Extraneuronal effects of 6-hydroxydopamine and extraneuronal uptake of noradrenaline

Cell Tiss. Res. 173, 4 5 - 6 9 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

Extraneuronal Effects of 6-Hydroxydopamine and Extraneuronal Uptake of Noradrenaline

In-v ivo and in-v i tro S t u d i e s on A d r e n o c o r t i c a l C e l l s o f L i z a r d s and R a t s *

K. Unsicker*, I.J. Allan and D.F. Newgreen Department of Zoology, University of Melbourne, Parkville, Australia

Summary. 6-hydroxydopamine (6-OHDA) was shown to cause ultrastructural changes in adrenocortical cells of lizards and rats. These changes comprised the formation of dense bodies with lamellar and crystalloid patterns, a de- crease in the number of mitochondria and structural alterations of mitochondria. Alterations in adrenocortical cells of lizards and rats differed in both qualitative and quantitative aspects. Adrenomedullary cells were not affected as a rule. Only in young animals did 6-OHDA cause deposits of an electron- dense material in medullary cells.

An attempt was made to obtain information on amine uptake into cortical cells using the Falck-Hillarp technique to analyse the in-vivo and in-vitro uptake of noradrenaline (NA) into the adrenal cortex in adult rats. Extraneu- ronal uptake into heart and spleen was studied as well. Our results suggest that NA is taken up into cortical cells, particularly into nuclei, after exposure to 10 .4 gm/ml in-vitro indicating that uptake of 6-OHDA is also likely. Investigations using labelled 6-OHDA are required for further elucidating its extraneuronal uptake.

K e y w o r d s : 6 - h y d r o x y d o p a m i n e - N o r a d r e n a l i n e - Extraneuronal uptake - A d r e n a l cortex - Falck-Hillarp technique -- Electron microscopy.

In troduct ion

Since the first report by Tranzer and Thoenen (1967) on the ultramorphological changes in adrenergic nerves caused by 6-hydroxydopamine (6-OHDA), this drug has become a widely used and almost indispensable tool in catecholamine research (for reviews see Malmfors and Thoenen, 1971 ; Kostrzewa and Jacobo-

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

* Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellow- ship of the University of Melbourne to K.U.

46 K. Unsicker et al.

witz, 1974; Sachs and Jonsson, 1975; Jonsson etal., 1975). The usefulness of 6 -OH DA as a specific marker of central and peripheral catecholaminergic neurons results from the high degree of selectivity of its uptake and concentration, leading eventually to degeneration of nerve terminals, the effect of which can be visualised by both fluorescence and electron microscopy.

During a study on the innervation of the reptilian adrenal gland (Unsicker, 1974, 1976) an effect of 6 -OHDA on interrenal (adrenocortical) cells in-vivo was observed ultrastructurally. To our knowledge there are only two published reports on the effect of 6 -OHDA on non-neuronal tissue (Malmfors and Thoenen, 1971, p. 353; Martin and Barlow, 1975). These concerned hemolysis and damage of kidney tubules in mammals and muscle and gland cell degeneration in the octopus posterior salivary gland. The following report demonstrates a similar action of 6 -OHDA on adrenocortical cells in lizards and rats.

The effect of 6 -OHDA on adrenocortical cells may follow its uptake or be mediated by other means, for example via catecholamines released from catecholamine (CA) storing cells to compensate for lost peripheral stores, or by toxic effects of 6 -OHDA metabolites and H20 2. Since extraneuronal uptake of 6 -O HDA cannot be visualised with the fluorescence microscope (Ljungdahl et al., 1971 ; Unsicker et al., 1976) an attempt will be made to study the possible uptake of a closely related catecholamine - noradrenaline (NA) - which can be observed with the method of Falck and Hillarp.

Materials and Methods

Electron Microscopy. Male adult lizards of the species Lacerta dugesi and both newborn (6-10 days old) and adult rats (weight 180-230 gm, Wistar strain) of either sex were treated with 6-OHDA. The compound was calculated as the base and dissolved in either 0.9% NaCI or Hanks ' Balanced Salt Solution, with 0.2 mg/ml ascorbic acid. The volume of vehicle used was 0.5 ml. The animals were treated according to the schedule given in Figure 1. Controls were treated with the vehicle only.

Between 4 and 6 h after the last injection the animals were anesthetised with Nembutal | or ether and perfused for 15 min via heart or aorta with 2.5% glutaraldehyde in phosphate buffer adjusted to pH 7.4-7.6. Subsequently, the vascular bed was rinsed with phosphate buffer, or slices of adrenal glands, 1 mm thick, were rinsed in phosphate buffer for at least 2 h. All material was postfixed in aqueous OsO4 for 2 h, dehydrated in graded series of ethanol and embedded in Araldite. Sections were stained with uranyl acetate in 70% methanol and lead citrate for 5 min each and viewed under a Siemens 101 or Jeol JEM 100 B.

In order to determine the possible effect of depleted adrenomedullary catecholamines on cortical cells a few lizards were treated with 5 mg/kg reserpine (Serpasil| i.p. and processed for EM 24 and 48 h later.

Fluorescence Microscopy. Adult rats of both sexes, weighing between 170 gm and 220gm, were used throughout these experiments. Animals were killed under ether anesthesia and tissues removed for further treatment. Both in-vivo and in-vitro treatments were used. Following drug treatment each piece of tissue was quenched in liquid propane in preparation for freeze-drying and the specific localization of catecholamines using the Falck and Hillarp technique (Falck, 1962; Falck and Owman, 1965) for formaldehyde-induced fluorescence (see review by Bj6rklund, Falck and Owman, 1972). The procedures employed and machinery used were those reported by McLean and Burnstock (1966) for embedded tissues. Following incubation with paraformaldehyde for 1 h

Extraneuronal Effects of 6 -OHDA I on Adrenocortical Cells 47

150-

~ IO0-

.S

~ 50-

=

= = =

,,

m

l 1

m

Ei:i: i%eo i;.;.

2

m

3

Fig. 1. Schedule of t reatment with 6 -OHDA in-vivo

i.p. ['--] lizards

i . p . [ ] young rats

i.p~ [ ] adult rats group T o r

i .v . t [ ] adu t rats group.K

# 7 8 d a y s

at 80 ~ C the tissue was vacuum embedded in Paraplast plus (Sherwood, Missouri, m.p. 56 ~ C 57 ~ C). Sections were cut at 10 lam, mounted in paraffin oil on heated glass slides and examined with a Leitz Ortholux fluorescence microscope, the stage of which was heated by warm air from a hair-dryer.

In-vivo treatment: NA in concentrations of 10 mg/kg or 100 mg/kg, dissolved in a 2 ml vehicle of Hanks ' Balanced-Salt Solution, was gradually introduced intravenously or intraperitoneally, over a 6 to 8 min period, into rats which had been treated with pargyline (100 mg/kg) 3 h previously. The adrenal glands along with selected control tissues f rom the heart and spleen were removed 1 min after delivery of the drug was completed.

