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Changes in monoamines and their metabolite levels in substantia nigra of aged rats
Mechanism of Ageing and Developing, 49 (1989)227--233
227
ElsevierScientificPublishersIrelandLtd.
CHANGES IN MONOAMINES AND THEIR METABOLITE LEVELS IN SUBSTANTIA NIGRA OF AGED RATS
J.L. VENERO, A. MACHADO and J. CANO* Departmento de Bioquimica, Facultadde Farmacia, 41012-Sevilla (Spain)
(ReceivedJanuary3rd, 1989)
SUMMARY A decrease in the monoamine and monoamine metabolite contents in the substantia nigra of aged rats vs. controls has been found. Hence, it can be speculated that this decrease may play a role in the multiple alterations in dopaminergic functions observed in aged rats.
Key words: Substantia nigra; Ageing; Biogenic amines; Basal ganglia; HPLC
INTRODUCTION Deficits in specific neurotransmitter systems occur in the basal ganglia during ageing [1]. Most of these deficits have been associated with age-related changes in dopaminergic neurotransmission [1--4]. Such age-related alterations in the basal ganglia are important because of the involvement of these structures in ageing disorders exemplified by Parkinson's disease. The elucidation of changes in particular well-identified neuronal systems is one way to begin a rational investigation into the causes of senile behavioral changes. One such system is the dopaminergic projection of the substantia nigra (SN) to the striatum which has been studied extensively in humans and rats. This pathway may be an appropriate one in which to study age-related alterations in dopamine (DA) metabolism. We have previously reported the age-related changes of biogenic amines and their metabolite content in the striatum of the rat [5]. Therefore, in the present investigation, the SN has been studied in order to establish the regional differences between the SN (cell bodies and dendrites) and striatum (axonal terminals) in DA metabolism during ageing. *To whomall correspondenceshouldbe addressed. 0047-6374/89/$03,50 Printed and Publishedin Ireland
© 1989ElsevierScientificPublishersIrelandLtd.
228 D A release from dendrites in SN has been reported to influence the release of serotonin (5-HT) in various structures of the basal ganglia [6]. A noradrenaline-dopamine interaction has also been suggested [7]. Thus, taking into account the possible interaction among different neurotransmitters monoamines (DA; noradrenaline, N A and 5-NT) and some o f their main metabolites (3-methoxytyramine, 3-MT; 3,4dihydroxyphenylacetic acid, D O P A C ; homovanillic acid, HVA; 5-hydroxyindolacetic acid, 5-HIAA; 5-hydroxytrytophol, 5-Htryptophol; 3-methoxy-4hydroxyphenylglycol, M H P G and normetanephrine, NMN) have been investigated using H P L C - E D . MATERIALSAND METHODS Male Wistar rats were used. They were studied at the age of 6, 12 and 24 months. Animals at all stages were decapitated between 1000 and 1100 h and the whole brain was quickly removed. The mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis o f mesencephalon exactly at the caudal border eminence. The two SN were then easily identified and freely dissected. The ventral tegmental area (A 10) was not included. The total time for the isolation o f the tissues was < 3 min. Analyses o f the measurements of the biogenic amines and their metabolites were performed as previously described [8,9]. Statistical analyses
Behavioural data were treated via analysis o f the variance (Snedecor's F-test). Observed mean differences were evaluated using Tukey's T-test.
TABLE I CONCENTRATIONOF DOPAMINE AND ITS METABOLITESIN THE SUBSTANTIANIGRAOF RATS DURINGAGEING PERIOD Age in months
DA
DOPA C
3-MT
HVA
6
478.3 ± 10.9 (6)
159.4 ± 3.7 (6)
43.9 ± 3.5 (4)
56.1 ± 3.6 (5)
12
473.1 ± 39.5 (5)
83.4 ± 16.1
52.3 ± 3.3
38.7 ± 3.4
(5)**"
(4)
(4)*'
24
328.3 ± 17.6
102.8 ± 6.0
25.9 ± 2.7
17.0 ± 2.1
(4) **'*b
(4)**"
(4)**"
(4)**'**b
Results are givenin ng/g wet tissue as mean ± S.E.M. for the number of animals shownin parentheses. Statistical significance(Tukey T-test): *P< 0.05; **P< 0.01, avs. 6 months; bvs.previous age.
