- Email: [email protected]
Pharmacological evidence for a stimulation of dopamine neurons by noradrenaline neurons in the brain
European Journal of Pharmacology, 39 (1976) 275--282 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
275
P H A R M A C O L O G I C A L E V I D E N C E F O R A S T I M U L A T I O N O F D O P A M I N E N E U R O N S BY N O R A D R E N A L I N E N E U R O N S IN T H E B R A I N NILS-ERIK ANDI~N and MARIA GRABOWSKA Department of Pharmacology, University of GSteborg, Fack, S-400 33 GSteborg 33, Sweden
Received 20 January 1976, revised MS received 13 April 1976, accepted 10 June 1976
N.-E. ANDl~N and M. GRABOWSKA, Pharmacological evidence for a stimulation of dopamine neurons by noradrenaline neurons in the brain, European J. Pharmacol. 39 (1976) 275--282. The effects of yohimbine (3 mg/kg i.p.), phenoxybenzamine (20 mg/kg i.p.) and clonidine (0.1 mg/kg i.p.) on the synthesis and the utilization of dopamine and noradrenaline in the central nervous system of rats were investigated. Dopa accumulation following decarboxylase inhibition and the ~-methyltyrosine-induced disappearance of the amines were used as the measure of these effects. The synthesis and the utilization of dopamine and noradrenaline were accelerated by yohimbine. Clonidine plus phenoxybenzamine inhibited the synthesis and utilization of dopamine and the combination also partly antagonized the effects of yohimbine on the turnover of dopamine. The effects of the three drugs on the synthesis and utilization of dopamine might be secondary to their actions on ~-adrenoreceptors of noradrenaline synapses since, at the doses used, yohimbine increases the release of noradrenaline, phenoxybenzamine blocks postsynaptic noradrenaline receptors and clonidine reduces the release of noradrenaline. This hypothesis is supported by the findings that yohimbine and phenoxybenzamine did not change the increased synthesis of dopamine in reserpine-treated rats and that clonidine did not inhibit the increased synthesis of dopamine after axotomy or treatment with reserpine. Receptor activity Phenoxybenzamine
Indirect effect Noradrenaline
Utilization Synthesis
1. I n t r o d u c t i o n Central, p o s t s y n a p t i c ~ - a d r e n o r e c e p t o r s can be s t i m u l a t e d b y clonidine (And4n et al., 1974; A n d 4 n et al., 1976) and t h e y can be b l o c k e d b y p h e n o x y b e n z a m i n e as well as b y high doses o f y o h i m b i n e (And~n et al., 1963; And~n et al., 1967; A n d 4 n and S t r S m b o m , 1974; A n d 4 n et al., 1976), judging f r o m effects o n f l e x o r reflex activity and o n m o t o r activity. T h e t u r n o v e r o f n o r a d r e n a l i n e (NA) in the central nervous s y s t e m is decreased b y clonidine and increased b y p h e n o x y b e n z a mine and b y y o h i m b i n e , p r o b a b l y due t o their effects on ~ - a d r e n o r e c e p t o r s (And~n et al., 1 9 6 7 ; A n d 4 n et al., 1970; A n d 4 n et al., 1976). These drugs can also change d o p a m i n e (DA) t u r n o v e r : the disappearance o f DA in the brain following synthesis i n h i b i t i o n is t h u s d e c e l e r a t e d b y clonidine as well as b y phe-
Dopamine
Yohimbine
Clonidine
n o x y b e n z a m i n e (And~n et al., 1970; And~n and S t r S m b o m , 1974). F u r t h e r m o r e , clonidine decreases and y o h i m b i n e increases t h e a c c u m u l a t i o n o f D o p a in the DA-rich c o r p u s striatum following d e c a r b o x y l a s e inhibition, indicating effects on DA synthesis (And~n et al., 1976; S t r S m b o m , 1 9 7 6 ) (cf. R o c h e t t e and Bralet, 1 9 7 5 ; Papeschi and Theiss, 1975). These findings p r o m p t e d us t o use the abovem e n t i o n e d drugs t o investigate in m o r e detail the possible regulation o f the synthesis and utilization o f brain DA t h r o u g h changes in NA n e u r o t r a n s m i s s i o n . 2. Materials and m e t h o d s 2.1. M e t h o d s
Male S p r a g u e - - D a w l e y rats ( 1 6 0 - - 2 7 0 g) were used. T h e utilization o f DA in the brain
/
276 and of the NA in the brain and in the spinal cord was investigated by measuring the disappearance of the amines for 1 h following treatment with the tyrosine hydroxylase inhibitor a-methyltyrosine {a-MT; 250 mg/kg i.p. of the D,L-form of the methyl ester HC1) (Spector et al., 1965; Corrodi and Hanson, 1966; And6n et at., 1966a). The synthesis of DA and NA was followed by measuring Dopa accumulation in different parts of the forebrain for 30 rain following injection of the aromatic amino acid decarboxylase inhibitor 3-hydroxybenzylhydrazine HC1 (NSD 1015; 100 mg/kg i.p.) {Carlsson et al., 1972). In the hemisection experiments, the rats were anaesthesized with pentobarbital sodium (about 40 mg/kg i.p.) and complete and almost frontal section of the brain on one side at the level of the caudal hypothalamus was performed with a blunt-edged spatula (Hassler and Bak, 1969; And~n et al., 1972). The NSD 1015 was injected immediately after the hemisection. The animals were decapitated 30 min later. Except for the hemisection experiments, the rats were killed by t h o r a c o t o m y and exsanguination under light chloroform anaesthesia. In the a-MT experiments, the brains and the spinal cords from two rats were pooled. In the Dopa experiments, the brain was divided bilaterally as described above for hemisection. The parts from two sides were pooled. The brain in front of the cut was separated into the corpus striatum, the limbic system and the rest of the forebrain (i.e., neocortex + hippocampus + thalamus + hypothalamus) by incisions through the outer borders of the corpus striatum, in the frontal part of the optic chiasm and through the rhinal fissures. The corpus striatum has many DA b u t few NA nerve terminals whereas the reverse holds true for the rest of the forebrain (see Results). The limbic system was discarded since it is rich in both DA and NA (And6n, 1967). DA, NA and Dopa were determined spectrofluorimetrically after homogenization in 0.4 N perchloric acid, cation exchange chromatography and oxidation (Atack and Magnusson, 1970; Atack,
N.-E. ANDEN, M. GRABOWSKA 1973; Bertler et al., 1958; H~ggendal, 1963; Kehr et al., 1972).
2.2. Drugs The following drugs were used: D,L-amethyltyrosine methyl ester HC1 (a-MT, H 44/68, *H~ssle, MSlndal), yohimbine HC1 (Merck, Darmstadt), clonidine HC1 (*Boehringer Ingelheim, Stockholm), phenoxybenzamine HC1 (*Leo, Helsingborg), 3-hydroxybenzylhydrazine HC1 (NSD 1015; synthesized in this department by Dr. Per Martinson), reserpine (*CIBA, MSlndal). Phenoxybenzamine and reserpine were dissolved in a few drops of glacial acetic acid and made up to volume with 5.5% glucose. The other drugs were dissolved in 0.9% NaC1, sometimes with a few drops of N HC1 and heating. The doses refer to the salts indicated.
3. Results
3.1. a-Methyltyrosine-induced disappearance of dopamine and noradrenaline Brain DA and NA, and the NA in the spinal cord were decreased by 38, 22 and 32% respectively, 1 h after the administration of aMT (table 1). Yohimbine (3 mg/kg i.p.) accelerated the a-MT-induced disappearance of DA as well as of NA. Combined treatment with clonidine and phenoxybenzamine slowed the utilization of both DA and NA. This treatment partly antagonized the stimulation by yohimbine of the a-MT-induced disappearance of DA, b u t not of NA.