In all instances control tissues were removed from both untreated animals and pargyline treated animals into which only the vehicle had been injected.

In-vitro treatment: Upon removal, tissues f rom the adrenal gland and control tissues from the heart and spleen were cut into 1-3 m m slices and immersed in a modified Krebs solution (Furness, 1969) following the procedures of Costa and Furness (1971). Tissues were vigorously oxygenated at 36 ~ C for 15 min in the Krebs solution containing pargyline (10-6 gm/ml) followed by periods of 1/2 h and 1 1 /2h in the Krebs solution containing pargyline (10 -6 gm/ml) plus NA (10 a, 1 0 - 4 o r 1 0 - 5 gm/ml). Following drug treatment tissues were bubbled in the Krebs solution for periods of 2 min or 30 min. Control tissues were bubbled in the Krebs solution for periods of 45 min and 1 1/2 h.

Adrenal tissues were also treated as above with the addition of ascorbic acid (0.2 mg/ml) to the NA containing solutions (10 -3gm/ml and 10-~gm/ml) and with pargyline-free solutions containing ~-methyl-noradrenaline (10-3 gm/ml and 10-4 gm/ml).

Drugs used: L-Noradrenaline bitartrate (levarterenol bitartrate as a salt f rom Winthrop). dl e-methyl-noradrenaline hydrochloride (Cobefrin as a salt from Sterling-Winthrop Research

Institute Rensselaer, N.Y.). Pargyline hydrochloide (Eutonyl, as the salt from Abbotts Laboratoires). 2,4,5-trihydroxyphenethylamine hydrochloride (6-OHDA), as a salt from Labkemi AB, Stock-

holm.


Results

ELECTRON MICROSCOPY

A. Lizards

1. Control Animals. Interrenal cells of Lacerta dugesi are arranged in distinct outer and inner zones. (Fig. 2A) shows a group of interrenal cells of the outer zone. Single cells were columnar with nuclei resting close to the basal lamina. There were large amounts of electron-translucent lipid droplets and mitochondria with tubular cristae. Smooth ER was scarce and only present in few short elongated or vesicular profiles. A moderate number of dense bodies was regularly observed. They ranged in size from about 300 nm to more than one micrometer. Usually they were membrane-limited and contained a material of high electron density, occasionally intermingled with even darker patches and membranes. Frequently a slighter material, apparently lipid, was found incorporated within dense bodies. Cells in the inner zone were more often polyhedral than columnar in shape. They contained less lipid droplets than cells in the outer zone, but smooth vesicles probably representing the ER were more readily seen. Mitochon- dria and dense bodies were the same both in respects to numbers and ultrastructure, as compared to cells in the outer zone.

2. 6-OHDA-Treated Animals. In adrenocortical cells of either zone severe ultrastructural changes occurred after three injections of 100-150 mg/kg 6-OHDA in 24-h intervals. No dose-dependent ultrastructural differences could be detected. The following changes were observed: (i) Mitochondria were altered in many ways. Profiles up to 15 times the area of normal mitochondria were observed (Fig. 5 A). Cup-shaped mitochondria, attached to lipid droplets (Fig. 5 B) were frequently seen. These were not observed in control animals. A few mitochondria were slightly enlarged and contained conspicuous tubular cristae dispersed in an electron-lucent matrix. Numerous mitochondria were encountered which were transformed into dense bodies frequently displaying a myelin-like internal structure (see below). The total number of mitochondria was consider- ably less than in controls.

(ii) Dense bodies occurred in large numbers. They showed three different types of patterns and were apparently derived from several sources. Most of them appeared to be altered mitochondria as judged by the decreased number of normal mitochondria and by the presence of transitional stages between normal and altered mitochondria. Most altered mitochondria appeared as onion- like, concentrically laminated structures (Fig. 3). In a few a delimiting unit membrane could be distinguished. Many exhibited a centrally situated dense core consisting of a granular material.

Fig. 2A and B. Adrenal gland of a lizard before A and after B treatment with 3 x 100 mg/kg 6-OHDA. A Interrenal (adrenocortical) cells of the outer zone containing numerous lipid droplets (L), tubular mitochondria (M) and a few dense bodies (D). x 7,800. B Ultrastructurally normal chromaffin cells (C) and interrenal cells with lamellated inclusion bodies (/). Swollen adrenergic axon (S). x 5,200

Extraneuronal Effects of 6-OHDA I on Adrenocortical Cells 49

50 K, Unsicker et al.

Fig. 3. Lamellated inclusion bodies (L) in lizard interrenal cells (3 x 100 mg/kg 6-OHDA). The formation of this type of dense body appears to be related to mitochondria, which are virtually absent in this picture. A few dense bodies, which could tentatively be described to represent intermedi- ate stages between mitochondria and fully developed lamellar inclusions, may also be observed (arrows). x 20,000. Inset: rhomboid inclusion body. x 12,000


Fig. 4. Membrane-delimited dense bodies with an internal crystalloid lattice in lizard interrenal cells (3 x 100 mg/kg 6-OHDA). x 60,000

Another type of dense body, less frequently observed, was characterised by an internal crystalloid lattice (Fig. 4), embedded into a matrix of medium electron density. High magnifications revealed a hexagonal array with center to center distance varying from 140 to 150 A. Pairs of parallel membranes with a thickness of about 50 X, could be seen in close proximity to the hexagons. By tilting the sections they both could be shown to be different aspects of the same structure. Both lamellated and erystalloid inclusions occasionally showed arbitrarily situated osmium deposits. Unaltered dense bodies comparable to those in the control material were lacking in adrenocortical cells of treated animals. Autophagic vacuoles which might have been expected to accompany the drastic reduction in the number of mitochondria were not visible.


A third type of dense body which was occasionally found in 6-OHDA treated animals is shown in Figure 3 (inset). These were rhomboid bodies containing membranes in a parallel array. A surrounding membrane was only rarely discernible.

Smooth membranes of the cisternal and the vesicular type were widely distrib- uted within the cells and present in larger amounts than in controls. No differences were observed concerning the effects of 6-OHDA on adrenocortical cells of the outer or inner zone of the interrenal gland. CA-storing cells (Unsicker, 1976) appeared ultrastructurally normal after treatment with 6-OHDA (Fig. 2B).

A few red and white blood cells which were occasionally seen attached to the wall of vessels did not display any changes comparable to those seen in adrenocortical cells.

3. Reserpinized Animals. 24 or 48 h after a single injection of 5 mg/kg reserpine, interrenal cells displayed large numbers of membrane-delimited dense bodies with only very few of the lamellated type (Fig. 6). The crystalloid type of inclusion body was not observed.

