Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 296, 2 5 - 30 (1976)
9 by Springer-Verlag 1976
Effect of (+)Amphetamine on Monoamine Synthesis and Metabolism after Axotomy in Rat Forebrain* W O L F G A N G SPECKENBACH** and W O L F G A N G K E H R Department fiir Neuropsychopharmakologie, Schering AG, Miillerstrasse 170-178, D-1000 Berlin 65
Summary. (+)Amphetamine is known to produce feedback inhibition of catecholamine-containing neurons in the central nervous system due to its indirect receptor stimulating properties. This feedback control may involve postsynaptic receptors and neuronal loops or presynaptic receptors and may be restricted to the catecholaminergic terminal system. In order to study the effect of (+)amphetamine on catecholamine synthesis and metabolism in the terminal system changes in impulse flow were eliminated by cutting the ascending monoaminergic axons. Axotomy of the ascending monoaminergic fibers by means of a complete transverse cerebral hemisection resulted in a 3-fold increase in Dopa formation in the lesioned forebrain during 30 min after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine HC1, 100 mg/kg i.p. (+)Amphetamine sulfate, 10 mg/kg i.p. antagonized the hemisection-induced increase in Dopa formation and reduced the formation of 5-hydroxytryptophan. Pretreatment with haloperidol, 5 mg/kg i.p. failed to counteract the effect of (+)amphetamine. In the intact forebrain the stimulation of Dopa accumulation was more than additive after combined treatment with haloperidol and (+)amphetamine. Hemitransection retarded the disappearance of dopamine and noradrenaline after administration of ~-methyl-p-tyrosine methylester HC1, 300 mg/kg. (+)Amphetamine, 10 mg/kg accelerated the utilization of dopamine on the lesioned side. Hemitransection reduced the formation of 3methoxytyramine during 1 h after pargyline, 75 mg/kg i.p. After (+)amphetamine 3-methoxytyramine formation in the intact forebrain was 3 times higher than in the lesioned forebrain. Send offprint requests to: W. Kehr at the above address * A preliminary report has been given recently (Kehr and Speckenbach, 1975) ** Data of this communication are part W. S.'s thesis for an M.D.
The action of (+)amphetamine on dopamine synthesis and release appears to be dependent on the firing rate in dopamine neurons.
Key words."(+)Amphetamine - Haloperidol - Monoamine synthesis hemitransection.
Dopamine release -
Cerebral
INTRODUCTION (+)Amphetamine is known to indirectly stimulate catecholamine receptors by releasing catecholamines from extragranular storage sites (Carlsson et al., 1966a; Besson et al., 1969) and in high doses by inhibiting the reuptake of catecholamines (Glowinski et al., 1966; Carlsson et al., 1966b). Stimulation of catecholamine receptors induced by (+)amphetamine is known to initiate a neuronal feedback inhibition of catecholamine-containing cells (Corrodi et al., 1967) as indicated by a diminished firing rate of e.g. dopaminergic neurons (Bunney et al., 1973). In order to study the direct effect of (+)amphetamine on monoamine synthesis and catecholamine turnover in the terminal system we eliminated the changes in impulse flow by cutting the ascending monoaminergic fibers. As an extension of a previous communication (Kehr, 1976b) the present study was carried out in order to clarify 1. whether (+)amphetamine could release catecholamines in the absence of impulse flow and 2. whether (+)amphetamine would induce a local feedback mechanism with regard to catecholamine synthesis involving presynaptic or autoreceptors, which have been described previously using a direct receptor agonist (Kehr et al., 1972b; Carlsson, 1975).
26 MATERIAL AND METHODS
A. Drugs and Solutions
Naunyn-Schmiedeberg's Arch. Pharmacol. 296 (1976) adrenaline was analyzed according to Bertler et al. (1958) as modified by Kehr et al. (1976) and dopamine according to Atack (1973).
e) DopamineRelease.Theformationof3-methoxytyramine(3-MT)
The following drugs were used: (+)amphetamine sulfate (Merck, Darmstadt, Germany), haloperidol (Janssen AG, Dfisseldorf, Germany), c~-methyl-p-tyrosine methylester hydrochloride (H44/68, Hhssle AB, Gothenburg, Sweden), pargyline hydrochloride (Abbott, Chicago, U.S.A.), and 3-hydroxybenzylhydrazine hydrochloride (NSD 1015, Sandew Ltd., Harlow, England). Haloperidol was dissolved in a few drops of glacial acetic acid and the final volume was made up with 5.5 ~ glucose solution. All other drugs were dissolved in 0.9 ~ saline solution.
