- Email: [email protected]
Effect of nigral and raphé lesions on the catechol-O-methyl transferase and monoamine oxidase activities in the rat striatum
EUROPEAN JOURNAL OF PHARMACOLOGY 19 (1972) 35-42. NORTH-HOLLAND PUBLISHING COMPANY
EFFECT OF NIGRAL AND RAPHI~ LESIONS ON THE CATECHOL-O-METHYL TRANSFERASE AND MONOAMINE OXIDASE ACTIVITIES IN THE RAT STRIATUM C.A. MARSDEN*, O.J. BROCH, JR. and H.C. GULDBERG Department of Pharmacology, University of Bergen Medical School, Bergen, Norway
Received 29 September 1971
Accepted 7 March 1972
C.A. MARSDEN, 03. BROCH, Jr. and H.C. GULDBERG, Effect ofnigral and raphdlesions on cateehol-O-methyl transferase and monoamine oxidase activities in the rat striatum, European J. Pharmacol. 19 (1972) 35-42. Dopamine concentration and catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO) activities were determined in the rat striatum 21 days after lesions had been placed in the left substantia nigra or the left substantia nigra and the medial raphe" nucleus. The results were paired with the right striatum, acting as a control. 5-Hydroxytryptamine (5-HT) concentration was measured in the forebrain after the different lesions. Changes in COMT activity were also studied after reserpine (5 mg/kg) and ~-methyl-p-tyrosine (a-MPT) (300 mg/kg X 2). MAO activity was measured after e~-MPT. Dopamine concentration in the left striatum was reduced about 70~ compared with the right striatum after successful lesions in the substantia nigra. 5-HT concentration of the forebrain was unaffected by lesions in the substantia nigra but severely reduced after successful lesions in the medial raph~ nucleus. Neither COMT nor MAO activities were significantly altered by any of the lesions. COMT activity showed a small significant rise 18 and 24 hr after reserpine, but was unaffected at 6 and 36 hr. a-MPT had not significant effect on either enzyme. The results are compared with previously published findings from the peripheral sympathetic system. Dissimilarities may reflect differences between central and peripheral COMT and MAO or differences between the metabolism of striatal dopamine and peripheral noradrenaline. Catechol-O-methyl transt'erase Monoamine oxidase Dopamine 1. INTRODUCTION The high dopamine concentration in the rat striaturn is reduced by stereotaxic lesions in the substantia nigra (Anddn et al., 1966). It has been shown histochemically that dopamine cell bodies are found in the zona compacta of the substantia nigra with the terminal system in the nuc. caudatus putamen (see review Fuxe et al., 1969). Lesions placed in the medial raphg nucleus result in a decrease in 5-hydroxytryptamine (5-HT) in the striatum and forebrain without affecting noradrenaline or dopamine concentrations (Kostowski et al., 1968; Gumulka et al., 1970). There *
Present address: Department of Neurochemistry, Institute of Neurology, Queen Square, London WCI 3BG, England.
5-Hydroxytryptamine Medial raph~ nucleus
Striatum Substantia nigra
is histochemical evidence that most of the brain 5-HT cell bodies are found in the medial and dorsal raph~ nuclei and that 5-HT terminals are found in the globus pallidus (Fuxe et al., 1969). In the brain of the monkey, ventromedial tegmental lesions that result in a fall in striatal dopamine, also reduce the activities of tyrosine hydroxylase and dopa decarboxylase but are without effect on MAO activity (Goldstein et al., 1969). After intraventricular injection of 6-hydroxydopamine, there is a long lasting reduction in brain dopamine but there is no consistent change in either the activity of MAO or COMT (Uretsky and Iversen, 1970). COMT is thought to be primarily located at an extraneuronal site (Axelrod, 1966), though the fall in activity f o l -
36
CA. Marsden et al., COMT and MAO in rat striatum
lowing sympathetic denervation of the vas deferens suggests that some of the COMT in this organ may have an intraneuronal localization (Jarrott and lversen, 1971). In the submaxillary gland of the rat, there is similar evidence that part of the COMT may be intraneuronal, but the activity appears to be dependent on the presence of adequate substrate since reserpine and a-methyl-p-tyrosine (a-MPT) reduce the activity of the enzyme (Marsden et al., 1971). MAO is widely distributed in the brain and is involved in the intraneuronal deamination of amines. In this study, the effects of monoaminergic denervation of the striatum and substrate depletion by reserpine and a-MPT on the COMT and MAO activities were compared with results previously obtained from the salivary gland where the activities of both enzymes were significantly reduced following postganglionic sympathectomy and substrate depletion (Marsden et at., 1971). The results are discussed so as to contrast some properties of the two enzymes in the peripheral and central nervous system.
2. MATERIALS AND METHODS
activities and dopamine concentration were determined in both striata. In rats with lesions in the substantia nigra and the medial raphd nucleus and, also in some rats with lesions only in the substantia nigra, the 5-HT concentrations were determined in both ipsi- and contralateral forebrain. The remainder ot the brain was frozen in solid carbon dioxide, sectioned coronally on a cryostat and the sections stained for the histochemical localization of acetylcholinesterase to verify the placing of the lesion (Storm-Mathisen, 1970).
2.2. Drug studies The following drugs were given to non-lesioned animals: reserpine (serpasil C1BA) in a single dose of 5 mg/kg s.c. and the animals killed 6, 18, 24 and 36 hr after administration; a-methyl-p-tyrosine methyl ester hydrochloride (a-MPT) (Kistner Labtj~inst AB), 300 mg/kg, i.p., twice at 12 hr intervals and the animals killed after 24hr. The a-MPT was dissolved in 0.9% NaCl. Drug-treated and control rats were kept at an environmental temperature of 30 ° . The other experimental rats were kept at a room temperature of 20 °.