B. Rats

1. Control Animals. The ultrastructure of cortical cells of both young and adult animals corresponded to previous descriptions (cf. Idelman, 1970). Since the adrenal cortex of the rat is structurally fully differentiated around the time of birth (Idelman, 1970), newborn and adult animals are not referred to separate- ly. The different kinds of dense bodies seen in the three cortical zones were identical to those mentioned in Idelman's (1970) review article. The most com- mon type present in the glomerulosa, fasciculata and reticularis zone were electron dense granules which were surrounded by a single membrane and were quite frequently found in the cell periphery. Similar corpuscles have been shown to possess acid phosphatase activity (Idelman, 1966); thus they are probably lysosomes. These corpuscles may be associated with lipid droplets or may incor- porate patches of highly electron-dense material, but never show myelin-like formations. The ultrastructural features of the adrenal medulla were completely identical with those described by Coupland (1965).

2. 6-OHDA Animals. Cellular alterations observed in rats were less severe than those seen in lizards. Two kinds of alterations were observed. Firstly, there were polymorphic mitochondria (Fig. 5C). Secondly, myelinated inclusion bodies were seen which seemed to originate from the periphery of lipid droplets (Fig. 7A). In NA-storing cells of young animals membrane-delimited deposits of a granular electron-dense material were found in a few instances (Fig. 7B).


Fig. 5A-C. Ultrastructural changes with mitochondria in adrenocortical cells of lizards A and B and rats C after 3 injections of 100 mg/kg 6-OHDA. A Giant form (G). Compare with mitochondria of normal size (M). Lamellar bodies (L). • 20,000. B Cup-shaped mitochondria associated with a lipid droplet, x 18,000. C Polymorphic mitochondria x 27,000


Fig. 6. Dense bodies in a lizard interrenal cell 48 h after a single injection of 5 mg]kg reserpine. Most inclusions differ from those seen after application of 6-OHDA (see Figs. 2 and 3). x 12,000

FLUORESCENCE HISTOCHEMISTRY

A. In-vivo Results

1. Adrenals

Normal. Both pargyline treated and unt rea ted controls were examined. No marked differences were noted other than a brighter fluorescence in perivascular nerves of the capsule of pargyline treated animals. (See no rma l heart controls.)


Fig. 7A and B. Ultrastructural changes in cortical A and medullary chromaffin B cells of adult rats after treatment with 3 x 100 mg]kg 6-OHDA. A Inclusion body with a myelin-like internal pattern (M). Another inclusion, which could be a myelin-like body in an early stage, is associated with a lipid droplet, x 20,000. B Deposits of an electron-dense material surrounded by a smooth membrane (arrows) in a NA-storing cell of a young rat. x 12,000

Cort ical cells in the three zones exhibited a dull backg round fluorescence (Fig. 8A). F luorescent fibres were seen a r o u n d b lood vessels in the serosa and benea th the capsule. A few fluorescent mast cells were also present in the serosa. The medul la included A and N A - c o n t a i n i n g cells showing a yellow-green and orange-yel low fluorescence, respectively. Some neurons , which were visible in a few instances, had a greenish appearance, bu t this could be due to diffusion from the su r round ing A and N A cells. Diffus ion could also account for the fluorescence seen in the reticularis zone in a few preparat ions .


Fig. 8A-D. Uptake of NA into rat adrenal cortex. A Pargyline treated control. Fluorescent nerve fibres are seen around blood vessels (V). Cortex cells exhibit a weak background fluorescence. x 80. B 10 mg/kg NA given i.v. There is specific fluorescence in the musculature of vessels (V) and along capillary beds (arrows) of the cortex. • 170. C 10 3 gm/ml ~-methyl-NA in vitro 1/2 h with short washout. Note specific fluorescence in the capsule (6"), in patches of cortical tissue, particularly in nuclei (arrows) and along the capillary beds. x 80. D 10 4 gm/ml NA in-vitro 1]2 h with short washout. Distinct fluorescence occurs in nuclei of cortical cells and along capillaries (arrows). x 425


NA- Treated. After applications of 10 mg/kg NA intravascularly an intense fluorescence was observed in the musculature of the serosal vessels and the capsule. In addition, the perivascular and intravascular material of the capillary beds of the cortical layers was fluorescent (Fig, 8B). There was no evidence of fluorescence in cortical cells. After 100 mg/kg intravascularly, the tissue overall had a weak yellow appearance. However, clearly demarked fluorescence was limited to the perivascular material. In the medullary region there was a very high fluorescent response in all cell types. A large axonal region assumed to contain predominantly preganglionic cholinergic fibres entering the medulla was also seen to have increased fluorescence.

The results of intraperitoneal treatment were similar to i.v. treated animals except that the loading was not as pronounced suggesting a slower penetration of the catecholamine throughout the cortical vascular beds.

2. Heart

Normal. There was a very faint background fluorescence in the atrial and ventricular striated musculature, a dense net of catecholaminergic nerve fibres in the atrium and less dense innervation of the ventricular tissue. As previously reported (Costa and Furness, 1971) there was an increased brightness of fluorescent nerve fibres in pargyline treated tissues.

NA-Treated. A clear uptake of NA (10 mg/kg i.v.) was observed in all forms of vascular muscle both surrounding and within heart tissues in addition to uptake into both atrial and ventricular muscle. After 100 mg/kg i.v. there was an even more pronounced uptake in all tissues mentioned above, The same dose administered intraperitoneally failed to produce the same effects. There was some loading in the ventricular muscle and an increase in the accompanying nerves, but this was absent from ventricular vascular tissue (Figs. 9C and D). Also, there was slight loading of atrial muscle.

3. Spleen

Normal. Specific formaldehyde-induced fluorescence was restricted to the nerve fibres surrounding the arteries within the trabeculae and white pulp, small follicular arteries of lymphoidal nodules, throughout the trabecular smooth muscle and around blood vessels of the serosa that surrounds the spleen's capsule (Fig. 10A). Increased intensity of fluorescence was once again observed in pargyline treated animals.

NA-Treated. Uptake into splenic tissues was not observed following in-vivo treatment intravenously although intense fluorescence within the trabeculae and capsule of the hilus region was noted in animals treated intraperitoneally. This was peripheral and was absent from tissues within the spleen.