3.2. Synthesis o f dopamine and noradrenaline 3.2.1. Normal animals In the group treated with only NSD 1015 and shown in table 2, the concentrations of DA and NA were 5.45 and 0.18 pg/g in the corpus striatum, whereas they were 0.05 and 0.40 t~g/g in the rest of the forebrain. The accumulation of Dopa in the corpus striatum
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
277
TABLE 1 Effects o f y o h i m b i n e (3 m g / k g i.p., 75 rain b e f o r e sacrifice), c l o n i d i n e (0.1 m g l k g i.p., 105 m i n b e f o r e sacrifice) a n d p h e n o x y b e n z a m i n e ( 2 0 m g / k g i.p., 9 0 m i n b e f o r e sacrifice) o n t h e d i s a p p e a r a n c e o f d o p a m i n e in w h o l e b r a i n a n d o f n o r a d r e n a l i n e in w h o l e b r a i n a n d in spinal c o r d following t r e a t m e n t o f rats w i t h D , L - a - m e t h y l t y r o s i n e m e t h y l e s t e r (a-MT, 250 m g / k g i.p., 60 rain). T h e values (/1g/g) are m e a n s ± S.E.M. w i t h t h e n u m b e r o f experim e n t s in p a r e n t h e s e s . Statistical significance was calculated w i t h S t u d e n t ' s t-test ***, **, * Treatment
Dopamine, whole brain
Noradrenaline, whole brain
Noradrenaline, spinal cord
No t r e a t m e n t ~-MT Y o h i m b i n e + ~-MT Clonidine + phenoxybenzamine + ~-MT Clonidine + phenoxybenzamine + y o h i m b i n e + ~-MT
0 . 5 5 8 ± 0 . 0 1 2 9 (6) a 0 . 3 4 8 -+ 0 . 0 0 9 5 (6) 0 . 2 8 9 ± 0 . 0 1 2 0 (6) d
0.330_+ 0 . 0 1 6 8 (6) a 0 . 2 6 2 ± 0 . 0 0 7 0 (6) 0 . 1 6 8 ± 0 . 0 0 4 3 (6) a
0 . 3 4 2 ± 0 . 0 1 9 6 (6) a 0 . 2 4 1 ± 0 . 0 1 2 7 (6) 0 . 1 3 4 ± 0 . 0 0 9 0 (6) g
0 . 4 4 0 _+ 0 . 0 1 3 4 (6) a
0 . 3 1 8 ± 0 . 0 1 4 6 (6) d
0 . 2 9 6 ± 0 . 0 1 5 9 (6) g
0 . 3 5 8 ± 0 . 0 1 4 0 (6) e,f
0 . 1 5 6 ± 0 . 0 0 8 9 (6) c
0 . 1 3 7 ± 0 . 0 1 4 2 (6) c
*** p < 0 . 0 0 1 , as c o m p a r e d to a ~-MT, b y o h i m b i n e + ~-MT, c c l o n i d i n e + p h e n o x y b e n z a m i n e + ~-MT; ** p < 0.01, as c o m p a r e d t o d ~-MT, e y o h i m b i n e + ~-MT, f c l o n i d i n e + p h e n o x y b e n z a m i n e + 0~-MT; * p < 0 . 0 2 5 , as c o m p a r e d t o g ~-MT, h y o h i m b i n e + q-MT, i c l o n i d i n e + p h e n o x y b e n z a m i n e + ~-MT.
and in the rest of the forebrain should thus reflect the synthesis of DA and of NA respectively. Yohimbine (3 mg/kg) markedly accelerated the formation of Dopa caused by NSD 1015 both in the corpus striatum and in the rest of the forebrain (table 2). Phenoxybenzamine
(20 mg/kg) only insignificantly decreased both the normal accumulation of Dopa in the corpus striatum and its enhancement by yohimbine. The accumulation of Dopa in the rest of the forebrain was only slightly elevated by phenoxybenzamine. Combined treatment with clonidine and phenoxybenzamine re-
TABLE 2 Effects o f y o h i m b i n e (3 m g / k g i.p., 45 m i n b e f o r e sacrifice), p h e n o x y b e n z a m i n e ( 2 0 m g / k g i.p., 60 m i n b e f o r e sacrifice) a n d c l o n i d i n e (0.1 m g / k g i.p., 75 m i n b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m a n d in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) f o l l o w i n g treatm e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 1 0 0 m g / k g i.p., 30 m i n ) . T h e values (pg/g) are m e a n s ± S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical significance was c a l c u l a t e d with S t u d e n t ' s ttest *** ** * Treatment
Dopa, c o r p u s s t r i a t u m
Dopa, rest o f t h e f o r e b r a i n
NSD Y o h i m b i n e + NSD P h e n o x y b e n z a m i n e + NSD P h e n o x y b e n z a m i n e + y o h i m b i n e + NSD C l o n i d i n e + p h e n o x y b e n z a m i n e + NSD Clonidine + phenoxybenzamine + yohimbine + NSD
1.04 2.08 0.82 2.01 0.72
0.096 0.162 0.124 0.171 0.066
+- 0 . 0 6 3 ± 0.175 ± 0.099 ± 0.260 ± 0.083
(13) (13) a (7) (7) g (6) e
1.38 ± 0 . 0 3 2 (6) k,d
+- 0 . 0 0 5 4 -+ 0 . 0 0 8 4 ± 0.0101 ± 0.0135 ± 0.0100
(12) (13) a (7) i (7) 1 (6) i
0 . 1 3 9 ± 0 . 0 0 9 2 (6) d
*** p < 0 . 0 0 1 , as c o m p a r e d t o a NSD, b y o h i m b i n e + NSD, c p h e n o x y b e n z a m i n e + NSD, d c l o n i d i n e + p h e n o x y b e n z a m i n e + NSD; ** p < 0.01, as c o m p a r e d t o e NSD, f y o h i m b i n e + NSD, g p h e n o x y b e n z a m i n e + NSD, h clonidine + p h e n o x y b e n z a m i n e + NSD; * p < 0 . 0 2 5 , as c o m p a r e d t o i NSD, k y o h i m b i n e + NSD, 1 p h e n o x y b e n z a m i n e + NSD, rn c l o n i d i n e + p h e n o x y b e n z a m i n e + NSD.
278
N.-E. ANDEN, M. G R A B O W S K A
TABLE 3 E f f e c t s o f d i f f e r e n t doses o f c l o n i d i n e (mg/kg i.p., 60 min b e f o r e sacrifice) and y o h i m b i n e (mg/kg i.p., 75 rain b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m and in t h e rest o f the f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) following t r e a t m e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 100 m g / k g i.p., 30 min). The values (pg/g) are m e a n s +_ S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in parentheses. Statistical significance was calculated w i t h S t u d e n t ' s t-test * * * , **, * Treatment
Dopa, c o r p u s s t r i a t u m
Dopa, rest o f the f o r e b r a i n
NSD C l o n i d i n e (0.1) + NSD C l o n i d i n e (0.3) + NSD Clonidine (1.0) + NSD Clonidine (3.0) + NSD Y o h i m b i n e (3) + NSD Y o h i m b i n e (3) + c l o n i d i n e (1.0) + NSD Y o h i m b i n e (10) + NSD Y o h i m b i n e (10) + c l o n i d i n e (1.0) + NSD
1.12 0.80 0.75 0.74 0.86 2.20 1.89 2.05 2.32
0.081 0.057 0.068 0,060 0.070 0.138 0.081 0.192 0.120
÷ ± t ± ~ + + ÷ ±
0.066 0.111 0.074 0.098 0.038 0.089 0.338 0.159 0.195
(18) (6) i (6) e (7) e (6) (12) a (4) f (4) a (4) b
~ 0.0041 t 0.0093 ± 0.0069 +- 0.0031 ± 0.0072 ÷ 0.0077 ± 0.0109 ÷ 0.0210 ~: 0.0167
(18) (5) i (5) (6) e (6) (12) a (4) g (4) a (4) f,m
* * * p < 0.001, as c o m p a r e d t o a NSD, b c l o n i d i n e (1.0) + NSD, c y o h i m b i n e (3) + NSD, d y o h i m b i n e (10) + N S D ; ** p < 0.01, as c o m p a r e d t o e NSD, f c l o n i d i n e (1.0) + NSD, g y o h i m b i n e (3) + NSD, h y o h i m b i n e (10) + NSD; * p < 0.05, as c o m p a r e d t o i NSD, k clonidine (1.0) + NSD, ! y o h i m b i n e (3) + NSD, m y o h i m b i n e (10) + NSD.
duced the normal Dopa accumulation in the corpus striatum as well as that after yohimbine (table 2). Clonidine inhibited Dopa accumulation both in the corpus striatum and in the rest of the forebrain (table 3). The effect did not appear to be appreciably stronger with doses higher than 0.1 mg/kg. The inhibition of the Dopa accumulation produced by clonidine
(1.0 mg/kg) in the corpus striatum was partly antagonized by yohimbine 3 mg/kg and completely by 10 mg/kg. 3.2.2. Reserpine-treated animals Reserpine enhanced the accumulation of DolJa after NSD 1015 both in corpus striatum and in the rest of the forebrain (table 4; cf. tables 2 and 3). Yohimbine (3 m g / k g ) a n d
TABLE 4 E f f e c t s o f y o h i m b i n e (3 m g / k g i.p., 45 rain b e f o r e sacrifice), p h e n o x y b e n z a m i n e (20 mg/kg i.p., 60 min before sacrifice) and c l o n i d i n e (0.1 m g / k g i.p., 75 min b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e corpus striat u m and in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) following t r e a t m e n t o f rats w i t h reserpine (R, 5 m g / k g i.p., 4 h b e f o r e sacrifice) and 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1015, 100 mg/kg, i.p., 30 min). The values (pg/g) are m e a n s ± S.E.M. w i t h the n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical significance was calculated w i t h S t u d e n t ' s t-test * Treatment
Dopa, corpus striatum
D o p a , rest o f t h e f o r e b r a i n
R R R R
3.41 2.94 3.57 3.68
0.165 0.160 0.173 0.101
+ + + +
NSD y o h i m b i n e + NSD p h e n o x y b e n z a m i n e + NSD c l o n i d i n e + NSD
• p < 0.025, as c o m p a r e d t o R + NSD.