2.1. Lesions
Male Wistar rats of between 250 300 g were used in all experiments. The animals were anaesthetized with sodium pentobarbital, 50 mg/kg, i.p., and placed in a David Kopf stereotaxic frame. Unilateral electrolytic lesions were made in the left substantia nigra with a teflon coated steel electrode of tip diameter 0.2 mm using a 2.0 mA anodal current for 20 sec. The parameters were: frontal 2.4mm, sagittal 1.4 mm and horizontal 2.6 ram. In one group of rats a lesion was placed in the substantia nigra and another in the medial raphe~ nucleus of the same rat. The parameters for the raphg lesions were frontal 0.5 ram, sagittal O.0mm and horizontal 2.5 mm (K6nig and Klippel, 1963). Sham operations were performed with the same frontal and sagittal parameters but with a horizontal parameter of 0.5 ram; no current was passed. After the operations, the rats were housed in individual cages and given food and water ad libitum. 21 days after the lesions had been made the rats were killed and the corpora striata from both sides removed and placed separately on ice. COMT and MAO
2.3. Dopamine and 5-HT assays
Striata were homogenised individually in 0.1 M phosphate buffer using a mechanical glass homogeniser. A constant volume of the homogenate was removed for the enzyme assays, then the final concentration of the original homogenate was adjusted to 0.4 N with respect to perchloric acid and the dopamine in the homogenate determined fluorometrically after acetylation and separation by paper chromatography (Crawford and Yates, 1970). The 5-HT was determined in the right and left sides of individual forebrains using the method of Ahtee et at. (1970). The final dopamine and 5-HT values were not corrected for the recoveries obtained of 87-+ 24% and 35 -+ 8% (S.D.) respectively. 2.4. E n z y m e assays For the COMT assay, a radioactive method based on that of McCaman (1965) was employed. A 0.4 ml sample of the phosphate buffer homogenate was used in the reaction mixture, which was 1 ml containing 80mM KH2PO4/Na2HPO4 buffer, pH 7.8, 1.2raM
CA. Marsden et al., COMT and MAO in rat striatum
3,4-dihydroxyphenylacetic acid, 5 mM MgC12, 150 mg/1 S-adenosyl methionine chloride (SAMeC1) (0.07 mM if pure) and 1/~1 3H.SAMe 500 Ci/mol, 0.5 mCi/ml. The mixture was incubated for 1 hr and the reaction stopped with one drop of concentrated HC1. After centrifugation, the supernatant was decanted and the radioactive product extracted twice with 2 ml ethyl acetate. The ethyl acetate was washed with 2 ml 0.1 N HC1 and then 2 ml was transferred to a scintillation vial to which 10 ml of scintillation fluid was added (2 parts toluene containing 0.5% PPO and 0.015% POPOP and one part of Triton-X-100) and the activity counted in a Tri-Carb scintillation spectrometer. The activity was obtained from some parallel calibration assays using a chemical method (Broch and Guldberg, 1971). In the study of the effect of reserpine and a-MPT on COMT activity, 0.4 ml of phosphate buffer homogenate was mixed with 0.02 ml of 10% Triton and this was then incubated with the reaction mixture as described. A sample not treated with Triton was always run through the procedure at the same time. MAO was determined according to Snyder and Hendly (1968). This method is based upon the coupling of H202, synthesized from the oxidative deamination, to homovanillic acid by means of peroxidase to make a fluorophore. All solutions were made up in 0.1 M KH2PO4/Na2 HPO4 buffer, pH 7.8: Tissue homogenate 5 mg/ml, peroxidase (Sigma horse radish) 0.05 mg/ml, tyramine hydrochloride (Sigma) 0.4 mM and homovanillic acid (Fluka) 1.6 raM. The standard H202 was made by diluting a 0.03% stock solution 1:200 (the exact molarity of the standard was determined by titration). After 10 min preincubation of 1 ml homogenate with 1 ml peroxidase, 1 ml homovanillic acid and 1 ml of tyramine solution (or 1 ml standard) were added, and the mixture was incubated for 30rain at 37 ° . The reaction was stopped by immersion in ice and water. The tubes were centrifuged at 1000 g for 20 min at 2 °, and the fluorescence determined in the supernatant at 320 nm excitation and 425 nm emission. 2.5. Calculations The results of the dopamine and 5-HT values and the enzyme activities are expressed as the mean + standard error of the mean (S.E.M.). Lord's test for the paired and Student's t test for the unpaired
37
results were used to calculate the significance. In all cases p<0.05 was considered significant.
3. RESULTS 3.1. Histology The site of the lesion was verified in each brain by histological examination. In 73% of the animals, the lesion was in the area of the substantia nigra. The greatest reduction in striatal dopamine (see later) was found in rats that had lesions in the dorso-medial area of the substantia nigra, this often included damage to the crus cerebi (fig. 1). The lesions in the medial raph6 nucleus were mainly confined to that area but there tended to be some lateral extension into the reticular formation (fig. 2).
3,2. Effect o f lesions in the substantia nigra on striatal dopamine There was a marked reduction in the dopamine concentration in the striatum ipsilateral to the lesion in the substantia nigra when compared with the contralateral striatum (tables 1 and 2). A similar reduction was found in the ipsilateral striatum of rats with a lesion in the left substantia nigra and another lesion in the medial raphct nucleus (table 3). 3.3. Effect o f lesions in the substantia nigra and the medial raphd nucleus on forebrain 5-HT There was no significant change in the concentra: tion of 5-HT in the ipsilateral forebrain after a unilateral lesion in the substantia nigra (table 4). Rats with lesions in the substantia nigra and the medial raph(nucleus showed marked reductions in the forebrain concentrations compared to sham operated animals (table 4). In this case there was no difference in the values obtained from the forebrain sides ipsi- and contralateral to the nigral lesion. The fall in forebrain 5-HT after a lesion in the medial raphe'nucleus probably reflects a similar fall in striatal 5-HT since Gumulka et al. (1970) showed that lesions in the medial raphe" nucleus produced a reduction in 5-HT in the striatum. 3.4. E f f e c t o f lesions on the COMT and MAO activities in the striatum Neither COMT nor MAO activities were signifi-
38
C A . Marsden et al., COMT and M A O in rat striatum
Fig. 1. Photomicrograph showing the site of a lesion in a substantia nigra of the rat (~). The substantia nigra (SN on the opposit~ side is unaffected (× 14).
Table 1 The effects of lesions in the left substantia nigra on the dopamine concentration and COMT activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the m e a n -+ S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (~ug/g)
Control (right) 4.30-+0.58 (8)* Lesioned (left) 1.44-+0.19 (8)**
'~ Change
COMT activity (nmol/g/hr)
Table 2 The effect of lesions in the left substantia nigra on the dopamine concentration and the MAO activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the m e a n _+ S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (ug/g)
% Change
MAO % activity Change (umol/g/hr)
--
74.4+_4.5 (8)
Control (right)
4.36-+0.29 (13)*
-
8.18-+0.65 (13)
-
-66
78.1-+7.3 (8)
Lesioned (left)
1.45+_0.19 (13)**
- 67
7.53-+0.6 (13)
- 8
* The figures in brackets refer to n u m b e r of experiments. ** Statistically significant (p < 0.001)
* The figures in brackets refer to the n u m b e r of experiments. ** Statistically significant (p < 0.001).
