In vivo conversion of 3H-L-tryptophan into 3H-serotonin in brain areas of adrenalectomized rats.

Rats were adrenalectomized 10 days before we estimated in vivo the conversion index of (3)H-tryptophan into radioactive serotonin in brainstem and telediencephalon. We found that the conversion index in the brainstem of adrenalectomized rats is smaller than in the same area of sham-operated rats. Conversely, the conversion index in the telediencephalon was similar in the two groups of rats. The serotonin concentrations were unchanged by adrenalectomy, which suggests that in brainstem the decrease of tryptophan hydroxylase is reflected by the conversion index estimation and not by measurement of serotonin steady-state concentrations.

Narcotic tolerance and dependence: lack of relationship with serotonin turnover in the brain.

Serotonin turnover was measured in mouse brain by means of the conversion of radioactivity from labeled tryptophan into serotonin. Animals with a high degree of tolerance to and physical dependence on morphine did not differ from control mice.

Lesions of central norepinephrine terminals with 6-OH-dopamine: biochemistry and fine structure.

Intracisternal injections of 6-hydroxydopamine produce rapid and long-lasting depletion of brain catecholamines without effects on serotonin concentrations. Depletion of norepinephrine is greatest in areas containing only nerve terminals and axons and least in areas containing monoamine cell bodies. The norepinephrine loss is accompanied by electron microscopic evidence of nerve terminal degeneration and decreased turnover. Dopamine loss is less marked and is not accompanied by degeneration or alteration of turnover rate.

Deterioration of spatial and nonspatial reference and working memory in aged rats: protective effect of life-long calorie restriction.

Two different aspects of learning (spatial and nonspatial) and two different types of memory (reference and working) were simultaneously measured in populations of 3- (young), 11- (adult), and 25-month-old (aged) rats fed ad libitum either a standard (ST) or a hypocaloric (HY) diet. All groups, regardless of age or diet, showed ability in learning all four versions of the task. However, old ST rats were significantly slower and less efficient at learning than the young and adult ST rats. In contrast, senescent HY rats' cognitive abilities did not differ from those of their young and adult counterparts. The decline in reference and working memory in the aged ST rats was more pronounced in the spatial than the nonspatial version of the task. This study confirms and extends to more specific aspects of memory our earlier finding that age-related cognitive deterioration in rats was antagonized by life-long calorie restriction.

Age-related ability of rats kept on a life-long hypocaloric diet in a spatial memory test. Longitudinal observations.

Cognitive behavior was assessed in a test specific for spatial memory, according to a longitudinal experimental model, in populations of 12-, 24- and 30-month-old rats which were fed ad lib either a standard (ST) or a hypocaloric (HY) diet, that had been shown to reduce age-related pathologies and to increase survival. Already at 12 months of age, some cognitive deficits were observed in ST but not in HY rats. When retested 12 and 18 months later, the animals performed better at the beginning of the test than when tested for the first time, indicating that some aspects of previous experience lead to a preservation of spatial memory. Deficits which had been previously observed in 2-year-old groups in a cross-sectional experimental model were not evident this time. Some of the differences between the two diet groups observed 12 months before disappeared. When testing was repeated for the third time at 30 months of age, ST fed rats presented very marked deficits in learning and in memory, which were not seen in the HY group. It thus appears that a dietary regimen in which part of the calorie-rich components, such as lipids and carbohydrates, are replaced with vegetable fibers, retards some of the age-related deteriorations of brain functions.

Effect of life-long hypocaloric diet on age-related changes in motor and cognitive behavior in a rat population.

Aged (24 months), adult (12 months), and young (4 months) rats kept on standard or hypocaloric diets from the age of three weeks old were tested in a range of behavioral tests to determine the effects of aging on sensory-motor and cognitive behavior and to assess whether such effects were prevented by life-long calorie restriction. An age-related deterioration of sensory-motor functions, motility and exploratory activity was observed in all the senescent animals independent of diet. Swimming ability did not deteriorate with age. Spatial memory, evaluated by the Morris water maze test, showed some deterioration in normally fed adult rats as indicated by the deceleration in the learning curve. In aged rats, not only was learning slowed down, but memory utilization was also impaired. These cognitive deficiencies were absent in rats fed the hypocaloric diet.

Subunit and region-specific decreases in nicotinic acetylcholine receptor mRNA in the aged rat brain.

