these types of dense bodies could be explained by the fact that the period covered by our experiments was too short for the development of these highly organized structures.

Both in-vivo and in-vitro studies have demonstrated the high susceptibility of adrenocortical cells for 6-OHDA. The reason for this pronounced susceptibility is unknown at present. It might be speculated that a commen metabolic mechanism typical of steroidogenic cells is operating because of the similar


reaction of other steroid-producing cells (Unsicker 1975). However it must be stressed that dense bodies arising in adrenocortical cells after 6-OHDA were ultrastructurally different under in-vivo and in-vitro conditions. This could mean that the attack of 6-OHDA occurs along two different lines and/or that cortical cells in-vivo and in-vitro differ with respect to their autophagic or heterophagic capacity.

The present study does not provide an answer as to the mechanism of action of 6-OHDA operating on adrenocortical cells. As has been discussed for the degenerative changes in adrenergic neurons covalent binding of 6-OHDA oxidation products to macromolecules (Saner and Thoenen, 1971; Thoenen, 1972) and/or H 2 0 2 formed during the oxidation of 6-OHDA (Heikkila and Cohen, 1971, 1972) could account for the degenerative changes in adrenocortical cells. Denatured macromolecules could fuse with lysosomes leading to the formation of the complex structures observed in this investigation. Whether an additional deleterious effect of H202 should be taken into account is unknown: adrenocortical cells of rats are known to possess peroxisomes (Beard, 1972; Magalhaes and Magalhaes, 1971) containing catalase which break down H 2 0 2.

Peroxides are particulary powerful if administered over a longer period. This is obvious from experiments showing that deficency of substances acting as "antioxidants" like Tocopheroles (Vitamin E) leads to an accumulation of lipofuscine-like particles in a variety of organs, amoungst others in rat adrenals (Weglicki, 1968). Since only the short-time effect of 6-OHDA was studied in our cultures, it does not seem very likely that H z O 2 could make a major contribution to the degenerative effect of 6-OHDA.

References

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Beard, M.E.: Identification of peroxisomes in the rat adrenal cortex. J. Histochem. Cytochem. 20, 173 179 (1972)

Bj6rklund, A., Lindvall, O., Svensson, L.A. : Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines. Histochemie 32, 113-131 (1972)

Chamley, J.H., Mark, G.E., Campbell, G.R., Burnstock, G.: Sympathetic ganglia in culture. I. Neurons. Z. Zellforsch. 135, 287-314 (1972)

Ehinger, B., Falck, B. : Fluorescence microscopical demonstration of 5-hydroxydopamine in adrenergic nerves. Histochemie 18, 1-7 (1969)

Furness, J.B., Costa, M. : The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts. Histoehemistry 41, 335 352 (1975)

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Lfillmann-Rauch, R. : Lipidosis-like alterations in spinal cord and cerebellar cortex of rats treated with chlorphentermine or tricyclic antidepressants. Acta neuropath. (Berl.) 29, 237-249 (1974)

Lfillmann-Rauch, R., Scheid, D. : Intraalveolar foam cells associated with lipidosis-like alteration in lung and liver of rats treated with tricyclic psychotropic drugs. Virchows Arch. Abt. B 19, 255 268 (1975)


Magalh~es, M.M., Magalh~es, M.C. : Microbodies (peroxisomes) in rat adrenal cortex. J. Ultrastruct. Res. 37, 563-573 (1971)

Malmfors, Y., Sachs, Ch.: Degeneration of adrenergic nerves produced by 6-hydroxydopamine. Europ. J. Pharmacol. 3, 89-92 (1968)

Sachs, Ch., Jonsson, G.: Mechanism of action of 6-hydroxydopamine. Biochem. Pharmacol. 24, 1-8 (1975)

Saner, A., Thoenen, H. : Model experiments on the molecular mechanism of action of 6-hydroxydopamine. Molec. Pharmacol. 7, 147-154 (1971)

Thoenen, H. : Surgical, immunological and chemical sympathectomy. In : Handbook of experimental pharmacology, Vol. 23, Catecholamines, ed. by H. Blaschko and E. Muscholl, pp. 813-841. Ber- lin-Heidelberg-New York: Springer 1972

Unsicker, K.: Fine structure of the male genital tract and kidney in the Anura Xenopus laevis Daudin, Rana temporaria L. and Bufo bufo L. under normal and experimental conditions. 1. Testicular interstitial tissue and seminal efferent ducts. Cell Tis.. Res. 158, 215-240 (1975)

Unsicker, K., Allan, I.J., Newgreen, D.F. : Extraneuronal effects of 6-hydroxydopamine and extraneuronal uptake of noradrenaline : In-vivo and in-vitro studies on adrenocortical cells of lizards and rats. Cell Tiss. Res. 173, 45 69 (1976)

Unsicker, K., Chamley, J.H. : Effects of dbcAMP and theophylline on rat adrenal medulla grown in tissue culture. Histochemistry 46, 197-201 (1976)

Unsicker, K., Chamley, J.H.: Growth characteristics of rat adrenal medulla in tissue culture: A study correlating phase contrast, microcinematography, histochemical and electron microscopical observations. In preparation

Weglicki, W.B. : Accumulation of lipofuscine-like pigment in the rat adrenal gland as a function of vitamin-E-deficiency. J. Geront. 23, 465-475 (1968)

Received April 15, 1976

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Extraneuronal effects of 6-hydroxydopamine