CA. Marsden et aL, COMT and MAO in rat striatum
39
Fig. 2. Photomicrograph showing the site of a lesion in the area of the medial raph6 nucleus of the rat (*--).There is some lateral and dorsal extension of the damage (× 14).
cantly altered in striata ipsilateral to lesions in the substantia nigra when compared with the contralateral side (tables 1 and 2). The MAO activity was also unaffected in rats with a lesion in the medial raph6 nucleus and the substantia nigra.
3.5. Effects o f reserpine and a-MPT on the COMT and M A O activities in the striatum 6 hr after reserpine administration COMT activity was unaffected b u t after 24 hr there was a small b u t significant increase; values were normal at 3 6 h r
40
C.A. Marsden et al., COMT and MA 0 in rat striatum
Table 3 The effect of lesions in both the left substantia nigra and the medial raphe nucleus on the dopamine concentration and the MAO activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the mean ± S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (tag/g)
% Change
MAO % activity Change (tamol/g/hr)
Control (right)
4.11±0.37 (5)*
-
9.04±0.23 (5)
-
Lesioned (left)
0.82_+0.09 (5)**
- 80
8.66±0.46 (5)
- 4
* The figures in brackets refer to the n u m b e r of experiments. ** Statistically significant (p < 0.001).
Table 4 The 5-HT concentration in the forebrain of rats with lesions in both the left substantia nigra and the medial raph~ nucleus. Each value represents the mean ± S.E.M. The differences between the sham operated and lesioned rats were analysed by Student's t test. Treatment
No. exp.
5-HT(ug/g)
(/~ Change
Sham operated
11
0.29±0.03
-
Lesion in substantia nigra only
6
0.27±0.04
-7
Lesion in substantia nigra and medial raph~ nucleus
5
0.12±0.01"
--58
* Statistically significant (p < 0.001)
Table 5 The effect of reserpine, 5 mg/kg, s.c., on the COMT activity and the dopamine concentration in the striatum of the rat 6, 18, 24 and 36 hr after adminsitration.
* ** a b
Treatment
Dopamine (tag/g)
Control Reserpine 6 hr Reserpine 18 hr Reserpine 24 hr Reserpine 36 hr
3.88 0.43 0.10 0.15 0.29
± ± ± ± ±
0.19 0.18 0.02 0.06 0.06
(4) b (4)** (4)** (4)** (4)**
COMT activity no triton (nmol/g/hr)
COMT activity triton a (nmol/g/hr)
80 72 84 91 -
158 149 180 180 163
± 1.2 +- 4.1 ± 2.4 ± 2.5
(8) (4) (4) (4)*
± 4.3 ± 5.3 ± 5.3 ± 5.8 -+ 1.9
(8) (4) (4)* (8)* (8)
Statistically significant (p < 0.05). Statistically significant (p < 0.001). The phosphate buffer h o m o g e n a t e was m i x e d with 0.02 ml of 10% Triton before incubation with the COMT mixture. Figures in brackets refer to the n u m b e r of experiments.
Table 6 The effect of a-MPT on the COMT and MAO activities and the dopamine concentration in the striatum of the rat 24 hr aftel the administration of the drug in two doses of 300 mg/kg at 12-hr intervals. Each value represents the mean +- S.E.M. The differences between the control and drug-treated animals analysed by Student's t test. Treatment
Dopaminc (tag/g)
COMT activity no triton (nmol/g/hr)
COMT activity triton a (nmol/g/hr)
MAO activity (umol/g/hr)
Control c~-MPT
3.53 ± 0.62 (3) b 0.28 -+ 0.02 (5)*
83 + 1.4 (4) 84 ± 2.2 (4)
177 + 8.0 (7) 192 ± 3.4 (9)
8.9 ± 0.53 (6) 9.4 +_ 0.41 (6)
* Statistically significant ( p < 0.001). a The phosphate buffer h o m o g e n a t e was m i x e d with 0.02 ml of 10% Triton before incubation with the COMT reaction mixture. b The figures in brackets refer to the n u m b e r of experiments.
CA. Marsden et aL, COMT and MAO in rat striatum
(table 5). Recently it has been shown that when brain homogenates are treated with Triton there is a two fold increase in COMT activity and it is suggested that Triton may act by releasing an occluded form of the enzyme (Broch and Fonnum, 1972). In order to show if the occluded form of COMT was affected by reserpine, homogenates were treated with Triton. This increased the COMT activity two fold but the relative activities 6, 18, 24 and 36 hr after reserpine were identical with those obtained in the absence of Triton (table 5). Dopamine concentration was reduced, following the administration of reserpine, at all the time intervals studied. Guldberg and Broch (1971) found a significant fall in MAO activity from 3 to 4 8 h r after reserpine, 5 mg/kg, s.c., with a return to normal values at 96 hr. a-MPT had no significant effect on COMT or MAO activities 24 hr after the first injection while the same experimental procedure produced a drop in the concentration of striatal dopamine of 90% (table 6). Triton treatment did not alter the relative COMT activities obtained after a-MPT administration.
