Franca Ponzio

Changes in monamines and their metabolite levels in some brain regions of aged rats

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-SO4, 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.

Significance of dopamine metabolites in the evaluation of drugs acting on dopaminergic neurones

The effect of various drugs was studied on 3-methoxytyramine (3-MT) concentrations in rat striatum. The drugs were chosen for their ability to interfere with the dopaminergic system at different levels. Dopamine (DA) acidic metabolites, i.e. homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), were also measured. Changes of 3-MT, unlike those of DOPAC and HVA, seem to reflect the functional activity of dopaminergic neurons. In fact drugs believed to increase or decrease DA content in the synaptic cleft produce predictable changes of striatal 3-MT. Thus cocaine, nomifensine and d-amphetamine increase 3-MT concentrations while gamma-butyrolactone, alpha-methyltyrosine and apomorphine decrease it.

Catecholamine synthesis in brain of ageing rat

SEVERAL lines of evidence indicate that in mammals there is a progressive deterioration of central nervous system function with ageing (BOWERS & GERBODE, 1968; BRIZZEE et al., 1975; DHOPESHWARKAR & MEAD, 1975 ; SCHEIBEL & SCHEIBEL, 1975). Many neurophysiological processes such as motor activity, thermoregulation, sleep and hormonal secretion do in fact deteriorate in old animals (FINCH et al., 1969; GOODRICK,

Changes in specific activity of dopamine metabolites as evidence of a multiple compartmentation of dopamine in striatal neurons.

The functional status of dopaminergic nerve terminals has been studied with a method that allows the simultaneous determination of the specific activities of dopamine (DM), tyrosine (Tyr), 3‐methoxytyramine (3‐MT) and 3,4‐dihydroxyphenylacetic acid (DOPAC), after administration of [3H]tyrosine ([3H]Tyr).

Dopaminergic effects of buspirone, a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methoxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [3H]spiroperidol from striatal binding sites, with an IC50 (1.8 x 10(-7) M), comparable to that of clozapine (IC50 = 1.4 x 10(-7) M) but considerably lower than that of haloperidol (4.7 x 10(-9) M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.

Application of the dual-cell coulometric detector: A method for assaying monoamines and their metabolites

A new HPLC assay technique for monoamines and their metabolites, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite through which the samples flow, is described in comparison with a classical amperometric detector equipped with a glassy carbon electrode. Different potentials can be applied at each cell of the coulometric detector to improve sample resolution and detection sensitivity. The signal-to-noise ratio (s/n) calculated in similar conditions was 10 times lower for the coulometric detector than for the amperometric one. The dual-coulometric detector does not undergo daily decay or variation, and needs no particular care or preparation. It is therefore possible to achieve stable routine sensitivity in a range of 10 fmol. This new technique has been applied for assaying monoamines and their precursors and metabolites by direct injection of clear supernatant after centrifugation and for determination of catecholamine turnover in rat pineal gland and neuro- and adenohypophysis in samples purified by Al2O3 adsorption.

Dihydropteridine reductase and tyrosine hydroxylase activities in rat brain during development and senescence: a comparative study