229 TABLE lI CONCENTRATION OF SEROTONIN AND ITS METABOLITES IN THE SUBSTANTIA NIGRA OF RATS DURING AGEING PERIOD Age in months
5-HT
J-HIAA
5-Htryptophol
6
1992.1 ± 57.2 (5)
614.8 ± 20.1 (6)
17.3 ± 1.1 (6)
12
1491.1 ± 84.0 (5)**"
499.9 ± 35.8 (5)*"
(4)
1298.2 ± 84.5 (4)**a
630.5 ± 14.5 (4)*b
20.8 ± 0.6 (4)
24
18.2 ± 2.4
Results are given in ng/g wet tissue as means ± S.E.M. for the number of animals shown in parentheses. Statistical significance(Tukey T-test); *P< 0.05; **P< 0.01. "vs. 6 months; bvs. previous age.
RESULTS Tables I, II a n d III show the levels o f c o n c e n t r a t i o n o f D A , N A a n d 5 - H T a n d their m a i n metabolites i n SN o f the rat at different ages. T h e D A c o n t e n t decreased ( a b o u t 30%) at 24 m o n t h s as c o m p a r e d with 6 a n d 12 m o n t h s (Table I). This c h a n g e shows a decrease o f D A m e t a b o l i s m with age since there is also a decrease i n D O P A C a n d H V A c o n c e n t r a t i o n s , the m a i n metabolites o f D A . A l t h o u g h it has b e e n suggested that D O P A C : D A ratio supports the relationship o f D O P A C to D A t u r n o v e r , o u r results seem to indicate that the fraction o f the released D A which is c o n v e r t e d into D O P A C is the same in aged (24 m o n t h s ) a n d in
TABLE III CONCENTRATION OF NORADRENALINE AND ITS METABOLITES IN THE SUBSTANTIA NIGRA OF RATS DURING AGEING PERIOD A ge in months
?CA
MHPG
202.2 ± 16.3
19.7 ± 0.7
(6)
(5)
(4)
12
245.9 :t: 16.5 (5)**"
167.5 + 12.5 (4)
16.1 ± 1.5 (4)
24
176.1 4- 13.5 (4)**'*b
159.3 ± 14.7 (4)
11.9 ± 1.2 (4)**"
6
376.4 4-
12.5
NMN
Results are given in ng/g wet tissue as means ± S.E.M. for the number of animals shown in parentheses. Statistical significance(Tukey T-test): *P< 0.05; **P< 0.01. "vs. 6 months; bVS. previous age.
230 T A B L E IV R A T I O S B E T W E E N D O P A M I N E A N D ITS M E T A B O L I T E S IN T H E S U B S T A N T I A N I G R A OF THE RAT Age in months
DOPAC:DA
3-MT:DA
HVA:DA
D O P A C + HVA: DA
6 12 24
0.33 O. 17 0.31
0.09 O. 11 0.07
0.11 0.08 0.05
0.45 0.25 0.36
adult rats (6 months). This is consistent with the constant D O PA C: D A ratios in both periods (0.3) (Table IV). On the other hand HVA: DA ratio underwent a linear decrease in all stages analyzed as shown by their correlation coefficient (r = 0.97; P < 0.01) (Table IV). The H V A : D A ratio in 24 months was about 56°70 of that in 6 months. Therefore, DOPAC + H V A ' D A ratio decreased from 6 to 24 months which suggests a decrease of DA turnover during ageing in SN (Table IV). The 3-MT:DA ratio decreased about 15070 in aged rats (24 months) with respect to the control (6 months) (Table IV). This seems to indicate that in aged rats there is a decrease of DA release. Thus, the nigral concentration of 3-MT is thought to reflect the level of DA in the synaptic cleft [10--12]. Changes in the levels of 3-MT rather than DOPAC, are therefore thought to be a more accurate index of changes in DA release. The decrease of DA and its metabolites in SN could be due either to failure of DA synthesis or to a decrease in the number of dopaminergic neurons with age, as a decrease (1307o) in the number of dopaminergic neurons in the SN of aged mice (30 months) has been recently reported (13). However, a third cause for DA decrease could be a mechanism for the local control of the neuronal metabolism since the DA released into the SN seems to have a physiological role. This amine inhibited the activity of dopaminergic ceils by acting on dopaminergic autoreceptors [14]. In the striatum, it has been reported [5] that there are few changes in DA metabolism in aged rats. But in SN we have found that there is a decrease of DA metabolism. Taken together, this opposite dopaminergic behavioral seems to suggest that the dopaminergic cells, by means of changes in the DA release in the body, control the DA metabolism in the terminals. These results are in agreement with previous reports that showed that the release of [3H]DA increased in the caudate nucleus during the nigral application of alpha-methyl-paratyrosine [15]. It can be speculated that during the ageing there could be a compensatory mechanism either of the decrease in number of neurons or of the failure of DA metabolism synthesis. These regional differences also support suggestions that the axonal and dendritic fields of SN neurons regulate DA metabolism differently [16--18]. The 5-HT concentration underwent at 12 and 24 months a decrease of 2507o and 3507o respectively when they were compared with 6 months (Table II). However, 5-HIAA: 5-HT and