4. DISCUSSION The decrease in striatal dopamine after successful lesions in the substantia nigra is in agreement with previous reports (And~n et al., 1966). Ventromedial tegmental lesions in the monkey produced falls in striatal dopamine, tyrosine hydroxylase and dopa decarboxylase but MAO was unchanged (Goldstein et al., 1969). In the present study, degeneration of the dopaminergic striatal nerve terminals produced no change in COMT or MAO activities. This is in contrast to results obtained after post-ganglionic sympathectomy of the submaxillary glands (Marsden et al., 1971) and the vas deferens (Jarrott and Iversen, 1971). In these cases both COMT and MAO activities were significantly reduced. There are various reasons why degeneration of striatal dopaminergic fibres and peripheral sympathetic fibres have different effects on the activities of MAO and COMT. Sympathectomy results in a total loss of the sympathetic innervation of the rat salivary gland (Marsden et al., 1971) while lesions in the substantia nigra produce about 70% decrease in striatal dopamine and thus probably a similar degree of re-
41
duction in the nerve terminals. It has been shown that the dopamine terminals in the striatum only constitute about 16% of the total terminal population (H~kfelt, 1968) and as MAO is a mitochondrial enzyme with a wide distribution in the brain these will contain only a small part of the total MAO in the striaturn. There is a considerable population of 5-HT terminals in the globus pallidus of the rat (Fuxe et al., 1969) and in unpublished work we have found that lesions in the medial raph6 nucleus reduce the 5-HT concentration in the striatum by 60-75%. When lesions were placed in both the left substantia nigra and the medial raphe" nucleus, resulting in degeneration of two monoaminergic pathways to the striatum, MAO activity remained unaffected. These results could indicate that MAO in the striatum is not confined to monoaminergic terminals but is also found in other types of terminals. Moreover MAO may be found in extraneuronal cells in the brain as has been shown to occur in the peripheral system (Snyder et al., 1965). It is probable that COMT has a wide distribution in the striatum since the enzyme is unaffected by degeneration of the dopaminergic terminals. It has been suggested that the transmitter, noradrenaline may have a role in determining the activities of MAO and COMT in the rat salivary gland (Marsden et al., 1971). In the striatum, reserpine caused a smaller reduction MAO activity (Guldberg and Broch, !971) than in the salivary gland (Marsden et al., 1971) and the fall in COMT activity in the salivary gland following reserpine could not be shown in the striatum. In fact there was a tendency for a rise in COMT activity 18 and 24 hr after reserpine administration. The differences between the effect of reserpine on COMT activity in salivary gland and striatum would appear not to be due to different binding of the enzyme in the two structures. Triton increased COMT activity in the striatum two fold, probably by releasing an occluded form of the enzyme, but did not affect COMT in the salivary glands. The results indicate that reserpine does not affect this occluded form. Finally a-MPT, which caused a pronounced fall in salivary gland COMT and a smaller fall in MAO activity (Marsden et al., 1971) lacked effect on either enzyme in the striatum. The results suggest that the properties of MAO and COMT in the striatum and salivary gland differ both in response to surgical destruction of monoaminergic
42
C A . Marsden et aL, COMT and MA 0 in rat striatum
nerves a n d m o n o a m i n e d e p l e t i o n b y drugs. F a c t o r s that There
might
explain
may
be
these
different
d i f f e r e n c e s include: forms
of
the
(a)
enzymes
( Y o u d i m et al., 1969; A x e l r o d a n d Vesell, 1 9 7 0 ) a n d w h i l e COMT in salivary gland is soluble ( M a r s d e n et al., 1 9 7 1 ) n o f u r t h e r increase is o b t a i n e d following t r e a t m e n t w i t h T r i t o n ; in the b r a i n only 51Y~ o f the activity can be m e a s u r e d w i t h o u t T r i t o n ( B r o c h a n d F o n n u m , 1972). ( b ) T h e r e m a y be d i f f e r e n c e s bet w e e n the m e t a b o l i s m o f d o p a m i n e in the s t r i a t u m and p e r i p h e r a l s y m p a t h e t i c n o r a d r e n a l i n e w h i c h m a y a f f e c t the activity o f the e n z y m e s .
ACKNOWLEDG EMENTS We thank Miss Randi SCraas, Miss Elf rid G~sdal and Mr. Halvard Bergesen for excellent technic',d assistance. The work was supported by research grants from the Norwegian Research Council for Science and the Humanities and from Norsk Medisinaldepot to whom we are grateful.
REFERENCES Ahtee, L., D.F. Sharman and M. Vogt, 1970, Acid metabolites of monoamines in avian brain, effects of probenecid and reserpine. Brit. J. Pharmac. 38, 7 2 - 8 5 . Ande'n. N.-E., A. DahlstrOm, K. Fuxe, L. Olson and U. Ungerstedt, 1966, Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Stand. 67, 313-326. Axelrod, J., 1966, Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines, Pharmacol. Rev. 18, 95-113. Axelrod, J. and E.S. Vesell, 1970, Heterogenity of N- and O-methyltransferases, Mol. Pharmacol. 6, 7 8 - 8 4 . Broch, O.J. Jr., and H.C. Guldberg, 1971, On the determination of catecbol-O-methyl transferase activity in tissue homogenates, Acta Pharmacol. Toxicol. 30,266 277. Broch, O.J. Jr., and F. Fonnunr, 1972. The regional and subcellular distribution of catechol-O-methyl transferase in the rat brain, J. Neurochem. (in press}. Crawford, T.B.B. and C.M. Yates, 1970, A method for the estimation of the catecholamines and their metabolites in brain tissue, Brit. J. Pharmacol. 38, 5 6 - 7 1 .
Fuxe, K., T. H~kfelt and U. Ungerstedt, 1969, Distribution of monoamines in the mammalian central nervous system by histochemical studies, in: Metabolism of Amines in the Brain, ed. G. Hooper (Macmillan, London) pp. 10-22. Goldstein, M., B. Anagnoste, A.t:. Battista, W.S. Owen and S. Nakatani, 1969, Studies of amines in the striatum in monkeys with nigral lesions. The disposition, biosynthesis and metabolites of (3H) Dopamine and (14C) Serotonin in the striatum, J. Neurochem. 16,645 653. Guldberg, H.C. and O.J. Broch, Jr., 1971, On the mode of action of reserpine on dopamine metabolism in the rat striatum. European J. Pharmacol. 13~ 155-167. Gumulka, W., A. Ramirez Del Angel, R. Samanin and L. Valzelli, 1970, Lesions of substantia nigra: Biochemical and behavioural effects in rats, European J. Pharmacol. 10, 7 9 - 8 2 . H~rkfelt, T., 1968, In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch. 91, 1- 74. Jarrott, B. and L.L. Iversen. 1971, Noradrenaline metabolizing enzymes in normal and sympathetically denervated vas deferens, J. Neurochem. 18, 1-6. K6nig, J.F.R. and R.A. Klippel, 1963, The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem (Williams and Wilkins, Baltimore). Kostowski, W., E. Giacalone, S. Garattini and L. Valzelli, 1968, Studies on behavioural and biochemical changes in rats after lesions of midbrain raph6, European J. Pharmacol. 4,371 376. McCaman, R.E., 1965, Microdetermination of catechol-Omethyl transferase, Life Sci. 4, 2353-2359. Marsden, C.A., O.J. Broch, Jr. and H.C. Guldberg, 1971, Catechol-O-methyl transferase and monoamine oxidase activities in rat submaxillary gland: Effects of ligation, sympathectomy and some drugs, European J. Pharmacol. 15,335 342. Snyder, S.H. and E.D. Henley, 1968, A simple and sensitive fluorescence assay for monoamine oxidase and diamine oxidase, J. Pharmacol. Exptl. Therap. 163,386 392. Snyder, S.H., J. Fischer and J. Axelrod, 1965, Evidence for the presence of monoamine oxidase in sympathetic nerve endings, Biochem. Pharmacol. 1 4 , 3 6 3 - 3 6 5 . Storm-Mathisen, J., 1970, Quantitative histochemistry of acetylcholine esterase in rat hippocampal region correlated to histochemical staining, J. Neurochem. 17, 739-750. Uretsky, N.J. and L i . lversen, 1970, Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neurochem. 17, 269 278. Youdim, M.B.H., G.G.S. Collins and M. Sandler, 1969, Multiple forms of rat brain monoamine oxidase, Nature 223, 626 628.
