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Changes in monamines and their metabolite levels in some brain regions of aged rats
Neurobiology of Aging, Vol. 3, pp. 23-29, 1982. Printed in the U.S.A.
Changes in Monamines and Their Metabolite Levels in Some Brain Regions of Aged Rats FRANCA PONZIO,* GABRIELLA CALDERINI,I" GIUSEPPE LOMUSCIO,* GUIDO VANTINI,* GINO TOFFANO~f AND SERGIO ALGER1.1
*Istituto di Ricerche Farmacologiche " M a r i o N e g r i , " Via Eritrea, 62-20157 Milan, Italy tFidia Research Laboratories, 35031 A b a n o Terrne, Italy R e c e i v e d 7 A u g u s t 1981 PONZIO, F., G. CALDERIN1, G. LOMUSCIO, G. VANTINI, G. TOFFANO AND S. ALGERI. Changes in monoamines and their metabolite levels in some brain regions of aged rats. NEUROBIOL. AGING 3(1) 23-29, 1982.The concentrations of dopamine (DA), norepinephrine (NE), serotonin (5HT) and their metabolites, HVA, DOPAC, MHPG-SO4 and 5HIAA were measured in several brain areas of rats aged 4, 18 and 29 months. Dopamine and its metabolites showed a decline, statistically correlated with age, in all the dopaminergic areas considered, indicating that this system is profoundly affected in the senescent rat. The changes in the noradrenergic system were more complex. This neurotransmitter was reduced in spinal cord and in limbic area, but was not modified in hippocampus, cerebellum, striatum and s. nigra. In cortex, MHPG-SO,, the main NE metabolite, showed a significantly age-related increase. Tyrosine hydroxylase (TH) activity was low in striatum, and brainstem but not in hypothalamus of aged rats. Neither 5HT nor its metabolites was affected by age. The results indicate that central catecholaminergic systems are markedly affected in senescent rats. Aging, effect in CNS Aging, effect on dopamine Aging, effect on norepinephrine Aging, effect on tyrosine hydroxylase Aging, effect on serotonin
A G I N G is associated with a progressive deterioriation of the nervous system. There are several physiological, cytological and biochemical parameters that are altered by aging processes and may therefore cause malfunctioning of the nervous system. Alterations affecting neurotransmission mechanisms are particularly important in view of the neuronal network's role in coordinating the body's relationships with its internal and external environment. Levels and metabolism of neurotransmitters are important biochemical parameters. Alteration of these parameters may indicate changes in neuronal firing or changes in neuron numbers. As noted in a recent review [19] age is accompanied by significant changes in the catecholaminergic and serotonergic systems. Brain catecholamine levels and/or synthesis have been reported to be decreased in striatum brainstem and hypothalamus [1,5, 12, 13] of senescent rats and mice [6, 7, 16, 21] and the activity of tyrosine hydroxylase, the enzyme controlling catecholamine synthesis, was low in homogenates prepared from brains of aged animals, including man [1, 3, 12, 13]. Histofluorescence intensity due to catecholamines is also reportedly low in the substantia nigra and locus coeruleus of aged monkeys [22] and Fisher 344 rats [23]. Serotonin synthesis, on the other hand, was enchanced in the hypothalamus of old rats [21].
Most of the data accumulated so far has been obtained in a limited number of cerebral regions and is generally based on the comparison of two age groups usually at the lower and upper ends of the iifenspan; information relating to intermediate ages is lacking. However, such information is very important in a phenomenon such as senescence which is probably the result of gradual rather than sudden changes. In the experiments presented here we measured simultaneously the levels of dopamine, norepinephrine and serotonin and/or their metabolites in several different brain areas of rats aged 4, 18 and 29 months. Many of these areas have not been investigated before. We checked whether there was any possible correlation between the observed changes and age. The activity of tyrosine hydroxylase was also studied in order to verify the age-dependent modification, of this enzyme that has been previously reported [1, 3, 13, 21]. METHOD
Animals Male Sprague Dawley rats of different ages were obtained from Bio Breeding Laboratories (Beaconsfield, Quebec, Canada). A first shipment of two groups of rats aged l and 12 months was received. These animals were kept for 12 months in our animal house under standard controlled con-
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24 ditions of temperature, humidity and dark-light cycles (12 hr of light each day from 7 a.m.-7 p.m.). They were housed in Makrolon cages (2--4 rats/cage) with free access to water and standard rat chow (Altromin pellets). Rats tend to accumulate fat with age. As we intended to use part of this rat colony to study the effect of cold exposure in the different classes of age (paper submitted for publication) we put them on a restricted diet in order to minimize the possible interference of subcutaneous adipose layers of different thickness. After 12 months both groups of rats were put on a restricted diet consisting of only 50% (15 g/day) of the chow they consumed when fed ad lib (30 g/day). They were kept on this dietary regimen for 5 months until the day of the experiment. During the first 3 months of restricted diet the rats gradually lost weight. In one group the mean body weight fell from 543___10 g at 24 to 467___14 at 27 months of age in the second group from 468-+10 at 13 to 445___10at 16 months of age. After this period until the day of the experiment, body weight remained constant. Two months before the experiment a group of 2month-old rats was received. They too were put on a restricted diet for 2 months. During this period their body weight rose from 302±9 g to 382+-10 g. At the time of the study, therefore, we could compare three age groups, one 4 months old (382_+10 g), one 18 months old (435+-10 g) and one 29 months old (446_+13 g). This enabled us to minimize differences