Male Sprague-Dawley rats were housed in a climate and light-controlled room (12:12 LD cycle; lights on at 08.00h) and allowed food and water ad libitum. At either 1, 3, 6 or 12 months of age, groups of five animals were killed by decapitation between 17.00h-19.00h. The brains were quickly removed and placed on a chilled glass plate on ice to be dissected in discrete brain areas. Brain tissues were frozen for latter assays.

The content of DA, DOPAC, NA, 5-HT and 5-hydroxy-3-indoleacetic acid (5-HIAA) in brain tissues were measured by HPLC. The chromatographic system consisted of a Waters M501 solvent delivery pump, a Spherisorb reversed-phase analytical column (3 µm particle size, 150 x 4 mm), and a Coulochem M5010A detector that included an analytical cell set at +50 mV (first electrode) and +300 mV (second electrode). The mobile phase, composed of a mixture of 0.1M potassium dihydrogen phosphate, 0.1mM di-sodium-EDTA, 0.8mM octanesulphonic acid and 18% methanol (final pH of 3.10), was pumped isocratically (0.8 ml/min) at room temperature. Tissues were sonicated in the mobile phase, centrifuged, and supernatants filtered. The concentration of monoamines and metabolites was estimated from data obtained from 20 µl sample aliquots injected into the chromatographic system by comparing peak heights to those of appropiate standards. To estimate procedural losses, an internal standard, 3,4-dihydroxybenzylamine, was added to each sample and data were corrected for recovery of the internal standard (95-100 % recovery).

Data were expressed as mean standard error of mean (S.E.M). The groups of ages were statistically compared by one way ANOVA followed by Duncan's multiple-range test.

a) Dopaminergic system. The DA and DOPAC concentrations showed a progressive increase from young (1 month-old) to adult rats (6 month-old) in the striatum but not in other brain regions with lower catecholamine content. In aged rats (24-month-old), a significant drop in the striatal contents of DA and DOPAC were observed. Decreased contents of DA and DOPAC were also found in the hippocampus from the 24 month-old rats. In the amygdala, the concentration of DA was significantly lower in the oldest rats than in the middle age animals (3-6 month-old). The concentrations of DA also fell significantly in the brainstem of aged rats (24-month-old) as compared to animals of one and six months of age.

b) Noradrenergic system. The concentration of NA in hippocampus, amygdala and frontal cortex increased significantly from the first to the third month of age; in these areas, the levels of catecholamines were not affected by senescence. Only the NA content in the pons-medulla decreased significantly in aged rats (24-month-old), showing concentrations 25-27% lower than in animals of 1, 3 or 6 months of age.

c) Serotonergic system. The concentrations of 5-HT in the rat hippocampus rise significantly from the first to the third month of age, and a progressive age-related increase in the concentration of 5-HT from the youngest (1 month-old) to the adult (12 month-old) rats was observed in all the brain regions studied. From middle age to 24 months of age, the 5-HT content showed a tendency to decline in all brain regions, although this reduction was significant only in frontal cortex. These changes in 5-HT levels were not accompanied by alterations in the content of the 5-HIAA in the 5-HT terminal regions, and a significant increase in the 5-HIAA/5-HT contents ratio was found in amygdala, striatum and frontal cortex. In addition, the levels of 5-HIAA were decreased by 28-32% in the brainstem of aged rats as compared to middle-aged ones, but no change was observed in the ratio of the contents of 5-HIAA to 5-HT in the mesencephalic area.

The present results indicate the existence of a depression in the functional status of brain biogenic amines during aging, which is characterized by a regional selectivity and a differential susceptibility according to the neurotransmitter system considered.