after inhibition of monoamine oxidase with pargyline was taken as an indicator of dopamine release in vivo (Kehr, 1976a). Rats were treated with H 44/68, 400 mg/kg 5 rain prior to pargyline, 75 mg/kg, which was given at the time of hemitransection and the animals were killed by decapitation after another 60 min. After dissection (see above) 3 forebrains were pooled and homogenized in perchloric acid. The time from decapitation until homogenization did not exceed 11/2 min. After cation exchange column chromatography (Atack and Magnusson, 1970; Kehr, 1974a) dopamine was analyzed according to Atack (1973) and 3-methoxytyramine according to Kehr (1974a).
B. Animals and Treatment Schedules
d) Statistics. For comparison of two experimental groups t-test was used. For more than two groups one-way analysis of variance was applied followed by Scheff6-test or Tukey-test.
Male Wistar rats weighing 180-220 g were used. The animals received a standard diet (Herilan | and water ad libitum. All solutions were administered by the intraperitoneal route in a volume of 0.5 ml/100 g bodyweight with randomized allocation of treatment. Doses of basic compounds refer to the salts. Control animals always received the same number of injections of the respective solvent at corresponding time intervals.
C. Animal Operations Axotomy of the ascending monoaminergic fiber system was performed under light ether anaesthesia by means of the complete transverse cerebral hemisection at the level of the caudal hypothalamus as described by Hassler and Bak (1969) and B6dard et al. ' (1972). In sham operated animals a transverse slot was drilled at the middle between lambda and bregma, and the dura mater was split.
D. Biochemical Analyses a) Monoamine Synthesis. The rate limiting steps in the synthesis of catecholamines and 5-HT, i.e. tyrosine and tryptophan hydroxylase, respectively, were evaluated by measuring Dopa and 5-hydroxytryptophan (5-HTP) accumulating after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine (Carlsson et al., 1972). Dopa formation in rat forebrain mainly reflects the synthesis of dopamine, since dopamine is the predominant catecholamine in this brain part (And6n et al., 1966). 3-Hydroxybenzylhydrazine, 100 mg/kg was injected at the time of hemitransection and the rats were killed by decapitation 30 rain later. The brains were quickly taken out, the lesions checked for completeness and correct location and extended symmetrically to the other side. Single left and right forebrains were homogenized in 10.3 ml ice-cold 0.4 N perchloric acid containing 5 mg Na2S205 and 20 mg EDTA. The extracts were passed through a strong cation exchange column (Dowex 50, X-4) according to Kehr et al. (1972a). The following spectrophotofluorimetric analyses were made: tyrosine (Waalkes and Udenfriend, 1957), Dopa (Kehr et al., 1972a), tryptophan (B~dard et al., 1972), 5-HTP (Atack and Lindqvist, 1973).
b) Catecholamine Disappearance after Synthesis Inhibition. The disappearance of dopamine and noradrenaline after inhibition of tyrosine hydroxylase with H 44/68 was taken as an indicator of catecholamine utilization. H 44/68, 300 mg/kg was injected at the time of hemitransection and rats were killed 90 min later. Single forebrains were dissected and homogenized as described above and the extracts were passed through a strong cation exchange column (Dowex 50, X-4) according to Atack and Magnusson (1970). Nor-
RESULTS
Effect of (+)Amphetamine and Haloperidol on Monoamine Synthesis a) Tyrosine Hydroxylation. A x o t o m y o f the a s c e n d i n g m o n o a m i n e r g i c fibers b y m e a n s o f a transverse c e r e b r a l h e m i s e c t i o n a n d s i m u l t a n e o u s i n h i b i t i o n o f the arom a t i c a m i n o acid d e c a r b o x y l a s e resulted in a p r o n o u n c e d a l m o s t 3-fold increase in the f o r m a t i o n o f D o p a in the lesioned f o r e b r a i n (Fig. 1 a). A l s o s h a m o p e r a t i o n c a u s e d a 20 ~o increase in D o p a o n the side o f o p e r a t i o n . (+)amphetamine at a d o s e o f 10 m g / k g a d m i n i s t e r e d 2 rain p r i o r to h e m i s e c t i o n a n t a g o n i z e d the increase in D o p a f o r m a t i o n o n