+ 0.621 ± 0.182 +- 0.279 +- 0.380
(7) (7) (7) (7)
± 0.0238 ± 0.0114 +- 0.0111 -+ 0.0070
(7) (7) (7) (7) *
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
279
TABLE 5 Effects o f c l o n i d i n e (0.1 m g ] k g i.p., 6 0 m i n b e f o r e sacrifice ) a n d unilateral a x o t o m y ( h e m i s e c t i o n , 30 m i n b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m a n d in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) f o l l o w i n g t r e a t m e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 100 m g / k g i.p., 30 min). T h e values (pg/g) are m e a n s _+ S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical s i g n i f i c a n c e w a s calculated w i t h S t u d e n t ' s t-test ***, **, * Drug t r e a t m e n t
NSD C l o n i d i n e + NSD
Dopa, c o r p u s s t r i a t u m
Dopa, rest of t h e f o r b r a i n
I n t a c t side
S e c t i o n e d side
I n t a c t side
S e c t i o n e d side
1.47 +0.085 (8) 1.04 _+ 0 . 1 2 2 (8) e
4.61 ~ 0.237 (8) b 4.41 ± 0.271 (8) b
0.110 ± 0 . 0 0 8 0 (8) 0 . 0 7 6 -+ 0 . 0 0 6 3 (8) c
0 . 1 8 0 +0.0156 (8) d 0.148 ± 0 . 0 1 2 8 (8) b
*** p < 0 . 0 0 1 , as c o m p a r e d to a NSD a n d b i n t a c t side; ** p < 0.01, as c o m p a r e d to c NSD a n d d i n t a c t side; * p < 0.05, as c o m p a r e d to e NSD a n d f i n t a c t side.
phenoxybenzamine (20 mg/kg) did n o t alter the increase in Dopa accumulation in these animals. Clonidine (0.1 mg/kg) did not influence the accumulation of Dopa in the corpus striatum but inhibited that in the rest of the forebrain. 3.2.3. Hemisection Cutting the ascending DA and NA axons to the forebrain increased Dopa accumulation markedly in the corpus striatum and moderately in the rest of the forebrain (table 5). Clonidine (0.1 mg/kg) did not change the enhanced formation of Dopa in the corpus striatum on the sectioned side b u t it slightly inhibited Dopa accumulation in the corpus striatum as well as in the rest of the forebrain on the intact side.
4. Discussion The synthesis and the utilization of DA in the corpus striatum were accelerated by yohimbine and slowed by clonidine given with phenoxybenzamine. The effects of yohimbine on the turnover of DA were antagonized by clonidine plus phenoxybenzamine. The synthesis of DA appeared to be more influenced by clonidine plus phenoxybenza-
mine than by either drug alone. The aMT-induced disappearance of brain DA can be inhibited by both clonidine and phenoxybenzamine (And~n et al., 1970; And~n and StrSmbom, 1974). The effect of the combination of the two drugs appears to be stronger in the rat brain (cf. And~n et al., 1976). Thus, the synthesis and the utilization of DA are changed in opposite directions by yohimbine and by clonidine or phenoxybenzamine. The synthesis of NA in the rest of the forebrain was markedly accelerated by yohimbine, less so by phenoxybenzamine and was slowed by clonidine (cf. And~n et al., 1976). The utilization of NA was markedly enhanced by yohimbine (cf. Papeschi and Theiss, 1975; And~n et al., 1976); it is known to be increased by phenoxybenzamine and decreased by clonidine (And~n et al., 1967; And~n et al., 1970; And~n et al., 1976). Thus, phenoxybenzamine can slow DA turnover but speeds up NA turnover whereas clonidine inhibits both DA and NA turnover and yohimbine stimulates the turnover of both amines. The effects of yohimbine, phenoxybenzamine and clonidine on NA turnover are in all probability caused by alterations in the activity of a-adrenoreceptors. In functional experiments on flexor reflex activity and on m o t o r
¢
280
activity, postsynaptic a-adrenoreceptors were stimulated by clonidine and blocked by phenoxybenzamine or high doses of yohimbine (And~n et al., 1963; And~n et al., 1967; And~n et al., 1970; And~n and StrSmbom, 1974; And~n et al., 1976). There are, however, certain differences between the functional and the biochemical responses (And~n et al., 1976). Phenoxybenzamine is a more effective blocker than yohimbine for the postsynaptic a-adrenoreceptors which regulate functions such as flexor reflex activity and m o t o r activity, whereas the reverse is true for their acceleration of NA turnover. Furthermore, higher doses of clonidine are needed to stimulate the functional postsynaptic aadrenoreceptors than to inhibit NA turnover. Therefore, the a-adrenoreceptors mediating the biochemical responses might be different from those responsible for the functional postsynaptic effects. The a-adrenoreceptors regulating NA turnover might occur on the NA neurons (presynaptic receptors or autoreceptors). The activity of the postsynaptic a-adrenoreceptors is markedly inhibited by the dose of phenoxybenzamine used (20 mg/ kg) b u t n o t directly influenced by the doses used for yohimbine (3 mg/kg) or clonidine (0.1 mg/kg) (And~n et al., 1976). Therefore, NA neurotransmission might be facilitated by yohimbine (3 mg/kg) due to increased release of NA without a simultaneous blockade of the postsynaptic NA receptors, whereas NA neurotransmission might be impaired by phenoxybenzamine (20 mg/kg) and clonidine (0.1 mg/kg) due to effects on a-receptors postsynaptically and presynaptically, respectively. Reserpine increased Dopa formation both in the corpus striatum and in the rest of the forebrain, most probably as a result of reduced catecholamine release and subsequently diminished receptor activation (cf. Carlsson, 1976). Since yohimbine and phenoxybenzamine did n o t cause an additional rise in Dopa accumulation after pretreatment with reserpine, their effects on the synthesis of DA and NA are probably also due to changes in the
N.-E. ANDEN, M. GRABOWSKA
activity of monoamine receptors. In the reserpine-treated rats, clonidine reduced the synthesis of NA, but not of DA indicating that it affects NA neurons directly and DA neurons indirectly. Further evidence for this view was obtained in the experiments with a x o t o m y when DA synthesis was inhibited by clonidine only on the intact side. The selective DA receptor stimulating agent, apomorphine, has been shown to affect the synthesis of both DA and NA normally while, following reserpine or a x o t o m y , only DA synthesis is influenced, indicating an indirect effect of apomorphine on the synthesis of NA (Carlsson et al., 1976). Since the DA mechanisms appear to be influenced indirectly by clonidine, the same may also hold for the effects of phenoxybenzamine and yohimbine. The turnover of DA was inhibited by clonidine and phenoxybenzamine and was stimulated by yohimbine, i.e., DA turnover was affected by these drugs in the same direction as was the activity of the postsynaptic a-adrenoreceptors of the NA synapses. Therefore, it is possible that DA turnover is modified by changes in the transmission of impulses through NA synapses: phenoxybenzamine decreases DA turnover by blocking postsynaptic NA receptors; clonidine decreases DA turnover by depressing NA release via stimulation of presynaptic a-receptors on the NA neurons; yohimbine increases DA turnover by facilitating NA release via blockade of presynaptic a-receptors without a simultaneous blockade of postsynaptic NA receptors. There are NA nerve terminals in the substantia nigra area where the cell bodies of the DA neurons ascending to the corpus striatum are localized (Fuxe, 1965). It is not known, however, if the NA from these nerve terminals can affect the nigro-neostriatal DA neurons directly. An indirect regulation of DA turnover in the corpus striatum through changes in the NA neurotransmission might explain why the drugs influencing the a-adrenoreceptors have less clear-cut effects on DA than on NA turnover. After a drug-induced change in the activ-
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
ity of the NA receptors, the turnover of DA can still be regulated via DA receptors whereas any a-receptor-mediated feedback regulation of the turnover of NA should be impaired. This difference might be responsible for the weak effect of yohimbine on the utilization of DA in contrast to the pronounced effect on the turnover of NA. Actually, the a-MT-induced disappearance of DA is accelerated by yohimbine only when a-MT is given shortly, but not 4 h before sacrifice (Papeschi and Theiss, 1975; And~n et al., 1976). With longer intervals, a-MT itself might accelerate DA turnover by successive reduction of the DA store, DA release and the activation of DA receptors (Persson and Waldeck, 1970). Combined treatment with clonidine plus phenoxybenzamine probably caused a complete blockade of the NA-mediated transmission but only partially inhibited the yohirnbine-induced stimulation of DA turnover. Therefore it is possible that yohimbine also influences the DA neurons in other ways than via increased release of NA, e.g., through its excitation of central 5-HT receptors (cf. And~n et al., 1976). The postsynaptic DA receptors in the corpus striatum are not directly influenced by phenoxybenzamine, clonidine or yohimbine, as seen in experiments on turning following unilateral striatectomy of rats treated with reserpine and ~-MT without, or in combination with apomorphine or Dopa (And~n et al., 1966b; And~n et al., 1970; And~n and StrSmborn, 1974; And~n and Grabowska, unpublished). There are, however, indications that the postsynaptic DA receptor activity in the corpus striatum can be decreased indirectly by phenoxybenzamine and increased by yohimbine. Phenoxybenzamine induces a slight turning to the side contralateral to a unilateral striatectomy provided the animals had not been pretreated with reserpine and ~-MT (And~n et al., 1966b; And~n and StrSmbom, 1974). Yohimbine given into the lateral cervical ventricle on one side causes contralateral turning of the head and tail of rats (Zebrowska-Zupina and Kleinrok, 1973).
