Dehydroepiandrosterone increases synaptosomal glutamate release and improves the performance in inhibitory avoidance task.

Dehydroepiandrosterone (DHEA) exerts multiple effects in the rodent central nervous system (CNS), mediated through its nongenomic actions on several neurotransmitter systems, increasing neuronal excitability, modulating neuronal plasticity and presenting neuroprotective properties. It has been demonstrated that DHEA is a potent modulator of GABA(A), NMDA and Sigma receptors. In the present study, we investigated the effect of DHEA on (i) basal- and K(+)-stimulated l-[(3)H]glutamate release from synaptosomes (both in vitro and ex vivo), (ii) synaptosomal l-[(3)H]glutamate uptake (in vitro), and (iii) an inhibitory avoidance task (in vivo). The results indicated that DHEA in vitro increased glutamate release by 57%, and its intracerebroventricular infusion increased the basal-[(3)H]glutamate release by 15%. After 30 min of intraperitoneal administration, DHEA levels in the serum or CSF increased 33 and 21 times, respectively. Additionally, DHEA, intraperitoneally administrated 30 min before training, improved memory for inhibitory avoidance task. Concluding, DHEA could improve memory on an inhibitory avoidance task, perhaps due to its ability to physiologically strength the glutamatergic tonus by increasing glutamate release.

Methylmercury increases glutamate release from brain synaptosomes and glutamate uptake by cortical slices from suckling rat pups: modulatory effect of ebselen.

During the early postnatal period the brain is extremely sensitive to external agents. Here, we examined the effect of subcutaneous injections of methylmercury (MeHg; 2 mg/kg) during the suckling period (postnatal days [PND] 3-10, 3-17, or 3-24) on glutamate release from brain synaptosomal preparations and on glutamate uptake by brain cortical slices of rat pups. The possible antagonist effect of ebselen against MeHg effect was also examined at PND 24. MeHg increased the basal (but not K+-stimulated) glutamate release and glutamate uptake at PND 24. A strong tendency of increase in the basal glutamate release from synaptosomes (p= 0.088) was observed at PND 17. Ebselen, which did not affect glutamate release and uptake per se, prevented both effects of MeHg. This study indicates that (1) the effect of MeHg on glutamate release could be involved in its toxicity; (2) the increase in the glutamate uptake could represent a pathophysiological response to MeHg-induced glutamate release; (3) the inhibitory effect of ebselen on MeHg-induced glutamate release could be related to its reported neuroprotective effects.

Ganglioside alterations in the central nervous system of rats chronically injected with methylmalonic and propionic acids.

Neurological dysfunction and structural cerebral abnormalities are commonly found in patients with methylmalonic and propionic acidemia. However, the mechanisms underlying the neuropathology of these disorders are poorly understood. We have previously demonstrated that methylmalonic and propionic acids induce a significant reduction of ganglioside N-acetylneuraminic acid in the brain of rats subjected to chronic administration of these metabolites. In the present study, we investigated the in vivo effects of chronic administration of methylmalonic (MMA) and propionic (PA) acids (from the 6th to the 28th day of life) on the distribution and composition of gangliosides in the cerebellum and cerebral cortex of rats. Control rats were treated with the same volumes of saline. It was first verified that MMA and PA treatment did not modify body, cerebellum, or cortical weight, nor the ganglioside concentration in the cerebral cortex of the animals. In contrast, a significant reduction in total ganglioside content in the cerebellum of approximately 20-30% and 50% of control levels occurred in rats injected with MMA and PA, respectively. Moreover, chronic MMA and PA administration did not interfere with the ganglioside pattern in the cerebral cortex, whereas the distribution of individual gangliosides was altered in the cerebellum of MMA- and PA-treated animals. Rats injected with MMA demonstrated a marked decrease in GM1 and GD3, whereas chronic PA treatment provoked a significant reduction of all ganglioside species, with the exception of an increase in GM2. Since gangliosides are closely related to the dendritic surface and other neural membranes, indirectly reflecting synaptogenesis, these ganglioside abnormalities may be associated with the brain damage found in methylmalonic and propionic acidemias.

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats.

Neurological dysfunction is common in patients with methylmalonic and propionic acidemias. However, the mechanisms underlying the neuropathology of these disorders are far from understood. In the present study we investigated the in vitro effects of methylmalonic (MMA) and propionic (PA) acids at various concentrations (1 microM-5 mM) on three parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomes, Na+-dependent L-[3H]glutamate uptake by synaptosomes and Na+-independent L-[3H]glutamate uptake by synaptic vesicles from cerebral cortex of male adult Wistar rats. The results showed that MMA significantly increased potassium-induced but not basal L-[3H]glutamate release from synaptosomes with no alteration in synaptosomal L-[3H]glutamate uptake. A significant reduction of L-[3H]glutamate incorporation into vesicles caused by MMA was also detected. In contrast, PA had no effect on these parameters. These findings indicate that MMA alters the glutamatergic system. Although additional studies are necessary to evaluate the importance of these observations for the neuropathology of methylmalonic acidemia, it is possible that the effects elicited by MMA may lead to excessive glutamate concentrations at the synaptic cleft, a fact that may explain previous in vivo and in vitro findings associating MMA with excitotoxicity.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 æmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 æmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children

Methylmalonate administration decreases Na+,K+-ATPase activity in cerebral cortex of rats.