We have investigated possible changes in the mRNA levels for several alpha and beta subunits of the nicotinic acetylcholine receptor (nAChR) and the level of binding for nicotinic ligands in 7- to 32-month-old rats. Alpha4 and beta2, and to a lesser extent alpha6 and beta3, mRNA levels showed decreases between 20 and 30% at 29 months of age which in some areas reached 50% at 32 months of age. Alpha7 showed a small increase from 7 to 14 months and then a progressive decrease from 14 to 32 months down to the 7-month levels. 3H-epibatidine binding did not significantly change from 7 to 32 months of age in rat tel- and diencephalon. Binding in the substantia nigra was exceptional in that it showed a significant decrease starting from 23 months of age. 125I-alpha-bungarotoxin binding showed a pattern of change which roughly paralleled that of alpha7 mRNA. These findings show that an alteration in some steps of nAChR biosynthesis takes place during aging, which may be related to functional changes in nicotinic transmission.

Aging and food restriction: effect on lipids of cerebral cortex.

In experimental animals dietary restriction reduces the body weight increase due to aging, increases longevity and delays the onset of age-related physiological deterioration, including age-related changes in serum lipids. Little is known about the influence of food restriction on brain lipids, whose concentration and composition have been shown to change with age. We studied whether some biochemical and biophysical parameters of rat brain membranes, known to be modified with age, were affected by a diet low in calories, in which 50% of lipids and 35% of carbohydrates have been replaced by fibers. The diet was started at weaning and maintained throughout the animal's entire life span. Animals fed the low calorie diet survived longer and gained less body weight than standard diet fed rats. Age-related increases in microviscosity, cholesterol/phospholipid and sphingomyelin/phosphatidylcholine ratios were reduced or restored to the levels of young animals in cortex membranes of 32 old rats fed the low calorie diet, while the age-related increase in mono- to polyunsaturated fatty acid ratios in phospholipids was further raised. In conclusion we have shown that a diet low in calories and high in fibers affects lipid composition in the rat brain, in a direction opposite to that normally believed to reduce age-related deterioration of brain functions.

Differential response to immobilization stress of striatal dopaminergic and hippocampal noradrenergic systems in aged rats.

The effect of restraint stress on synthesis of central norepinephrine (NE) and dopamine (DA) was studied in adult and old rats. The rate of in vivo synthesis of the two catecholamines was determined in hippocampus (a prevalently noradrenergic area) and in striatum (a prevalently dopaminergic area) by measuring the accumulation of DOPA for 60 min after decarboxylase inhibition. NE synthesis was stimulated by stress in the first 30 min, after which the accumulation of DOPA declined. The stimulation was much greater in old rats. In striatum, endogenous DOPA concentration was significantly lower in old rats. Stress significantly enhanced DOPA accumulation in the first 30 min in both age groups but after this interval accumulation continued linearly only in young rats. These results indicate that in aged rats the response to stress of some noradrenergic and dopaminergic systems may be altered in opposite directions.

Age-related changes in metabolic responses to chronic monoamine depletion in central dopaminergic and serotonergic systems of rats treated with reserpine.

Groups of 5, 15, and 27-month-old rats were treated with reserpine (5 mg/kg IP) and killed after 1, 7, 14, and 21 days. Reserpine's effects on dopamine (DA) and serotonin (5HT) metabolism were studied in the striata and limbic areas, on the basis of changes in metabolite concentrations and a calculated index [delta(C/M)] expressing changes in the molar ratio between metabolite and parent monoamine. Twenty-four hours after drug treatment, when the two monoamines reached a new steady-state, about 10% of the normal concentration, there was evident enhancement in the metabolism of striatal DA, indicated by HVA levels and delta(C/M) changes. This increase was significantly lower in the senescent rats. In contrast with HVA, the levels of DOPAC were lowered in reserpinized rats. This effect, present in all three age groups, lasted up to 21 days in young and 14 days in adult and old rats. However, for this metabolite too, the delta(C/M) indicated an increase in its formation which was lower in 27-month-old rats. Similar effects were observed in limbic areas, but here the age-related differences were less evident. Serotonin metabolism was also increased by reserpine as indicated by the delta(C/M). This parameter was lower in young than in adult and old rats, at least in limbic area. Similarly, 5HIAA increased only in the senescent rats. These age-related differences were not evident in striata. In this rat population, aging apparently modified the compensatory metabolic responses of some dopaminergic and serotonergic systems. The direction of these changes, however, depends on the system and the brain region considered.

Changes in monoamines 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.

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.

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.

A reduced calorie-high fiber diet retards age-associated decreases in muscarinic receptor sensitivity.