281
Furthermore, unilateral lesions of the NA cell bodies in the locus coeruleus result in an increased sensitivity of the striatal postsynaptic DA receptors on the operated side in turning experiments, perhaps due to interruption of the NA neurons from the locus coeruleus normally facilitating the nerve impulse flow in the nigro-neostriatal DA neurons (Pycock et al., 1975; Donaldson et al., 1975). Such an interaction between the NA and DA neuron systems might also be of importance for the changes in motor activity induced in normal animals by clonidine, phenoxybenzamine and yohimbine.
Acknowledgements This work was supported by the Swedish Medical Research Council (project number 502). M.G. is on leave of absence from the Institute of Pharmacology, Polish Academy of Sciences, PL-31-344 Krak6w, Poland, and is a recipient of a fellowship from the Polish and Swedish Academies of Sciences. The companies indicated by an asterisk above generously donated drugs. Maria Lindb~ck and Inger Oscarsson gave excellent technical assistance.
References And~n, N.-E., 1967, Effects of reserpine and a tyrosine hydroxylase inhibitor on the monoamine levels in different regions of the rat central nervous system, European J. Pharmacol. 1, 1. And4n, N.-E., P. B4dard, K. Fuxe and U. Ungerstedt, 1972, Early and selective increase in brain dopamine levels after axotomy, Experientia (Basel) 38, 300. And~n, N.-E., H. Corrodi, A. DahlstrSm, K. Fuxe and T. H6kfelt, 1966a, Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons, Life Sci. 5,561. And~n, N.-E., H. Corrodi, K, Fuxe, B. HSkfelt, T. H6kfelt, C. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9 (1), 513. And~n, N.-E., H. Corrodi, K. Fuxe and T. HSkfelt, 1967, Increased impulse flow in bulbospinal noradrenaline neurons produced by catecholamine receptor blocking agents, European J. Pharmacol. 2, 59.
/
282 And~n, N.-E., A. Dahlstr6m, K. Fuxe and K. Larsson, 1966b, Functional role of the nigro-neostriatal dopamine neurons, Acta Pharmacol. (Kbh.) 2 4 , 2 6 3 . And~n, N.-E., M. Grabowska and U. StrSmbom, 1976, Different a-adrenoreceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents, Naunyn-Schmiedeb. Arch. Pharmacol. 292, 43. And~n, N.-E., A. Lundberg, E. Rosengren and L. Vyklick:~, 1963, The effect of DOPA on spinal reflexes from the FRA (flexor reflex afferents), Experientia (Basel) 1 9 , 6 5 4 . And~n, N.-E. and U. StrSmbom, 1974, Adrenergic receptor blocking agents: effects on central noradrenaline and dopamine receptors and on motor activity, Psychopharmacologia (Berlin) 38, 91. Atack, C.V., 1973, The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay, Brit. J. Pharmacol. 4 8 , 6 9 9 . Atack, C.V. and T. Magnusson, 1970, Individual elution or noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures, J. Pharm. Pharmacol. 2 2 , 6 2 5 . Bertler, A., A. Carlsson and E. Rosengren, 1958, A method for the fluorimetric determination of adrenaline and noradrenaline in tissues, Acta Physiol. Scand. 4 4 , 2 7 3 . Carlsson, A., 1976, The effect of neuroleptic drugs on brain catecholamine metabolism, in: Antipsychotic Drugs, Pharmacodynamics and Pharmacokinetics, eds. G. Sedvall, B. Uvn~/s and Y. Zotterman (Pergamon Press, Oxford--New York-Toronto--Sydney) p. 99. Carlsson, A., J.N. Davis, W. Kehr, M. Lindqvist and C.V. Atack, 1972, Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in vivo using an inhibitor of the aromatic amino acid decarboxylase, Naunyn-Schmiedeb. Arch. Pharmacol. 2 7 5 , 1 5 3 . Carlsson, A., W. Kehr and M. Lindqvist, 1976, Agonist--antagonist interaction on dopamine receptors in brain, as reflected in the rates of tyrosine and tryptophan hydroxylation, in: Advances in Parkinsonism. Biochemistry, Physiology, Treatment, eds. W. Birkmayer and O. Hornykiewicz (Editiones Roche, Basle) (in press). Corrodi, H. and L.C.F. Hanson, 1966, Central effects
N.-E. ANDEN, M. GRABOWSKA of an inhibitor of tyrosine hydroxylation, Psychopharmacologia (Berlin) 10,116. Donaldson, I., A. Dolphin, P. Jenner, C.D. Marsden and C. Pycock, 1975, The role of dopamine in rotational behaviour produced by unilateral lesions of the locus coeruleus, Brit. J. Pharmacol. 55, 290P. Fuxe, K., 1965, The distribution of monoamine terminals in the central nervous system, Acta Physiol. Scand. 64, Suppl. 247, 37. H//ggendal, J., 1963, An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues, Acta Physiol. Scand. 5 9 , 2 4 2 . Hassler, R. and I.J. Bak, 1969, Unbalanced ratios of striatal dopamine and serotonin after experimental interruption of strionigral connections in rat, in: Third symposium on Parkinson's disease, eds. F.J. Gillingham and I.M.L. Donaldson (Livingstone, Edinburgh--London) p. 29. Kehr, W., A. Carlsson and M. Lindqvist, 1972, A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain, Naunyn-Schmiedeb. Arch. Pharmacol. 274,273. Papeschi, R. and P. Theiss, 1975, The effect of yohimbine on the turnover of brain catecholamines and serotonin, European J. Pharmacol. 33, 1. Persson, T. and B. Waldeck, 1970, Further studies on the possible interaction between dopamine and noradrenaline containing neurons in the brain, European J. Pharmacol. 11,315. Pycock, C.J., I. Mac G. Donaldson and C.D. Marsden, 1975, Circling behaviour produced by unilateral lesions in the region of the locus coeruleus in rats, Brain Res. 9 7 , 3 1 7 . Rochette, L, and J. Bralet, 1975, Effect of clonidine on the synthesis of cerebral dopamine, Biochem. Pharmacol. 24,303. Spector, S., A. Sjoerdsma and S. Udenfriend, 1965, Blockade of endogenous norepinephrine synthesis by a-methyltyrosine, an inhibitor of tyrosine hydroxylase, J. Pharmacol. Exptl. Therap. 147, 86. StrSmbom, U., 1976, Catecholamine receptor agonists: effects on motor activity and rate of tyrosine hydroxylation in mouse brain, NaunynSchmiedeb. Arch. Pharmacol. 292,167. Zebrowska-Lupina, I. and Z. Kleinrok, 1973, Behavioural effects of yohimbine administered intraventricularly in the rat, Psychopharmacologia (Berlin) 33,267.