in body weight due to adipose tissue, without affecting normal body growth. The difference in body weight between the young and the two groups of aged rats reached the level of statistical significance (p <0.01) but as it still only amounted to about+- 15% it should not have any biological relevance. The rats lifespan may also have been prolonged as reported by some investigators [8,26]. In a previous pilot experiment we had assessed the possible effects of restricted diet on organs wet weight and on the concentrations of monoamine and their metabolites and on tyrosine hydroxylase (TH) activity. No differences were found in these parameters in restricted and ad lib feed groups (data not reported). Rats with visible tumors (lipomas and adenomas) were discarded. The percentage of survival was 100 in the 4-months group, 91 for the 18-month and 55 for the 29-month animals. Rats were killed by guillotine, alternating the three age groups. Brains were rapidly excised, put on a cold plate and dissected into limbic area (olfactory tubercola, n. accumbens), striata, hippocampus, s. nigra, cerebral cortex, lower brainstem) Pons and medulla), hypothalamus and cerebellum. The spinal cord was taken by opening the spinal column. The samples were immediately frozen on dry ice and kept at -80°C until assayed. Biochemical Methods Catecholamine assay. Endogenous norepinephrine ('NE) and dopamine (DA) were determined using high pressure liquid chromatography with electrochemical detection (LCEC) according to Keller et al. [10]. The tissue was extracted with 320/zl 0.1 M perchloric acid containing 27-520 pmoles of a-methyl-dopamine (internal standard) using a Branson B30 sonifier. After an AlcOa prepuritication step, the extracted catecholamines were determined by LCEC. The values were expressed as ng/g wet weight of the tissue, based on internal standard measurements. 5-Hydrox?,'tryptamine (5-HT) assay. 5-HT was deter-
PONZIO £ 7 A~' mined using the LCEC-technique according to Ponzio and Jonsson [18]. Frozen tissue samples were homogenized in 6 vol (w/v) acidified n-butanol and centrifuged. Then 80/.tt of the supernatant was added to 200 tzl heptane and 25 ¢zl of 0. I M perchloric acid. This mixture was shaken and after removal of the organic phase and washing with chloroform, 10 /xl of the acid phase was injected into the liquid chromatograph. The values obtained were corrected for 5-HT recovery (80--90%), determined in separate samples, and expressed as ng/g wet weight of the tissue. Monoamine metabolite assay. Endogenous homovanillic acid (HVA), dihydroxy-phenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIAA) levels were determined using high pressure liquid chromatography with electrochemical detection (LCEC) according to Ponzio and Jonsson [17]. Frozen tissue samples were homogenized in 10 vol (w/v) 0.4 M perchioric acid and centrifuged. The supernatant was extracted with ethylacetate. The final extraction was with 0.35 M sodium acetate (pH 8.1-8.2) and 10 ~1 of the aqueous layer were utilized for the assay. The values obtained were corrected for HVA, DOPAC, 5HIAA recoveries (60-75%), determined in separate samples, and expressed as ng/g wet weight of the tissue. Norepinephrine metabolite assay. Endogenous 3-methoxy4-hydroxy-phenyl glycol-sulphate (MHPG-SO4) levels were determined spectrophotofluorometrically according to Meek and Neff [15]. Frozen tissues were homogenized in 4 volumes of 0.2 M ZnSO4 and 4 volumes of 0.2 M Ba(OH)2 and centrifuged. The supernatant was poured onto a DEAE Sephadex column to isolate MHPG-SO4, which was eluted with 5.5 ml of 0.06 N HCI. Fluorescence was measured at 465 nm after adding ethylene-diamine. Tyrosine hydroa3'lase activity assay. Tyrosine hydroxylase activity was measured according to the method of Waymire eta/. [25] based on the production, trapping and determination of ~4CO2from [carboxyl 14C] L-DOPA formed from p4C] l-tyrosine and developed by DOPA decarboxytase added to the medium. Enzyme activity was expressed per unit of protein. RESULTS Modification of DA Metabolism in Some Brain Areas of Aging Rats HVA levels in the striatum were reduced but not significantly so, but otherwise concentrations of DA and its metabolites HVA and DOPAC were significantly reduced with age in the three brain areas examined (Fig. 1). Generally the decrease was more pronounced in the group of oldest rats, and for striata and limbic area a significant correlation was found between age and decline of DA and both its metabolites. In the s. nigra the decline of DA and DOPAC was already maximal at 18 months of age (Fig. 1). Modifications of NE Metabolism in Some Areas o f Aging Rats Age-related changes in NE concentration were studied in the limbic area, hippocampus, cerebellum, spinal cord, striata and s. nigra. Changes in MHPG-SO, in cortex were also determined (Fig. 3). All these areas receive adrenergic innervation whose extent varies according to the region considered. Unlike DA and its metabolites, whose levels were constantly decreased with age, NE showed a more complex pattern of variation. In the limbic area and spinal
BRAIN M O N O A M I N E S IN A G E D RAT BRAIN
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FIG. 1. The existence of a concentration-age correlation was tested by a linear regression analysis. In the cases this is present the degrees of the significance for the F or correlation, the r values, and the degree of significance of the linearity are given. Statistical significance of the differences between groups was analyzed by Duncan's test. **p<0.01 vs 4 months; O p<0.01 vs 18 months.
cord there was a lowering in NE concentration, significantly correlated with age in the first area (Fig. 2); in the hippocampus, striata, s. nigra and cerebellum no modification was evident. MHPG-SO~, an index of NE turnover, tended to be significantly increased with age, the levels of this metabolite in the cortex of 29-month-old rats being 51% and 30% higher respectively than in 4- and 18-month-old rats.
