Effect of 6-hydroxydopamine on hypothalamic norepinephrine and dopamine content, ultrastructure of the median eminence, and plasma corticosterone

Brain Research, 78 (1974) 57-69 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

57

E F F E C T OF 6 - H Y D R O X Y D O P A M I N E O N H Y P O T H A L A M I C N O R E P I N E P H R I N E A N D D O P A M I N E C O N T E N T , U L T R A S T R U C T U R E OF T H E MEDIAN EMINENCE, AND PLASMA CORTICOSTERONE

A. C. CUELLO*, W. J. SHOEMAKER** ANDW. F. GANONG Department of Physiology, University of California, San Francisco, Calif. 94143 (U.S.A.)

(Accepted April 30th, 1974)

SUMMARY The effects of 6-hydroxydopamine on the catecholamine content of the hypothalamus and other tissues, the morphology of the median eminence of the hypothalamus, and plasma corticosterone were studied after intraperitoneal injection of the drug. The effect of injecting 6-hydroxydopamine into the third ventricle of the brain in unanesthetized animals was also studied. One day after injection by either route, hypothalamic norepinephrine was reduced and plasma corticosterone was elevated, suggesting increased secretion of A C T H from the pituitary. There were degenerative changes in a small number of neurons ending in the external layer of the median eminence, while other neurons in the median eminence were unaffected. Fifteen days after injection, hypothalamic norepinephrine and plasma corticosterone had returned to normal in the animals given 6-hydroxydopamine intraperitoneally. In the rats injected intraventricularly, hypothalamic norepinephrine was still reduced but plasma corticosterone was normal. In both groups at 15 days, neuronal debris had accumulated in phagocytic cells, but most neurons appeared normal. The data provide evidence that norepinephrine-containing neurons end in the external layer of the median eminence. They are also consistent with the hypothesis that a central adrenergic system inhibits A C T H secretion. Fifteen days after injection, an as yet unknown mechanism compensates for the decrease in norepinephrine content and A C T H secretion returns to normal.

* Present address: Orientacion Histologie Humana, Facultad de Farmacia y Bioquimica, Universidad Nacional de Buenos Aires, Junin 956, Buenos Aires, Argentina. ** Present address: Laboratory of Neuropharmacology, National Institute of Mental Health, W. A. White Building, St. Elizabeth's Hospital, Washington, D.C. 20032, U.S.A.

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INTRODUCTION

6-Hydroxydopamine is known to selectively destroy adrenergic neurons in the peripheral nervous system, and upon intracisternal administration, in the CNS t8,'-''-'. In the hypothalamus, the tubero-infundibular dopaminergic system of neurons is resistant to the action of the drug, but it causes marked depletion of hypothalamic norepinephrineT,')L We have therefore observed the morphological changes produced in the ventral hypothalamus by 6-hydroxydopamine to determine the distribution and morphology of the norepinephrine-containing neurons in this part of the brain. Since the drug does not cross the blood-brain barrier to any appreciable degree, we have compared its effects upon intraperitoneal administration with those upon intraventricular administration to determine whether norepinephrine-containing neurons in the median eminence end outside the blood-brain barrier. In addition, since there is considerable evidence that the norepinephrine-containing neurons in the ventral hypothalamus are involved in the regulation of ACTH secretion, we have studied the effect of 6-hydroxydopamine on ACTH secretion, using plasma corticosterone as the indicator. Part of the results have previously been reported in two abstracts aHJ. MATERIALS A N D M E T H O D S