231 TABLE V RATIOS BETWEEN SEROTONIN AND EITHER ITS METABOLITES OR DOPAMINE IN THE SUBSTANTIA NIGRA OF THE RAT Age in months
5-HIAA : 5-HI
5-Htryptophol:5-HT
DA :5-HT
6 12 24
0.30 0.33 0.48
0.008 0.012 0.016
0.24 0.31 0.25
5-Htryptophol: HT ratios increased during ageing (Table V). The increase of the 5HT metabolite indexes could indicate an increase either of the MAO activity in the brain with age or a decreased elimination of 5-HIAA from the brain. DISCUSSION
The role of the 5-HT in the SN is not fully understood. Neurons containing 5-HT have been localized within SN by retrogade labelling after injection of [3H]5-HT into the caudate-putamen [19]. Anatomical studies have clearly shown the existence of projections to the SN from the dorsal raphe and median raphe nuclei [20]. In the SN inmunohistochemical studies [21] have reported that the labeled varicosities form either conventional type synapses or non-synaptic varicosities. The symmetrical synapses generally seemed to be associated with inhibitory action. Stimulation of the dorsal raphe nucleus predominantly produces inhibition of spontaneous activity in single neurons of the SN [22]. If 5-HT is released non synaptically it affects surrounding neurons as a modulator. The SN has been reported to be densely innervated with 5-HT fibres [23,24], thus the serotoninergic neuron system may at least partially regulate the SN by modulating the function of its output. There are some authors who have pointed out the importance of the DA: 5-HT ratio in the striatum since motor disturbances in experimental animals may result from a modification of this ratio [25]. In our results the DA:5-HT ratio did not change during the period studied (Table V). An age-related decline in DA and 5-HT, if present in humans and progressive into senescence, could be responsible for the increased risk of developing Parkinson's disease and drug-induced Parkinsonian-like extrapyramidal side effects with age. NA content decreased at 12 and 24 months 35070 and 53070 respectively as compared with 6 months but few changes were found in its metabolites (Table III). Utilization of the micropunch technique has for the first time demonstrated a possible noradrenergic-dopaminerglc interaction in the dopamine cell body-containing region of the substantia nigra pars compacta (A9 group) [26]. In addition, an interesting reciprocal interaction of the cell body v s . the terminal innervation regions has
232
been revealed [26]. A decreased NA concentration in the SN region was correlated with an increased turnover and steady state level of DA in the striatum. This apparent inhibitory noradrenergic - - dopaminergic interaction has been suggested in a variety of experiments [27]. In addition, lessions of the ventral noradrenergic pathway have revealed changes in the DA concentration in several organs [27]. In conclusion, findings of the present study strongly suggest that the monoaminergic system plays an essential role in regulation of the SN. Therefore, due to decreasing of the monoamines during ageing, the SN will be more vulnerable to changes, increasing the risk of disease with age. ACKNOWLEDGEMENTS
This work was supported by CAICYT 1029/84. J.L.V. was a recipient of a predoctoral fellowship from Junta de Andalucia. REFERENCES 1 2 3 4 5 6
7 8 9 10
11
12 13
14
C.E. Finch, P.K. Randall and J.F. Marshall, Aging and basal ganglia functions. Annu. Rev. Gerontol. Geriatr., 2 (1981) 49--87. S. Govoni, P. Loddo, P.F. Spano and M. Trabucchi, Dopamine receptor sensitivity in brain and retina of rats during aging. Brain Res., 138 (1977) 565--570. E.G. McGeer, H.C. Fibiger, P.L. McGeer and V. Wickson, Aging and brain enzymes. Exp. Gerontol., 6 (1971) 391--396. J.P. Walker and J. Boas-Walker, Properties of adenyl cyclase from senescent rat brain. Brain Res., 54 (1973) 391--396. A. Machado, J. Carlo and M. Santiago, The change with age in biogenic amines and their metabolites in the striatum of the rat. Arch. Gerontol. Geriatr., 