EFFECT OF NIGRAL AND RAPHI~ LESIONS ON THE CATECHOL-O-METHYL TRANSFERASE AND MONOAMINE OXIDASE ACTIVITIES IN THE RAT STRIATUM C.A. MARSDEN*, O.J. BROCH, JR. and H.C. GULDBERG Department of Pharmacology, University of Bergen Medical School, Bergen, Norway
Received 29 September 1971
Accepted 7 March 1972
C.A. MARSDEN, 03. BROCH, Jr. and H.C. GULDBERG, Effect ofnigral and raphdlesions on cateehol-O-methyl transferase and monoamine oxidase activities in the rat striatum, European J. Pharmacol. 19 (1972) 35-42. Dopamine concentration and catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO) activities were determined in the rat striatum 21 days after lesions had been placed in the left substantia nigra or the left substantia nigra and the medial raphe" nucleus. The results were paired with the right striatum, acting as a control. 5-Hydroxytryptamine (5-HT) concentration was measured in the forebrain after the different lesions. Changes in COMT activity were also studied after reserpine (5 mg/kg) and ~-methyl-p-tyrosine (a-MPT) (300 mg/kg X 2). MAO activity was measured after e~-MPT. Dopamine concentration in the left striatum was reduced about 70~ compared with the right striatum after successful lesions in the substantia nigra. 5-HT concentration of the forebrain was unaffected by lesions in the substantia nigra but severely reduced after successful lesions in the medial raph~ nucleus. Neither COMT nor MAO activities were significantly altered by any of the lesions. COMT activity showed a small significant rise 18 and 24 hr after reserpine, but was unaffected at 6 and 36 hr. a-MPT had not significant effect on either enzyme. The results are compared with previously published findings from the peripheral sympathetic system. Dissimilarities may reflect differences between central and peripheral COMT and MAO or differences between the metabolism of striatal dopamine and peripheral noradrenaline. Catechol-O-methyl transt'erase Monoamine oxidase Dopamine 1. INTRODUCTION The high dopamine concentration in the rat striaturn is reduced by stereotaxic lesions in the substantia nigra (Anddn et al., 1966). It has been shown histochemically that dopamine cell bodies are found in the zona compacta of the substantia nigra with the terminal system in the nuc. caudatus putamen (see review Fuxe et al., 1969). Lesions placed in the medial raphg nucleus result in a decrease in 5-hydroxytryptamine (5-HT) in the striatum and forebrain without affecting noradrenaline or dopamine concentrations (Kostowski et al., 1968; Gumulka et al., 1970). There *
Present address: Department of Neurochemistry, Institute of Neurology, Queen Square, London WCI 3BG, England.
5-Hydroxytryptamine Medial raph~ nucleus
Striatum Substantia nigra
is histochemical evidence that most of the brain 5-HT cell bodies are found in the medial and dorsal raph~ nuclei and that 5-HT terminals are found in the globus pallidus (Fuxe et al., 1969). In the brain of the monkey, ventromedial tegmental lesions that result in a fall in striatal dopamine, also reduce the activities of tyrosine hydroxylase and dopa decarboxylase but are without effect on MAO activity (Goldstein et al., 1969). After intraventricular injection of 6-hydroxydopamine, there is a long lasting reduction in brain dopamine but there is no consistent change in either the activity of MAO or COMT (Uretsky and Iversen, 1970). COMT is thought to be primarily located at an extraneuronal site (Axelrod, 1966), though the fall in activity f o l -
36
CA. Marsden et al., COMT and MAO in rat striatum
lowing sympathetic denervation of the vas deferens suggests that some of the COMT in this organ may have an intraneuronal localization (Jarrott and lversen, 1971). In the submaxillary gland of the rat, there is similar evidence that part of the COMT may be intraneuronal, but the activity appears to be dependent on the presence of adequate substrate since reserpine and a-methyl-p-tyrosine (a-MPT) reduce the activity of the enzyme (Marsden et al., 1971). MAO is widely distributed in the brain and is involved in the intraneuronal deamination of amines. In this study, the effects of monoaminergic denervation of the striatum and substrate depletion by reserpine and a-MPT on the COMT and MAO activities were compared with results previously obtained from the salivary gland where the activities of both enzymes were significantly reduced following postganglionic sympathectomy and substrate depletion (Marsden et at., 1971). The results are discussed so as to contrast some properties of the two enzymes in the peripheral and central nervous system.
2. MATERIALS AND METHODS
activities and dopamine concentration were determined in both striata. In rats with lesions in the substantia nigra and the medial raphd nucleus and, also in some rats with lesions only in the substantia nigra, the 5-HT concentrations were determined in both ipsi- and contralateral forebrain. The remainder ot the brain was frozen in solid carbon dioxide, sectioned coronally on a cryostat and the sections stained for the histochemical localization of acetylcholinesterase to verify the placing of the lesion (Storm-Mathisen, 1970).
2.2. Drug studies The following drugs were given to non-lesioned animals: reserpine (serpasil C1BA) in a single dose of 5 mg/kg s.c. and the animals killed 6, 18, 24 and 36 hr after administration; a-methyl-p-tyrosine methyl ester hydrochloride (a-MPT) (Kistner Labtj~inst AB), 300 mg/kg, i.p., twice at 12 hr intervals and the animals killed after 24hr. The a-MPT was dissolved in 0.9% NaCl. Drug-treated and control rats were kept at an environmental temperature of 30 ° . The other experimental rats were kept at a room temperature of 20 °.