58 K, Unsicker et al.

Fig. 9A-D. Uptake of NA into rat heart (ventricle). A Pargyline treated control. Adrenergic nerve fibres (arrows.) x 80. B 10-3 gm/ml NA in-vitro 1/2 h with short washout. Note differences in fluorescence intensity between central and peripheral ventricular regions, x 80. C 100 mg/kg NA given i.v. There is a clear uptake into vascular smooth muscle (S) and ventricular muscle (V). x 80. D 100 mg/kg NA given i.p. Compare the effects with C! Vascular smooth muscle (S), ventricular muscle (V). x 80

Extraneuronal Effects o f 6 -OHDA I on Adrenocortical Cells 59

Fig. 10A-C. Uptake of NA into rat spleen. A Pargyline treated control. Specific fluorescence is seen in nerve fibres running with trabeculae (arrows). Capsule (C). z95 . B 1 0 - 4 g m / m l NA in-vitro 11/2 h with long washout. There are no marked differences f rom the untreated spleen other than an increased intensity of normally fluorescent nerves (arrows). x 95. C 10 3 gm/ml NA in-vitro 1/2 h with long washout. Uptake has occurred to reticular cells, which exhibit a striking nuclear-bound fluorescence, x 510


B. In-vitro Results

1. Adrenal

Normal. The results corresponded largely with those of the in-vivo experiments. 10-5 gm/ml NA : There was no evidence of loading into cortex areas, al-

though some dull loading in the serosal vascular muscle was noted. 10 -4 gm/ml NA (Fig. 8D): There was a loading of nuclei in patches of

the cortex, which was particularly evident in the glomerulosa and outer fasciculata region. Fluorescent nuclei were clearest in cells of the fasciculata. In some regions fluorescent endothelial cells could also be observed. When ascorbic acid was included with NA all fluorescent features were observed although of reduced intensity. Results with e-methyl NA were similar to those of NA without ascorbic acid.

A long wash-out removed most of the fluorescence in the cortex, without markedly affecting that of the vascular smooth muscle.

10-3 gm/ml NA (Fig. 8C): There was a general yellow appearance of the cortical tissues, but distinct fluorescence was seen in

1. the nuclei, particularly in patches of the outer fasciculata 2. the endothelial cells 3. intra- and perivascular material. While there was a small reduction in the intensity of the above fluorescence

in those preparations washed out for a longer period, there was still a relatively clear presentation of all features. The vascular muscle of the capsule was observed to be highly fluorescent and not noticeably reduced with longer wash- outs. However, the fluorescence of surrounding capsular tissue was reduced by longer wash-outs.

2. Heart

Normal. The results resembled those of the in-vivo experiments. 10- 5 gm/ml NA : There was a clear loading of ventricular muscle peripherally,

but not in the centre of the tissue. Vascular muscle exhibited fluorescence only in the periphery, while there was an overall increase in brightness of nerve fibres.

10- 4 gm/ml NA : Atrial and peripheral ventricular tissues were clearly loaded. Inner ventricular areas were still dull and the same was true for the muscle walls of blood vessels in this region. The patchy fluorescence, resistant to wash out in heart muscle loaded in vitro, showed a clear line of demarcation and is difficult to explain by diffusion (Wright, 1972; Burnstock et al., 1972).

10- 3 gm/ml NA (Fig. 9B) : A general fluorescence of all elements was noted with distinct differences in fluorescence intensity between central and outer ventricular regions. This regional variation of heart tissue treated in-vitro has previously been recorded (Wright, 1972), where it was explained by regional variations in COMT activity, the variation being absent in those tissues previous-

Extraneuronal Effects of 6-OHDA I on Adrenocortica[ Cells 61

ly treated with catechol, a COMT inhibitor. While variations in COMT activity may occur within a tissue no simple relationship could be demonstrated between COMT or MAO activity and the capacity for extraneuronal uptake by a variety of smooth muscle tissues (Burnstock et al., 1972).

3. Spleen

Normal. The results resembled those of the normal in-vivo spleens. NA-treated: Incubation in concentrations up to 10 - 4 gm/ml showed only

an increased intensity of normally fluorescent nervous tissues (Fig. 10B). Treat- ment with 10 -3 gm/ml resulted in uptake by the capsule, trabecular smooth muscle, arterial smooth muscle, vascular endothelium and reticular cells of the lymphoidal white pulp regions (Fig. 10C). Those tissues washed out in a NA-free Krebs solution for longer periods showed a reduction in fluorescent intensity which was most marked in the capsule. These results are similar to those previously reported for the cat (Gillespie et al., 1970) where extraneuronal uptake was revealed at lower concentrations. A previous report on the similarly treated rat spleen (Gillespie and Muir, 1970) has indicated minimal uptake at concentrations of 3x 10 -4 gm/ml. Our results demonstrate that extraneuronal uptake by splenic tissues of the rat does occur at higher concentrations.

Discussion

6-OHDA is known to cause a long-lasting depletion of NA in peripheral and central adrenergic nerves (Porter et al., 1965; Thoenen and Tranzer, 1968; Uretsky and Iversen, 1970). The morphological correlates of this phenomenon are a reduction of the number of adrenergic terminals and an increase in transmitter at the non-terminal parts, as judged by fluorescence histochemistry according to the Falck-Hillarp method (Malmfors and Sachs, 1968; H6kfelt et al., 1972). On the ultrastructural level adrenergic terminals have been shown to undergo a series of dose- and time-dependent changes. Low doses cause initial loading of large and small dense-cored vesicles with an electron-dense material followed by degranulation (Kostrzewa and Jacobowitz, 1974, chapter VI). If higher doses are applied, these effects are followed by the appearance of various types of dense bodies and damaged mitochondria showing swelling and densifica- tion. Finally axons become swollen and the organelles disappear (Bennett et al., 1970; Furness et al., 1970). In the present study the ultramorphological alterations reported to occur in adrenocortical cells were similar in some respects to those seen in adrenergic nerves. The most spectacular changes in adrenocortical cells of lizards and rats concerned mitochondria and dense bodies. Mitochon- dria showed a clear polymorphism in both of the species. In lizards they were transformed into inclusion bodies with a lamellar pattern, whilst crystalloid bodies also occurred. Since the normally occurring dense bodies were absent in treated lizards it seems likely that these can give rise to either or both profiles.


The morphological changes per se are certainly not specific in any way. A host of agents, diseases, and mechanical lesions are known to cause the formation of dense bodies with myelin-like internal patterns. For example, lamellar bodies occur in the rat epididymis after vasectomy (Alexander, 1973), in cultured human glial cells as a response to aging (Brunk et al., 1973), or in dorsal root ganglia after application of chloroquine (Tischner, 1974). Both lamellar and crystalloid inclusions occur in various organs of rats, mice and hamsters after administration of cholesterol inhibitors such as Triparanol (MER-29) or AY 9944 (Hruban et al., 1965; Yates, 1966; Arai et al., 1967; Chen and Yates, 1967; Yates et al., 1967; Dietert and Scallen, 1969; Suzuki et al., 1973).