5 (1986) 333--342. F. H&y, P. Soubri~, S. Bourgoin, J.L. Montastruc, F. Artaud and J. Glowinski, The nigral dendritic release of dopamine controls the local and striatal release of serotonin. Brain Res., 193 (1980) 143--151. S.M. Antelman and A.R. Caggiula, Norepinephrine-dopamine interaction and behavior. Science, 195 (1977) 646--653. M. Santiago, A. Machado and J. Cano, Age-related changes of catecholamines and their metabolires content in the visual system of the rat. Mech. Ageing Dev., 47 (1989) 77--84. M. Santiago, J. Cano, F. Reinoso-Suarez and A. Machado, Age-related changes of serotonin and its metabolites content in the visual system of the rat. Mech. Ageing Dev., 38 (1987) 157--165. B.H.C. Westerink, Effects of drugs on the formation of 3-methoxytyramine, a dopamine metabolite, in the substantia nigra, striatum, nucleus accumbens and tuberculum oifactorium. J. Pharm. Pharmacol., 31 (1979)94--99. B.H.C. Westerink and S.J. Spaan, On the significance of endogenous 3-methoxytyramine for the effects of centrally acting drugs on dopamine release in the rat brain. J. Neurochem., 38 (1982) 680 --686. P.L. Wood, N.P.V. Nair and W. Bozarath, Striatal 3-methoxytyramine as an index of dopamine release: Effects of electrical stimulation. Neurosci. Lett., 32 0982) 291--294. T.H. McNeill, L. Koek and S,N. Haber, Differential effects of aging on neurotransmitter cell loss of the substantia nigra and striatum in mouse and human brain. Soc. Neurosci. Abstr., 14 0988) 1292. A. Nieoullon, A. Cheramy and J. Glowinski, Release of Dopamine in vivo from cat substantia nigra. Nature, 266 (1977) 375--377.
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V. Leviel, A. Cheramy and J. Glowinski, Role of the dendritic release of dopamine in the reciprocal control of the two nigro-striatal dopaminergic pathways. Nature, 280 (1979) 236--239. F. Hefti and W. Lichteusteiger, Dendritic dopamine: studies on the release of endogenous dopamine from subcellular particles derived from dendrites of rat nigro-striatal neurons. Neurosci. Lett., 10 (1978) 65--7O. B.H.C. Westeilnk and J, Korf, Comparison of effects of drugs on dopamine in the substantia nigra and the corpus striatum of rat brain. Eur. J. Pharmacol., 40 (1976) 131-- 136. S.M. Wuerthele, N.M. Friedle and K.E. Moore, Effects of dopaminergic antagonists on striatal DOPAC concentrations and a-methyl-p-tyrosine-induced decline of dopamine following intrastriatal injections of kainic acid. J. Pharm. Pharmacol., 31 (1979) 180--182. P. Streit, Selective retrograde labelling indicating the transmitter of neuronal pathways. J. Comp. Neurol., 191 (1980) 429--463. D. Van der Kooy and T. Hattori, Dorsal raphe cells with collateral projections to the caudate-putamen and substantia nigra: A fluorescent retrogade double labelling study in the rat. Brain Res., 186 (1980) 1--7. S. Moil, T. Matsuura, T. Takino and Y. Sano, Light and electron microscopic immunohistochemical studies of serotonin nerve fibers in the substantia nigra of the rat, cat and monkey. Anat. EmbryoL, 176(1987) 13--18. A. Dray, T.J. Gonye, N.R. Oakley and T. Tanner, Evidence for the existence of a raphe projection to the substantia nigra in rat. Brain Res., 113 (1976) 45--57. S. Moil, S. Ueda, H. Yamada, T. Takino and Y. Sano, Immunohistochemical demonstration of serotonin nerve fibers in the corpus striatum of the rat, cat and monkey. Anat. Embryoi., 173 (1985) 1--5. S. Moil, T. Takino, H. Yamada and Y. Sano, Immunohistochemical demonstration of serotonin nerve fibers in the subthaiamic nucleus of the rat, cat and monkey. Neurosci. Lett., 62 (1985) 305-309. R. Hassler and I.J. Bak, Unbalanced ratios of stilatal dopamine and serotonin after experimental interruption of strionigral connections. In F.J. Gillinghan and I.M.L. Donaldson (eds.), Third Symposium on Parkinson "s Disease, Churchill Livingstone, Edinburgh, London, 1969, pp. 29--37. Y. Tizabi, J. Massari and D.M. Jacobowitz, Isolation induced aggression and catecholamine variations in discrete brain areas of the mouse. Brain Res. Bull., 5 (1980) 81--86. T.L. O'Donohue, W.R. Crowley and D.M. Jacobowitz, Biochemical mapping of the noradrenergic ventral bundle projection sites: evidence for a noradrenergic-dopaminergic interaction. Brain Res., 172 (1979) 87--100.