2.1. Lesions
Male Wistar rats of between 250 300 g were used in all experiments. The animals were anaesthetized with sodium pentobarbital, 50 mg/kg, i.p., and placed in a David Kopf stereotaxic frame. Unilateral electrolytic lesions were made in the left substantia nigra with a teflon coated steel electrode of tip diameter 0.2 mm using a 2.0 mA anodal current for 20 sec. The parameters were: frontal 2.4mm, sagittal 1.4 mm and horizontal 2.6 ram. In one group of rats a lesion was placed in the substantia nigra and another in the medial raphe~ nucleus of the same rat. The parameters for the raphg lesions were frontal 0.5 ram, sagittal O.0mm and horizontal 2.5 mm (K6nig and Klippel, 1963). Sham operations were performed with the same frontal and sagittal parameters but with a horizontal parameter of 0.5 ram; no current was passed. After the operations, the rats were housed in individual cages and given food and water ad libitum. 21 days after the lesions had been made the rats were killed and the corpora striata from both sides removed and placed separately on ice. COMT and MAO
2.3. Dopamine and 5-HT assays
Striata were homogenised individually in 0.1 M phosphate buffer using a mechanical glass homogeniser. A constant volume of the homogenate was removed for the enzyme assays, then the final concentration of the original homogenate was adjusted to 0.4 N with respect to perchloric acid and the dopamine in the homogenate determined fluorometrically after acetylation and separation by paper chromatography (Crawford and Yates, 1970). The 5-HT was determined in the right and left sides of individual forebrains using the method of Ahtee et at. (1970). The final dopamine and 5-HT values were not corrected for the recoveries obtained of 87-+ 24% and 35 -+ 8% (S.D.) respectively. 2.4. E n z y m e assays For the COMT assay, a radioactive method based on that of McCaman (1965) was employed. A 0.4 ml sample of the phosphate buffer homogenate was used in the reaction mixture, which was 1 ml containing 80mM KH2PO4/Na2HPO4 buffer, pH 7.8, 1.2raM
CA. Marsden et al., COMT and MAO in rat striatum
3,4-dihydroxyphenylacetic acid, 5 mM MgC12, 150 mg/1 S-adenosyl methionine chloride (SAMeC1) (0.07 mM if pure) and 1/~1 3H.SAMe 500 Ci/mol, 0.5 mCi/ml. The mixture was incubated for 1 hr and the reaction stopped with one drop of concentrated HC1. After centrifugation, the supernatant was decanted and the radioactive product extracted twice with 2 ml ethyl acetate. The ethyl acetate was washed with 2 ml 0.1 N HC1 and then 2 ml was transferred to a scintillation vial to which 10 ml of scintillation fluid was added (2 parts toluene containing 0.5% PPO and 0.015% POPOP and one part of Triton-X-100) and the activity counted in a Tri-Carb scintillation spectrometer. The activity was obtained from some parallel calibration assays using a chemical method (Broch and Guldberg, 1971). In the study of the effect of reserpine and a-MPT on COMT activity, 0.4 ml of phosphate buffer homogenate was mixed with 0.02 ml of 10% Triton and this was then incubated with the reaction mixture as described. A sample not treated with Triton was always run through the procedure at the same time. MAO was determined according to Snyder and Hendly (1968). This method is based upon the coupling of H202, synthesized from the oxidative deamination, to homovanillic acid by means of peroxidase to make a fluorophore. All solutions were made up in 0.1 M KH2PO4/Na2 HPO4 buffer, pH 7.8: Tissue homogenate 5 mg/ml, peroxidase (Sigma horse radish) 0.05 mg/ml, tyramine hydrochloride (Sigma) 0.4 mM and homovanillic acid (Fluka) 1.6 raM. The standard H202 was made by diluting a 0.03% stock solution 1:200 (the exact molarity of the standard was determined by titration). After 10 min preincubation of 1 ml homogenate with 1 ml peroxidase, 1 ml homovanillic acid and 1 ml of tyramine solution (or 1 ml standard) were added, and the mixture was incubated for 30rain at 37 ° . The reaction was stopped by immersion in ice and water. The tubes were centrifuged at 1000 g for 20 min at 2 °, and the fluorescence determined in the supernatant at 320 nm excitation and 425 nm emission. 2.5. Calculations The results of the dopamine and 5-HT values and the enzyme activities are expressed as the mean + standard error of the mean (S.E.M.). Lord's test for the paired and Student's t test for the unpaired
37
results were used to calculate the significance. In all cases p<0.05 was considered significant.
3. RESULTS 3.1. Histology The site of the lesion was verified in each brain by histological examination. In 73% of the animals, the lesion was in the area of the substantia nigra. The greatest reduction in striatal dopamine (see later) was found in rats that had lesions in the dorso-medial area of the substantia nigra, this often included damage to the crus cerebi (fig. 1). The lesions in the medial raph6 nucleus were mainly confined to that area but there tended to be some lateral extension into the reticular formation (fig. 2).
3,2. Effect o f lesions in the substantia nigra on striatal dopamine There was a marked reduction in the dopamine concentration in the striatum ipsilateral to the lesion in the substantia nigra when compared with the contralateral striatum (tables 1 and 2). A similar reduction was found in the ipsilateral striatum of rats with a lesion in the left substantia nigra and another lesion in the medial raphct nucleus (table 3). 3.3. Effect o f lesions in the substantia nigra and the medial raphd nucleus on forebrain 5-HT There was no significant change in the concentra: tion of 5-HT in the ipsilateral forebrain after a unilateral lesion in the substantia nigra (table 4). Rats with lesions in the substantia nigra and the medial raph(nucleus showed marked reductions in the forebrain concentrations compared to sham operated animals (table 4). In this case there was no difference in the values obtained from the forebrain sides ipsi- and contralateral to the nigral lesion. The fall in forebrain 5-HT after a lesion in the medial raphe'nucleus probably reflects a similar fall in striatal 5-HT since Gumulka et al. (1970) showed that lesions in the medial raphe" nucleus produced a reduction in 5-HT in the striatum. 3.4. E f f e c t o f lesions on the COMT and MAO activities in the striatum Neither COMT nor MAO activities were signifi-
38
C A . Marsden et al., COMT and M A O in rat striatum
Fig. 1. Photomicrograph showing the site of a lesion in a substantia nigra of the rat (~). The substantia nigra (SN on the opposit~ side is unaffected (× 14).
Table 1 The effects of lesions in the left substantia nigra on the dopamine concentration and COMT activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the m e a n -+ S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (~ug/g)
Control (right) 4.30-+0.58 (8)* Lesioned (left) 1.44-+0.19 (8)**
'~ Change
COMT activity (nmol/g/hr)
Table 2 The effect of lesions in the left substantia nigra on the dopamine concentration and the MAO activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the m e a n _+ S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (ug/g)
% Change
MAO % activity Change (umol/g/hr)
--
74.4+_4.5 (8)
Control (right)
4.36-+0.29 (13)*
-
8.18-+0.65 (13)
-
-66
78.1-+7.3 (8)
Lesioned (left)
1.45+_0.19 (13)**
- 67
7.53-+0.6 (13)
- 8
* The figures in brackets refer to n u m b e r of experiments. ** Statistically significant (p < 0.001)
* The figures in brackets refer to the n u m b e r of experiments. ** Statistically significant (p < 0.001).
CA. Marsden et aL, COMT and MAO in rat striatum
39
Fig. 2. Photomicrograph showing the site of a lesion in the area of the medial raph6 nucleus of the rat (*--).There is some lateral and dorsal extension of the damage (× 14).
cantly altered in striata ipsilateral to lesions in the substantia nigra when compared with the contralateral side (tables 1 and 2). The MAO activity was also unaffected in rats with a lesion in the medial raph6 nucleus and the substantia nigra.