Tyrosine Hydroxylase Activity in Some Brain Areas of Aging Rats TH activity measured in striata, hypothalamus and brainstem of rats in the three age groups is summarized in Fig. 4. The activity of this enzyme in hypothalamus prepara-
tions did not differ in the different age groups but it was lower in striata and brainstems of 18 and 29 month-old rats. In striata of the 18-months group the enzyme activity was reduced to 68% of the activity present in the tissue of young animals, but was not any lower in the oldest group. In brainstem the decline was more gradual and was lowest in the 29-month-old rats where it was 69% of that measured in young rats.
Modification of 5-HT Metabolism in Some Brain Areas of Aging Rats The effect of age on serotonin was studied determining the concentration of 5HT and its metabolite 5HIAA in hip-
26
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FIG. 2. Concentrations of NA in some noradrenergic brain areas of young and old rats. Data are the mean±S.E. of 30 samples. The significance of the concentration-age correlation was tested by linear regression analysis~ The correlation is significant in spinal cord and limbic area. Statistical significance of the differences between groups was analyzed by Duncan's new multiple test. **p<0.01 vs 4 months; *p<0.05 vs 18 months.
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FIG. 3. Concentration of MHPG-SO, in cerebral cortex of young and old rats. Data are the mean-S.E, of 30 samples. The significance of the concentration-age correlation was tested by linear regression analysis. The significance of the differences between groups was analyzed by Duncan's new multiple test. **p<0.0l vs 4 months; *p<0.05 vs 18 months.
pocampus, cerebellum and spinal cord. Levels of 5HIAA were also determined in limbic area and s. nigra. None of the areas considered presented age-dependent modifications in the levels of 5HT or its metabolite (Fig. 5).
The results in this sereis of experiments confirm that aging does cause modifications to the rat central nervous system. The concentrations of some monoamines and their metabolites in different brain areas of three different age groups of rats indicate that senescence has complex effects on the normal state of some neurotransmitters causing quantitative differences that vary according the neurotransmitter and the area considered. Dopamine was the neurotransmitter most affected by aging. In all areas considered, this monoamine and its acid metabolites were decreased at 18 and 29 months of age compared to 4-month-old rats. This decrease was generally sig, nificantly correlated with age. Only in s. nigra was DA maximally decreased already at 18 months of age. In previous experiments with Wistar rats [2,16] we found no change in DA concentration, but there was a significant reduction in the synthesis of this amine in striata and hypothalamus of 36-month-old rats [2,16]. In contrast, in the rat population we studied this time, DA concentration showed a clear age-related decrease in all the brain areas investigated. DA hystofiuorescence was also reduced in perikarya of old Fisher 344 rats [23] and a similar phenomenon has been observed in the monkey brain [22]. These conflicting results underline the importance of the
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FIG. 4. Activity of TH tested in some brain areas of young and old rats. Data are the mean ~ S.E. of 6 samples. Statistical significance was analyzed by Duncan's new multiple test. *,o<0.05 vs controls; **p<0.01 vs controls. possible role of interspecific and inter-strain differences, in the age-related decline of normal steadystate level of DA, suggesting that the vulnerability to aging of certain neuronal systems may be under genetic control. Here together with the decline in the parent monoamine a parallel decline of the acid metabolites was evident. It is difficult to establish whether we are dealing with decreased synthesis and turnover of the neurotransmitter or with degeneration of dopaminergic neurons. If there is a degeneration of dopaminergic neurons, then the parallel decrease of HVA and DOPAC suggests that the remaining neurons cannot compensate this loss by increased turnover of the transmitter. Aging would therefore result in a less efficient dopaminergic tonus. In these rats we have observed that in conditions of increased DA turnover, as in the case of stress [4, 11, 24]. striatal HVA rose much less in old than in young rats, indicating lower turnover of DA [3], on the same lines as results from a different methodological approach, in a different rat strain [2,16], or in a different rodent species [6,7]. An indication of depressed dopaminergic synthesis was also derived from TH activity values in the striata of aged" rats (Fig. 3); in agreement with similar results obtained previously in our and other laboratories [l, 12, 14], this was lower than in young controls. As reported by Reis e t a / . [20] aging seems to affect this enzyme differently in CNS and in peripheral adrenergic tissues. While TH activity is very markedly increased in sympathetic ganglia [20] and in adrenal medulla [3,20] it is only slight decreased ( - 3 0 % ) in striata. In contrast with Reis' findings in Fisher 344 rats [20] we detected a significant reduction in TH activity in the brainstem an area where cell bodies of catecholaminergic neurons are located. In this case too strain differences are at the basis of these different i'mdings. While a constant decline of DA and its metabolite was found in all the brain areas of old rats, the changes brought about by age in the noradrenergic system were more corn-
plex. In most of the areas considered, such as hippocampus, cerebellum, striata and s. nigra, the levels of this amine were not changed, but they were significantly reduced in limbic area and spinal cord (Fig. 2). In another rat strain we have previously found a decrease of N E levels in pons and medulla oblungata of aged rats [2,16]. Tyrosine hydroxylase in brainstem, but not in hypothalamus was also found to be reduced by age, as the present results confirm (Fig. 4). The noradrenergic nuclei from which the dorsal and ventral bundles projecting to the spinal cord and the various telencephalic structures originate are in the brainstem [9]. As our data indicate, it is possible that agining causes degeneration or reduces the activity of the fibers in some selected noradrenergic pathways. It was interesting to find that aging did not always lower the concentrations of catecholamine metabolites. In the cortex in fact we noted an age-related increase in MHPG-SO4, but whether this is an indication of increased N E turnover has yet to be clarified by different methodological approaches. The biological significance of this phenomenon is also still obscure. Simpkins et al. reported that 5HT and 5 H I A A concentrations were not changed in the brain of aged rats except in the hypothalamus where there was a slight but significant increase in 5 H I A A [21]. In the experiment reported here we measured 5HT and its metabolite in several brain areas (but not the hypothalamus which was used for TH assay) and we also found no changes. The same authors reported however, and we have confirmed in other experiments (paper in preparation), that the turnover of this monoamine appears to be increased in the brain of aged rats. It is thus a possibility that in balance between dopaminergic and serotonergic system may be one aspect of aging of the brain. The lack of modification in 5 H I A A suggests that the turnover rate of 5HT also is not changed. However in this regard the parameter must be studied in conditions of changed serotonergic functionality to give a better insight into the questions of the effect of age on the serotonergic system.