Enzymatic hydroxylation of the ring of the aromatic amino acids tyrosine, tryptophan and phenylalanine requires the availability of reduced tetrahydrobiopterin, a non-protein cofactor which functions as the donor of reducing equivalents in the hydroxylating reaction4,S,% 1°. In turn, reduction of the pterine cofactor is accomplished by a pyridine-dependent enzyme coupled with the hydroxylase: dihydropteridine reductase (DHPR). This enzyme is present in various tissues including the brainS, xS. As the reduced cofactor is essential for the hydroxylase reaction, the hypothesis has been put forward that D H P R may in certain circumstances control the synthesis of biogenic amines11,16. A previous paper 19 investigated the regional distribution of D H P R in rat brain, and its activity in some experimental conditions in which tyrosine hydroxylase (TH), the enzyme controlling catecholamine biosynthesis 17, was induced or lowered by different pharmacological treatments. No correlation between the two enzymes was found. To further investigate this problem we studied brain D H P R at different stages of life in the rat, comparing it with TH. The results provide some new information about D H P R and also give a better insight into its possible relationship with tyrosine hydroxylase. Brains of male and female Wistar rats from birth to 30 months of age were used; foetuses were removed after 13, 15,17, 19 or 20 days of gestation and their brains were excised under a dissection microscope. For regional studies in adult rats, the different brain areas were dissected according to the procedures of Glowinski and Iversen 6. All specimens were kept frozen at -30 °C until assay. Tissues were homogenized in 3 vol. of ice-cold distilled water in a motor driven all-glass homogenizer and enzyme assays were performed on the supernatant obtained after centrifugation of the homogenate (22,000 × g × 20 min at 4 °C). DHPR activity was measured spectrophotometrically by the decrease in absorbance at 340 nm as described by Craine et al. 4; artificial pteridine 6,7-dimethyl-5,6, 7,8-tetrahydropteridine (DMPH4) was used as substrate. TH activity was measured

Depletion and recovery of neuronal monoamine storage in rats of different ages treated with reserpine.

The effect of reserpine on dopamine, noradrenaline, adrenaline and serotonin concentrations in different brain regions, and the recovery of normal levels of these monoamines after such treatment were studied in rats aged 5, 15 and 27 months. In a preliminary experiment we found that distribution of the drug was not altered in the aged rats. Then we observed that a single dose of reserpine (5 mg/kg IP) had a similar depleting effect on all the brain monoamines, in all the brain regions considered in all three age groups. The curves expressing recovery of monoamine storage in all the nerve terminals, several days after treatment, were superimposable. These results suggest that in the rat, age does not influence the effect of reserpine on the storage mechanism of brain monoamines. Moreover, as restoration of this mechanism depends on the synthesis of new vesicles, the similarity in the rates of recovery in adult, old and very old rats indicated indirectly that synthesis of these neuronal organelles is not affected by aging.

Does acutel-DOPA increase active release of dopamine from dopaminergic neurons?

L-DOPA is believed to be decarboxylated by the residual striatal dopaminergic presynaptic terminals with formation of the putative neurotransmitter dopamine (DA) and with increased availability of DA at post-synaptic receptors. However there is no direct evidence that the DA formed is released into the synaptic cleft. We therefore investigated the biochemical modifications occurring in the dopaminergic system after acute administration of L-DOPA. After acute L-DOPA (100 mg/kg plus 25 mg/kg of benserazide p.o.) the levels of 3-methoxytyramine (3-MT), a metabolite reflecting release of the neurotransmitter DA, were significantly raised, following the same pattern as DA levels, indicating that DA release from DA nerve terminals is increased after L-DOPA administration. The increased DA release and 3-MT formation were not reduced by pretreatment with direct DA agonists such as apomorphine (5 mg/kg i.p.) or piribedil (120 mg/kg p.o.). Thus in this case DA release is not under the control of the compensatory mechanisms induced by post-synaptic receptor hyperstimulation.

Changes with age in rat central monoaminergic system responses to cold stress

Changes with age in responses to stress of certain central monoaminergic systems were investigated. Three groups of rats, 4, 18 and 29 months old, were exposed to cold and the effect of this stress on hypothalamic tyrosine hydroxylase, and on the metabolism of DA and 5HT in different brain regions was evaluated. Senescent rats were unable for several hours to compensate the loss of body heat. Corticosterone secretion however was equally stimulated. Hypothalamic tyrosine hydroxylase activity was enhanced in the young rats but not in the old ones. However, the two groups of senescent rats did not show the increase in HVA levels noted in striata of young rats 2 hours after cold exposure. In contrast, the 18 and 29-month-old rats presented enhanced serotonergic tonus, indicated by the greater increase in 5HIAA determined by stress.