275
P H A R M A C O L O G I C A L E V I D E N C E F O R A S T I M U L A T I O N O F D O P A M I N E N E U R O N S BY N O R A D R E N A L I N E N E U R O N S IN T H E B R A I N NILS-ERIK ANDI~N and MARIA GRABOWSKA Department of Pharmacology, University of GSteborg, Fack, S-400 33 GSteborg 33, Sweden
Received 20 January 1976, revised MS received 13 April 1976, accepted 10 June 1976
N.-E. ANDl~N and M. GRABOWSKA, Pharmacological evidence for a stimulation of dopamine neurons by noradrenaline neurons in the brain, European J. Pharmacol. 39 (1976) 275--282. The effects of yohimbine (3 mg/kg i.p.), phenoxybenzamine (20 mg/kg i.p.) and clonidine (0.1 mg/kg i.p.) on the synthesis and the utilization of dopamine and noradrenaline in the central nervous system of rats were investigated. Dopa accumulation following decarboxylase inhibition and the ~-methyltyrosine-induced disappearance of the amines were used as the measure of these effects. The synthesis and the utilization of dopamine and noradrenaline were accelerated by yohimbine. Clonidine plus phenoxybenzamine inhibited the synthesis and utilization of dopamine and the combination also partly antagonized the effects of yohimbine on the turnover of dopamine. The effects of the three drugs on the synthesis and utilization of dopamine might be secondary to their actions on ~-adrenoreceptors of noradrenaline synapses since, at the doses used, yohimbine increases the release of noradrenaline, phenoxybenzamine blocks postsynaptic noradrenaline receptors and clonidine reduces the release of noradrenaline. This hypothesis is supported by the findings that yohimbine and phenoxybenzamine did not change the increased synthesis of dopamine in reserpine-treated rats and that clonidine did not inhibit the increased synthesis of dopamine after axotomy or treatment with reserpine. Receptor activity Phenoxybenzamine
Indirect effect Noradrenaline
Utilization Synthesis
1. I n t r o d u c t i o n Central, p o s t s y n a p t i c ~ - a d r e n o r e c e p t o r s can be s t i m u l a t e d b y clonidine (And4n et al., 1974; A n d 4 n et al., 1976) and t h e y can be b l o c k e d b y p h e n o x y b e n z a m i n e as well as b y high doses o f y o h i m b i n e (And~n et al., 1963; And~n et al., 1967; A n d 4 n and S t r S m b o m , 1974; A n d 4 n et al., 1976), judging f r o m effects o n f l e x o r reflex activity and o n m o t o r activity. T h e t u r n o v e r o f n o r a d r e n a l i n e (NA) in the central nervous s y s t e m is decreased b y clonidine and increased b y p h e n o x y b e n z a mine and b y y o h i m b i n e , p r o b a b l y due t o their effects on ~ - a d r e n o r e c e p t o r s (And~n et al., 1 9 6 7 ; A n d 4 n et al., 1970; A n d 4 n et al., 1976). These drugs can also change d o p a m i n e (DA) t u r n o v e r : the disappearance o f DA in the brain following synthesis i n h i b i t i o n is t h u s d e c e l e r a t e d b y clonidine as well as b y phe-
Dopamine
Yohimbine
Clonidine
n o x y b e n z a m i n e (And~n et al., 1970; And~n and S t r S m b o m , 1974). F u r t h e r m o r e , clonidine decreases and y o h i m b i n e increases t h e a c c u m u l a t i o n o f D o p a in the DA-rich c o r p u s striatum following d e c a r b o x y l a s e inhibition, indicating effects on DA synthesis (And~n et al., 1976; S t r S m b o m , 1 9 7 6 ) (cf. R o c h e t t e and Bralet, 1 9 7 5 ; Papeschi and Theiss, 1975). These findings p r o m p t e d us t o use the abovem e n t i o n e d drugs t o investigate in m o r e detail the possible regulation o f the synthesis and utilization o f brain DA t h r o u g h changes in NA n e u r o t r a n s m i s s i o n . 2. Materials and m e t h o d s 2.1. M e t h o d s
Male S p r a g u e - - D a w l e y rats ( 1 6 0 - - 2 7 0 g) were used. T h e utilization o f DA in the brain
/
276 and of the NA in the brain and in the spinal cord was investigated by measuring the disappearance of the amines for 1 h following treatment with the tyrosine hydroxylase inhibitor a-methyltyrosine {a-MT; 250 mg/kg i.p. of the D,L-form of the methyl ester HC1) (Spector et al., 1965; Corrodi and Hanson, 1966; And6n et at., 1966a). The synthesis of DA and NA was followed by measuring Dopa accumulation in different parts of the forebrain for 30 rain following injection of the aromatic amino acid decarboxylase inhibitor 3-hydroxybenzylhydrazine HC1 (NSD 1015; 100 mg/kg i.p.) {Carlsson et al., 1972). In the hemisection experiments, the rats were anaesthesized with pentobarbital sodium (about 40 mg/kg i.p.) and complete and almost frontal section of the brain on one side at the level of the caudal hypothalamus was performed with a blunt-edged spatula (Hassler and Bak, 1969; And~n et al., 1972). The NSD 1015 was injected immediately after the hemisection. The animals were decapitated 30 min later. Except for the hemisection experiments, the rats were killed by t h o r a c o t o m y and exsanguination under light chloroform anaesthesia. In the a-MT experiments, the brains and the spinal cords from two rats were pooled. In the Dopa experiments, the brain was divided bilaterally as described above for hemisection. The parts from two sides were pooled. The brain in front of the cut was separated into the corpus striatum, the limbic system and the rest of the forebrain (i.e., neocortex + hippocampus + thalamus + hypothalamus) by incisions through the outer borders of the corpus striatum, in the frontal part of the optic chiasm and through the rhinal fissures. The corpus striatum has many DA b u t few NA nerve terminals whereas the reverse holds true for the rest of the forebrain (see Results). The limbic system was discarded since it is rich in both DA and NA (And6n, 1967). DA, NA and Dopa were determined spectrofluorimetrically after homogenization in 0.4 N perchloric acid, cation exchange chromatography and oxidation (Atack and Magnusson, 1970; Atack,
N.-E. ANDEN, M. GRABOWSKA 1973; Bertler et al., 1958; H~ggendal, 1963; Kehr et al., 1972).
2.2. Drugs The following drugs were used: D,L-amethyltyrosine methyl ester HC1 (a-MT, H 44/68, *H~ssle, MSlndal), yohimbine HC1 (Merck, Darmstadt), clonidine HC1 (*Boehringer Ingelheim, Stockholm), phenoxybenzamine HC1 (*Leo, Helsingborg), 3-hydroxybenzylhydrazine HC1 (NSD 1015; synthesized in this department by Dr. Per Martinson), reserpine (*CIBA, MSlndal). Phenoxybenzamine and reserpine were dissolved in a few drops of glacial acetic acid and made up to volume with 5.5% glucose. The other drugs were dissolved in 0.9% NaC1, sometimes with a few drops of N HC1 and heating. The doses refer to the salts indicated.
3. Results
3.1. a-Methyltyrosine-induced disappearance of dopamine and noradrenaline Brain DA and NA, and the NA in the spinal cord were decreased by 38, 22 and 32% respectively, 1 h after the administration of aMT (table 1). Yohimbine (3 mg/kg i.p.) accelerated the a-MT-induced disappearance of DA as well as of NA. Combined treatment with clonidine and phenoxybenzamine slowed the utilization of both DA and NA. This treatment partly antagonized the stimulation by yohimbine of the a-MT-induced disappearance of DA, b u t not of NA.
