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Effects of intravenously administered 6-hydroxydopamine on the content of monoamines in the median eminence and neurointermediate lobe of the rat
Neuroscience Letters, 55 (1985) 141-144
141
Elsevier Scientific Publishers Ireland Ltd. NSL 03218
EFFECTS OF I N T R A V E N O U S L Y A D M I N I S T E R E D 6 - H Y D R O X Y D O P A M I N E O N T H E C O N T E N T OF M O N O A M I N E S IN T H E M E D I A N E M I N E N C E A N D N E U R O I N T E R M E D I A T E LOBE OF T H E RAT
W. JOHN SHEWARD', ALAN G. WATTS I, GEORGE F1NKI'* and GRAEME C. SMITH 2 ~MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ (U.K.) and 2Department of Psychological Medicine, Monash University, Prince Henry's Hospital, St. Kilda Road, Melbourne, Vic. (Australia)
(Received December 28th, 1984; Accepted January 15th, 1984)
Key words: 6-hydroxydopamine - desipramine - amines- median eminence - neurointermediate lobe -
high-performance liquid chromatography
The effects of intravenous administration of 6-hydroxydopamine (6-OHDA) on the content of catecholamines in the median eminence and neurointermediate lobe was studied in male rats. The results confirmed previous findings of a depletion of catecholamines in both regions as a result of this lesion, although the magnitude of the effect was less than that revealed by fluorescence histochemistry. Administration of desipramine before 6-OHDA injection produced a more specific lesion, depleting dopamine but leaving noradrenaline unaffected. I n t r a v e n o u s (i.v.) injection o f 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) p r o d u c e s a deplet i o n o f c a t e c h o l a m i n e s in the m e d i a n e m i n e n c e (ME) a n d n e u r o i n t e r m e d i a t e lobe ( N I L ) while leaving the r e m a i n d e r o f the h y p o t h a l a m u s s u b s t a n t i a l l y u n a f f e c t e d [3]. This p r e p a r a t i o n has p r e v i o u s l y been used to e x a m i n e the effects o f such a lesion o n the secretion o f p i t u i t a r y h o r m o n e s in male rats [4]. W h i l e the lesion has been s h o w n b y fluorescence h i s t o c h e m i s t r y to be c o m p l e t e after 24 h, the tissue c o n t e n t o f the c a t e c h o l a m i n e s has not been m e a s u r e d by assay. H e r e we have d e t e r m i n e d the c o n t e n t o f n o r a d r e n a l i n e ( N A ) , d o p a m i n e ( D A ) t o g e t h e r with d i h y d r o x y p h e n y l a c e t i c acid ( D O P A C ) , 5 - h y d r o x y t r y p t a m i n e (5-HT) a n d 5 - h y d r o x y i n d o l e acetic acid ( 5 - H I A A ) in the M E a n d N I L o f a n i m a l s t r e a t e d with 6 - O H D A a n d in a n i m a l s in which the lesion h a d been m o d i f i e d b y p r e t r e a t m e n t with d e s i p r a m i n e ( D M I ) in o r d e r to p r o d u c e , p r e s u m a b l y , a m o r e selective d e p l e t i o n o f D A [3]. T h e a n i m a l s used were a d u l t m a l e W i s t a r rats (180-240 g b o d y wt.), which were s u p p l i e d by the E d i n b u r g h U n i v e r s i t y C e n t r e for L a b o r a t o r y A n i m a l s , m a i n t a i n e d u n d e r c o n t r o l l e d lighting c o n d i t i o n s (14 h light, 10 h d a r k ) a n d t e m p e r a t u r e (22°C) a n d a l l o w e d free access to diet 41B ( O x o i d ; B a s i n g s t o k e ) a n d t a p water. T h e a n i m a l s *Author for correspondence. 0304-3940/85/$ 03.50 © 1985 Elsevier Scientific Publishers Ireland Ltd.