the lesioned side. H o w e v e r , the D o p a level was still 2 times t h a t o f the i n t a c t side. H a l o p e r i d o l , 5 m g / k g , a d m i n i s t e r e d i.p. 90 rain p r i o r to h e m i s e c t i o n led to a 2-fold increase in D o p a o n the i n t a c t side b u t d i d n o t alter the elevated D o p a f o r m a t i o n i n d u c e d b y h e m i s e c t i o n in the lesioned f o r e b r a i n . W h e n given in c o m b i n a t i o n , h a l o p e r i d o l c o u l d n o t c o u n t e r a c t the ( + ) a m p h e t a m i n e - i n d u c e d red u c t i o n o f D o p a in the lesioned f o r e b r a i n . O n the i n t a c t side, the c o m b i n e d a p p l i c a t i o n m a r k e d l y enh a n c e d the D o p a f o r m a t i o n , the increase being 50 ~o h i g h e r t h a n after h a l o p e r i d o l alone. C o n c o m i t a n t d e t e r m i n a t i o n o f t y r o s i n e levels d i d n o t reveal m a r k e d c h a n g e s b y a n y o f the t r e a t m e n t s ( d a t a n o t shown). I n m o s t cases t y r o s i n e levels were slightly higher in the lesioned as c o m p a r e d to the intact forebrain. b) Tryptophan
Hydroxylation. Hemitransection c a u s e d a small (13 ~ ) b u t statistically significant red u c t i o n o f t r y p t o p h a n h y d r o x y l a t i o n in the lesioned f o r e b r a i n as c o m p a r e d to the i n t a c t f o r e b r a i n (Fig. I b). ( + ) A m p h e t a m i n e , 10 m g / k g , led to a further decrease in 5 - H T P f o r m a t i o n o n the lesioned side w h e n given a l o n e o r in c o m b i n a t i o n with h a l o p e r i d o l . H a l o -
W. Speckenbach and W. Kehr: (+)Amphetamine and Monoamines after Axotomy
a
lesioned side
27
intact side
10 ~
I Sham operation 10 ] I
L,~
+
lesionedside
14 ~--~[14
-L
~mp-'~h ~ x-~r~ \
Hal +
t
•L•
,'Y,-
800
600
7 4;0
Hal 5 mg/kg i.p. /
,/
200 Dopa ng/g 200
:~
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 296, 2 5 - 30 (1976)
9 by Springer-Verlag 1976
Effect of (+)Amphetamine on Monoamine Synthesis and Metabolism after Axotomy in Rat Forebrain* W O L F G A N G SPECKENBACH** and W O L F G A N G K E H R Department fiir Neuropsychopharmakologie, Schering AG, Miillerstrasse 170-178, D-1000 Berlin 65
Summary. (+)Amphetamine is known to produce feedback inhibition of catecholamine-containing neurons in the central nervous system due to its indirect receptor stimulating properties. This feedback control may involve postsynaptic receptors and neuronal loops or presynaptic receptors and may be restricted to the catecholaminergic terminal system. In order to study the effect of (+)amphetamine on catecholamine synthesis and metabolism in the terminal system changes in impulse flow were eliminated by cutting the ascending monoaminergic axons. Axotomy of the ascending monoaminergic fibers by means of a complete transverse cerebral hemisection resulted in a 3-fold increase in Dopa formation in the lesioned forebrain during 30 min after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine HC1, 100 mg/kg i.p. (+)Amphetamine sulfate, 10 mg/kg i.p. antagonized the hemisection-induced increase in Dopa formation and reduced the formation of 5-hydroxytryptophan. Pretreatment with haloperidol, 5 mg/kg i.p. failed to counteract the effect of (+)amphetamine. In the intact forebrain the stimulation of Dopa accumulation was more than additive after combined treatment with haloperidol and (+)amphetamine. Hemitransection retarded the disappearance of dopamine and noradrenaline after administration of ~-methyl-p-tyrosine methylester HC1, 300 mg/kg. (+)Amphetamine, 10 mg/kg accelerated the utilization of dopamine on the lesioned side. Hemitransection reduced the formation of 3methoxytyramine during 1 h after pargyline, 75 mg/kg i.p. After (+)amphetamine 3-methoxytyramine formation in the intact forebrain was 3 times higher than in the lesioned forebrain. Send offprint requests to: W. Kehr at the above address * A preliminary report has been given recently (Kehr and Speckenbach, 1975) ** Data of this communication are part W. S.'s thesis for an M.D.
The action of (+)amphetamine on dopamine synthesis and release appears to be dependent on the firing rate in dopamine neurons.