PONZIO EY At.
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FIG. 5. Concentration of 5 H T and 5 H I A A in some serotonergic areas of young and old rats. 5 H T data are the mean ± S.E. of 30 samples for each group of age. 5 H I A A data are the mean ± S.E. of 6 samples for each group of age. N o significant difference is present (Duncan's test).
BRAIN MONOAMINES
IN AGED RAT BRAIN
29
ACKNOWLEDGEMENTS This work was supported by grants from the Gustavus and
Louise Pfeiffer Foundation, Los Angeles, CA; CARIPLO, Milan, Italy; Fidia Res. Lab., Abano Terme, Padua, Italy and by Contract No. 79.02355.65 from Italian National Research Council (CNR).
REFERENCES i. Algeri, S., M. Bonati, N. BruneUo and F. Ponzio. Dihydropteridine reduetase and tyrosine hydroxylase activities in rat brain during development and senescence: A comparative study. Brain Res. 132: 569-574, 1977. 2. Algeri, S., M. Bonati, N. Brunello, F. Ponzio, G. Stramentinoli and M. Gualano. Biochemical changes in central catecholaminergic neurons of the senescent rats. In: NeuroPsychopharmacology, edited by P. Deniker, C. RadoucoThomas and A. Villeneuve. Oxford: Pergamon Press, 1978, pp. 1647-1654. 3. Algeri, S., G. Calderini, G. Lomuscio, G. Toffano and F. Ponzio. Catecholamines and adaptive mechanisms in senescent rats. Presented at Proceedings in Clinical Pharmacology of Apomorphine and other Dopaminomimetics. Villasimius, Cagliari, Sept. 28-Oct. 3, 1980. 4. Avakian, E. V. and S. M. Horvath. Plasma catecholamine responses to tyrosine hydroxylase inhibition and cold exposure. Life Sci. 26: 1691-1696, 1980. 5. Estes, K. S. and J. W, Simpkins. Age-related alterations in catecholamine concentrations in discrete preoptic area and hypothalamic regions in the male rat. Brain Res. 194: 556--569, 1980. 6. Finch, C. E. Catecholamine metabolism in the brains of ageing male mice. Brain Res. 52: 261-276, 1973. 7. Finch, C. E. Age-related changes in brain catecholamines; A synopsis of findings in C57BL/6J mice and other rodent models. Adv. exp. Med. Biol. 113: 15-39, 1978. 8. Forbes, W. B. Dietary restriction, longevity and CNS aging. In: The Psychobiology of Aging: Problems and Perspective, edited by E. D. Stein. New York: Elsevier, 1980, pp. 145-160. 9. Fuxe, K. Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol. scand. 64: Suppl. 247, 37, 1965. 10. Keller, R., A. Oke, I. Mefford and R. N. Adams. Liquid chromatographic analysis of catecholamines routine assay for regional brain mapping. Life Sci. 19: 995--1004, 1976. I1. Kvetnansky, R., M. Palkovits, A. Mitro, T, Torda and L. Mikulaj. Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats. Neuroendocrinology 23: 257-267, 1977. 12. McGeer, E. G., P. L. McGeer and J. A. Wada. Distribution of tyrosine hydroxylase in human and animal brain. J. Neurochem, 18: 1647-1658, 1971.
13. McGeer, P. L. and E. G. McGeer. Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. J. Neurochem. 26: 65-76, 1976. 14. McNamara, M. C., A. T. Miller, Jr., V. A. Benignus and J. N. Davis. Age related changes in the effect of electroconvulsive shock (ECS) on the in vivo hydroxylation of tyrosine and tryptophan in rat brain. Brain Res. 131: 313-320, 1977. 15. Meek, J. L. and N. H. Neff. Fluorometric estimation of 4-hydroxy-3-methoxy-phenylethyleneglycol sulphate in brain. Br. £. Pharmac. 45: 435-441, 1972. 16. Ponzio, F., N. Bruneilo and S. Algeri. Catecholamine-synthesis in brain of ageing rat. J. Neurochem. 30: 161%1620, 1978. 17. Ponzio, F. and G. Jonsson. Effects of neonatal 5,7dihydroxytryptamine treatment on the development of serotonin neurons and their transmitter metabolism. Devl Neurosci. 1: 80-89, 1978. 18. Ponzio, F. and G. Jonsson. A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection, d. Neurochem. 32: 129-132, 1979. 19. Pradhan, S. N. Central neurotransmitters and aging. Life Sci. 26: 1643--1656, 1980. 20. Reis, D. J., R. A. Ross and T. H. Joh. Changes in the activity and amounts of enzymes synthesizing catecholamines and acetylcholine in brain, adrenal medulla, and sympathetic ganglia of aged rat and mouse. Brain Res. 136: 465--474, 1977. 21. Simpkins, J. W., G. P. MueUer, H. H. Huang and J. Meites. Evidence for depressed catecholamine and enhanced serotonin metabolism in aging male rats: Possible relation to gonadotropin secretion. Endocrinology 100: 1672-1678, 1977. 22. Sladek, J. R. and C. D. Sladek. Relative quantitation of monoamine histofluorescence in young and old non-human primates. Adv. exp. Med. Biol. 113: 231-'240, 1979. 23. Sladek, J. R. and B. C. Blanchard. Age-related declines in perikaryal monoamine histofluorescence in the Fischer 344 rat. ln: Brain Neurotransmitters and Receptors in Aging and AgeRelated Disorders, edited by S. J. Enna, T. Samorajski and B. Beer. New York: Raven Press, 1981, pp. 13-21. 24. Thoenen, H. Induction of tyrosine hydroxylase in peripheral and central neurones by cold-exposure of rats. Nature 228: 861-862, 1970. 25. Waymire, J. C., R. Bjur and N. Weiner. Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from l-~4C-L-tyrosine. Analyt. Biochem. 43: 588-600, 1971. 26. Young, V. R. Diet as a modulator of aging and longevity. Fedn Proc. 38: 1994-2000, 1979.