142 were injected i.v., under light ether anaesthesia, with either 150 m g / k g 6-OHDAHCI (Sigma U.K.; 50 m g / m l in 0.1°70 ascorbic acid-0.9% saline solution) or with 0.1% ascorbic acid-0.9% saline (vehicle). One group of animals was also injected with 30 m g / k g DMI-HC1 (Geigy, Macclesfield; 12 m g / m l in 0.9% saline i.p.) administered 30 rain before the injection of 6-OHDA. A further group of untreated control animals were not injected with either vehicle or drugs. Forty-eight hours after drug treatment, animals were killed by decapitation and the brain rapidly removed, placed on ice and the pituitary stalk-median eminence (SME) dissected out. The SME was removed by gently holding the stalk in watchmakers forceps and undercutting an area of the ME about 2 m m wide, from the stalk-ME junction, and as deep as the floor of the third ventricle. The mean (_+ S.E.M.) weight of the ME tissue was 0.44 +_0.07 mg. The pituitary was removed from its dural capsule, and the NIL dissected from the anterior pituitary. The mean (_+S.E.M.) weight of the NIL was 1.39_+0.39 rag. NIL and SME samples were stored in liquid nitrogen until the contents of amines were determined by highperformance liquid chromatography with electrochemical detection ( H P L C - E C D ) . The samples were homogenized in 80 #1 of 0.2 M perchloric acid in 1 0 4 M cysteine solution to which had been added 400 pg/20 t*l of n-acetyl dopamine (n-AcDA; Sigma U.K.) as internal standard (IS). Homogenates were centrifuged at 2000 g for 5 rain, the supernatant removed and filtered through 0.2 #m regenerated cellulose filters (BAS Inc., West Lafayette, IN). Twenty microliters of the filtered homogenate were applied onto a 250x 4.6 mm analytical column of Ultrasphere ODS (Altex, Berkley, CA) with a 50 × 4.6 mm Ultrasphere ODS pre-column. The mobile phase consisted of 0.1 M acetate-0.1 M citrate buffer, methanol and tetrahydrofuran (95:5:2, v/v) adjusted to pH 5.0 by the addition of 6.5 g sodium hydroxide/litre and with the addition of 100 m g / l of octanesulphonic acid (Fisons, Loughborough) as ion-pair reagent. The flow-rate was 1.0 ml/min. The eluant from the column was passed through an LC-5A glassy carbon transducer cell (BAS) connected to an LC-4A amperometric detector (BAS). The working potential of the electrode was maintained at + 0.70 V, and the generated current was displayed on a Tarkan 600 chart recorder. Fresh standard solutions of NA, DA, 5-HT, 5-HIAA and D O P A C (Sigma U.K.) were prepared in 0.2 M perchloric acid. Standard curves were linear over a range of 20 pg to 2 ng per 20-t*l injection. Concentrations of amines in homogenates were calculated by peak-height ratio with respect to 400 pg per 20-t,1 injection of IS added to all samples and standards. The significance of differences between means was calculated using unpaired t-tests. Table I shows the amine and metabolite content of the SEM and NIL after drug treatment. Forty-eight hours after 6 - O H D A administration there was a significant decrease in the contents of both NA ( P < 0.005 and P < 0.02, SME and NIL, respectively) and DA (P<0.001 and P < 0 . 0 0 5 ) compared with the contents in untreated or vehicle-treated control groups. In animals treated with DMI 30 rain before
143 TABLE I CONTENT OF AMINES AND METABOLITES IN STALK MEDIAN EMINENCE (SME) AND NEUROINTERMEDIATE LOBE (NIL) 48 h AFTER DRUG TREATMENT Values=pg/area dissected (mean±S.E.M.). n, number of animals per treatment group; ND, not determined. Treatment group
n
NIL 48 h after injections untreated ascorbic acid 6-OHDA 6-OHDA+DMI
6 6 6 7
SME 48 h after injections untreated ascorbic acid 6-OHDA 6-OHDA+DM!
