Brain Research, 557 (1991) 109-114 ~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116893X
109
BRES 16893
Age effects on monoamine turnover of the rat substantia nigra J.L. Venero, A. Machado and J. Cano Departamento de Bioqufmica, Bromatologfa y Toxicologla, Universidad de SevUla, Sevilla (Spain)
(Accepted 26 March 1991) Key words: Substantia nigra; Aged; Monoamine; Turnover; Rat
Measurement of turnover of dopamine (DA), noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)), and their metabolites has been performed in 6- and 24-month-old rats. Dopamine synthesis in 24-month-old rats did not show any change with respect to 6-month-old rats. However, our results seem to indicate decreased DA release in 24-month-old rats. This hypothesis could be supported by the changes found in the 3-methoxytyramine (3-MT) accumulation rate after monoamine oxidase inhibition with pargyfine. The turnover of NA and its main metabolite, 3-methoxy-4-hydroxyphenylglycol(MHPG) decreased in 24-month-old rats compared with 6-month-old rats. Serotonin synthesis did not change in 24-month-old rats with respect to 6-month-old rats. However, the metabolism of 5-HT quantified as turnover of 5-hydroxy-3-indoleacetic acid (5-HIAA) increased in 24-month-old rats with respect to 6-month-old rats. The monoamine oxidaseB:monoamine oxidase-A ratio increased in 24-month-old rats. The significance of these changes is discussed. INTRODUCTION A large body of experimental evidence indicates that multiple age-related changes occur in different neurotransmitter systems of the mammalian brain 9. Deficiencies in one specific neurotransmitter system occur in the basal ganglia during ageing 1°. It is well established that ageing is associated with changes in striatal dopamine metabolism 3°. The levels of dopamine (DA) and its metabolites have previously been studied in the entire substantia nigra as well as in the pars compacta and the pars reticulata of the substantia nigra in adult rats 12'16'27"29. However, little is known about age-related changes of the neurotransmitters in the substantia nigra. Our laboratory has investigated monoamine levels in the substantia nigra of aged rats 36. These studies show an age-related decline in DA, noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)). As a further step in this analysis, we have studied the turnover rate of these amines, since changes in turnover rate may give a better understanding of the functional activity of monoaminergic neurons than changes in concentration of the amines, which might remain constant or even decline despite an increased rate of synthesis. While there are many studies dealing with the turnover of biogenic amines and their metabolites in the rat corpus striatum, the substantia nigra has been studied much less.
Our work offers more data regarding the turnover of dopamine and its metabolites in the substantia nigra, and supplies additional data on other neurotransmitter systems. However, the main aim of the study is to draw conclusions of the adaptive changes in the biogenic amine turnover taking place during ageing, since an age-related neuronal degenerative process has been reported to occur in the rat substantia nigra 1'2°'22. Turnover of the catecholamines in aged nigrostriatal mice has previously been studied 31. However, this study used another method and considered only DA, N A and 3,4-dihydroxyphenylacetic acid (DOPAC), after injection of methyl-p-tyrosine. We have undertaken a study of the turnover of DA, N A and their metabolites along with the turnover of 5-HT and its main metabofite 5-hydroxy-3-indoleacetic acid (5-HIAA). This approach has been applied to the rat substantia nigra to investigate the metabolism of D A in dendrites in ageing and to compare the results with those obtained in the striatum 3s. We have used non-isotopic methods to estimate the turnover rate of DA, NA and 5-HT as follows: (1) measurement of the rate of biosynthesis of D A and 5-HT based on the accumulation of 3,4-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP) after central decarboxylase inhibition by NSD-10155, or the rate of accumulation of DA, N A and 5-HT after inhibition of monoamine oxidase by pargyline25; (2) determination of D A release as 3-methoxytyramine
Correspondence: J. Cano, Departamento de Bioqu/mica, Bromatologia y Toxicologia, Facultad de Farmacia, C/Prof. Garcia Gonzalez s/n, 41012 Sevilla, Spain. Fax: (34) (5) 4233765.
110 (3-MT)
accumulation
(MAO)
i n h i b i t i o n w i t h p a r g y l i n e ; (3) d e t e r m i n a t i o n o f
rate
after
monoamine
oxidase ! A
D A , N A a n d 5 - H T m e t a b o l i s m as t u r n o v e r o f t h e i r acid metabolites:
DOPAC,
homovanillic
methoxy-4-hydroxyphenylglycol after MAO
acid
(MHPG)
(HVA),
3-
and 5-HIAA
i n h i b i t i o n w i t h p a r g y l i n e 34.