227
ElsevierScientificPublishersIrelandLtd.
CHANGES IN MONOAMINES AND THEIR METABOLITE LEVELS IN SUBSTANTIA NIGRA OF AGED RATS
J.L. VENERO, A. MACHADO and J. CANO* Departmento de Bioquimica, Facultadde Farmacia, 41012-Sevilla (Spain)
(ReceivedJanuary3rd, 1989)
SUMMARY A decrease in the monoamine and monoamine metabolite contents in the substantia nigra of aged rats vs. controls has been found. Hence, it can be speculated that this decrease may play a role in the multiple alterations in dopaminergic functions observed in aged rats.
Key words: Substantia nigra; Ageing; Biogenic amines; Basal ganglia; HPLC
INTRODUCTION Deficits in specific neurotransmitter systems occur in the basal ganglia during ageing [1]. Most of these deficits have been associated with age-related changes in dopaminergic neurotransmission [1--4]. Such age-related alterations in the basal ganglia are important because of the involvement of these structures in ageing disorders exemplified by Parkinson's disease. The elucidation of changes in particular well-identified neuronal systems is one way to begin a rational investigation into the causes of senile behavioral changes. One such system is the dopaminergic projection of the substantia nigra (SN) to the striatum which has been studied extensively in humans and rats. This pathway may be an appropriate one in which to study age-related alterations in dopamine (DA) metabolism. We have previously reported the age-related changes of biogenic amines and their metabolite content in the striatum of the rat [5]. Therefore, in the present investigation, the SN has been studied in order to establish the regional differences between the SN (cell bodies and dendrites) and striatum (axonal terminals) in DA metabolism during ageing. *To whomall correspondenceshouldbe addressed. 0047-6374/89/$03,50 Printed and Publishedin Ireland
© 1989ElsevierScientificPublishersIrelandLtd.
228 D A release from dendrites in SN has been reported to influence the release of serotonin (5-HT) in various structures of the basal ganglia [6]. A noradrenaline-dopamine interaction has also been suggested [7]. Thus, taking into account the possible interaction among different neurotransmitters monoamines (DA; noradrenaline, N A and 5-NT) and some o f their main metabolites (3-methoxytyramine, 3-MT; 3,4dihydroxyphenylacetic acid, D O P A C ; homovanillic acid, HVA; 5-hydroxyindolacetic acid, 5-HIAA; 5-hydroxytrytophol, 5-Htryptophol; 3-methoxy-4hydroxyphenylglycol, M H P G and normetanephrine, NMN) have been investigated using H P L C - E D . MATERIALSAND METHODS Male Wistar rats were used. They were studied at the age of 6, 12 and 24 months. Animals at all stages were decapitated between 1000 and 1100 h and the whole brain was quickly removed. The mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis o f mesencephalon exactly at the caudal border eminence. The two SN were then easily identified and freely dissected. The ventral tegmental area (A 10) was not included. The total time for the isolation o f the tissues was < 3 min. Analyses o f the measurements of the biogenic amines and their metabolites were performed as previously described [8,9]. Statistical analyses
Behavioural data were treated via analysis o f the variance (Snedecor's F-test). Observed mean differences were evaluated using Tukey's T-test.