3.2. Synthesis o f dopamine and noradrenaline 3.2.1. Normal animals In the group treated with only NSD 1015 and shown in table 2, the concentrations of DA and NA were 5.45 and 0.18 pg/g in the corpus striatum, whereas they were 0.05 and 0.40 t~g/g in the rest of the forebrain. The accumulation of Dopa in the corpus striatum
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
277
TABLE 1 Effects o f y o h i m b i n e (3 m g / k g i.p., 75 rain b e f o r e sacrifice), c l o n i d i n e (0.1 m g l k g i.p., 105 m i n b e f o r e sacrifice) a n d p h e n o x y b e n z a m i n e ( 2 0 m g / k g i.p., 9 0 m i n b e f o r e sacrifice) o n t h e d i s a p p e a r a n c e o f d o p a m i n e in w h o l e b r a i n a n d o f n o r a d r e n a l i n e in w h o l e b r a i n a n d in spinal c o r d following t r e a t m e n t o f rats w i t h D , L - a - m e t h y l t y r o s i n e m e t h y l e s t e r (a-MT, 250 m g / k g i.p., 60 rain). T h e values (/1g/g) are m e a n s ± S.E.M. w i t h t h e n u m b e r o f experim e n t s in p a r e n t h e s e s . Statistical significance was calculated w i t h S t u d e n t ' s t-test ***, **, * Treatment
Dopamine, whole brain
Noradrenaline, whole brain
Noradrenaline, spinal cord
No t r e a t m e n t ~-MT Y o h i m b i n e + ~-MT Clonidine + phenoxybenzamine + ~-MT Clonidine + phenoxybenzamine + y o h i m b i n e + ~-MT
0 . 5 5 8 ± 0 . 0 1 2 9 (6) a 0 . 3 4 8 -+ 0 . 0 0 9 5 (6) 0 . 2 8 9 ± 0 . 0 1 2 0 (6) d
0.330_+ 0 . 0 1 6 8 (6) a 0 . 2 6 2 ± 0 . 0 0 7 0 (6) 0 . 1 6 8 ± 0 . 0 0 4 3 (6) a
0 . 3 4 2 ± 0 . 0 1 9 6 (6) a 0 . 2 4 1 ± 0 . 0 1 2 7 (6) 0 . 1 3 4 ± 0 . 0 0 9 0 (6) g
0 . 4 4 0 _+ 0 . 0 1 3 4 (6) a
0 . 3 1 8 ± 0 . 0 1 4 6 (6) d
0 . 2 9 6 ± 0 . 0 1 5 9 (6) g
0 . 3 5 8 ± 0 . 0 1 4 0 (6) e,f
0 . 1 5 6 ± 0 . 0 0 8 9 (6) c
0 . 1 3 7 ± 0 . 0 1 4 2 (6) c
*** p < 0 . 0 0 1 , as c o m p a r e d to a ~-MT, b y o h i m b i n e + ~-MT, c c l o n i d i n e + p h e n o x y b e n z a m i n e + ~-MT; ** p < 0.01, as c o m p a r e d t o d ~-MT, e y o h i m b i n e + ~-MT, f c l o n i d i n e + p h e n o x y b e n z a m i n e + 0~-MT; * p < 0 . 0 2 5 , as c o m p a r e d t o g ~-MT, h y o h i m b i n e + q-MT, i c l o n i d i n e + p h e n o x y b e n z a m i n e + ~-MT.
and in the rest of the forebrain should thus reflect the synthesis of DA and of NA respectively. Yohimbine (3 mg/kg) markedly accelerated the formation of Dopa caused by NSD 1015 both in the corpus striatum and in the rest of the forebrain (table 2). Phenoxybenzamine
(20 mg/kg) only insignificantly decreased both the normal accumulation of Dopa in the corpus striatum and its enhancement by yohimbine. The accumulation of Dopa in the rest of the forebrain was only slightly elevated by phenoxybenzamine. Combined treatment with clonidine and phenoxybenzamine re-
TABLE 2 Effects o f y o h i m b i n e (3 m g / k g i.p., 45 m i n b e f o r e sacrifice), p h e n o x y b e n z a m i n e ( 2 0 m g / k g i.p., 60 m i n b e f o r e sacrifice) a n d c l o n i d i n e (0.1 m g / k g i.p., 75 m i n b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m a n d in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) f o l l o w i n g treatm e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 1 0 0 m g / k g i.p., 30 m i n ) . T h e values (pg/g) are m e a n s ± S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical significance was c a l c u l a t e d with S t u d e n t ' s ttest *** ** * Treatment
Dopa, c o r p u s s t r i a t u m
Dopa, rest o f t h e f o r e b r a i n
NSD Y o h i m b i n e + NSD P h e n o x y b e n z a m i n e + NSD P h e n o x y b e n z a m i n e + y o h i m b i n e + NSD C l o n i d i n e + p h e n o x y b e n z a m i n e + NSD Clonidine + phenoxybenzamine + yohimbine + NSD
1.04 2.08 0.82 2.01 0.72
0.096 0.162 0.124 0.171 0.066
+- 0 . 0 6 3 ± 0.175 ± 0.099 ± 0.260 ± 0.083
(13) (13) a (7) (7) g (6) e
1.38 ± 0 . 0 3 2 (6) k,d
+- 0 . 0 0 5 4 -+ 0 . 0 0 8 4 ± 0.0101 ± 0.0135 ± 0.0100
(12) (13) a (7) i (7) 1 (6) i
0 . 1 3 9 ± 0 . 0 0 9 2 (6) d
*** p < 0 . 0 0 1 , as c o m p a r e d t o a NSD, b y o h i m b i n e + NSD, c p h e n o x y b e n z a m i n e + NSD, d c l o n i d i n e + p h e n o x y b e n z a m i n e + NSD; ** p < 0.01, as c o m p a r e d t o e NSD, f y o h i m b i n e + NSD, g p h e n o x y b e n z a m i n e + NSD, h clonidine + p h e n o x y b e n z a m i n e + NSD; * p < 0 . 0 2 5 , as c o m p a r e d t o i NSD, k y o h i m b i n e + NSD, 1 p h e n o x y b e n z a m i n e + NSD, rn c l o n i d i n e + p h e n o x y b e n z a m i n e + NSD.
278
N.-E. ANDEN, M. G R A B O W S K A
TABLE 3 E f f e c t s o f d i f f e r e n t doses o f c l o n i d i n e (mg/kg i.p., 60 min b e f o r e sacrifice) and y o h i m b i n e (mg/kg i.p., 75 rain b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m and in t h e rest o f the f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) following t r e a t m e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 100 m g / k g i.p., 30 min). The values (pg/g) are m e a n s +_ S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in parentheses. Statistical significance was calculated w i t h S t u d e n t ' s t-test * * * , **, * Treatment
Dopa, c o r p u s s t r i a t u m
Dopa, rest o f the f o r e b r a i n
NSD C l o n i d i n e (0.1) + NSD C l o n i d i n e (0.3) + NSD Clonidine (1.0) + NSD Clonidine (3.0) + NSD Y o h i m b i n e (3) + NSD Y o h i m b i n e (3) + c l o n i d i n e (1.0) + NSD Y o h i m b i n e (10) + NSD Y o h i m b i n e (10) + c l o n i d i n e (1.0) + NSD
1.12 0.80 0.75 0.74 0.86 2.20 1.89 2.05 2.32
0.081 0.057 0.068 0,060 0.070 0.138 0.081 0.192 0.120
÷ ± t ± ~ + + ÷ ±
0.066 0.111 0.074 0.098 0.038 0.089 0.338 0.159 0.195
(18) (6) i (6) e (7) e (6) (12) a (4) f (4) a (4) b
~ 0.0041 t 0.0093 ± 0.0069 +- 0.0031 ± 0.0072 ÷ 0.0077 ± 0.0109 ÷ 0.0210 ~: 0.0167
(18) (5) i (5) (6) e (6) (12) a (4) g (4) a (4) f,m
* * * p < 0.001, as c o m p a r e d t o a NSD, b c l o n i d i n e (1.0) + NSD, c y o h i m b i n e (3) + NSD, d y o h i m b i n e (10) + N S D ; ** p < 0.01, as c o m p a r e d t o e NSD, f c l o n i d i n e (1.0) + NSD, g y o h i m b i n e (3) + NSD, h y o h i m b i n e (10) + NSD; * p < 0.05, as c o m p a r e d t o i NSD, k clonidine (1.0) + NSD, ! y o h i m b i n e (3) + NSD, m y o h i m b i n e (10) + NSD.
duced the normal Dopa accumulation in the corpus striatum as well as that after yohimbine (table 2). Clonidine inhibited Dopa accumulation both in the corpus striatum and in the rest of the forebrain (table 3). The effect did not appear to be appreciably stronger with doses higher than 0.1 mg/kg. The inhibition of the Dopa accumulation produced by clonidine
(1.0 mg/kg) in the corpus striatum was partly antagonized by yohimbine 3 mg/kg and completely by 10 mg/kg. 3.2.2. Reserpine-treated animals Reserpine enhanced the accumulation of DolJa after NSD 1015 both in corpus striatum and in the rest of the forebrain (table 4; cf. tables 2 and 3). Yohimbine (3 m g / k g ) a n d
TABLE 4 E f f e c t s o f y o h i m b i n e (3 m g / k g i.p., 45 rain b e f o r e sacrifice), p h e n o x y b e n z a m i n e (20 mg/kg i.p., 60 min before sacrifice) and c l o n i d i n e (0.1 m g / k g i.p., 75 min b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e corpus striat u m and in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) following t r e a t m e n t o f rats w i t h reserpine (R, 5 m g / k g i.p., 4 h b e f o r e sacrifice) and 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1015, 100 mg/kg, i.p., 30 min). The values (pg/g) are m e a n s ± S.E.M. w i t h the n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical significance was calculated w i t h S t u d e n t ' s t-test * Treatment
Dopa, corpus striatum
D o p a , rest o f t h e f o r e b r a i n
R R R R
3.41 2.94 3.57 3.68
0.165 0.160 0.173 0.101
+ + + +
NSD y o h i m b i n e + NSD p h e n o x y b e n z a m i n e + NSD c l o n i d i n e + NSD
• p < 0.025, as c o m p a r e d t o R + NSD.