We observed a reduction in DA and/or DOPAC levels in DA terminal regions (striatum, amygdala, hippocampus) which agrees with previous studies in the striatum and mesolimbic areas of rats (Demarest et al., 1980; Ponzio et al., 1980; Strong et al., 1982; Estes and Simpkins, 1984; Machado et al., 1986; Moretti et al., 1987; Venero et al., 1991) and humans (McGeer, 1981; Arranz et al., 1996). In addition, a decrease in the striatal DA turnover (Ponzio et al., 1978; Carfagna, 1985; Venero et al,. 1991) and high affinity DA uptake (Strong et al., 1984) was found in aged rats, which seems to be related to the well-known loss of dopaminergic neurons projecting to the striatum (Brizee et al., 1981; Kish et al., 1992). It is noteworthy that age affects hippocampal and amydaloid DA content in a similar way to that in the striatum, this suggesting that both the nigrostriatal and the mesolimbic dopaminergic systems might be simultaneously changed by aging. However, the metabolism of DA was unchanged in other brain areas, such as the frontal or the remaning cortex, which receive also mesolimbic fibers, thus suggesting a selective age-dependent decline of DA content among the mesotelencephalic dopaminergic neurons.

From the first to the third month of life period, the concentration of NA increased significantly in hippocampus, amygdala and frontal cortex, as well as the concentration of 5-HT in most of the terminal regions of the serotonergic system, and that of DA in the striatum. These results suggest that development of NA, 5-HT and possibly DA in the striatum might be predominantly a postnatal event, as previously reported in other structures of the rat brain (Santiago et al., 1988).

In contrast to DA, the NA concentration in brain regions containing NA projections was scarcely affected by aging, whereas it was severely reduced in the pons-medullary area, region which includes the locus coeruleus, with most of the NA cell bodies projecting to the diencephalic and telecencephalic structures. Our results confirm those of authors who showed a reduction in NA activity in the rat locus coeruleus (Olpe and Steinmann, 1982) as well as absence of changes in the NA projection regions. Decrease of NA content was not associated with a similar tendency in DA content in the pons-medulla, suggesting that the mechanism involved in the deficit of NA speciphically affects the synthesis or the storage of this neurotransmitter.

Our results show a decreased 5-HT content in the frontal cortex of 24-month-old rats, although a tendency to decrease was also observed in all 5-HT terminal areas examined. This decline in amine content in brain areas of old rats could be explained because of a lower 5-HT synthesis rate in serotonergic terminal regions (Meek et al., 1977; Venero et al., 1991). The levels of 5-HIAA, the main 5-HT oxidative metabolite, were found to be decreased in the brainstem of 24-month-old rats. This area contains the raphe nuclei, where most neuronal bodies of the ascending serotonergic system are found (McGeer et al., 1987). Since the levels of 5-HT observed in the brainstem did not change with aging, the deficit of this amine in the serotonergic terminals of the aged rats could be related with the decrease in the catabolism of 5-HT to 5-HIAA (Strolin-Benedetti and Keane, 1980). These results are indicative of a different behavior for 5-HT in senescence, between the regions containing the 5-HT cell bodies and those containing the 5-HT terminals. In support of this hypothesis is the fact that the 5-HIAA / 5-HT ratio (considered an index of the metabolic utilization of 5-HT) increased with age in the amygdala, striatum and frontal cortex, whereas it was unchanged in the brainstem. These data reflect that an age-dependent increased oxidative deamination occurs in the 5-HT terminal areas in spite of a lower synthesis of the amine and agree with previous results showing either increases in 5-HIAA content or in 5-HIAA / 5-HT ratio in the striatum and mesolimbic areas of the old rats (Moretti et al., 1987; Venero 1993).

The present results illustrate well that aging has complex effects on the neurotransmitter systems, and that a deficit in the biochemistry of DA, NE and 5-HT was manifested at various levels of their functional organization. The hippocampus, amygdala, cortex and striatum are brain areas actively involved in age-related neurodegenerative processes, including motor malfunction, emotive and cognitive disorders, or learning deficits associated with dementia, Parkinson's or Alzheimer diseases (McGeer, 1981; Burchinsky, 1985; Kish et al., 1992). These areas show an important deficit of DA and 5-HT, while NA and also 5-HT appeared to be reduced in the lower brainstem, an area that has been related with sleep disturbances and depression in the ederly (Olpe and Steinmann, 1982; Burchinsky et al., 1985). Therefore, changes in the function of single neurotransmitters in specific brain areas or the imbalance between various neurotransmitter systems must be considered to understand the mechanisms of aging and to explain some of the age-related impairment of neurophysiological functions.

Acknowledgments The work was supported by the European Commission contract number C11*-CT94-0036, and a grants from the TMR program of the European Commission (J.M.M).

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