Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures

Background: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. Methods: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. Results: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival)...(AU)

Response of Azospirillum brasilense Cd to sodium chloride stress.

Growth of Azospirillum brasilense Cd in the presence of different NaCl concentrations showed that it tolerates up to 200 mM NaCl in the medium, without appreciable decline in growth rate. At 300 mM NaCl, a decrease of 66% in growth was observed at 24 h of culture. At 48 h of culture, bacteria in the presence of 300 mM NaCl reached the maximum optical density value that was attained at 12 h by control cultures. This investigation was designed to elucidate the effect of saline stress on Azospirillum brasilense Cd and the physiologic mechanism involved in its possible salinity tolerance. For this reason, studies of other osmolytes, as well as of putrescine metabolism and protein patterns were done with bacteria grown with this NaCl concentration in the medium, at 24 and at 48 hours. A. brasilense responded to saline stress elevating the intracellular concentration of glutamate at 24 h, and of K+ at 48 h. Glucan pattern, putrescine metabolism, and total and periplasmic protein patterns of the treated group showed several changes with respect to the control. In spite of the several cellular functions affected by saline stress, the results imply that A. brasilense Cd shows salinity tolerance in these experimental conditions.

In vivo monitoring of glutamate in the brain by microdialysis and capillary electrophoresis with laser-induced fluorescence detection.

Glutamic acid, an excitatory neurotransmitter, was monitored in vivo in the corpus striatum of freely moving rats by brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection. A procedure to derivatize glutamate in complex matrices was developed. Capillary electrophoresis in 12 microns I.D. capillaries was performed to determine glutamate with a migration time of 195 s. Laser-induced fluorescence detection with 488-nm radiation from an argon ion laser and with colinear geometry was used. An injection of haloperidol decreased the concentration of glutamic acid in the dialysates. These experiments support the hypothesis that dopamine receptor blockade decreases glutamate release. The potential of these techniques for the study of chemicals in biomedical experiments is discussed.

Colinear laser-induced fluorescence detector for capillary electrophoresis. Analysis of glutamic acid in brain dialysates.

Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a colinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10(-10) M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10(-12) M) of this compound in biological samples should be possible. Fourth, narrow-bore capillaries (5-10 microm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.

Effects of ovarian steroids on the gonadotropin response to N-methyl-D-aspartate and on hypothalamic excitatory amino acid levels during sexual maturation in female rats.

In order to evaluate the involvement of estrogen-progesterone (EP) in the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on gonadotropin secretion during sexual development in female rats, NMDA (30 mg/kg sc) was administered to 16- and 30-day-old female rats pretreated with EP. NMDA administration induced increases in plasma LH concentration that were 13.6-fold and 94.5-fold higher, respectively, than those found after NMDA alone. The increase of LH levels induced by NMDA was accompanied by a significant enhancement of the content of GnRH in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus (APOA/MBH). EP potentiated this increase of GnRH induced by NMDA. NMDA increased plasma FSH levels at 16 days of age, and this increase was inhibited by EP treatment. In 30-day-old rats EP induced FSH release in response to NMDA. This release was not observed in rats treated only with NMDA. In 16-day-old rats EP induced an increase in the concentrations of aspartate, glutamate, and glycine in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus, the excitatory amino acids involved in NMDA neurotransmission. This effect was not observed in rats of 30 days of age. In summary, the present results show that during sexual maturation ovarian steroids potentiated the LH-releasing response to NMDA probably by acting at the hypothalamic level; furthermore, during sexual maturation there are changes in the response to EP of the hypothalamic concentrations of excitatory amino acids. These findings could be related to the neuroendocrine mechanisms regulating the onset of puberty and the sexual cycle in female rats.

Microiontophoresis of biogenic amines on cortical neurons: amounts of NA, DA and 5-HT ejected, compared with tissue content.

The present series of studies were carried out to quantify the amounts of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) ejected from iontophoresis micropipettes and that produce inhibitory and modulatory effects on cortical neurons, in the frontoparietal cortex of the rat and in the occipital cortex of the cat. Using radioactive isotopes of the biogenic amines the effective transport number (n) was found to be 0.08 for DA; 0.13 for NA, and 0.22 for 5-HT. In addition, similar determinations were made, for comparison purposes, of the transport numbers of the neurotransmitters acetylcholine (ACh; n = 0.44), gamma-amino-n-butyrate (GABA, n = 13), and glutamate (GLU; n = 0.27). The quantities ejected under in vivo conditions were then estimated using Faraday's formula and fell in the pmol range (10(-12) mol). The effects of DA, NA and 5-HT on cortical units were studied; the amounts ejected were compared with the endogenous tissue content of these amines, determined by means of specific and sensitive radioenzymatic assays in the regions where the microiontophoretic experiments were performed. These results are discussed in the light of the anatomical, biochemical and electrophysiological data suggesting a modulatory role for the biogenic amines in the cerebral neocortex.