+ 0.621 ± 0.182 +- 0.279 +- 0.380
(7) (7) (7) (7)
± 0.0238 ± 0.0114 +- 0.0111 -+ 0.0070
(7) (7) (7) (7) *
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
279
TABLE 5 Effects o f c l o n i d i n e (0.1 m g ] k g i.p., 6 0 m i n b e f o r e sacrifice ) a n d unilateral a x o t o m y ( h e m i s e c t i o n , 30 m i n b e f o r e sacrifice) o n t h e a c c u m u l a t i o n o f D o p a in t h e c o r p u s s t r i a t u m a n d in t h e rest o f t h e f o r e b r a i n ( n e o c o r t e x + h i p p o c a m p u s + t h a l a m u s + h y p o t h a l a m u s ) f o l l o w i n g t r e a t m e n t o f rats w i t h 3 - h y d r o x y b e n z y l h y d r a z i n e (NSD 1 0 1 5 , 100 m g / k g i.p., 30 min). T h e values (pg/g) are m e a n s _+ S.E.M. w i t h t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Statistical s i g n i f i c a n c e w a s calculated w i t h S t u d e n t ' s t-test ***, **, * Drug t r e a t m e n t
NSD C l o n i d i n e + NSD
Dopa, c o r p u s s t r i a t u m
Dopa, rest of t h e f o r b r a i n
I n t a c t side
S e c t i o n e d side
I n t a c t side
S e c t i o n e d side
1.47 +0.085 (8) 1.04 _+ 0 . 1 2 2 (8) e
4.61 ~ 0.237 (8) b 4.41 ± 0.271 (8) b
0.110 ± 0 . 0 0 8 0 (8) 0 . 0 7 6 -+ 0 . 0 0 6 3 (8) c
0 . 1 8 0 +0.0156 (8) d 0.148 ± 0 . 0 1 2 8 (8) b
*** p < 0 . 0 0 1 , as c o m p a r e d to a NSD a n d b i n t a c t side; ** p < 0.01, as c o m p a r e d to c NSD a n d d i n t a c t side; * p < 0.05, as c o m p a r e d to e NSD a n d f i n t a c t side.
phenoxybenzamine (20 mg/kg) did n o t alter the increase in Dopa accumulation in these animals. Clonidine (0.1 mg/kg) did not influence the accumulation of Dopa in the corpus striatum but inhibited that in the rest of the forebrain. 3.2.3. Hemisection Cutting the ascending DA and NA axons to the forebrain increased Dopa accumulation markedly in the corpus striatum and moderately in the rest of the forebrain (table 5). Clonidine (0.1 mg/kg) did not change the enhanced formation of Dopa in the corpus striatum on the sectioned side b u t it slightly inhibited Dopa accumulation in the corpus striatum as well as in the rest of the forebrain on the intact side.
4. Discussion The synthesis and the utilization of DA in the corpus striatum were accelerated by yohimbine and slowed by clonidine given with phenoxybenzamine. The effects of yohimbine on the turnover of DA were antagonized by clonidine plus phenoxybenzamine. The synthesis of DA appeared to be more influenced by clonidine plus phenoxybenza-
mine than by either drug alone. The aMT-induced disappearance of brain DA can be inhibited by both clonidine and phenoxybenzamine (And~n et al., 1970; And~n and StrSmbom, 1974). The effect of the combination of the two drugs appears to be stronger in the rat brain (cf. And~n et al., 1976). Thus, the synthesis and the utilization of DA are changed in opposite directions by yohimbine and by clonidine or phenoxybenzamine. The synthesis of NA in the rest of the forebrain was markedly accelerated by yohimbine, less so by phenoxybenzamine and was slowed by clonidine (cf. And~n et al., 1976). The utilization of NA was markedly enhanced by yohimbine (cf. Papeschi and Theiss, 1975; And~n et al., 1976); it is known to be increased by phenoxybenzamine and decreased by clonidine (And~n et al., 1967; And~n et al., 1970; And~n et al., 1976). Thus, phenoxybenzamine can slow DA turnover but speeds up NA turnover whereas clonidine inhibits both DA and NA turnover and yohimbine stimulates the turnover of both amines. The effects of yohimbine, phenoxybenzamine and clonidine on NA turnover are in all probability caused by alterations in the activity of a-adrenoreceptors. In functional experiments on flexor reflex activity and on m o t o r
¢
280
activity, postsynaptic a-adrenoreceptors were stimulated by clonidine and blocked by phenoxybenzamine or high doses of yohimbine (And~n et al., 1963; And~n et al., 1967; And~n et al., 1970; And~n and StrSmbom, 1974; And~n et al., 1976). There are, however, certain differences between the functional and the biochemical responses (And~n et al., 1976). Phenoxybenzamine is a more effective blocker than yohimbine for the postsynaptic a-adrenoreceptors which regulate functions such as flexor reflex activity and m o t o r activity, whereas the reverse is true for their acceleration of NA turnover. Furthermore, higher doses of clonidine are needed to stimulate the functional postsynaptic aadrenoreceptors than to inhibit NA turnover. Therefore, the a-adrenoreceptors mediating the biochemical responses might be different from those responsible for the functional postsynaptic effects. The a-adrenoreceptors regulating NA turnover might occur on the NA neurons (presynaptic receptors or autoreceptors). The activity of the postsynaptic a-adrenoreceptors is markedly inhibited by the dose of phenoxybenzamine used (20 mg/ kg) b u t n o t directly influenced by the doses used for yohimbine (3 mg/kg) or clonidine (0.1 mg/kg) (And~n et al., 1976). Therefore, NA neurotransmission might be facilitated by yohimbine (3 mg/kg) due to increased release of NA without a simultaneous blockade of the postsynaptic NA receptors, whereas NA neurotransmission might be impaired by phenoxybenzamine (20 mg/kg) and clonidine (0.1 mg/kg) due to effects on a-receptors postsynaptically and presynaptically, respectively. Reserpine increased Dopa formation both in the corpus striatum and in the rest of the forebrain, most probably as a result of reduced catecholamine release and subsequently diminished receptor activation (cf. Carlsson, 1976). Since yohimbine and phenoxybenzamine did n o t cause an additional rise in Dopa accumulation after pretreatment with reserpine, their effects on the synthesis of DA and NA are probably also due to changes in the
N.-E. ANDEN, M. GRABOWSKA
activity of monoamine receptors. In the reserpine-treated rats, clonidine reduced the synthesis of NA, but not of DA indicating that it affects NA neurons directly and DA neurons indirectly. Further evidence for this view was obtained in the experiments with a x o t o m y when DA synthesis was inhibited by clonidine only on the intact side. The selective DA receptor stimulating agent, apomorphine, has been shown to affect the synthesis of both DA and NA normally while, following reserpine or a x o t o m y , only DA synthesis is influenced, indicating an indirect effect of apomorphine on the synthesis of NA (Carlsson et al., 1976). Since the DA mechanisms appear to be influenced indirectly by clonidine, the same may also hold for the effects of phenoxybenzamine and yohimbine. The turnover of DA was inhibited by clonidine and phenoxybenzamine and was stimulated by yohimbine, i.e., DA turnover was affected by these drugs in the same direction as was the activity of the postsynaptic a-adrenoreceptors of the NA synapses. Therefore, it is possible that DA turnover is modified by changes in the transmission of impulses through NA synapses: phenoxybenzamine decreases DA turnover by blocking postsynaptic NA receptors; clonidine decreases DA turnover by depressing NA release via stimulation of presynaptic a-receptors on the NA neurons; yohimbine increases DA turnover by facilitating NA release via blockade of presynaptic a-receptors without a simultaneous blockade of postsynaptic NA receptors. There are NA nerve terminals in the substantia nigra area where the cell bodies of the DA neurons ascending to the corpus striatum are localized (Fuxe, 1965). It is not known, however, if the NA from these nerve terminals can affect the nigro-neostriatal DA neurons directly. An indirect regulation of DA turnover in the corpus striatum through changes in the NA neurotransmission might explain why the drugs influencing the a-adrenoreceptors have less clear-cut effects on DA than on NA turnover. After a drug-induced change in the activ-
NORADRENALINE NEURONS AND DOPAMINE TURNOVER
ity of the NA receptors, the turnover of DA can still be regulated via DA receptors whereas any a-receptor-mediated feedback regulation of the turnover of NA should be impaired. This difference might be responsible for the weak effect of yohimbine on the utilization of DA in contrast to the pronounced effect on the turnover of NA. Actually, the a-MT-induced disappearance of DA is accelerated by yohimbine only when a-MT is given shortly, but not 4 h before sacrifice (Papeschi and Theiss, 1975; And~n et al., 1976). With longer intervals, a-MT itself might accelerate DA turnover by successive reduction of the DA store, DA release and the activation of DA receptors (Persson and Waldeck, 1970). Combined treatment with clonidine plus phenoxybenzamine probably caused a complete blockade of the NA-mediated transmission but only partially inhibited the yohirnbine-induced stimulation of DA turnover. Therefore it is possible that yohimbine also influences the DA neurons in other ways than via increased release of NA, e.g., through its excitation of central 5-HT receptors (cf. And~n et al., 1976). The postsynaptic DA receptors in the corpus striatum are not directly influenced by phenoxybenzamine, clonidine or yohimbine, as seen in experiments on turning following unilateral striatectomy of rats treated with reserpine and ~-MT without, or in combination with apomorphine or Dopa (And~n et al., 1966b; And~n et al., 1970; And~n and StrSmborn, 1974; And~n and Grabowska, unpublished). There are, however, indications that the postsynaptic DA receptor activity in the corpus striatum can be decreased indirectly by phenoxybenzamine and increased by yohimbine. Phenoxybenzamine induces a slight turning to the side contralateral to a unilateral striatectomy provided the animals had not been pretreated with reserpine and ~-MT (And~n et al., 1966b; And~n and StrSmbom, 1974). Yohimbine given into the lateral cervical ventricle on one side causes contralateral turning of the head and tail of rats (Zebrowska-Zupina and Kleinrok, 1973).