3.5. Effects o f reserpine and a-MPT on the COMT and M A O activities in the striatum 6 hr after reserpine administration COMT activity was unaffected b u t after 24 hr there was a small b u t significant increase; values were normal at 3 6 h r
40
C.A. Marsden et al., COMT and MA 0 in rat striatum
Table 3 The effect of lesions in both the left substantia nigra and the medial raphe nucleus on the dopamine concentration and the MAO activity in the left striatum. The results are paired with the right striatum acting as control. Each value represents the mean ± S.E.M. The differences between the striata on the lesioned and non-lesioned sides were analysed using Lord's test. Treatment
Dopamine (tag/g)
% Change
MAO % activity Change (tamol/g/hr)
Control (right)
4.11±0.37 (5)*
-
9.04±0.23 (5)
-
Lesioned (left)
0.82_+0.09 (5)**
- 80
8.66±0.46 (5)
- 4
* The figures in brackets refer to the n u m b e r of experiments. ** Statistically significant (p < 0.001).
Table 4 The 5-HT concentration in the forebrain of rats with lesions in both the left substantia nigra and the medial raph~ nucleus. Each value represents the mean ± S.E.M. The differences between the sham operated and lesioned rats were analysed by Student's t test. Treatment
No. exp.
5-HT(ug/g)
(/~ Change
Sham operated
11
0.29±0.03
-
Lesion in substantia nigra only
6
0.27±0.04
-7
Lesion in substantia nigra and medial raph~ nucleus
5
0.12±0.01"
--58
* Statistically significant (p < 0.001)
Table 5 The effect of reserpine, 5 mg/kg, s.c., on the COMT activity and the dopamine concentration in the striatum of the rat 6, 18, 24 and 36 hr after adminsitration.
* ** a b
Treatment
Dopamine (tag/g)
Control Reserpine 6 hr Reserpine 18 hr Reserpine 24 hr Reserpine 36 hr
3.88 0.43 0.10 0.15 0.29
± ± ± ± ±
0.19 0.18 0.02 0.06 0.06
(4) b (4)** (4)** (4)** (4)**
COMT activity no triton (nmol/g/hr)
COMT activity triton a (nmol/g/hr)
80 72 84 91 -
158 149 180 180 163
± 1.2 +- 4.1 ± 2.4 ± 2.5
(8) (4) (4) (4)*
± 4.3 ± 5.3 ± 5.3 ± 5.8 -+ 1.9
(8) (4) (4)* (8)* (8)
Statistically significant (p < 0.05). Statistically significant (p < 0.001). The phosphate buffer h o m o g e n a t e was m i x e d with 0.02 ml of 10% Triton before incubation with the COMT mixture. Figures in brackets refer to the n u m b e r of experiments.
Table 6 The effect of a-MPT on the COMT and MAO activities and the dopamine concentration in the striatum of the rat 24 hr aftel the administration of the drug in two doses of 300 mg/kg at 12-hr intervals. Each value represents the mean +- S.E.M. The differences between the control and drug-treated animals analysed by Student's t test. Treatment
Dopaminc (tag/g)
COMT activity no triton (nmol/g/hr)
COMT activity triton a (nmol/g/hr)
MAO activity (umol/g/hr)
Control c~-MPT
3.53 ± 0.62 (3) b 0.28 -+ 0.02 (5)*
83 + 1.4 (4) 84 ± 2.2 (4)
177 + 8.0 (7) 192 ± 3.4 (9)
8.9 ± 0.53 (6) 9.4 +_ 0.41 (6)
* Statistically significant ( p < 0.001). a The phosphate buffer h o m o g e n a t e was m i x e d with 0.02 ml of 10% Triton before incubation with the COMT reaction mixture. b The figures in brackets refer to the n u m b e r of experiments.
CA. Marsden et aL, COMT and MAO in rat striatum
(table 5). Recently it has been shown that when brain homogenates are treated with Triton there is a two fold increase in COMT activity and it is suggested that Triton may act by releasing an occluded form of the enzyme (Broch and Fonnum, 1972). In order to show if the occluded form of COMT was affected by reserpine, homogenates were treated with Triton. This increased the COMT activity two fold but the relative activities 6, 18, 24 and 36 hr after reserpine were identical with those obtained in the absence of Triton (table 5). Dopamine concentration was reduced, following the administration of reserpine, at all the time intervals studied. Guldberg and Broch (1971) found a significant fall in MAO activity from 3 to 4 8 h r after reserpine, 5 mg/kg, s.c., with a return to normal values at 96 hr. a-MPT had no significant effect on COMT or MAO activities 24 hr after the first injection while the same experimental procedure produced a drop in the concentration of striatal dopamine of 90% (table 6). Triton treatment did not alter the relative COMT activities obtained after a-MPT administration.