TABLE I CONCENTRATIONOF DOPAMINE AND ITS METABOLITESIN THE SUBSTANTIANIGRAOF RATS DURINGAGEING PERIOD Age in months
DA
DOPA C
3-MT
HVA
6
478.3 ± 10.9 (6)
159.4 ± 3.7 (6)
43.9 ± 3.5 (4)
56.1 ± 3.6 (5)
12
473.1 ± 39.5 (5)
83.4 ± 16.1
52.3 ± 3.3
38.7 ± 3.4
(5)**"
(4)
(4)*'
24
328.3 ± 17.6
102.8 ± 6.0
25.9 ± 2.7
17.0 ± 2.1
(4) **'*b
(4)**"
(4)**"
(4)**'**b
Results are givenin ng/g wet tissue as mean ± S.E.M. for the number of animals shownin parentheses. Statistical significance(Tukey T-test): *P< 0.05; **P< 0.01, avs. 6 months; bvs.previous age.
229 TABLE lI CONCENTRATION OF SEROTONIN AND ITS METABOLITES IN THE SUBSTANTIA NIGRA OF RATS DURING AGEING PERIOD Age in months
5-HT
J-HIAA
5-Htryptophol
6
1992.1 ± 57.2 (5)
614.8 ± 20.1 (6)
17.3 ± 1.1 (6)
12
1491.1 ± 84.0 (5)**"
499.9 ± 35.8 (5)*"
(4)
1298.2 ± 84.5 (4)**a
630.5 ± 14.5 (4)*b
20.8 ± 0.6 (4)
24
18.2 ± 2.4
Results are given in ng/g wet tissue as means ± S.E.M. for the number of animals shown in parentheses. Statistical significance(Tukey T-test); *P< 0.05; **P< 0.01. "vs. 6 months; bvs. previous age.
RESULTS Tables I, II a n d III show the levels o f c o n c e n t r a t i o n o f D A , N A a n d 5 - H T a n d their m a i n metabolites i n SN o f the rat at different ages. T h e D A c o n t e n t decreased ( a b o u t 30%) at 24 m o n t h s as c o m p a r e d with 6 a n d 12 m o n t h s (Table I). This c h a n g e shows a decrease o f D A m e t a b o l i s m with age since there is also a decrease i n D O P A C a n d H V A c o n c e n t r a t i o n s , the m a i n metabolites o f D A . A l t h o u g h it has b e e n suggested that D O P A C : D A ratio supports the relationship o f D O P A C to D A t u r n o v e r , o u r results seem to indicate that the fraction o f the released D A which is c o n v e r t e d into D O P A C is the same in aged (24 m o n t h s ) a n d in
TABLE III CONCENTRATION OF NORADRENALINE AND ITS METABOLITES IN THE SUBSTANTIA NIGRA OF RATS DURING AGEING PERIOD A ge in months
?CA
MHPG
202.2 ± 16.3
19.7 ± 0.7
(6)
(5)
(4)
12
245.9 :t: 16.5 (5)**"
167.5 + 12.5 (4)
16.1 ± 1.5 (4)
24
176.1 4- 13.5 (4)**'*b
159.3 ± 14.7 (4)
11.9 ± 1.2 (4)**"
6
376.4 4-
12.5
NMN
Results are given in ng/g wet tissue as means ± S.E.M. for the number of animals shown in parentheses. Statistical significance(Tukey T-test): *P< 0.05; **P< 0.01. "vs. 6 months; bVS. previous age.
230 T A B L E IV R A T I O S B E T W E E N D O P A M I N E A N D ITS M E T A B O L I T E S IN T H E S U B S T A N T I A N I G R A OF THE RAT Age in months
DOPAC:DA
3-MT:DA
HVA:DA
D O P A C + HVA: DA
6 12 24
0.33 O. 17 0.31
0.09 O. 11 0.07
0.11 0.08 0.05
0.45 0.25 0.36
adult rats (6 months). This is consistent with the constant D O PA C: D A ratios in both periods (0.3) (Table IV). On the other hand HVA: DA ratio underwent a linear decrease in all stages analyzed as shown by their correlation coefficient (r = 0.97; P < 0.01) (Table IV). The H V A : D A ratio in 24 months was about 56°70 of that in 6 months. Therefore, DOPAC + H V A ' D A ratio decreased from 6 to 24 months which suggests a decrease of DA turnover during ageing in SN (Table IV). The 3-MT:DA ratio decreased about 15070 in aged rats (24 months) with respect to the control (6 months) (Table IV). This seems to indicate that in aged rats there is a decrease of DA release. Thus, the nigral concentration of 3-MT is thought to reflect the level of DA in the synaptic cleft [10--12]. Changes in the levels of 3-MT rather than DOPAC, are therefore thought to be a more accurate index of changes in DA release. The decrease of DA and its metabolites in SN could be due either to failure of DA synthesis or to a decrease in the number of dopaminergic neurons with age, as a decrease (1307o) in the number of dopaminergic neurons in the SN of aged mice (30 months) has been recently reported (13). However, a third cause for DA decrease could be a mechanism for the local control of the neuronal metabolism since the DA released into the SN seems to have a physiological role. This amine inhibited the activity of dopaminergic ceils by acting on dopaminergic autoreceptors [14]. In the striatum, it has been reported [5] that there are few changes in DA metabolism in aged rats. But in SN we have found that there is a decrease of DA metabolism. Taken