Changes in Monamines and Their Metabolite Levels in Some Brain Regions of Aged Rats FRANCA PONZIO,* GABRIELLA CALDERINI,I" GIUSEPPE LOMUSCIO,* GUIDO VANTINI,* GINO TOFFANO~f AND SERGIO ALGER1.1
*Istituto di Ricerche Farmacologiche " M a r i o N e g r i , " Via Eritrea, 62-20157 Milan, Italy tFidia Research Laboratories, 35031 A b a n o Terrne, Italy R e c e i v e d 7 A u g u s t 1981 PONZIO, F., G. CALDERIN1, G. LOMUSCIO, G. VANTINI, G. TOFFANO AND S. ALGERI. Changes in monoamines and their metabolite levels in some brain regions of aged rats. NEUROBIOL. AGING 3(1) 23-29, 1982.The concentrations of dopamine (DA), norepinephrine (NE), serotonin (5HT) and their metabolites, HVA, DOPAC, MHPG-SO4 and 5HIAA were measured in several brain areas of rats aged 4, 18 and 29 months. Dopamine and its metabolites showed a decline, statistically correlated with age, in all the dopaminergic areas considered, indicating that this system is profoundly affected in the senescent rat. The changes in the noradrenergic system were more complex. This neurotransmitter was reduced in spinal cord and in limbic area, but was not modified in hippocampus, cerebellum, striatum and s. nigra. In cortex, MHPG-SO,, the main NE metabolite, showed a significantly age-related increase. Tyrosine hydroxylase (TH) activity was low in striatum, and brainstem but not in hypothalamus of aged rats. Neither 5HT nor its metabolites was affected by age. The results indicate that central catecholaminergic systems are markedly affected in senescent rats. Aging, effect in CNS Aging, effect on dopamine Aging, effect on norepinephrine Aging, effect on tyrosine hydroxylase Aging, effect on serotonin
A G I N G is associated with a progressive deterioriation of the nervous system. There are several physiological, cytological and biochemical parameters that are altered by aging processes and may therefore cause malfunctioning of the nervous system. Alterations affecting neurotransmission mechanisms are particularly important in view of the neuronal network's role in coordinating the body's relationships with its internal and external environment. Levels and metabolism of neurotransmitters are important biochemical parameters. Alteration of these parameters may indicate changes in neuronal firing or changes in neuron numbers. As noted in a recent review [19] age is accompanied by significant changes in the catecholaminergic and serotonergic systems. Brain catecholamine levels and/or synthesis have been reported to be decreased in striatum brainstem and hypothalamus [1,5, 12, 13] of senescent rats and mice [6, 7, 16, 21] and the activity of tyrosine hydroxylase, the enzyme controlling catecholamine synthesis, was low in homogenates prepared from brains of aged animals, including man [1, 3, 12, 13]. Histofluorescence intensity due to catecholamines is also reportedly low in the substantia nigra and locus coeruleus of aged monkeys [22] and Fisher 344 rats [23]. Serotonin synthesis, on the other hand, was enchanced in the hypothalamus of old rats [21].