Male adult Sprague-Dawley rats, weighing 250-300 g, were used in this study. The animals were kept under controlled lighting with 12 h light and 12 h dark per day. The rats received food and water ad libitum. Four experiments were performed. In experiments 1 and 2, 6-hydroxydopamine hydrobromide (Regis), 50 mg/kg body weight, was administered intraperitoneally on two occasions 12 h apart. It was dissolved in normal saline containing 0.1% ascorbic acid. The rats were sacrificed 24 h after the first injection in experiment 1 and 15 days after the first injection in experiment 2. Control animals received only the vehicle. For experiments 3 and 4, permanent cannulae were implanted stereotaxically in the third ventricle, using de Groot's atlas ~. The tip of the cannula was introduced to a position 1 mm above the floor of the third ventricle in the arcuate-median eminence area (Fig. 1). The cannulas were pieces of 22-gauge stainless steel tubing 18 mm long. They were implanted through a skull window, and a piece of Gelfoam (Upjohn) was used to cover the window after implantation. Fast-curing dental cement held the cannulas anchored on small screws placed in the skull. When the guide was properly placed, cerebrospinal fluid spontaneously flowed out of it. An inner plug of 28-gauge stainless steel wire was placed in the cannula. The animals received antibiotics for 1 week after the operation. Ten to 15 days after cannula implantation, half of the animals were injected with 6-hydroxydopamine. Each received two injections, 48 h apart. Each injection contained 200 #g of 6-hydroxydopamine hydrobromide and 25 #1 of distilled water containing 0.1 }/o ascorbic acid. Control animals received two injections of an equal volume of solution containing 0.1 ~ ascorbic acid and sufficient sodium bromide to procude a solution with an osmolarity analogous to the 6-hydroxydopamine solution.

6-HYDROXYDOPAM1NE AND

__

ACTH SECRETION

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Cement. ~;-CANNULA ~ Gel .

foam

/

THAL

ME8 CERE8

VENTRC I LE Fig. 1. Chronic cannula implant in the third ventricle in the rat. Amyg, amygdala; Arc, arcuate nucleus; CC, corpus callosum; Cereb, cerebellum; CI, internal capsule; CPu, caudate-putamen; DM, dorsomedial nucleus; Fx, fornix; Hypoth, hypothalamus; Mes, mesencephalon; OT, optic tract; Pirif, piriform cortex; Telenceph, telencephalon; Thal, thalamus; VM, ventromedial nucleus.

All injections were carried out through a 28-gauge stainless steel needle attached to a Hamilton syringe which was inserted in place of the plug in the implanted cannula. The animals were not anesthetized but were held in a towel during the injection procedure. Control and treated animals were sacrificed 24 h after the second injection in experiment 3 and 15 days after the second injection in experiment 4. In all cases, the animals were killed by decapitation between 0800 and 0830 h. A few of the brains in each group were saved for electron microscopy, and the remainder analyzed for catecholamine content. Trunk blood was collected for corticosterone determination by the method of Givener and Rochefort 11. The brains and spleens which were analyzed for catecholamine content were quickly removed and placed on dry ice. The hypothalamus and a sample of the telencephalon (a piece of tissue 2 mm wide from the region above the anterior hypothalamus, with its caudal face tangential to the nucleus anterior thalami) were separated from the rest of the brain. These samples and the spleen were homogenized and centrifuged in 5 ~ trichloroacetic acid. Aliquots of the supernatant were analyzed for catecholamine content. The method of Anton and Sayre ~ was used in experiments 3 and 4. In experiments 1 and 2, most of the determinations were made by the method of Udenfriend and Zaltzman-Nirenberg 19, as modified by Weiner 24, although a few samples were assayed by the method of Anton and Sayre. For catecholamine determinations, brain tissues from 3 animals were pooled in some cases. In the case of the animals in which electron microscopy was carried out, fixative was administered through the foramen magnum after decapitationlL The brain was removed from the skull and the median eminence of the hypothalamus dissected,