The effects of a reduced calorie-high fiber diet (RCHF) were examined on three cholinergic signal transduction (ST) parameters: (a) oxotremorine enhancement of K(+)-evoked dopamine release and (b) carbachol-stimulated low KM GTPase activity [an indicator of muscarinic receptor (mAChR)-G protein coupling/uncoupling], and (c) [3H]Quinuclidinyl benzilate (QNB) autoradiography. Comparisons were made among: young control (6 months), old normal control, old reduced calorie high fiber [both 24 months)]. The results indicated that old reduced calorie high fiber rats (1900 kcal/kg/day, 2.4%, lipids 2.4%, fiber 28%, carbohydrates 40.7%) as compared to the old normal control rats (3000 kcal/kg/day, 4.8% lipids, 4.2% fiber, carbohydrates 61.5%) showed a retardation of age-related deficits in dopamine release (a above) and GTPase activity (b above). These parameters were 25% higher in the old reduced calorie high fiber rats as compared to old normal controls and did not differ from young controls, even though there was no increase in mAChR concentration in the restricted group. Thus, these results indicate that a reduced calorie high fiber diet as utilized in these experiments was effective in retarding the age-related decrements in two of three signal transduction parameters. They are discussed in terms of the induction of membrane changes (e.g., fluidity) or related decreases in oxidative stress by the restricted diet that may be involved in these signal transduction effects.

Cross tolerance between ketamine and morphine to some pharmacological and biochemical effects.

Whether morphine and ketamine induced cross-tolerance to some of their common pharmacological and biochemical effects, namely analgesia and enhancement of metabolites of dopamine (DA) in the striatum and limbic area of the rat was analysed. Ketamine was given at the dose of 100 mg/kg, twice a day for 8 days. After this treatment, a challenge dose of morphine (15 mg/kg, i.p.) still induced analgesia comparable to that induced by morphine alone, showing no cross-tolerance to this effect. In contrast, the challenge dose of morphine given to ketamine-tolerant rats no longer enhanced metabolism of DA, indicating the appearance of cross-tolerance to this effect. A high degree of tolerance to morphine was obtained after the subcutaneous implantation of rats with pellets of morphine; a challenge dose of ketamine to morphine-tolerant rats induced marked analgesia, with no cross-tolerance to this pharmacological effect, while cross-tolerance was present to the biochemical effect. The existence of a high degree of reciprocal cross-tolerance in both areas and on both metabolites of DA is consistent with the hypothesis of action at a common receptor; the lack of cross-tolerance to the analgesic effect indicates that analgesia is achieved by a different mechanism for the two drugs.

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.

Stimulation of serotonin synthesis in rat brain after antiepilepsirine, an antiepileptic piperine derivative.

Piperine and two of its derivatives, antiepilepsirine (AE or 3,4-methylendioxycynnamoylpiperine) and compound 7448 (N-isopropyl 3 (4 chloro-phenyl) propenoylamide) are very effective in stimulating serotonin (5HT) synthesis. AE raises the ratio of free-to-bound tryptophan (TP) in plasma and induces a long-lasting increase of this aminoacid in brain. At the same time in striatum and limbic area it causes a lasting increase in 5 hydroxyindolacetic acid (5HIAA) a 5HT metabolite and to a lesser extent, an increase in the levels of the monoamine itself. Together with this action on 5HT metabolism we found that AE caused release of 3H-5HT from an in vitro synaptosomal preparation. It thus appears that piperine and its derivatives AE and compound 7148 affect the central serotonergic system.

The ACTH/MSH (4-9) analog Org2766 improves retrieval of information after a fimbria fornix transection.

The fimbria fornix of male Wistar rats was transected unilaterally after they had been successfully trained in the Morris maze and the passive avoidance task. Sham-operated and lesioned animals were treated either with Org2766 or saline for two weeks. Subsequently, the performance of all groups was tested again starting two days after the last treatment. The lesioned animals showed a deficit in performance in both tasks, indicating interference of the lesion with retrieval of information. Org2766 improved the poor performance of the lesioned animals in the Morris maze, but not in the passive avoidance task.

Miniaturized optoelectronic system for telemetry of in vivo voltammetric signals.

In vivo voltammetry is an electrochemical technique that uses carbon fiber microelectrodes stereotaxically implanted in brain areas to monitor monoamine metabolism and release continuously, in freely moving animals. Electric wires connect the polarograph to the animal. A wire-less transmission system (optoelectronic transmission, OPT) of voltammetric signals is described here. It uses infrared diffused light, exploiting the diffusion of the transmitted light over walls and ceiling towards a receiver. The transmission system consists of a main unit and a satellite unit (40 x 30 x 5 mm) positioned on the animal's back. Voltammetric recordings obtained by the classical system (with wires) and by OPT are well defined and almost identical in shape. The power supply is provided by two thin lithium batteries (+/- 3V) that can record for up to 20 h. OPT permits detailed behavioral observations since the animal can be left free to move in a spacious environment. Voltammetry using OPT allows simultaneous recording of neuronal firing activity as well as electroencephalographic recordings (EEG) since there is no cross-talk between the circuits used. The results illustrate the reliability and usefulness of this wire-less transmission system for studying relationships between neurochemical, behavioral and electrophysiological activities.

Does acute L-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.