Most of the data accumulated so far has been obtained in a limited number of cerebral regions and is generally based on the comparison of two age groups usually at the lower and upper ends of the iifenspan; information relating to intermediate ages is lacking. However, such information is very important in a phenomenon such as senescence which is probably the result of gradual rather than sudden changes. In the experiments presented here we measured simultaneously the levels of dopamine, norepinephrine and serotonin and/or their metabolites in several different brain areas of rats aged 4, 18 and 29 months. Many of these areas have not been investigated before. We checked whether there was any possible correlation between the observed changes and age. The activity of tyrosine hydroxylase was also studied in order to verify the age-dependent modification, of this enzyme that has been previously reported [1, 3, 13, 21]. METHOD
Animals Male Sprague Dawley rats of different ages were obtained from Bio Breeding Laboratories (Beaconsfield, Quebec, Canada). A first shipment of two groups of rats aged l and 12 months was received. These animals were kept for 12 months in our animal house under standard controlled con-
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C o p y r i g h t © 1982 A N K H O I n t e r n a t i o n a l Inc.m0197-4580/82/010023-07503.00/0
24 ditions of temperature, humidity and dark-light cycles (12 hr of light each day from 7 a.m.-7 p.m.). They were housed in Makrolon cages (2--4 rats/cage) with free access to water and standard rat chow (Altromin pellets). Rats tend to accumulate fat with age. As we intended to use part of this rat colony to study the effect of cold exposure in the different classes of age (paper submitted for publication) we put them on a restricted diet in order to minimize the possible interference of subcutaneous adipose layers of different thickness. After 12 months both groups of rats were put on a restricted diet consisting of only 50% (15 g/day) of the chow they consumed when fed ad lib (30 g/day). They were kept on this dietary regimen for 5 months until the day of the experiment. During the first 3 months of restricted diet the rats gradually lost weight. In one group the mean body weight fell from 543___10 g at 24 to 467___14 at 27 months of age in the second group from 468-+10 at 13 to 445___10at 16 months of age. After this period until the day of the experiment, body weight remained constant. Two months before the experiment a group of 2month-old rats was received. They too were put on a restricted diet for 2 months. During this period their body weight rose from 302±9 g to 382+-10 g. At the time of the study, therefore, we could compare three age groups, one 4 months old (382_+10 g), one 18 months old (435+-10 g) and one 29 months old (446_+13 g). This enabled us to minimize differences in body weight due to adipose tissue, without affecting normal body growth. The difference in body weight between the young and the two groups of aged rats reached the level of statistical significance (p <0.01) but as it still only amounted to about+- 15% it should not have any biological relevance. The rats lifespan may also have been prolonged as reported by some investigators [8,26]. In a previous pilot experiment we had assessed the possible effects of restricted diet on organs wet weight and on the concentrations of monoamine and their metabolites and on tyrosine hydroxylase (TH) activity. No differences were found in these parameters in restricted and ad lib feed groups (data not reported). Rats with visible tumors (lipomas and adenomas) were discarded. The percentage of survival was 100 in the 4-months group, 91 for the 18-month and 55 for the 29-month animals. Rats were killed by guillotine, alternating the three age groups. Brains were rapidly excised, put on a cold plate and dissected into limbic area (olfactory tubercola, n. accumbens), striata, hippocampus, s. nigra, cerebral cortex, lower brainstem) Pons and medulla), hypothalamus and cerebellum. The spinal cord was taken by opening the spinal column. The samples were immediately frozen on dry ice and kept at -80°C until assayed. Biochemical Methods Catecholamine assay. Endogenous norepinephrine ('NE) and dopamine (DA) were determined using high pressure liquid chromatography with electrochemical detection (LCEC) according to Keller et al. [10]. The tissue was extracted with 320/zl 0.1 M perchloric acid containing 27-520 pmoles of a-methyl-dopamine (internal standard) using a Branson B30 sonifier. After an AlcOa prepuritication step, the extracted catecholamines were determined by LCEC. The values were expressed as ng/g wet weight of the tissue, based on internal standard measurements. 5-Hydrox?,'tryptamine (5-HT) assay. 5-HT was deter-
PONZIO £ 7 A~' mined using the LCEC-technique according to Ponzio and Jonsson [18]. Frozen tissue samples were homogenized in 6 vol (w/v) acidified n-butanol and centrifuged. Then 80/.tt of the supernatant was added to 200 tzl heptane and 25 ¢zl of 0. I M perchloric acid. This mixture was shaken and after removal of the organic phase and washing with chloroform, 10 /xl of the acid phase was injected into the liquid chromatograph. The values obtained were corrected for 5-HT recovery (80--90%), determined in separate samples, and expressed as ng/g wet weight of the tissue. Monoamine metabolite assay. Endogenous homovanillic acid (HVA), dihydroxy-phenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIAA) levels were determined using high pressure liquid chromatography with electrochemical detection (LCEC) according to Ponzio and Jonsson [17]. Frozen tissue samples were homogenized in 10 vol (w/v) 0.4 M perchioric acid and centrifuged. The supernatant was extracted with ethylacetate. The final extraction was with 0.35 M sodium acetate (pH 8.1-8.2) and 10 ~1 of the aqueous layer were utilized for the assay. The values obtained were corrected for HVA, DOPAC, 5HIAA recoveries (60-75%), determined in separate samples, and expressed as ng/g wet weight of the tissue. Norepinephrine metabolite assay. Endogenous 3-methoxy4-hydroxy-phenyl glycol-sulphate (MHPG-SO4) levels were determined spectrophotofluorometrically according to Meek and Neff [15]. Frozen tissues were homogenized in 4 volumes of 0.2 M ZnSO4 and 4 volumes of 0.2 M Ba(OH)2 and centrifuged. The supernatant was poured onto a DEAE Sephadex column to isolate MHPG-SO4, which was eluted with 5.5 ml of 0.06 N HCI. Fluorescence was measured at 465 nm after adding ethylene-diamine. Tyrosine hydroa3'lase activity assay. Tyrosine hydroxylase activity was measured according to the method of Waymire eta/. [25] based on the production, trapping and determination of ~4CO2from [carboxyl 14C] L-DOPA formed from p4C] l-tyrosine and developed by DOPA decarboxytase added to the medium. Enzyme activity was expressed per unit of protein. RESULTS Modification of DA Metabolism in Some Brain Areas of Aging Rats HVA levels in the striatum were reduced but not significantly so, but otherwise concentrations of DA and its metabolites HVA and DOPAC were significantly reduced with age in the three brain areas examined (Fig. 1). Generally the decrease was more pronounced in the group of oldest rats, and for striata and limbic area a significant correlation was found between age and decline of DA and both its metabolites. In the s. nigra the decline of DA and DOPAC was already maximal at 18 months of age (Fig. 1). Modifications of NE Metabolism in Some Areas o f Aging Rats Age-related changes in NE concentration were studied in the limbic area, hippocampus, cerebellum, spinal cord, striata and s. nigra. Changes in MHPG-SO, in cortex were also determined (Fig. 3). All these areas receive adrenergic innervation whose extent varies according to the region considered. Unlike DA and its metabolites, whose levels were constantly decreased with age, NE showed a more complex pattern of variation. In the limbic area and spinal
BRAIN M O N O A M I N E S IN A G E D RAT BRAIN
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.ONTHS
FIG. 1. The existence of a concentration-age correlation was tested by a linear regression analysis. In the cases this is present the degrees of the significance for the F or correlation, the r values, and the degree of significance of the linearity are given. Statistical significance of the differences between groups was analyzed by Duncan's test. **p<0.01 vs 4 months; O p<0.01 vs 18 months.