In a series of papers, Ltillmann-Rauch and co-workers (see, e.g., Ltillmann et al., 1973; Ltillmann-Rauch and Pietschmann, 1974; Lfillmann-Rauch and Reil, 1974; Ltillmann-Rauch, 1974a, b, c) have shown that many drugs including some of those mentioned above, which all share the amphiphilic I character induce the formation of cytoplasmic inclusion bodies with lamellar and crystalloid internal patterns in a variety of tissues, amongst others the steroidogenic cells of adrenal gland and testis. This was interpreted as the ultramorphological manifestion of a drug-induced phospholipidosis, caused by an impairment of phospholipid degradation leading to an intralysosomal accumulation of lipids. As far as steroid producing cells are concerned Ltillmann-Rauch and Reil (1975) questioned whether an especially high rate of phospholipid turnover in these cells could account for their pronounced susceptibility. Although the final morphological results after administration of amphiphilic drugs and the non- amphiphilic drug 6-OHDA are very similar, there could be distinct differences as to the origin of lamellar and crystalloid inclusion bodies. There is reasonably good evidence for the lysosomal origin of lamellar and crystalloid bodies in tissues of animals treated with amphiphilic drugs. In our material we have failed to observe transitory stages between dense (lysosomal) bodies and lamellar or crystalloid inclusions. The disappearance of mitochondria and lamellar bodies exhibiting remnants of mitochondrial structures in the centre rather favor the view of a mitochondrial origin of lamellar bodies in interrenal cells of lizards. However, in the absence of appropriate histochemical data, in particular the demonstration of the state of acid phosphatase activity in altered mitochondria, it is difficult to further support this view. The formation of polymorphic and giant mitochondria was another effect observed after 6-OHDA treatment. These alterations have not been seen in animals treated with amphiphilic drugs. The pronounced effects of 6-OHDA on mitochondria which have been described previously in testicular interstitial cells of amphibians as well (Unsicker, 1975) might be ascribed to the action of 6-OHDA as a potent uncoupler of oxidative phosphorylation (Wagner and Trendelenburg, 1971) as well as to the degenerative action of H 2 0 2 o r 6-OHDA oxidation products (see below). Another differ- ence concerning the effect of amphiphilic drugs and 6-OHDA is a certain selectivity as to the kinds of tissues affected by 6-OHDA. In the adrenals only cortical cells were altered, while the medullary moiety, which is severely affected by

1 ,,amphiphilic" means that a highly hydrophobic and a hydrophilic moiety occur in close proximity within the same molecule


Chlorphentermine and Triparanol (Lfillmann et al., 1973), showed normal ultrastructural features, apart from electron-dense deposits found in a few NA-storing cells of young rats.

The effects of 6-OHDA leading to the alterations described in this paper result from either or both direct or indirect routes of actions for 6-OHDA.

An indirect action of 6-OHDA (or its metabolites) may result from the compensatory release of CA from the adrenal medulla. This is known to take place after the administration of 6-OHDA as an effort to compensate for CA lost from adrenergic terminals. In 6-OHDA treated dogs the adrenals have been shown to be largely responsible for maintaining blood pressure (Gauthier et al., 1972). Mueller et al., (1969) have demonstrated that 6-OHDA may induce a reflex increase in nerve impulse flow to the adrenals, which is accompanied by an induction of adrenal tyrosine hydroxylase, dopamine/%hydroxylase (Moli- noff et al., 1970) and dopamine levels (Snider, 1974). The different extent of damage caused by 6-OHDA in adrenocortical cells of lizards and rats could then be explained by the different arrangement of cortex and medullary cells in these species, which facilitates contact of freshly released CA with adrenocortical cells in the lizards whereas in rats CA would leave the gland via the medullary vein and recirculate prior to entering the cortex again. However, the hypothesis, that CA alone are responsible for the morphological alterations in cortex cells is not supported by our results in reserpinized lizards. When administered to lizards, doses of reserpine sufficient to deplete CA stores in mammals did not mimic the effect of 6-OHDA. Crystalloid bodies were absent whilst lamellar bodies occurred only in small numbers. In addition, compact dense bodies not present in 6-OHDA-treated animals were found in reserpinized animals. However, these changes could also be due to a direct effect of reserpine, which possesses a high lipid solubility and, hence, affinity to all kinds of membranes (Seeman, 1966).

A direct action of 6-OHDA (or its metabolites) on the cortical cells may provide an alternative or supplementary explanation for our results. Several suggestions have been made concerning the direct mechanisms of action of 6-OHDA. Two theories seem to be of major importance. Both are based on the fact that 6-OHDA is readily autooxidised. Saner and Thoenen (1971) suggested that the oxidation products would undergo covalent binding with nucleophilic groups of biological macromolecules leading to a functional impairment of the structures involved. On the other hand, Heikkila and Cohen (1971, 1972) assumed that H 2 0 2 which is formed during the oxidation of 6-OHDA could account for the degenerative effects as well.

The complex reaction of 6-OHDA leading to the formation of several cyto- toxic products does not allow a clear answer as to which of them actually initiates the degeneration. Nevertheless, the neurotoxic potency of 6-OHDA is associated with its uptake by the neuronal membrane, since inhibition of the uptake mechanism prevents its action upon the adrenergic neuron (Malmfors and Sachs, 1968; Jonsson and Sachs, 1970). Furthermore, the octopus posterior salivary gland (P.S.G.), the one well documented case of extraneuronal degeneration following 6-OHDA treatment (Martin and Barlow, 1975), demonstrates


3H-NA located about myofilaments of muscle cells and to a lesser extent over the secretory cells in the tubules suggesting that the selective degeneration in these tissues follows uptake into cells that possess a high affinity uptake mechanism for catecholamines. It could be of considerable interest phylogenetically that, in the case of the octopus P.S.G., the high affinity uptake mechanism appears to be located extraneuronally.

It is therefore a crucial question whether 6-OHDA is also taken up into adrenocortical cells. Since (1) it does not form a fluorophore similar to that of biogenic monoamines after exposure to paraformaldehyde or glyoxylic acid (Ljungdahl et al., 1971; Unsicker et al., 1976) and (2) labelled 6-OHDA was not immediately available, uptake studies using NA were used as a suitable method for further elucidating this question. Extraneuronal uptake of NA has been demonstrated, with considerable species variation, in smooth and cardiac muscle, endothelial cells, fibroblasts chondroblasts, collagen and elastin in a variety of tissues such as heart, blood vessels, salivary gland, nictitating membrane, vas deferens, trachea, colon, bladder, spleen and connective tissues (for reviews see Iversen, 1971; Trendelenburg, 1972; Gillespie, 1973; Burnstock and Costa, 1975). Our in-vivo and in-vitro experiments using NA for an evaluation of a possibly occurring amine uptake into adrenocortical cells suggest that such an uptake occurs in adrenocortical cells after exposure to 10 -4 gm/ml in-vitro, although not in-vivo. The lack of uptake in-vivo may be due to inade- quate delivery of NA to the adrenocortical cells. Intravenous injections of high doses of NA have been reported to cause a reduction in blood flow through peripheral organs as a result of peripheral vasoconstriction whereas coronary blood flow is increased probably due to coronary dilatation and raised blood pressure (Goodman and Gilman, 1965 ; Gillespie and Muir, 1967). In the present study the uptake of NA in-vivo by heart tissues at all concentrations accompanied by the simultaneous absence of any extraneuronal loading in the spleen, is consistent with this view. Furthermore, masking of the cardiovascular effects of high doses of NA in-vivo by phentolamine (Udenfriend et al., 1959; Crout et al., 1961) has been used to demonstrate in-vivo uptake into the spleen as well as the heart (Sivaramakrishna and Gulati, 1975).