6 5 6 7
NA
229_+ 204_+ 124_+ 265_+
25 20 17 34
1161 _+141 913_+ 70 584_+ 38 1130_+206
DA
DOPAC
5-HT
5-HIAA
2558_+157 2407_+130 1479_+177 1457_+153
530+111 785_+ 100 ND ND
632_+246 385_+ 32 403_+ 83 420_+ 24
251_+56 248_+52 219_+51 314_+65
5600_+289 5042_+375 2397_+191 2314_+281
1860_+237 2088_+500 899_+ 89 1085_+ 90
483_+172 280_+ 58 369_+ 97 275_+ 28
356_+86 301_+50 235_+44 239+49
6 - O H D A a d m i n i s t r a t i o n there was also a significant d e p l e t i o n o f D A after 48 h ( P < 0.001, b o t h areas), but N A c o n t e n t r e m a i n e d similar to the values in the c o n t r o l a n i m a l s . D O P A C was significantly r e d u c e d in the S M E in b o t h 6 - O H D A - t r e a t e d a n d D M I + 6 - O H D A - t r e a t e d g r o u p s c o m p a r e d with the values in c o n t r o l s ( P < 0.05 t o P < 0 . 0 0 5 ) . 5 - H T a n d 5 - H I A A c o n t e n t s r e m a i n e d u n c h a n g e d in all t r e a t m e n t groups. These results p r o v i d e direct evidence that c o n f i r m s the p r e v i o u s fluorescence h i s t o c h e m i c a l findings o f a d e p l e t i o n o f N A a n d D A in the M E a n d N I L after the i n t r a v e n o u s a d m i n i s t r a t i o n o f 6 - O H D A . T h e m a g n i t u d e o f the d e p l e t i o n (-~ 50%), h o w e v e r , was m u c h less t h a n suggested b y p r e v i o u s findings with fluorescence h i s t o c h e m i s t r y [3], which s h o w e d a c o m p l e t e d e p l e t i o n o f c a t e c h o l a m i n e s in these t w o areas in the first two d a y s a f t e r 6 - O H D A t r e a t m e n t . T h e m o s t likely e x p l a n a t i o n for the d i s c r e p a n c y in the results is that in the present s t u d y the extent o f the lesion was u n d e r e s t i m a t e d b y the inclusion in the dissected areas o f regions in which c a t e c h o l a m i n e s a c c u m u l a t e d b e h i n d the lesioned nerve t e r m i n a l s [5]. Cuello et al. [1] s h o w e d a 37% r e d u c t i o n in the h y p o t h a l a m i c c o n t e n t o f N A but no change in D A c o n t e n t 24 b after systemic 6 - O H D A a d m i n i s t r a t i o n . H o w e v e r , their s t u d y differs in two respects f r o m the present investigation in which the a m i n e c o n t e n t has been m e a s u r e d in m o r e discrete areas a n d a higher dose o f 6 - O H D A was adm i n i s t e r e d ( N A t e r m i n a l s are m o r e susceptible t h a n D A t e r m i n a l s to the effects o f 6 - O H D A [5]). T h e c o m b i n e d t r e a t m e n t with 6 - O H D A a n d D M I has been used in a previous s t u d y to p r o v i d e evidence for the i n h i b i t i o n o f g o n a d o t r o p i n release by the t u b e r o i n -
144
fundibular DA system because this treatment enhanced the luteinizing hormone surge induced by administration of pregnant mare serum gonadotropin [2]. The present results provide direct confirmation of the fact that a selective lesion of D A neurons can be produced by administration o f DMI before the 6 - O H D A injection, and this model may be of further use in studying aminergic-peptidergic interactions in the hypothalamic control o f hormone release from the anterior pituitary gland. 1 Cuello, A.C., Shoemaker, W.J. and Ganong, W.F., Effect of 6-hydroxydopamine on hypothalamic norepinephrine and dopamine content, ultrastructure of the median eminence, and plasma corticosterone, Brain Res., 78 (1974) 57-69. 2 Sarkar, D.K., Smith, G.C. and Fink, G., Effect of manipulating central catecholamines on puberty and the surge of luteinizing hormone and gonadotropin releasing hormone induced by pregnant mare serum gonadotropin in female rats, Brain Res., 213 (1981) 335-349. 3 Smith, G.C., Courtney, P.G., Wreford, N.G.M. and Walker, M.McD., Further studies on the effects of intravenously administered 6-hydroxydopamine on the median eminence of the rat, Brain Res., 234 (1982) 101-110. 4 Smith, G.C., Sheward, W.J. and Fink, G., Effect of 6-hydroxydopamine lesions of the median eminence and neurointermediate lobe on the secretion of pituitary hormones in the male rat, Brain Res., 246 (1982) 330-333. 5 Ungerstedt, U., Histochemical studies on the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in the rat brain. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, North-Holland, Amsterdam, 1971, pp. 101-127.