cord there was a lowering in NE concentration, significantly correlated with age in the first area (Fig. 2); in the hippocampus, striata, s. nigra and cerebellum no modification was evident. MHPG-SO~, an index of NE turnover, tended to be significantly increased with age, the levels of this metabolite in the cortex of 29-month-old rats being 51% and 30% higher respectively than in 4- and 18-month-old rats.
Tyrosine Hydroxylase Activity in Some Brain Areas of Aging Rats TH activity measured in striata, hypothalamus and brainstem of rats in the three age groups is summarized in Fig. 4. The activity of this enzyme in hypothalamus prepara-
tions did not differ in the different age groups but it was lower in striata and brainstems of 18 and 29 month-old rats. In striata of the 18-months group the enzyme activity was reduced to 68% of the activity present in the tissue of young animals, but was not any lower in the oldest group. In brainstem the decline was more gradual and was lowest in the 29-month-old rats where it was 69% of that measured in young rats.
Modification of 5-HT Metabolism in Some Brain Areas of Aging Rats The effect of age on serotonin was studied determining the concentration of 5HT and its metabolite 5HIAA in hip-
26
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FIG. 2. Concentrations of NA in some noradrenergic brain areas of young and old rats. Data are the mean±S.E. of 30 samples. The significance of the concentration-age correlation was tested by linear regression analysis~ The correlation is significant in spinal cord and limbic area. Statistical significance of the differences between groups was analyzed by Duncan's new multiple test. **p<0.01 vs 4 months; *p<0.05 vs 18 months.
nglg
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DISCUSSION
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FIG. 3. Concentration of MHPG-SO, in cerebral cortex of young and old rats. Data are the mean-S.E, of 30 samples. The significance of the concentration-age correlation was tested by linear regression analysis. The significance of the differences between groups was analyzed by Duncan's new multiple test. **p<0.0l vs 4 months; *p<0.05 vs 18 months.
pocampus, cerebellum and spinal cord. Levels of 5HIAA were also determined in limbic area and s. nigra. None of the areas considered presented age-dependent modifications in the levels of 5HT or its metabolite (Fig. 5).
The results in this sereis of experiments confirm that aging does cause modifications to the rat central nervous system. The concentrations of some monoamines and their metabolites in different brain areas of three different age groups of rats indicate that senescence has complex effects on the normal state of some neurotransmitters causing quantitative differences that vary according the neurotransmitter and the area considered. Dopamine was the neurotransmitter most affected by aging. In all areas considered, this monoamine and its acid metabolites were decreased at 18 and 29 months of age compared to 4-month-old rats. This decrease was generally sig, nificantly correlated with age. Only in s. nigra was DA maximally decreased already at 18 months of age. In previous experiments with Wistar rats [2,16] we found no change in DA concentration, but there was a significant reduction in the synthesis of this amine in striata and hypothalamus of 36-month-old rats [2,16]. In contrast, in the rat population we studied this time, DA concentration showed a clear age-related decrease in all the brain areas investigated. DA hystofiuorescence was also reduced in perikarya of old Fisher 344 rats [23] and a similar phenomenon has been observed in the monkey brain [22]. These conflicting results underline the importance of the
BRAIN M O N O A M I N E S IN A G E D R A T BRAIN
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FIG. 4. Activity of TH tested in some brain areas of young and old rats. Data are the mean ~ S.E. of 6 samples. Statistical significance was analyzed by Duncan's new multiple test. *,o<0.05 vs controls; **p<0.01 vs controls. possible role of interspecific and inter-strain differences, in the age-related decline of normal steadystate level of DA, suggesting that the vulnerability to aging of certain neuronal systems may be under genetic control. Here together with the decline in the parent monoamine a parallel decline of the acid metabolites was evident. It is difficult to establish whether we are dealing with decreased synthesis and turnover of the neurotransmitter or with degeneration of dopaminergic neurons. If there is a degeneration of dopaminergic neurons, then the parallel decrease of HVA and DOPAC suggests that the remaining neurons cannot compensate this loss by increased turnover of the transmitter. Aging would therefore result in a less efficient dopaminergic tonus. In these rats we have observed that in conditions of increased DA turnover, as in the case of stress [4, 11, 24]. striatal HVA rose much less in old than in young rats, indicating lower turnover of DA [3], on the same lines as results from a different methodological approach, in a different rat strain [2,16], or in a different rodent species [6,7]. An indication of depressed dopaminergic synthesis was also derived from TH activity values in the striata of aged" rats (Fig. 3); in agreement with similar results obtained previously in our and other laboratories [l, 12, 14], this was lower than in young controls. As reported by Reis e t a / . [20] aging seems to affect this enzyme differently in CNS and in peripheral adrenergic tissues. While TH activity is very markedly increased in sympathetic ganglia [20] and in adrenal medulla [3,20] it is only slight decreased ( - 3 0 % ) in striata. In contrast with Reis' findings in Fisher 344 rats [20] we detected a significant reduction in TH activity in the brainstem an area where cell bodies of catecholaminergic neurons are located. In this case too strain differences are at the basis of these different i'mdings. While a constant decline of DA and its metabolite was found in all the brain areas of old rats, the changes brought about by age in the noradrenergic system were more corn-