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using a dissecting microscope. This tissue was transferred to fresh fixative a n d processed for electron m i c r o s c o p y . F r o n t a l l y oriented sections t h a t included the full thickness o f the m e d i a n eminence were cut with a P o r t e r - B l u m M - 2 u l t r a m i c r o t o m e a n d m o u n t e d on c o a t e d grids. T h e sections were stained with l e a d citrate 2a a n d e x a m i n e d with an H i t a c h i HS8 electron microscope. I n experiments 1 a n d 2, the brains o f 9 o f the a n i m a l s in each g r o u p were used for c a t e c h o l a m i n e d e t e r m i n a t i o n and those o f 3 o f the animals in each g r o u p used for electron m i c r o s c o p y . I n experiments 3 a n d 4, the brains o f 15 animals in each g r o u p were used for c a t e c h o l a m i n e d e t e r m i n a t i o n a n d the brains o f 3 animals for electron microscopy. RESULTS (Table I). In rats given two doses o f 6 - h y d r o x y d o p a m i n e intraperitoneally a n d sacrificed 24 h after the first dose, there was a m a r k e d r e d u c t i o n in the n o r e p i n e p h r i n e c o n c e n t r a t i o n in the spleen, a small b u t significant r e d u c t i o n in the n o r e p i n e p h r i n e c o n c e n t r a t i o n in the h y p o t h a l a m u s , a n d no significant r e d u c t i o n in the n o r e p i n e p h r i n e c o n c e n t r a t i o n o f the telencephalon. There was no significant r e d u c t i o n o f the d o p a m i n e c o n c e n t r a t i o n in the h y p o t h a l a m u s or telencephalon. P l a s m a c o r t i c o s t e r o n e was significantly elevated. In these animals, a small n u m b e r o f axons located in the external layer o f the m e d i a n eminence o f the h y p o t h a l a m u s showed alterations which were n o t seen in the Experiment

1

TABLE I TISSUE CATECHOLAMINES DOPAMINE

AND PLASMA CORTICOSTERONE

24 h

AFTER INTRAPER1TONEAL

6-HYDROXY-

Values in parentheses are numbers of animals*. NE, norepinephrine; DA, dopamine. Control* *

Plasma corticosterone (~g/100 ml) Hypothalamus (~g/g) NE DA Telencephalon (btg/g) NE DA Spleen (/~g/g) NE

4.4 4- 1.0 2.21 2_ 0.24 0.50 ~ 0.01 0.34 ± 0.02 0.75 4- 0.08 0.73 4- 0.042

(19) (5) (3) (5) (3) (4)

Treated* *

P* **

19.0 4- 3.2 (19) 1.44 4- 0.04 (5) 0.46 4- 0.03 (3) 0.29 4- 0.03 (6) 0.65 4- 0.02 (3) 0.24 4- 0.062 (4)

< 0.001 < 0.01 NS§ NS§ NS§ < 0.001

* Some of these values represent pooled tissues. Each pool is considered as one animal. ** Values are given as 4- standard error of the mean. *** P values using Student's t-test of treated versus controls. § Not significant: used when P value is > 0.05.

Fig. 2. Contact zone of median eminence of an animal treated with 6-hydroxydopamine intraperitoneally 24 h previously, a: degenerating axon (arrows) containing multilamellar bodies and elongated synaptic vesicles located among normal nerve terminals (a). × 66,000. b: degenerating axon (arrow) containing abundant dense bodies, ct, connective tissue space. × 33,000.

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control animals. M o s t o f these axons were in close p r o x i m i t y to portal capillary vessels. The alterations included the presence of small, m u l t i l a m e l l a r bodies, dense bodiey,, aggregation o f g r a n u l a r vesicles, and elongation of the small a g r a n u i a r vesicles to an elliptical shape (Fig. 2). In some o f the e p e n d y m a l cells there was proliferation o f the r o u g h e n d o p l a s m i c reticulum, occasionally resulting in whorls o f membrane. E x p e r i m e n t 2 (Table II). Fifteen days after t r e a t m e n t with two intraperitoneal doses o f 6 - h y d r o x y d o p a m i n e , the c o n c e n t r a t i o n o f n o r e p i n e p h r i n e in the spleen was still reduced. However, h y p o t h a l a m i c n o r e p i n e p h r i n e c o n c e n t r a t i o n was the same in the treated as it was in the control animals. Plasma c o r t i c o s t e r o n e was also essentially the same in the two groups. In these animals the axons o f the m e d i a n eminence d i s p l a y e d a relatively n o r m a l a p p e a r a n c e . However, m a c r o p h a g e s were observed in the connective tissue space a r o u n d the portal vessels, aligned in the b o r d e r o f the neural tissue. Similar cells were f o u n d in smaller n u m b e r s in the interior of the pallisade zone o f the median eminence. There were a few very dilated axons c o n t a i n i n g vesicular and g r a n u l a r structures and dense bodies (Fig. 3). In these animals the changes in the e p e n d y m a l cells had disappeared. E x p e r i m e n t 3 (Table III). T w e n t y - f o u r hours after injecting 6 - h y d r o x y d o p a m i n e in the third ventricle, there was a m a r k e d depletion o f h y p o t h a l a m i c norepinephrine. Telencephalic n o r e p i n e p h r i n e was reduced to a lesser extent. T h r o u g h a technical