141
Elsevier Scientific Publishers Ireland Ltd. NSL 03218
EFFECTS OF I N T R A V E N O U S L Y A D M I N I S T E R E D 6 - H Y D R O X Y D O P A M I N E O N T H E C O N T E N T OF M O N O A M I N E S IN T H E M E D I A N E M I N E N C E A N D N E U R O I N T E R M E D I A T E LOBE OF T H E RAT
W. JOHN SHEWARD', ALAN G. WATTS I, GEORGE F1NKI'* and GRAEME C. SMITH 2 ~MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ (U.K.) and 2Department of Psychological Medicine, Monash University, Prince Henry's Hospital, St. Kilda Road, Melbourne, Vic. (Australia)
(Received December 28th, 1984; Accepted January 15th, 1984)
Key words: 6-hydroxydopamine - desipramine - amines- median eminence - neurointermediate lobe -
high-performance liquid chromatography
The effects of intravenous administration of 6-hydroxydopamine (6-OHDA) on the content of catecholamines in the median eminence and neurointermediate lobe was studied in male rats. The results confirmed previous findings of a depletion of catecholamines in both regions as a result of this lesion, although the magnitude of the effect was less than that revealed by fluorescence histochemistry. Administration of desipramine before 6-OHDA injection produced a more specific lesion, depleting dopamine but leaving noradrenaline unaffected. I n t r a v e n o u s (i.v.) injection o f 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) p r o d u c e s a deplet i o n o f c a t e c h o l a m i n e s in the m e d i a n e m i n e n c e (ME) a n d n e u r o i n t e r m e d i a t e lobe ( N I L ) while leaving the r e m a i n d e r o f the h y p o t h a l a m u s s u b s t a n t i a l l y u n a f f e c t e d [3]. This p r e p a r a t i o n has p r e v i o u s l y been used to e x a m i n e the effects o f such a lesion o n the secretion o f p i t u i t a r y h o r m o n e s in male rats [4]. W h i l e the lesion has been s h o w n b y fluorescence h i s t o c h e m i s t r y to be c o m p l e t e after 24 h, the tissue c o n t e n t o f the c a t e c h o l a m i n e s has not been m e a s u r e d by assay. H e r e we have d e t e r m i n e d the c o n t e n t o f n o r a d r e n a l i n e ( N A ) , d o p a m i n e ( D A ) t o g e t h e r with d i h y d r o x y p h e n y l a c e t i c acid ( D O P A C ) , 5 - h y d r o x y t r y p t a m i n e (5-HT) a n d 5 - h y d r o x y i n d o l e acetic acid ( 5 - H I A A ) in the M E a n d N I L o f a n i m a l s t r e a t e d with 6 - O H D A a n d in a n i m a l s in which the lesion h a d been m o d i f i e d b y p r e t r e a t m e n t with d e s i p r a m i n e ( D M I ) in o r d e r to p r o d u c e , p r e s u m a b l y , a m o r e selective d e p l e t i o n o f D A [3]. T h e a n i m a l s used were a d u l t m a l e W i s t a r rats (180-240 g b o d y wt.), which were s u p p l i e d by the E d i n b u r g h U n i v e r s i t y C e n t r e for L a b o r a t o r y A n i m a l s , m a i n t a i n e d u n d e r c o n t r o l l e d lighting c o n d i t i o n s (14 h light, 10 h d a r k ) a n d t e m p e r a t u r e (22°C) a n d a l l o w e d free access to diet 41B ( O x o i d ; B a s i n g s t o k e ) a n d t a p water. T h e a n i m a l s *Author for correspondence. 0304-3940/85/$ 03.50 © 1985 Elsevier Scientific Publishers Ireland Ltd.