Alternatively it is possible that the local concentration of corticosteroids may be responsible for an uptake inhibition which would be greater in the in-vivo experiments than those performed in-vitro where a prolonged washout effect may have occurred. The inhibitory action of corticosterone on extraneu- tonal uptake has been demonstrated through both the direct blocking of uptake, (Iversen and Salt, 1970) and by an enhanced uptake of applied NA after adrenal- ectomy (Sivaramakrishna and Gulati, 1975). Even so, corticosterone may be compartmentalised in adrenocortical tisses and as a result may be unable to block adequately perfused NA. Clearly, NA uptake following prior treatment with phentolamine is needed to clarify the differences between in-vivo and in-vitro uptake.

While a dose 10-4 gm/ml NA is a particularly high concentration for neuronal uptake, this is not so for extraneuronal uptake. Gillespie et al. (1970) have shown, in the cat spleen, that 10-s gm/ml NA is required for fluorescence in arterial smooth muscle and endothelial cells. With the same concentration

Extraneuronal Effects of 6-OHDA 1 on Adrenocortical Cells 65

we had a clear loading of vascular smooth muscle and perivascular spaces in the rat adrenal and uptake into heart muscle cells in the rat, but not into steroid producing cells or into splenic tissues. However, Gillespie and Muir (1970), in their comparative study, have shown the difficulty of extraneuronal uptake into splenic tissues of species other than the rabbit and the cat. This is particularly evident in the results for the spleen treated in-vitro with 3x 10 -4 gm/ml. We have shown that the rat spleen can be loaded in-vitro at concentrations higher than this (10-3 gm/ml).

The nuclear-bound fluorescence of the adrenocortical parenchyma (10-4 gin/ ml and above in-vitro) resembles that reported for the reticular cells of the spleen by Gillespie et al. (1970) and also observed in the present study. Similar nuclear binding of NA has also been observed after a long wash out in smooth muscle and ectodermal cells (Burnstock et al., 1971).

It must be clearly emphasised that uptake of NA into cortical cells at 10 .4 gm/ml does not necessarily mean that 6-OHDA is taken up at the same rate or concentration or is similarly retained. Although neuronal uptake of 6-OHDA is slower than that of NA it is retained more efficiently (Jonsson, 1971). Extraneuronal uptake of 3H-6-OHDA into the denervated atrium has been shown to increase with time and concentration (Jonsson, 1971). The most pronounced extraneuronal uptake occurred at 10-4 M, at which concentration there was a greater extraneuronal than neuronal uptake. Furthermore 3H-6- OHDA uptake to denervated atrium is relatively higher than that of 3H-NA. In addition, extraneuronal uptake of 3H-6-OHDA by the rat iris was less than for the mouse atrium, suggesting tissue and or species differences similar to those with extraneuronal uptake of NA. Given these results and the fluorescence microscopy reported in this paper it seems quite likely that uptake of 6-OHDA into the adrenal cortex does occur. Studies using labelled 6-OHDA for directly examining its extraneuronal uptake properties in adrenocortical tissues are in progress.

In view of the possibility of a phylogenetically primitive high affinity uptake mechanism in extraneuronal tissues of the octopus P.S.G. (Martin and Barlow, 1975) it would be of considerable ontogenetic interest to explore the effects of 6-OHDA on those developing vertebrate tissues where extraneuronal uptake of NA has been demonstrated (Kirby and Gilmore, 1973; Lawrence and Burden, 1973; Newgreen and Allan, 1976).

A somewhat surprising result of this study is the occurrence of deposits of electron-dense material in chromaffin cells of newborn rats following the administration of 6-OHDA. The adrenal medulla has been repeatedly shown not to be directly affected by 6-OHDA (cf. Cheah et al., 1971; Angeletti and Levi-Montalcini, 1970), which is consistent with the observation that, after almost complete chemical sympathectomy, the CA content of the adrenals is not reduced (Thoenen and Tranzer, 1968). Since fine structural changes caused by 6-OHDA have been well documented ir~ SIF-cells of the paracervical ganglion of the rat (Kanerva et al., 1974), it is possible that these cells resemble immature adrenal chromaffin cells in some respects.


References Alexander, N.J.: Ultrastructural changes in rat epididymis after vasectomy, Z. Zellforsch. 136,

177-192 (1973) Angeletti, P.U., Levi-Montatcini, R.: Sympathetic nerve cell destruction in newborn mammals

by 6-hydroxydopamine. Proc. nat. Acad. Sci. (Wash.) 65, 114-121 (1970) Arai, K., Yates, R.D., Rappoport, D.A.: Fine structure and chemical composition of opaque

cytoplasmic bodies of Triparanol treated Syrian hamsters. II. Phospholipid analysis of opaque bodies from adrenal glands. Tex. Rep. Biol. Med, 25, 350 359 (1967)

Bennett, T., Burnstock, Cobb, J.US., Malfors, T.: An ultrastructural and histocbemical study of adrenergic nerves in the domestic fowl. Brit. J. Pharmacol. 38, 802-809 (1970)

Bj6rklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterisation of biogenic amines. In: Methods in investigative endocrinology, Vol. 1, The thyroid and biogenic amines. Ed. J.E. Rail and I.S. Kopen. North Holland Publ. Co. (1972)

Brunck, U., Ericsson, J.L.E., Pont6n, J., Westermark, B. : Residual bodies and "aging" in cultured human gila cells. Exp. Cell Res. 79, 1-14 (1973)

Burnstock, G., Costa, M. : Adrenergic neurons. London: Chapman and Hall (1975) Burnstock, G., McCulloch, M.W., Story, O.F., Wright, M.E.: Factors affecting the extraneuronal

inactivation of noradrenaline in cardiac and smooth muscle. Brit. J. Pharmaco[. 46, 243 253 (1972)

Burnstock, G., McLean, J.R., Wright, M.E.: Noradrenaline uptake by non-innervated smooth muscle. Brit. J. Pharmacol. 43, 180-189 (1971)