4. DISCUSSION The decrease in striatal dopamine after successful lesions in the substantia nigra is in agreement with previous reports (And~n et al., 1966). Ventromedial tegmental lesions in the monkey produced falls in striatal dopamine, tyrosine hydroxylase and dopa decarboxylase but MAO was unchanged (Goldstein et al., 1969). In the present study, degeneration of the dopaminergic striatal nerve terminals produced no change in COMT or MAO activities. This is in contrast to results obtained after post-ganglionic sympathectomy of the submaxillary glands (Marsden et al., 1971) and the vas deferens (Jarrott and Iversen, 1971). In these cases both COMT and MAO activities were significantly reduced. There are various reasons why degeneration of striatal dopaminergic fibres and peripheral sympathetic fibres have different effects on the activities of MAO and COMT. Sympathectomy results in a total loss of the sympathetic innervation of the rat salivary gland (Marsden et al., 1971) while lesions in the substantia nigra produce about 70% decrease in striatal dopamine and thus probably a similar degree of re-
41
duction in the nerve terminals. It has been shown that the dopamine terminals in the striatum only constitute about 16% of the total terminal population (H~kfelt, 1968) and as MAO is a mitochondrial enzyme with a wide distribution in the brain these will contain only a small part of the total MAO in the striaturn. There is a considerable population of 5-HT terminals in the globus pallidus of the rat (Fuxe et al., 1969) and in unpublished work we have found that lesions in the medial raph6 nucleus reduce the 5-HT concentration in the striatum by 60-75%. When lesions were placed in both the left substantia nigra and the medial raphe" nucleus, resulting in degeneration of two monoaminergic pathways to the striatum, MAO activity remained unaffected. These results could indicate that MAO in the striatum is not confined to monoaminergic terminals but is also found in other types of terminals. Moreover MAO may be found in extraneuronal cells in the brain as has been shown to occur in the peripheral system (Snyder et al., 1965). It is probable that COMT has a wide distribution in the striatum since the enzyme is unaffected by degeneration of the dopaminergic terminals. It has been suggested that the transmitter, noradrenaline may have a role in determining the activities of MAO and COMT in the rat salivary gland (Marsden et al., 1971). In the striatum, reserpine caused a smaller reduction MAO activity (Guldberg and Broch, !971) than in the salivary gland (Marsden et al., 1971) and the fall in COMT activity in the salivary gland following reserpine could not be shown in the striatum. In fact there was a tendency for a rise in COMT activity 18 and 24 hr after reserpine administration. The differences between the effect of reserpine on COMT activity in salivary gland and striatum would appear not to be due to different binding of the enzyme in the two structures. Triton increased COMT activity in the striatum two fold, probably by releasing an occluded form of the enzyme, but did not affect COMT in the salivary glands. The results indicate that reserpine does not affect this occluded form. Finally a-MPT, which caused a pronounced fall in salivary gland COMT and a smaller fall in MAO activity (Marsden et al., 1971) lacked effect on either enzyme in the striatum. The results suggest that the properties of MAO and COMT in the striatum and salivary gland differ both in response to surgical destruction of monoaminergic
42
C A . Marsden et aL, COMT and MA 0 in rat striatum
nerves a n d m o n o a m i n e d e p l e t i o n b y drugs. F a c t o r s that There
might
explain
may
be
these
different
d i f f e r e n c e s include: forms
of
the
(a)
enzymes
( Y o u d i m et al., 1969; A x e l r o d a n d Vesell, 1 9 7 0 ) a n d w h i l e COMT in salivary gland is soluble ( M a r s d e n et al., 1 9 7 1 ) n o f u r t h e r increase is o b t a i n e d following t r e a t m e n t w i t h T r i t o n ; in the b r a i n only 51Y~ o f the activity can be m e a s u r e d w i t h o u t T r i t o n ( B r o c h a n d F o n n u m , 1972). ( b ) T h e r e m a y be d i f f e r e n c e s bet w e e n the m e t a b o l i s m o f d o p a m i n e in the s t r i a t u m and p e r i p h e r a l s y m p a t h e t i c n o r a d r e n a l i n e w h i c h m a y a f f e c t the activity o f the e n z y m e s .
ACKNOWLEDG EMENTS We thank Miss Randi SCraas, Miss Elf rid G~sdal and Mr. Halvard Bergesen for excellent technic',d assistance. The work was supported by research grants from the Norwegian Research Council for Science and the Humanities and from Norsk Medisinaldepot to whom we are grateful.
REFERENCES Ahtee, L., D.F. Sharman and M. Vogt, 1970, Acid metabolites of monoamines in avian brain, effects of probenecid and reserpine. Brit. J. Pharmac. 38, 7 2 - 8 5 . Ande'n. N.-E., A. DahlstrOm, K. Fuxe, L. Olson and U. Ungerstedt, 1966, Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Stand. 67, 313-326. Axelrod, J., 1966, Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines, Pharmacol. Rev. 18, 95-113. Axelrod, J. and E.S. Vesell, 1970, Heterogenity of N- and O-methyltransferases, Mol. Pharmacol. 6, 7 8 - 8 4 . Broch, O.J. Jr., and H.C. Guldberg, 1971, On the determination of catecbol-O-methyl transferase activity in tissue homogenates, Acta Pharmacol. Toxicol. 30,266 277. Broch, O.J. Jr., and F. Fonnunr, 1972. The regional and subcellular distribution of catechol-O-methyl transferase in the rat brain, J. Neurochem. (in press}. Crawford, T.B.B. and C.M. Yates, 1970, A method for the estimation of the catecholamines and their metabolites in brain tissue, Brit. J. Pharmacol. 38, 5 6 - 7 1 .
Fuxe, K., T. H~kfelt and U. Ungerstedt, 1969, Distribution of monoamines in the mammalian central nervous system by histochemical studies, in: Metabolism of Amines in the Brain, ed. G. Hooper (Macmillan, London) pp. 10-22. Goldstein, M., B. Anagnoste, A.t:. Battista, W.S. Owen and S. Nakatani, 1969, Studies of amines in the striatum in monkeys with nigral lesions. The disposition, biosynthesis and metabolites of (3H) Dopamine and (14C) Serotonin in the striatum, J. Neurochem. 16,645 653. Guldberg, H.C. and O.J. Broch, Jr., 1971, On the mode of action of reserpine on dopamine metabolism in the rat striatum. European J. Pharmacol. 13~ 155-167. Gumulka, W., A. Ramirez Del Angel, R. Samanin and L. Valzelli, 1970, Lesions of substantia nigra: Biochemical and behavioural effects in rats, European J. Pharmacol. 10, 7 9 - 8 2 . H~rkfelt, T., 1968, In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch. 91, 1- 74. Jarrott, B. and L.L. Iversen. 1971, Noradrenaline metabolizing enzymes in normal and sympathetically denervated vas deferens, J. Neurochem. 18, 1-6. K6nig, J.F.R. and R.A. Klippel, 1963, The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem (Williams and Wilkins, Baltimore). Kostowski, W., E. Giacalone, S. Garattini and L. Valzelli, 1968, Studies on behavioural and biochemical changes in rats after lesions of midbrain raph6, European J. Pharmacol. 4,371 376. McCaman, R.E., 1965, Microdetermination of catechol-Omethyl transferase, Life Sci. 4, 2353-2359. Marsden, C.A., O.J. Broch, Jr. and H.C. Guldberg, 1971, Catechol-O-methyl transferase and monoamine oxidase activities in rat submaxillary gland: Effects of ligation, sympathectomy and some drugs, European J. Pharmacol. 15,335 342. Snyder, S.H. and E.D. Henley, 1968, A simple and sensitive fluorescence assay for monoamine oxidase and diamine oxidase, J. Pharmacol. Exptl. Therap. 163,386 392. Snyder, S.H., J. Fischer and J. Axelrod, 1965, Evidence for the presence of monoamine oxidase in sympathetic nerve endings, Biochem. Pharmacol. 1 4 , 3 6 3 - 3 6 5 . Storm-Mathisen, J., 1970, Quantitative histochemistry of acetylcholine esterase in rat hippocampal region correlated to histochemical staining, J. Neurochem. 17, 739-750. Uretsky, N.J. and L i . lversen, 1970, Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neurochem. 17, 269 278. Youdim, M.B.H., G.G.S. Collins and M. Sandler, 1969, Multiple forms of rat brain monoamine oxidase, Nature 223, 626 628.