142 were injected i.v., under light ether anaesthesia, with either 150 m g / k g 6-OHDAHCI (Sigma U.K.; 50 m g / m l in 0.1°70 ascorbic acid-0.9% saline solution) or with 0.1% ascorbic acid-0.9% saline (vehicle). One group of animals was also injected with 30 m g / k g DMI-HC1 (Geigy, Macclesfield; 12 m g / m l in 0.9% saline i.p.) administered 30 rain before the injection of 6-OHDA. A further group of untreated control animals were not injected with either vehicle or drugs. Forty-eight hours after drug treatment, animals were killed by decapitation and the brain rapidly removed, placed on ice and the pituitary stalk-median eminence (SME) dissected out. The SME was removed by gently holding the stalk in watchmakers forceps and undercutting an area of the ME about 2 m m wide, from the stalk-ME junction, and as deep as the floor of the third ventricle. The mean (_+ S.E.M.) weight of the ME tissue was 0.44 +_0.07 mg. The pituitary was removed from its dural capsule, and the NIL dissected from the anterior pituitary. The mean (_+S.E.M.) weight of the NIL was 1.39_+0.39 rag. NIL and SME samples were stored in liquid nitrogen until the contents of amines were determined by highperformance liquid chromatography with electrochemical detection ( H P L C - E C D ) . The samples were homogenized in 80 #1 of 0.2 M perchloric acid in 1 0 4 M cysteine solution to which had been added 400 pg/20 t*l of n-acetyl dopamine (n-AcDA; Sigma U.K.) as internal standard (IS). Homogenates were centrifuged at 2000 g for 5 rain, the supernatant removed and filtered through 0.2 #m regenerated cellulose filters (BAS Inc., West Lafayette, IN). Twenty microliters of the filtered homogenate were applied onto a 250x 4.6 mm analytical column of Ultrasphere ODS (Altex, Berkley, CA) with a 50 × 4.6 mm Ultrasphere ODS pre-column. The mobile phase consisted of 0.1 M acetate-0.1 M citrate buffer, methanol and tetrahydrofuran (95:5:2, v/v) adjusted to pH 5.0 by the addition of 6.5 g sodium hydroxide/litre and with the addition of 100 m g / l of octanesulphonic acid (Fisons, Loughborough) as ion-pair reagent. The flow-rate was 1.0 ml/min. The eluant from the column was passed through an LC-5A glassy carbon transducer cell (BAS) connected to an LC-4A amperometric detector (BAS). The working potential of the electrode was maintained at + 0.70 V, and the generated current was displayed on a Tarkan 600 chart recorder. Fresh standard solutions of NA, DA, 5-HT, 5-HIAA and D O P A C (Sigma U.K.) were prepared in 0.2 M perchloric acid. Standard curves were linear over a range of 20 pg to 2 ng per 20-t*l injection. Concentrations of amines in homogenates were calculated by peak-height ratio with respect to 400 pg per 20-t,1 injection of IS added to all samples and standards. The significance of differences between means was calculated using unpaired t-tests. Table I shows the amine and metabolite content of the SEM and NIL after drug treatment. Forty-eight hours after 6 - O H D A administration there was a significant decrease in the contents of both NA ( P < 0.005 and P < 0.02, SME and NIL, respectively) and DA (P<0.001 and P < 0 . 0 0 5 ) compared with the contents in untreated or vehicle-treated control groups. In animals treated with DMI 30 rain before
143 TABLE I CONTENT OF AMINES AND METABOLITES IN STALK MEDIAN EMINENCE (SME) AND NEUROINTERMEDIATE LOBE (NIL) 48 h AFTER DRUG TREATMENT Values=pg/area dissected (mean±S.E.M.). n, number of animals per treatment group; ND, not determined. Treatment group
n
NIL 48 h after injections untreated ascorbic acid 6-OHDA 6-OHDA+DMI
6 6 6 7
SME 48 h after injections untreated ascorbic acid 6-OHDA 6-OHDA+DM!