Key words."(+)Amphetamine - Haloperidol - Monoamine synthesis hemitransection.
Dopamine release -
Cerebral
INTRODUCTION (+)Amphetamine is known to indirectly stimulate catecholamine receptors by releasing catecholamines from extragranular storage sites (Carlsson et al., 1966a; Besson et al., 1969) and in high doses by inhibiting the reuptake of catecholamines (Glowinski et al., 1966; Carlsson et al., 1966b). Stimulation of catecholamine receptors induced by (+)amphetamine is known to initiate a neuronal feedback inhibition of catecholamine-containing cells (Corrodi et al., 1967) as indicated by a diminished firing rate of e.g. dopaminergic neurons (Bunney et al., 1973). In order to study the direct effect of (+)amphetamine on monoamine synthesis and catecholamine turnover in the terminal system we eliminated the changes in impulse flow by cutting the ascending monoaminergic fibers. As an extension of a previous communication (Kehr, 1976b) the present study was carried out in order to clarify 1. whether (+)amphetamine could release catecholamines in the absence of impulse flow and 2. whether (+)amphetamine would induce a local feedback mechanism with regard to catecholamine synthesis involving presynaptic or autoreceptors, which have been described previously using a direct receptor agonist (Kehr et al., 1972b; Carlsson, 1975).
26 MATERIAL AND METHODS
A. Drugs and Solutions
Naunyn-Schmiedeberg's Arch. Pharmacol. 296 (1976) adrenaline was analyzed according to Bertler et al. (1958) as modified by Kehr et al. (1976) and dopamine according to Atack (1973).
e) DopamineRelease.Theformationof3-methoxytyramine(3-MT)
The following drugs were used: (+)amphetamine sulfate (Merck, Darmstadt, Germany), haloperidol (Janssen AG, Dfisseldorf, Germany), c~-methyl-p-tyrosine methylester hydrochloride (H44/68, Hhssle AB, Gothenburg, Sweden), pargyline hydrochloride (Abbott, Chicago, U.S.A.), and 3-hydroxybenzylhydrazine hydrochloride (NSD 1015, Sandew Ltd., Harlow, England). Haloperidol was dissolved in a few drops of glacial acetic acid and the final volume was made up with 5.5 ~ glucose solution. All other drugs were dissolved in 0.9 ~ saline solution.
after inhibition of monoamine oxidase with pargyline was taken as an indicator of dopamine release in vivo (Kehr, 1976a). Rats were treated with H 44/68, 400 mg/kg 5 rain prior to pargyline, 75 mg/kg, which was given at the time of hemitransection and the animals were killed by decapitation after another 60 min. After dissection (see above) 3 forebrains were pooled and homogenized in perchloric acid. The time from decapitation until homogenization did not exceed 11/2 min. After cation exchange column chromatography (Atack and Magnusson, 1970; Kehr, 1974a) dopamine was analyzed according to Atack (1973) and 3-methoxytyramine according to Kehr (1974a).
B. Animals and Treatment Schedules
d) Statistics. For comparison of two experimental groups t-test was used. For more than two groups one-way analysis of variance was applied followed by Scheff6-test or Tukey-test.
Male Wistar rats weighing 180-220 g were used. The animals received a standard diet (Herilan | and water ad libitum. All solutions were administered by the intraperitoneal route in a volume of 0.5 ml/100 g bodyweight with randomized allocation of treatment. Doses of basic compounds refer to the salts. Control animals always received the same number of injections of the respective solvent at corresponding time intervals.
C. Animal Operations Axotomy of the ascending monoaminergic fiber system was performed under light ether anaesthesia by means of the complete transverse cerebral hemisection at the level of the caudal hypothalamus as described by Hassler and Bak (1969) and B6dard et al. ' (1972). In sham operated animals a transverse slot was drilled at the middle between lambda and bregma, and the dura mater was split.