281
Furthermore, unilateral lesions of the NA cell bodies in the locus coeruleus result in an increased sensitivity of the striatal postsynaptic DA receptors on the operated side in turning experiments, perhaps due to interruption of the NA neurons from the locus coeruleus normally facilitating the nerve impulse flow in the nigro-neostriatal DA neurons (Pycock et al., 1975; Donaldson et al., 1975). Such an interaction between the NA and DA neuron systems might also be of importance for the changes in motor activity induced in normal animals by clonidine, phenoxybenzamine and yohimbine.
Acknowledgements This work was supported by the Swedish Medical Research Council (project number 502). M.G. is on leave of absence from the Institute of Pharmacology, Polish Academy of Sciences, PL-31-344 Krak6w, Poland, and is a recipient of a fellowship from the Polish and Swedish Academies of Sciences. The companies indicated by an asterisk above generously donated drugs. Maria Lindb~ck and Inger Oscarsson gave excellent technical assistance.
References And~n, N.-E., 1967, Effects of reserpine and a tyrosine hydroxylase inhibitor on the monoamine levels in different regions of the rat central nervous system, European J. Pharmacol. 1, 1. And4n, N.-E., P. B4dard, K. Fuxe and U. Ungerstedt, 1972, Early and selective increase in brain dopamine levels after axotomy, Experientia (Basel) 38, 300. And~n, N.-E., H. Corrodi, A. DahlstrSm, K. Fuxe and T. H6kfelt, 1966a, Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons, Life Sci. 5,561. And~n, N.-E., H. Corrodi, K, Fuxe, B. HSkfelt, T. H6kfelt, C. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9 (1), 513. And~n, N.-E., H. Corrodi, K. Fuxe and T. HSkfelt, 1967, Increased impulse flow in bulbospinal noradrenaline neurons produced by catecholamine receptor blocking agents, European J. Pharmacol. 2, 59.
/
282 And~n, N.-E., A. Dahlstr6m, K. Fuxe and K. Larsson, 1966b, Functional role of the nigro-neostriatal dopamine neurons, Acta Pharmacol. (Kbh.) 2 4 , 2 6 3 . And~n, N.-E., M. Grabowska and U. StrSmbom, 1976, Different a-adrenoreceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents, Naunyn-Schmiedeb. Arch. Pharmacol. 292, 43. And~n, N.-E., A. Lundberg, E. Rosengren and L. Vyklick:~, 1963, The effect of DOPA on spinal reflexes from the FRA (flexor reflex afferents), Experientia (Basel) 1 9 , 6 5 4 . And~n, N.-E. and U. StrSmbom, 1974, Adrenergic receptor blocking agents: effects on central noradrenaline and dopamine receptors and on motor activity, Psychopharmacologia (Berlin) 38, 91. Atack, C.V., 1973, The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay, Brit. J. Pharmacol. 4 8 , 6 9 9 . Atack, C.V. and T. Magnusson, 1970, Individual elution or noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures, J. Pharm. Pharmacol. 2 2 , 6 2 5 . Bertler, A., A. Carlsson and E. Rosengren, 1958, A method for the fluorimetric determination of adrenaline and noradrenaline in tissues, Acta Physiol. Scand. 4 4 , 2 7 3 . Carlsson, A., 1976, The effect of neuroleptic drugs on brain catecholamine metabolism, in: Antipsychotic Drugs, Pharmacodynamics and Pharmacokinetics, eds. G. Sedvall, B. Uvn~/s and Y. Zotterman (Pergamon Press, Oxford--New York-Toronto--Sydney) p. 99. Carlsson, A., J.N. Davis, W. Kehr, M. Lindqvist and C.V. Atack, 1972, Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in vivo using an inhibitor of the aromatic amino acid decarboxylase, Naunyn-Schmiedeb. Arch. Pharmacol. 2 7 5 , 1 5 3 . Carlsson, A., W. Kehr and M. Lindqvist, 1976, Agonist--antagonist interaction on dopamine receptors in brain, as reflected in the rates of tyrosine and tryptophan hydroxylation, in: Advances in Parkinsonism. Biochemistry, Physiology, Treatment, eds. W. Birkmayer and O. Hornykiewicz (Editiones Roche, Basle) (in press). Corrodi, H. and L.C.F. Hanson, 1966, Central effects
N.-E. ANDEN, M. GRABOWSKA of an inhibitor of tyrosine hydroxylation, Psychopharmacologia (Berlin) 10,116. Donaldson, I., A. Dolphin, P. Jenner, C.D. Marsden and C. Pycock, 1975, The role of dopamine in rotational behaviour produced by unilateral lesions of the locus coeruleus, Brit. J. Pharmacol. 55, 290P. Fuxe, K., 1965, The distribution of monoamine terminals in the central nervous system, Acta Physiol. Scand. 64, Suppl. 247, 37. H//ggendal, J., 1963, An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues, Acta Physiol. Scand. 5 9 , 2 4 2 . Hassler, R. and I.J. Bak, 1969, Unbalanced ratios of striatal dopamine and serotonin after experimental interruption of strionigral connections in rat, in: Third symposium on Parkinson's disease, eds. F.J. Gillingham and I.M.L. Donaldson (Livingstone, Edinburgh--London) p. 29. Kehr, W., A. Carlsson and M. Lindqvist, 1972, A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain, Naunyn-Schmiedeb. Arch. Pharmacol. 274,273. Papeschi, R. and P. Theiss, 1975, The effect of yohimbine on the turnover of brain catecholamines and serotonin, European J. Pharmacol. 33, 1. Persson, T. and B. Waldeck, 1970, Further studies on the possible interaction between dopamine and noradrenaline containing neurons in the brain, European J. Pharmacol. 11,315. Pycock, C.J., I. Mac G. Donaldson and C.D. Marsden, 1975, Circling behaviour produced by unilateral lesions in the region of the locus coeruleus in rats, Brain Res. 9 7 , 3 1 7 . Rochette, L, and J. Bralet, 1975, Effect of clonidine on the synthesis of cerebral dopamine, Biochem. Pharmacol. 24,303. Spector, S., A. Sjoerdsma and S. Udenfriend, 1965, Blockade of endogenous norepinephrine synthesis by a-methyltyrosine, an inhibitor of tyrosine hydroxylase, J. Pharmacol. Exptl. Therap. 147, 86. StrSmbom, U., 1976, Catecholamine receptor agonists: effects on motor activity and rate of tyrosine hydroxylation in mouse brain, NaunynSchmiedeb. Arch. Pharmacol. 292,167. Zebrowska-Lupina, I. and Z. Kleinrok, 1973, Behavioural effects of yohimbine administered intraventricularly in the rat, Psychopharmacologia (Berlin) 33,267.