6 5 6 7
NA
229_+ 204_+ 124_+ 265_+
25 20 17 34
1161 _+141 913_+ 70 584_+ 38 1130_+206
DA
DOPAC
5-HT
5-HIAA
2558_+157 2407_+130 1479_+177 1457_+153
530+111 785_+ 100 ND ND
632_+246 385_+ 32 403_+ 83 420_+ 24
251_+56 248_+52 219_+51 314_+65
5600_+289 5042_+375 2397_+191 2314_+281
1860_+237 2088_+500 899_+ 89 1085_+ 90
483_+172 280_+ 58 369_+ 97 275_+ 28
356_+86 301_+50 235_+44 239+49
6 - O H D A a d m i n i s t r a t i o n there was also a significant d e p l e t i o n o f D A after 48 h ( P < 0.001, b o t h areas), but N A c o n t e n t r e m a i n e d similar to the values in the c o n t r o l a n i m a l s . D O P A C was significantly r e d u c e d in the S M E in b o t h 6 - O H D A - t r e a t e d a n d D M I + 6 - O H D A - t r e a t e d g r o u p s c o m p a r e d with the values in c o n t r o l s ( P < 0.05 t o P < 0 . 0 0 5 ) . 5 - H T a n d 5 - H I A A c o n t e n t s r e m a i n e d u n c h a n g e d in all t r e a t m e n t groups. These results p r o v i d e direct evidence that c o n f i r m s the p r e v i o u s fluorescence h i s t o c h e m i c a l findings o f a d e p l e t i o n o f N A a n d D A in the M E a n d N I L after the i n t r a v e n o u s a d m i n i s t r a t i o n o f 6 - O H D A . T h e m a g n i t u d e o f the d e p l e t i o n (-~ 50%), h o w e v e r , was m u c h less t h a n suggested b y p r e v i o u s findings with fluorescence h i s t o c h e m i s t r y [3], which s h o w e d a c o m p l e t e d e p l e t i o n o f c a t e c h o l a m i n e s in these t w o areas in the first two d a y s a f t e r 6 - O H D A t r e a t m e n t . T h e m o s t likely e x p l a n a t i o n for the d i s c r e p a n c y in the results is that in the present s t u d y the extent o f the lesion was u n d e r e s t i m a t e d b y the inclusion in the dissected areas o f regions in which c a t e c h o l a m i n e s a c c u m u l a t e d b e h i n d the lesioned nerve t e r m i n a l s [5]. Cuello et al. [1] s h o w e d a 37% r e d u c t i o n in the h y p o t h a l a m i c c o n t e n t o f N A but no change in D A c o n t e n t 24 b after systemic 6 - O H D A a d m i n i s t r a t i o n . H o w e v e r , their s t u d y differs in two respects f r o m the present investigation in which the a m i n e c o n t e n t has been m e a s u r e d in m o r e discrete areas a n d a higher dose o f 6 - O H D A was adm i n i s t e r e d ( N A t e r m i n a l s are m o r e susceptible t h a n D A t e r m i n a l s to the effects o f 6 - O H D A [5]). T h e c o m b i n e d t r e a t m e n t with 6 - O H D A a n d D M I has been used in a previous s t u d y to p r o v i d e evidence for the i n h i b i t i o n o f g o n a d o t r o p i n release by the t u b e r o i n -
144
fundibular DA system because this treatment enhanced the luteinizing hormone surge induced by administration of pregnant mare serum gonadotropin [2]. The present results provide direct confirmation of the fact that a selective lesion of D A neurons can be produced by administration o f DMI before the 6 - O H D A injection, and this model may be of further use in studying aminergic-peptidergic interactions in the hypothalamic control o f hormone release from the anterior pituitary gland. 1 Cuello, A.C., Shoemaker, W.J. and Ganong, W.F., Effect of 6-hydroxydopamine on hypothalamic norepinephrine and dopamine content, ultrastructure of the median eminence, and plasma corticosterone, Brain Res., 78 (1974) 57-69. 2 Sarkar, D.K., Smith, G.C. and Fink, G., Effect of manipulating central catecholamines on puberty and the surge of luteinizing hormone and gonadotropin releasing hormone induced by pregnant mare serum gonadotropin in female rats, Brain Res., 213 (1981) 335-349. 3 Smith, G.C., Courtney, P.G., Wreford, N.G.M. and Walker, M.McD., Further studies on the effects of intravenously administered 6-hydroxydopamine on the median eminence of the rat, Brain Res., 234 (1982) 101-110. 4 Smith, G.C., Sheward, W.J. and Fink, G., Effect of 6-hydroxydopamine lesions of the median eminence and neurointermediate lobe on the secretion of pituitary hormones in the male rat, Brain Res., 246 (1982) 330-333. 5 Ungerstedt, U., Histochemical studies on the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in the rat brain. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, North-Holland, Amsterdam, 1971, pp. 101-127.