TABLE 1I TISSUE NOREPINEPHRINE AND PLASMA CORT1COSTERONE 15 DAYS AFTER INTRAPERITONEAL 6-HYDROXYDOPAMINE

See footnotes in Table i. Control* *

Plasma corticosterone (#g/100 ml) Hypothalamus (pg/g) NE Telencephalon (#g/g) NE Spleen (b~g/g) NE

3.8 ± 0 . 4 2.90 ± 0.20 0.48 g_. 0.04 0.71 ± 0.44

Treated*"

(21) (5) (5) (5)

4.3 ~ 1.0 2.92 :L 0.08 0.41 ± 0.03 0.32 ± 0.44

P* * *

(21) (5) (5) (5)

NS§ NS§ NS§ < 0.001

TABLE llI 24 h AFTER THE 3rd VENTRICLEVIA CHRONICALLYIMPLANTEDCANNULA

TISSUE NOREPINEPHR1NE AND PLASMA CORTICOSTERONE

INJECTING 6-HYDROXYDOPAMINE IN

See footnotes in Table I.

Plasma corticosterone (/tg/100 ml) Hypothalamus (/~g/g) NE Telencephalon (/~g/g) NE

Control**

Treated**

P* * *

11.2 J- 2.1 (9) 01.94 ± 0.15 (4) 00.34 ± 0.03 (4)

28.8 ± 6.3 (9) 00.22 3- 0.07 (5) 00.18 ± 0.05 (5)

< 0.05 < 0.001 < 0.05

6-HYDROXYDOPAMINE AND ACTH SECRETION

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Fig. 3. Contact zone of median eminence from an animal treated with 6-hydroxydopamine intraperitoneally 15 days previously. Enlarged axonal element containing a large variety of vesicles which contain different amounts of electron dense material, ep, ependymal process, x 13,500.

error, hypothalamic and telencephalic dopamine concentration was not measured. Plasma corticosterone was significantly elevated compared to controls. The control animals in this group, which had received an intraventricular injection, had higher plasma corticosterone levels than the controls in experiment 1, which had not. In these animals, there were dramatic changes in a limited number of axons terminating in the external layer of the median eminence. Most of the affected axons were reduced to a mass of homogeneous material (Fig. 4). Tanycytes showed various degrees of reaction from the injection (Fig. 5). Some were filled with large secondary lysosomes. Most of the tanycytes showed an enhanced number of dense bodies when compared to the tanycytes in the control animals. However, most of the axons of the external layer were normal. In addition, the magnocellular neurosecretory system of hypothalamic-neurohypophysial fibers showed no structural alterations (Fig. 6)

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TABLE IV HYPOTHALAMIC CATECHOLAM1NESAND PLASMACORTICOSTERONE 15 DAYSAFTERINJECTING 6-HYDROXY DOPAMINEIN THE 3rd VENTRICLE

See footnotesin Table I. Control* *

Plasma corticosterone (/~g/100 ml) Hypothalamus NE DA Telencephalon (pg/g) NE

5.5 1.13 0.41 0.16

4:_ 0.8 =k 0.09 ± 0.03 4- 0.02

(8) (6) (6) (6)