b o
~:
[
,I
d
o
1
MATERIALS AND METHODS
53
Male Wistar rats were studied at the age of 6 and 24 months. The animals were kept under controlled environmental conditions. Food and tap water were allowed ad libitum. The rats were decapitated between 10.00 and 11.00 a.m. and the brains were quickly removed. The mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis of mesencephalon exactly at the caudal border eminence. The two substantiae nigrae were then easily identified and freely dissected. The ventral tegmental area (A10) was not included. The total time for the isolation of the tissues was
109
BRES 16893
Age effects on monoamine turnover of the rat substantia nigra J.L. Venero, A. Machado and J. Cano Departamento de Bioqufmica, Bromatologfa y Toxicologla, Universidad de SevUla, Sevilla (Spain)
(Accepted 26 March 1991) Key words: Substantia nigra; Aged; Monoamine; Turnover; Rat
Measurement of turnover of dopamine (DA), noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)), and their metabolites has been performed in 6- and 24-month-old rats. Dopamine synthesis in 24-month-old rats did not show any change with respect to 6-month-old rats. However, our results seem to indicate decreased DA release in 24-month-old rats. This hypothesis could be supported by the changes found in the 3-methoxytyramine (3-MT) accumulation rate after monoamine oxidase inhibition with pargyfine. The turnover of NA and its main metabolite, 3-methoxy-4-hydroxyphenylglycol(MHPG) decreased in 24-month-old rats compared with 6-month-old rats. Serotonin synthesis did not change in 24-month-old rats with respect to 6-month-old rats. However, the metabolism of 5-HT quantified as turnover of 5-hydroxy-3-indoleacetic acid (5-HIAA) increased in 24-month-old rats with respect to 6-month-old rats. The monoamine oxidaseB:monoamine oxidase-A ratio increased in 24-month-old rats. The significance of these changes is discussed. INTRODUCTION A large body of experimental evidence indicates that multiple age-related changes occur in different neurotransmitter systems of the mammalian brain 9. Deficiencies in one specific neurotransmitter system occur in the basal ganglia during ageing 1°. It is well established that ageing is associated with changes in striatal dopamine metabolism 3°. The levels of dopamine (DA) and its metabolites have previously been studied in the entire substantia nigra as well as in the pars compacta and the pars reticulata of the substantia nigra in adult rats 12'16'27"29. However, little is known about age-related changes of the neurotransmitters in the substantia nigra. Our laboratory has investigated monoamine levels in the substantia nigra of aged rats 36. These studies show an age-related decline in DA, noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)). As a further step in this analysis, we have studied the turnover rate of these amines, since changes in turnover rate may give a better understanding of the functional activity of monoaminergic neurons than changes in concentration of the amines, which might remain constant or even decline despite an increased rate of synthesis. While there are many studies dealing with the turnover of biogenic amines and their metabolites in the rat corpus striatum, the substantia nigra has been studied much less.
Our work offers more data regarding the turnover of dopamine and its metabolites in the substantia nigra, and supplies additional data on other neurotransmitter systems. However, the main aim of the study is to draw conclusions of the adaptive changes in the biogenic amine turnover taking place during ageing, since an age-related neuronal degenerative process has been reported to occur in the rat substantia nigra 1'2°'22. Turnover of the catecholamines in aged nigrostriatal mice has previously been studied 31. However, this study used another method and considered only DA, N A and 3,4-dihydroxyphenylacetic acid (DOPAC), after injection of methyl-p-tyrosine. We have undertaken a study of the turnover of DA, N A and their metabolites along with the turnover of 5-HT and its main metabofite 5-hydroxy-3-indoleacetic acid (5-HIAA). This approach has been applied to the rat substantia nigra to investigate the metabolism of D A in dendrites in ageing and to compare the results with those obtained in the striatum 3s. We have used non-isotopic methods to estimate the turnover rate of DA, NA and 5-HT as follows: (1) measurement of the rate of biosynthesis of D A and 5-HT based on the accumulation of 3,4-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP) after central decarboxylase inhibition by NSD-10155, or the rate of accumulation of DA, N A and 5-HT after inhibition of monoamine oxidase by pargyline25; (2) determination of D A release as 3-methoxytyramine
Correspondence: J. Cano, Departamento de Bioqu/mica, Bromatologia y Toxicologia, Facultad de Farmacia, C/Prof. Garcia Gonzalez s/n, 41012 Sevilla, Spain. Fax: (34) (5) 4233765.
110 (3-MT)
accumulation
(MAO)
i n h i b i t i o n w i t h p a r g y l i n e ; (3) d e t e r m i n a t i o n o f
rate
after
monoamine
oxidase ! A
D A , N A a n d 5 - H T m e t a b o l i s m as t u r n o v e r o f t h e i r acid metabolites:
DOPAC,
homovanillic
methoxy-4-hydroxyphenylglycol after MAO
acid
(MHPG)
(HVA),
3-
and 5-HIAA
i n h i b i t i o n w i t h p a r g y l i n e 34.
b o
~:
[
,I
d
o
1
MATERIALS AND METHODS
53
Male Wistar rats were studied at the age of 6 and 24 months. The animals were kept under controlled environmental conditions. Food and tap water were allowed ad libitum. The rats were decapitated between 10.00 and 11.00 a.m. and the brains were quickly removed. The mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis of mesencephalon exactly at the caudal border eminence. The two substantiae nigrae were then easily identified and freely dissected. The ventral tegmental area (A10) was not included. The total time for the isolation of the tissues was