plex. In most of the areas considered, such as hippocampus, cerebellum, striata and s. nigra, the levels of this amine were not changed, but they were significantly reduced in limbic area and spinal cord (Fig. 2). In another rat strain we have previously found a decrease of N E levels in pons and medulla oblungata of aged rats [2,16]. Tyrosine hydroxylase in brainstem, but not in hypothalamus was also found to be reduced by age, as the present results confirm (Fig. 4). The noradrenergic nuclei from which the dorsal and ventral bundles projecting to the spinal cord and the various telencephalic structures originate are in the brainstem [9]. As our data indicate, it is possible that agining causes degeneration or reduces the activity of the fibers in some selected noradrenergic pathways. It was interesting to find that aging did not always lower the concentrations of catecholamine metabolites. In the cortex in fact we noted an age-related increase in MHPG-SO4, but whether this is an indication of increased N E turnover has yet to be clarified by different methodological approaches. The biological significance of this phenomenon is also still obscure. Simpkins et al. reported that 5HT and 5 H I A A concentrations were not changed in the brain of aged rats except in the hypothalamus where there was a slight but significant increase in 5 H I A A [21]. In the experiment reported here we measured 5HT and its metabolite in several brain areas (but not the hypothalamus which was used for TH assay) and we also found no changes. The same authors reported however, and we have confirmed in other experiments (paper in preparation), that the turnover of this monoamine appears to be increased in the brain of aged rats. It is thus a possibility that in balance between dopaminergic and serotonergic system may be one aspect of aging of the brain. The lack of modification in 5 H I A A suggests that the turnover rate of 5HT also is not changed. However in this regard the parameter must be studied in conditions of changed serotonergic functionality to give a better insight into the questions of the effect of age on the serotonergic system.
PONZIO EY At.
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FIG. 5. Concentration of 5 H T and 5 H I A A in some serotonergic areas of young and old rats. 5 H T data are the mean ± S.E. of 30 samples for each group of age. 5 H I A A data are the mean ± S.E. of 6 samples for each group of age. N o significant difference is present (Duncan's test).
BRAIN MONOAMINES
IN AGED RAT BRAIN
29
ACKNOWLEDGEMENTS This work was supported by grants from the Gustavus and
Louise Pfeiffer Foundation, Los Angeles, CA; CARIPLO, Milan, Italy; Fidia Res. Lab., Abano Terme, Padua, Italy and by Contract No. 79.02355.65 from Italian National Research Council (CNR).
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13. McGeer, P. L. and E. G. McGeer. Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. J. Neurochem. 26: 65-76, 1976. 14. McNamara, M. C., A. T. Miller, Jr., V. A. Benignus and J. N. Davis. Age related changes in the effect of electroconvulsive shock (ECS) on the in vivo hydroxylation of tyrosine and tryptophan in rat brain. Brain Res. 131: 313-320, 1977. 15. Meek, J. L. and N. H. Neff. Fluorometric estimation of 4-hydroxy-3-methoxy-phenylethyleneglycol sulphate in brain. Br. £. Pharmac. 45: 435-441, 1972. 16. Ponzio, F., N. Bruneilo and S. Algeri. Catecholamine-synthesis in brain of ageing rat. J. Neurochem. 30: 161%1620, 1978. 17. Ponzio, F. and G. Jonsson. Effects of neonatal 5,7dihydroxytryptamine treatment on the development of serotonin neurons and their transmitter metabolism. Devl Neurosci. 1: 80-89, 1978. 18. Ponzio, F. and G. Jonsson. A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection, d. Neurochem. 32: 129-132, 1979. 19. Pradhan, S. N. Central neurotransmitters and aging. Life Sci. 26: 1643--1656, 1980. 20. Reis, D. J., R. A. Ross and T. H. Joh. Changes in the activity and amounts of enzymes synthesizing catecholamines and acetylcholine in brain, adrenal medulla, and sympathetic ganglia of aged rat and mouse. Brain Res. 136: 465--474, 1977. 21. Simpkins, J. W., G. P. MueUer, H. H. Huang and J. Meites. Evidence for depressed catecholamine and enhanced serotonin metabolism in aging male rats: Possible relation to gonadotropin secretion. Endocrinology 100: 1672-1678, 1977. 22. Sladek, J. R. and C. D. Sladek. Relative quantitation of monoamine histofluorescence in young and old non-human primates. Adv. exp. Med. Biol. 113: 231-'240, 1979. 23. Sladek, J. R. and B. C. Blanchard. Age-related declines in perikaryal monoamine histofluorescence in the Fischer 344 rat. ln: Brain Neurotransmitters and Receptors in Aging and AgeRelated Disorders, edited by S. J. Enna, T. Samorajski and B. Beer. New York: Raven Press, 1981, pp. 13-21. 24. Thoenen, H. Induction of tyrosine hydroxylase in peripheral and central neurones by cold-exposure of rats. Nature 228: 861-862, 1970. 25. Waymire, J. C., R. Bjur and N. Weiner. Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from l-~4C-L-tyrosine. Analyt. Biochem. 43: 588-600, 1971. 26. Young, V. R. Diet as a modulator of aging and longevity. Fedn Proc. 38: 1994-2000, 1979.