Treated* *

P* * *

8.3 4- 1.5 (8) 0.19 ± 0.03 (6) 0.42 ± 0.03 (6) < 0.08 (6)

NS§ < 0.001 NS§ §§

§§ No telencephalic norepinephrine was detected. The limit of the assay was 0.08/~g/g. However, a standard error and t-test could not be performed.

even though they were in the internal layer of the median eminence and therefore closer to the tip of the cannula than the affected neurons. Experiment 4 (Table IV). Fifteen days after intraventricular 6-hydroxydopamine, hypothalamic norepinephrine concentration was still markedly reduced. Dopamine concentration was normal and plasma corticosterone was not significantly elevated. In these animals, most of the signs of axonal injury had disappeared. However, there was considerable evidence of removal of axonal debris. There were numerous macrophages along the border between the neural tissue and the connective tissue space (Fig. 7). These cells, which contained large dense bodies that were apparently secondary lysosomes, were also found in the interior of the neural tissue. The tanycytes appeared partially recovered, with less marked cytological alterations. DISCUSSION

In the present experiments, the intraperitoneal or intraventricular administration of 6-hydroxydopamine led to a decrease in hypothalamic norepinephrine concentration and to destructive changes in some of the neurons ending in the external layer of the median eminence without destruction of neighboring neurons. In those instances in which it was measured, hypothalamic dopamine concentration was unaffected. There was some fluctuation in the control values for hypothalamic norepinephrine concentration in the various experiments due apparently to the use of 2 catecholamine methods and different batches of alumina. However, in each experiment controls and treated animals were analyzed at the same time. In the case of intraperitoneal injection, the neuronal changes were mild, whereas they were marked following intraventricular injection. However, in both instances, they were remarkably

Fig. 4. Contact zone of median eminence 24 h after intraventricular 6-hydroxydopamine. Completely degenerated axons (arrows) in vicinity of normal nerve endings (a). × 14,700. Fig. 5. Group of degenerated axons inside an ependymal process 24 h after intraventricular 6-hydroxydopamine. × 15,300.

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6-HYDROXYDOPAMINE AND

ACTH

SECREI'ION

67

selective, with normal-appearing neurons located next to damaged neurons. In addition, in the intraventricular injection experiments, hypothalamo-hypophysial axons coursing from the supraoptic and paraventricular nuclei to the posterior lobe in the internal layer of the median eminence were not damaged, even though they were closer to the cannula. The selectivity of the changes combined with the data on catecholamine content suggests that the damaged neurons were norepinephrine-containing neurons. Since some of them ended in the external layer of the median eminence, this is additional evidence for such terminations of norepinephrine-secreting neurons. We have reported ~ that knife cuts along the lateral aspects of the mediobasal hypothalamus cause a similar pattern of degeneration and a decrease in hypothalamic norepinephrine concentration, suggesting that the cell bodies of the neurons that end in the median eminence are extrahypothalamic in location. The norepinephrine content of the median eminence is high ~. Morphological evidence that neurons with their cell bodies in the locus subcoeruleus terminate in the ventral hypothalamus has been published, but many of these neurons were reported to end in the internal layer of the median eminence 2. While this paper was in preparation, it was reported that axons containing dopamine-fl-hydroxylase end in the external layer of the median eminence 12. Additional evidence that norepinephrine-secreting neurons terminate in the ventral hypothalamus is provided by the observation that hypothalamic norepinephrine concentration was reduced following systemic administration o f 6-hydroxydopamine. Furthermore, the depletion was not as marked as it was after intraventricular 6-hydroxydopamine. This would be expected, since in this situation only the ventral hypothalamus would be exposed to appreciable quantities of 6-hydroxydopamine because of the blood-brain barrier. The entire hypothalamus was analyzed for norepinephrine content, and the median eminence makes up only a small part of the total tissue analyzed. A decrease in total hypothalamic norepinephrine following intraperitoneal administration of 6-hydroxydopamine has previously been reported in the kitten 14. It has also been observed by Scapagnini in the rat (unpublished observation). The apparent stimulation of the tanycytes in the 6-hydroxydopamine-treated rats deserves comment. This effect was more marked in the animals receiving intraventricular injections. It need not be due to a direct stimulation of cells by 6-hydroxydopamine, and could be secondary to phagocytic activity of these cells in response to the destruction of neurons produced by 6-hydroxydopamine. There is considerable evidence that there is a central noradrenergic system that inhibits pituitary secretion of ACTH, in this system 9. In the present studies, increased plasma corticosterone concentration was seen 24 h after 6-hydroxydopamine injec-