together, this opposite dopaminergic behavioral seems to suggest that the dopaminergic cells, by means of changes in the DA release in the body, control the DA metabolism in the terminals. These results are in agreement with previous reports that showed that the release of [3H]DA increased in the caudate nucleus during the nigral application of alpha-methyl-paratyrosine [15]. It can be speculated that during the ageing there could be a compensatory mechanism either of the decrease in number of neurons or of the failure of DA metabolism synthesis. These regional differences also support suggestions that the axonal and dendritic fields of SN neurons regulate DA metabolism differently [16--18]. The 5-HT concentration underwent at 12 and 24 months a decrease of 2507o and 3507o respectively when they were compared with 6 months (Table II). However, 5-HIAA: 5-HT and
231 TABLE V RATIOS BETWEEN SEROTONIN AND EITHER ITS METABOLITES OR DOPAMINE IN THE SUBSTANTIA NIGRA OF THE RAT Age in months
5-HIAA : 5-HI
5-Htryptophol:5-HT
DA :5-HT
6 12 24
0.30 0.33 0.48
0.008 0.012 0.016
0.24 0.31 0.25
5-Htryptophol: HT ratios increased during ageing (Table V). The increase of the 5HT metabolite indexes could indicate an increase either of the MAO activity in the brain with age or a decreased elimination of 5-HIAA from the brain. DISCUSSION
The role of the 5-HT in the SN is not fully understood. Neurons containing 5-HT have been localized within SN by retrogade labelling after injection of [3H]5-HT into the caudate-putamen [19]. Anatomical studies have clearly shown the existence of projections to the SN from the dorsal raphe and median raphe nuclei [20]. In the SN inmunohistochemical studies [21] have reported that the labeled varicosities form either conventional type synapses or non-synaptic varicosities. The symmetrical synapses generally seemed to be associated with inhibitory action. Stimulation of the dorsal raphe nucleus predominantly produces inhibition of spontaneous activity in single neurons of the SN [22]. If 5-HT is released non synaptically it affects surrounding neurons as a modulator. The SN has been reported to be densely innervated with 5-HT fibres [23,24], thus the serotoninergic neuron system may at least partially regulate the SN by modulating the function of its output. There are some authors who have pointed out the importance of the DA: 5-HT ratio in the striatum since motor disturbances in experimental animals may result from a modification of this ratio [25]. In our results the DA:5-HT ratio did not change during the period studied (Table V). An age-related decline in DA and 5-HT, if present in humans and progressive into senescence, could be responsible for the increased risk of developing Parkinson's disease and drug-induced Parkinsonian-like extrapyramidal side effects with age. NA content decreased at 12 and 24 months 35070 and 53070 respectively as compared with 6 months but few changes were found in its metabolites (Table III). Utilization of the micropunch technique has for the first time demonstrated a possible noradrenergic-dopaminerglc interaction in the dopamine cell body-containing region of the substantia nigra pars compacta (A9 group) [26]. In addition, an interesting reciprocal interaction of the cell body v s . the terminal innervation regions has
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been revealed [26]. A decreased NA concentration in the SN region was correlated with an increased turnover and steady state level of DA in the striatum. This apparent inhibitory noradrenergic - - dopaminergic interaction has been suggested in a variety of experiments [27]. In addition, lessions of the ventral noradrenergic pathway have revealed changes in the DA concentration in several organs [27]. In conclusion, findings of the present study strongly suggest that the monoaminergic system plays an essential role in regulation of the SN. Therefore, due to decreasing of the monoamines during ageing, the SN will be more vulnerable to changes, increasing the risk of disease with age. ACKNOWLEDGEMENTS
This work was supported by CAICYT 1029/84. J.L.V. was a recipient of a predoctoral fellowship from Junta de Andalucia. REFERENCES 1 2 3 4 5 6
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