Cheah, T.B., Geffen, L.B., Jarrot, B., Ostberg, A. : Action of 6-hydroxydopamine on lamb sympathetic ganglia, vas deferens and adrenal medulla: a combined histochemical, ultrastructural and biochemical comparison with the effects of reserpine. Brit. J. Pharmacol. 42, 543-557 (1971)

Chen, I-Li, Yates, R.D.: An ultrastructural study of opaque cytoplasmic inclusions induced by Triparanol treatment. Amer. J. Anat. 121,705-726 (1967)

Costa, M., Furness, J.B. : Storage, uptake and synthesis of catecholamines in the intrinsic adrenergic neurones in the proximal colon of the guinea pig. Z. Zellforsch. 120, 364-385 (1971)

Coupland, R.E.: Electron microscopic observations on the structure of the rat adrenal medulla. I. The ultrastructure and organisation of chromaffin cells in the normal rat adrenal medulla. J. Anat. (Lond.) 99, 231-254 (1965)

Crout, J.R., Creveling, C.R., Udenfriend, S.: Norepinephrine metabolism in rat brain and heart. J. Pharmacol. exp. Ther. 132, 269-277 (1961)

Dietert, S.F., Scallen, T.J. : An ultrastructural and biochemical study of the effect of three inhibitors of cholesterol biosynthesis upon murine adrenal gland and testis: histochemical evidence for a lysosomal response. J. Cell Biol. 40, 44-60 (1969)

Drask6czy, P.R., Trendelenburg, U. : Intraneuronal and extraneuronal accumulation of sympathomi- metic amines in the isolated nictitating membrane of the cat. J. Pharmacol. exp. Ther. 174, 290-306 (1970)

Falck, B. : Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56, Suppl. 191, 1 (1962)

Falck, B., Owman, Ch. : A detailed description of the fluorescence method for the cellular localization of biogenic monoamines. Acta Univ. Lurid., Sect. II, Nr. 7 (1965)

Furness, J.B. : An electrophysiological study of the innervation of the smooth muscle of the colon. J. Physiol. (Lond.) 205, 549-562 (1969)

Furness, J.B., Campbell, G.R., Gillard, S.M., Malmfors, T., Cobb, J.L.S., Burnstock, G. : Cellular studies of sympathetic denervation produced by 6-hydroxydopamine in the vas deferens. J. Pharmacol. exp. Ther. 174, 111-122 (1970)

Gauthier, P., Nadeau, R., De Champlain, J. : Acute and chronic cardiovascular effects of 6-hydroxydopamine in dogs. Circulat. Res. 31, 207-217 (1972)

Gillespie, J.S.: Uptake of noradrenaline by smooth muscle. Brit. med. Bull. 29, 136-141 (1973) Gillespie, J.S., Hamilton, O.N.H., Hosie, R.S.: The extraneuronal uptake and localization of nor-

adrenaline in the cat spleen and the effect on this of some drugs, of cold and of denervation. J. Physiol. (Lond.) 206, 563-590 (1970)

Gillespie, J.S., Muir, T.C. : The origin of the decline in the vasopressor response to infused noradrenaline in the pithed rat. Brit. J. Pharmacol. 30, 88-98 (1967)


Gillespie, J.S., Muir, T.S. : Species and tissue variation in extranenronal accumulation of noradrenaline. J. Physiol. (Lond.) 206, 591-604 (1970)

Goodman, L.S., Gilman, A.: The pharmacological basis of therapeutics, 3rd edit. New York: Macmillan 1965

Heikkila, R., Cohen, G. : Further studies on the generation of hydrogen peroxide by 6-hydroxydopamine. Molec. Pharmacol. 8, 241-248 (1972)

Heikkila, R., Cohen, G. : Further studies on the generation of hydrogen peroxide by 6-hydroxydopamine. Molec. Pharmacol. 8, 241-248 (1972)

H6kfelt, T., Jonsson, G., Sachs, Ch.: Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro. Z. Zellforsch. 131, 529-541 (1972)

Hruban, Z., Swift, H., Slesers, A. : Effect of Triparanol and Diethanolamine on the fine structure of hepatocytes and pancreatic acinar cells. Lab. Invest. 14, 1653-1672 (1965)

Idelman, S.: In: Ann. Sci. Nat. Zool. Biol. Animale 8, 205-362 (1966). Quoted after Idelman (1970)

Idelman, S. : Ultrastructure of the mammalian adrenal cortex. Int. Rev. Cytol. 27, 181~81 (1970) Iversen, L.L. : Role of transmitter uptake mechanisms in synaptic neurotransmission. Brit. J. Pharma-

col. 41, 571-591 (1971) Iversen, L.L., Salt, P.J.: Inhibition of catecholamine uptake/ by steroids in the isolated rat heart.

Brit. J. Pharmacol. 40, 528-530 0970) Jonsson, G.: Studies on the uptake and accumulation of 3H-6-hydroxydopamine in adrenergic

nerves. In: Malmfors, T. and Thoenen, H. (Eds.), 6-hydroxydopamine and catecholamine neurons, pp. 243-256. London: North-Holland 1971

Jonsson, G., Malmfors, T., Sachs, Ch. : 6-hydroxydopamine as a denervation tool in catecholamine research. Proceedings of a conference held in G6teborg, Sweden, July 17-19, 1965. Amsterdam Oxford: North-Holland Publ. Co. 1975

Jonsson, G., Sachs, Ch. : Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons. Europ. J. Pharmacol. 9, 141-155 (1970)

Kanerva, L., Hervonen, A., Er/ink6, O., Lietzbn, R.: Fine structural changes caused by 6-OH- dopamine in the small intensely fluorescent cells of the paracervical ganglion of the rat. Cell Tiss. Res. 152, 437-447 (1974)

Kirby, M.L., Gilmore, S.A.: A fluorescence study on the ability of the notochord to synthesize and store catecholamines in early chick embryos. Anat. Rec. 173, 469-478 (1973)

Kostrzewa, R.M., Jacohowitz, D.M.: Pharmacological actions of 6-hydroxydopamine. Pharmacol. Rev. 26, 199-288 (1974)

Laverty, R., Sharman, D.E., Vogt, M. : Action of 2,4,5-trihydroxyphenylethylamine on the storage and release of noradrenaline. Brit. J. Pharmacol. 24, 549-560 (1965)

Lawrence Jr., I.E., Burden, H.W.: Catecholamines and morphogenesis of the chick neural tube and notochord. Amer. J. Anat. 137, 19%208 (1973)

Ljungdahl, A., H6kfelt, T., Jonsson, G., Sachs, Ch.: Autoradiographic demonstration of uptake and accumulation of 3H-6-hydroxydopamine in adrenergic nerves. Experientia (Basel) 27, 297 299 (1971)