D. Biochemical Analyses a) Monoamine Synthesis. The rate limiting steps in the synthesis of catecholamines and 5-HT, i.e. tyrosine and tryptophan hydroxylase, respectively, were evaluated by measuring Dopa and 5-hydroxytryptophan (5-HTP) accumulating after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine (Carlsson et al., 1972). Dopa formation in rat forebrain mainly reflects the synthesis of dopamine, since dopamine is the predominant catecholamine in this brain part (And6n et al., 1966). 3-Hydroxybenzylhydrazine, 100 mg/kg was injected at the time of hemitransection and the rats were killed by decapitation 30 rain later. The brains were quickly taken out, the lesions checked for completeness and correct location and extended symmetrically to the other side. Single left and right forebrains were homogenized in 10.3 ml ice-cold 0.4 N perchloric acid containing 5 mg Na2S205 and 20 mg EDTA. The extracts were passed through a strong cation exchange column (Dowex 50, X-4) according to Kehr et al. (1972a). The following spectrophotofluorimetric analyses were made: tyrosine (Waalkes and Udenfriend, 1957), Dopa (Kehr et al., 1972a), tryptophan (B~dard et al., 1972), 5-HTP (Atack and Lindqvist, 1973).
b) Catecholamine Disappearance after Synthesis Inhibition. The disappearance of dopamine and noradrenaline after inhibition of tyrosine hydroxylase with H 44/68 was taken as an indicator of catecholamine utilization. H 44/68, 300 mg/kg was injected at the time of hemitransection and rats were killed 90 min later. Single forebrains were dissected and homogenized as described above and the extracts were passed through a strong cation exchange column (Dowex 50, X-4) according to Atack and Magnusson (1970). Nor-
RESULTS
Effect of (+)Amphetamine and Haloperidol on Monoamine Synthesis a) Tyrosine Hydroxylation. A x o t o m y o f the a s c e n d i n g m o n o a m i n e r g i c fibers b y m e a n s o f a transverse c e r e b r a l h e m i s e c t i o n a n d s i m u l t a n e o u s i n h i b i t i o n o f the arom a t i c a m i n o acid d e c a r b o x y l a s e resulted in a p r o n o u n c e d a l m o s t 3-fold increase in the f o r m a t i o n o f D o p a in the lesioned f o r e b r a i n (Fig. 1 a). A l s o s h a m o p e r a t i o n c a u s e d a 20 ~o increase in D o p a o n the side o f o p e r a t i o n . (+)amphetamine at a d o s e o f 10 m g / k g a d m i n i s t e r e d 2 rain p r i o r to h e m i s e c t i o n a n t a g o n i z e d the increase in D o p a f o r m a t i o n o n the lesioned side. H o w e v e r , the D o p a level was still 2 times t h a t o f the i n t a c t side. H a l o p e r i d o l , 5 m g / k g , a d m i n i s t e r e d i.p. 90 rain p r i o r to h e m i s e c t i o n led to a 2-fold increase in D o p a o n the i n t a c t side b u t d i d n o t alter the elevated D o p a f o r m a t i o n i n d u c e d b y h e m i s e c t i o n in the lesioned f o r e b r a i n . W h e n given in c o m b i n a t i o n , h a l o p e r i d o l c o u l d n o t c o u n t e r a c t the ( + ) a m p h e t a m i n e - i n d u c e d red u c t i o n o f D o p a in the lesioned f o r e b r a i n . O n the i n t a c t side, the c o m b i n e d a p p l i c a t i o n m a r k e d l y enh a n c e d the D o p a f o r m a t i o n , the increase being 50 ~o h i g h e r t h a n after h a l o p e r i d o l alone. C o n c o m i t a n t d e t e r m i n a t i o n o f t y r o s i n e levels d i d n o t reveal m a r k e d c h a n g e s b y a n y o f the t r e a t m e n t s ( d a t a n o t shown). I n m o s t cases t y r o s i n e levels were slightly higher in the lesioned as c o m p a r e d to the intact forebrain. b) Tryptophan
Hydroxylation. Hemitransection c a u s e d a small (13 ~ ) b u t statistically significant red u c t i o n o f t r y p t o p h a n h y d r o x y l a t i o n in the lesioned f o r e b r a i n as c o m p a r e d to the i n t a c t f o r e b r a i n (Fig. I b). ( + ) A m p h e t a m i n e , 10 m g / k g , led to a further decrease in 5 - H T P f o r m a t i o n o n the lesioned side w h e n given a l o n e o r in c o m b i n a t i o n with h a l o p e r i d o l . H a l o -
W. Speckenbach and W. Kehr: (+)Amphetamine and Monoamines after Axotomy
a
lesioned side
27
intact side
10 ~
I Sham operation 10 ] I
L,~
+
lesionedside
14 ~--~[14
-L
~mp-'~h ~ x-~r~ \
Hal +
t
•L•
,'Y,-
800
600
7 4;0
Hal 5 mg/kg i.p. /
,/
200 Dopa ng/g 200
:~