Buffered methylmalonate (MMA) was injected s.c. into rats twice a day at 8 h intervals from 5 to 25 days of age (chronic treatment), or into 10-day-old rats three times a day at 1 h intervals (acute treatment). Control rats received saline in the same volumes. Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from cerebral cortex of rats. Na+,K+-ATPase activity was reduced by 30-40% in MMA-treated rats, whereas Mg2+-ATPase activity was not. In contrast, MMA at final concentrations ranging from 0.1 to 2.0 mM had no in vitro effect on these enzyme activities. However, when brain homogenates were incubated with 2 mM MMA before membrane preparation, Na+,K+-ATPase activity was decreased by 44%. Furthermore, this reduction was totally prevented by the simultaneous addition of glutathione and MMA, suggesting that oxidation of thiol groups or other oxidative damage to the enzyme could be responsible for this effect.

Effect of chemically induced propionic acidemia on neurobehavioral development of rats.

High levels of propionic acid (PPA) comparable to those of human propionic acidemia were achieved in blood (1-5 mmol/l) and brain (1 micromol/g) of rats by administering saline-buffered propionate (pH 7.4) subcutaneously twice a day from the 6th to the 28th day of life. PPA doses ranged from 1.44 to 1.92 micromol/g body weight as a function of animal age. Control rats were treated with saline in the same volumes. Growth and development of physical landmarks were assessed by monitoring the following parameters daily: body weight, upper incisor eruption, eye opening, and hair coat. Development of some reflexes was also monitored, and a specific subset of motor skills was evaluated at days 14 and 21 of life by the free-fall righting test and the spontaneous alternation test. Chronic PPA administration had no effect on body weight, cerebral cortex weight, or cerebellum weight, but caused slight but significant delays in the day of appearance of hair coat and eye opening, indicating an effect of PPA on the development of physical parameters. Free-fall righting was impaired in PPA-treated animals. On the other hand, PPA administration had no effect on the performance of the animals in the spontaneous alternation tests. Long-term effects of early PPA administration were investigated by assessing animal performance in an aversive task (two-way shuttle avoidance task) and in a nonaversive (open-field task) behavioral task at 60 days of age. PPA-treated rats did not habituate to the open field, and presented a lack of retention of the shuttle-avoidance task. Our results suggest that early postnatal PPA administration to rats alters normal development and induces long-term behavioral deficits in aversive and nonaversive tasks.

Chronic early leucine administration induces behavioral deficits in rats.

Sustained levels of leucine comparable to those of human Maple Syrup Urine Disease (MSUD) were achieved in blood and brain of rats by subcutaneous leucine administration twice a day from the 6th to the 28th day of life. Control rats were treated with saline in the same volumes. Behavioral studies using aversive and nonaversive tasks were performed during adult age. Chronic early leucine treatment impaired acquisition of a two-way shuttle avoidance task and altered habituation to an open field. Our results suggest that early postnatal leucine administration induces long-lasting behavioral deficits.

Inhibition of rat brain lipid synthesis in vitro by 4-hydroxybutyric acid.

4-Hydroxybutyric acid (4HB) is accumulated in succinic semialdehyde dehydrogenase deficiency, an inherited metabolic disease severely affecting the CNS during postnatal development. Thus, the present study was designed to evaluate the in vitro influence of 4HB on lipid synthesis and CO2 production from [U-14C] acetate in cerebral cortex of 30-day-old Wistar rats. In the presence of 4HB, there was an inhibition of lipid synthesis in cerebral cortex prisms and homogenates. However, no inhibition of lipid synthesis occurred in the homogenates free of nuclei and mitochondria. In addition, CO2 production was inhibited by 4HB in cerebral cortex prisms, and homogenates and in the mitochondrial fraction. These results might possibly be explained by an impairment of mitochondrial metabolism by 4HB which may secondarily inhibit lipid synthesis. The results reported here may help to better understand the neuropathophysiology of 4-hydroxybutyric aciduria.

Chronic administration of propionic acid reduces ganglioside N-acetylneuraminic acid concentration in cerebellum of young rats.

Elevated levels of propionate comparable to those of human propionic acidaemia were achieved in the blood of young rats by injecting subcutaneously buffered propionic acid (PPA) twice a day at 8-h intervals from the 6th to the 28th day of life. A matched group of animals (controls) was treated with the same volumes of saline. The animals were weighed and sacrificed by decapitation at 28, 35 or 60 days of age. Cerebellum and cerebrum were weighed and their protein and ganglioside N-acetylneuraminic acid (G-NeuAc) contents determined. Body, cerebral and cerebellar weights were similar in both groups, suggesting that PPA per se neither alters the appetite of the rats nor causes malnutrition. Brain protein concentration was also not affected by chronic administration of PPA, in contrast to G-NeuAc concentration which was significantly reduced in the cerebellum. Since ganglioside concentration is closely related to the dendritic surface and indirectly reflects synaptogenesis, our results of an important ganglioside deficit in the brain of PPA-treated animals may be related to the neurologic dysfunction characteristic of propionic acidaemic patients.

Inhibition of Na+,K+-ATPase from rat brain cortex by propionic acid.

Buffered propionic acid was injected s.c. into rats twice a day at 8 h intervals from the 6 to 21 days of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation at 23 days. Whole brain and cerebral cortex were weighed and synaptic plasma membranes were prepared from cortex for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups, suggesting that propionic acid does not cause malnutrition in rats. Na+,K+-ATPase activity was significantly reduced by 30% in membranes from the propionate-treated group, whereas Mg2+-ATPase activity was not. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 23-day-old rats and incubated with propionic acid at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 22-32%. Since propionic acid concentrations in plasma of chronically treated rats and of propionic acidemic children are of the same order of magnitude as those tested in vitro, the results suggest that the inhibition of Na+,K+-ATPase activity may be related to the neurological dysfunction of patients affected by propionic acidaemia.