Liillmann, H., Ltillmann-Rauch, R., Reil, G.-H. : A comparative ultrastructural study of the effects of Chlorophentermine andTriparanol in rat lung and adrenal gland. Virchows Arch. Aht. B 12, 91 103 (1973)

Liillmann-Rauch, R.: Lipidosis-like alterations in dorsal root ganglion cells of rats treated with tricyclic antidepressants. Nannyn-Schmiedebergs Arch. Pharmacol. 283, 219-222 (1974a)

Lfillmann-Rauch, R.: Retinal lesions in rats after treatment with Chlorphentermine or with tricyclic antidepressants. Virchows Arch. Abt. B 15, 309-312 (1974b)

Lfillmann-Rauch, R. : Lipidosis-like alterations in spinal cord and cerebellar cortex of rats treated with Chlorphentermine or tricyclic antidepressants. Acta neuropath. (Bed.) 29, 237~49 (1974c)

Lfillmann-Rauch, R., Pietschmann, N.: Lipidosis-like cellular alterations in lymphatic tissues of Chlorphentermine-treated animals. Vircbows Arch. Abt. B 15, 295-308 (1974)

Lfillmann-Rauch, R., Reil, G.-H.: Fenfluramine-induced ultrastructural alterations in tissues of rats and guinea-pigs. Naunyn-Schmiedeberg's Arch. Pharmacol. 285, 175-184 (1974)

Liillmann-Rauch, R., Reil, G.-H.: Chlorphentermine-induced lipidosis-like nltrastructural alterations in testicular Leydig and Sertoli cells. Exp. molec. Path. 22, 98-108 (1975)

Malmfors, T.: The influence of drugs on the effect of 6-hydroxydopamine. In: Malmfors, T.


and Thoenen, H. (Eds.), 6-hydroxydopamine and catecholamine neurons pp. 239-242 Amster- dam: North Holland 1971

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

Malmfors, T., Thoenen, H.: 6-hydroxydopamine and catecholamine neurons. Amsterdam: North Holland 1971

Martin, R., Barlow, J.J. : Muscle and gland cell degeneration in the octopus posterior salivary gland after 6-hydroxydopamine administration. J. Ultrastruct. Res. 52, 167-178 (1975)

McLean, J.R., Burnstock, G.: Histochemical localization of catecholamines in the urinary bladder of the toad (Bufo marinus). J. Histocbem. Cytochem. 14, 538-548 (1966)

Molinoff, P.B., Brimijoin, S., Weinshilboum, R., Axelrod, J.: Neurally mediated increase in dopamine beta-hydroxylase activity. Proc. nat. Acad. Sci. (Wash.) 66, 453 458 (1970)

Mueller, R.A., Thoenen, H., Axelrod, J.: Adrenal tyrosine hydroxylase compensatory increase in activity after chemical sympathectomy. Science 163, 468 469 (1969)

Newgreen, D.F., Allan, i.J. : A procedure for the rapid freezing of whole embryos. To be submitted to Experientia

Porter, C.C., Totaro, J.A., Stone, C.A. : Effect of 6-hydroxydopamine and some other compounds on the concentration of norepinephrine in the hearts of mice. J. Pharmacoh exp. Ther. 140, 308-316 (1963)

Sachs, Ch., Jonsson, G.: Mechanisms 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)

Seeman, P.M.: Membrane stabilization by drugs: transquilizers, steroids, and anesthetics. Int. Rev. Neurobiol. 9, 145-221 (1966)

Sivaramakrishna, N., Gulati, O.D.: Accumulation of norepinephrine (NE) by rat organs in vivo. Pharmacology 13, 365 377 (1975)

Snider, S.R. : Increase in adrenal dopamine following 6-hydroxydopamine. J. Pharmac. Pharmacol. 26, 647-649 (1974)

Suzuki, K., Zagoren, J.L., Gonates, J., Suzuki, K,: Ultrastructural study of neuronal cytoplasmic inclusions produced by hypocholesterolemic drug, AY 9944. Acta neuropath. (Berh) 26, 185-197 (1973)

Tischner, K.: Effects of chloroquine on neurons of long-term cultures of peripheral and central nervous system. Acta neuropath. (Bed.) 28, 233 242 (1974)

Thoenen, H., Tranzer, J.P.: Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 261, 271- 288 (1968)

Tranzer, J.P., Thoenen, H. : Ultra-morphologische Ver~nderungen der sympathischen Nervenendi- gungen der Katze nach Vorbehandlnng mit 5- und 6-hydroxy-dopamin. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 257, 343-344 (1967)

Trendelenburg, U. : Factors influencing the concentration of catecholamines at the receptors. In: Handbook of exp. pharmacol., Vol. XXXlll (H. Blaschko and E. Muscholl, Eds.), pp. 726-761. Berlin-Heidelberg-New York: Springer 1972

Udenfriend, S., Creveling, C.R., Ozaki, M., Oaly, J.W., Witkop, B.: Inhibitors of norepinephrine metabolism in vivo. Arch. Biochem. Biophys. 84, 249-251 (1959)

Unsicker, K.: Innervation of adrenal cells in the lizards Lacerta dugesi and Lacerta pityusensis. Gen. comp. Endocr. 24, 409-412 (1974)

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. I. Testicular interstitial tissue and seminal efferent ducts. Cell Tiss. Res. 158, 215 240 (1975)

Unsicker, K.: Chromaffin, small granule-containing and ganglion cells in the adrenal gland of reptiles. A comparative ultrastructural study. Cell Tiss. Res. 165, 477-508 (1976)

Unsicker, K., Chamley, J.H., McLean, J.R.: Extraneuronal effects of 6-hydroxydopamine: tissue culture studies on adrenocortical cells of rats. (In press) Cell Tiss. Res. (1976)

Uretsky, N.J., Iversen, L.L.: Effects of 6-hydroxydopamine on catecholamine containing neurons in the rat brain. J. Neurochem. 17, 269-278 (1970)


Wagner, K., Trendelenburg, U.: Effect of 6-hydroxydopamine on oxidative phosphorylation and on monoamine oxidase activity. Naunyn-Schmiedebergs Arch. Pharmak. 269, II0 116 (1971)

Wright, M.E.: Histochemical and biochemical studies of noradrenaline accumulation in smooth and cardiac muscle. M.Sc. Thesis. University of Melbourne, Department of Zoology (1972)

Yates, R.D.: The effects of Triparanol on adrenocortical cells of the zona fascicutata of Syrian hamsters. Z. Zellforsch. 71, 41-52 (1966)

Yates, R.D., Arai, K., Rappoport, D.A.: Fine structure and chemical composition of opaque cytoplasmic bodies of Triparanol treated Syrian hamsters. Exp. Cell Res. 47, 459-478 (1967)

Received March 23, 1976

Documents

Extraneuronal effects of 6-hydroxydopamine and extraneuronal uptake of noradrenaline