Fig. 6. Internal layer of median eminence 24 h after intraventricular 6-hydroxydopamine. The axons of the magnocellular neurosecretory cells are well preserved. × 27,500. Fig. 7. Fifteen days after intraventricular treatment with 6-hydroxydopamine. Macrophages (mp) containing large lysosomes are seen lining the contact zone of the external layer (el) of the median eminence near a portal vessel (pv). × 12,200.

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tion, indicating that ACTH secretion was increased. This was not due to the injection because there was only a slight elevation in the control animals injected with sodium bromide. It could have been due to a nonspecific stress effect of the 6-hydroxydopamine. However, destruction of the norepinephrine-secreting neurons in the hypothalamus would be expected to be associated with increased ACTH secretion, so the data are also consistent with the hypothesis of central aminergic inhibition. A transient increase in the ACTH response to stress after intracerebral 6-hydroxydopamine has been reported by Fuxe and associates s. On the other hand, the plasma corticosterone concentration had returned to normal 15 days after 6-hydroxydopamine treatment. In the animals that received 6-hydroxydopamine intraperitoneally, the hypothalamic norepinephrine concentration had returned to normal. However, in the intraventricularly treated animals, there is still a marked depletion of norepinephrine. Others s,ls,2° have also reported normal resting plasma corticosterone values several days to severa'~ weeks after intracerebral 6-hydroxydopamine. At present, one can only speculate about the explanation of the normal level of ACTH secretion in the presence of marked norepinephrine depletion. One possibility is that after initial functional disraption, some of the adrenergic neurons in the hypothalamus recovered, and these neurons were capable of maintaining ACTH secretion at normal levels. These remaining neurons might be relatively hyperactive. Regrowth of adrenergic neurons is another possibility. These neurons have been shown to sprout new terminals when damaged 16, and hypothalamic norepinephrine concentration in the animals injected intraperitoneally had returned to normal in the hypothalamus in 15 days. Another possibility is that the denervated receptors on the membranes of the hypothalamic cells that .,ecrete the hypothalamic factor that stimulates ACTH secretion developed denervation hypersensitivity, and that in this situation, small amounts of norepinephrine held secretion of corticotropin releasing factor in check. There is other evidence for the development of hypersensitivity in receptors denervated by 6-hydroxydopamine treatment ~1. Another unsettled question is the site at which the central adrenergic neurons act to inhibit ACTH secretion. The data in the dog suggest that the site is 'inside the blood-brain barrier', and hence above the basal median eminence 9. The present data suggest that in the rat, the site of action may be the median eminence, since intraperitoneal 6-hydroxydopamine increased plasma corticosterone. However, 6-hydroxydopa, a compound which enters the brain and is converted to 6-hydroxydopamine in neurons in the central nervous system la, has a greater effect on plasma corticosterone in rats than an equal dose of 6-hydroxydopamine 1°. This suggests that in this species as well, the inhibitory effect of adrenergic discharge is at least in part exerted 'inside the blood-brain barrier'. p e r se

ACKNOWLEDGEMENTS

This investigation was supported by a USPHS Grant AM 06704. Dr. Cuello was supported by a USPHS International Postdoctoral Fellowship TW 01599 and Dr. Shoemaker by a USPHS Training Grant AM 05613.

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