Brain oxidative stress mediates anxiety-like behavior induced by indomethacin in zebrafish: protective effect of alpha-tocopherol.

RATIONALE: Indomethacin (INDO) is a widely utilized non-steroidal anti-inflammatory drug (NSAID) with recognized effect on the central nervous system. Although previous reports demonstrate that prolonged treatment with indomethacin can lead to behavioral alterations such as anxiety disorder, the biochemical effect exerted by this drug on the brain are not fully understood. OBJECTIVES: The aim of present study was to evaluate if anxiety-like behavior elicited by indomethacin is mediated by brains oxidative stress as well as if alpha-tocopherol, a potent antioxidant, is able to prevent the behavioral and biochemical alterations induced by indomethacin treatment. METHODS: Zebrafish were utilized as experimental model and subdivided into control, INDO 1 mg/Kg, INDO 2 mg/Kg, INDO 3 g/Kg, α-TP 2 mg/Kg, α-TP 2 mg/Kg + INDO 1 mg/Kg and α-TP + INDO 2 mg/Kg groups. Vertical distributions elicited by novelty and brain oxidative stress were utilized to determinate behavioral and biochemical alterations elicited by indomethacin treatment, respectively. RESULTS: Our results showed that treatment with indomethacin 3 mg/kg induces animal death. No changes in animal survival were observed in animals treated with lower doses of indomethacin. Indomethacin induced significant anxiogenic-like behavior as well as intense oxidative stress in zebrafish brain. Treatment with alpha-tocopherol was able to prevent anxiety-like behavior and brain oxidative stress induced by indomethacin. CONCLUSIONS: Data presented in current study demonstrated for the first time that indomethacin induces anxiety-like behavior mediated by brain oxidative stress in zebrafish as well as that pre-treatment with alpha-tocopherol is able to prevent these collateral effects.

Putative Activation of Cannabinoid Receptor Type 1 Prevents Brain Oxidative Stress and Inhibits Aggressive-Like Behavior in Zebrafish.

Background: Aggression is a set of complex behaviors commonly described in different neurological disorders, such as schizophrenia, autistim spectrum disorder, and anxiety. Previous studies have described that some changes in the redox status of the brain are closely associated with aggressive behavior in different species. In addition, the endocannabinoid system acts as a neuromodulator of the central nervous system, however, its participation in aggressive behavior needs to be elucidated. Danio rerio (zebrafish) is an important model in the study of aggression, in this context, the present study investigated whether the activation of type 1 cannabinoid receptors (CB1r) alters the cerebral redox state and aggressive behavior in zebrafish. Materials and Methods: We performed pharmacological manipulations with the CB1r agonist (ACEA) and antagonist (AM-251) to assess the role of this receptor in aggressive behavior. Individuals were isolated in pairs, without physical contact for 24 h, treated with the drugs of interest, and after 30 minutes of pharmacokinetics, the fights were filmed for 30 min, and the individuals were identified as dominant or subordinate. Results: A consistent decrease in the strike and bite aggressive behavior was observed in the group treated with the ACEA agonist compared with that in the control and AM-251 groups. When evaluating the cerebral redox state, we observed that treatment with the ACEA agonist reduced malondialdehyde (MDA) levels and increased the levels of sulfhydryl groups compared with those in the control group. These results indicate that the activation of CB1r by the ACEA agonist inhibited aggressiveness and attenuated the levels of oxidative stress in both subjects (dominant or subordinate) in the treated group. Conclusion: Thus, we suggest that zebrafish is an alternative model to study common aggressive behavior disorders among species and that CB1r represent a potential target for the development of treatments for aggressive disorders.

High performance liquid chromatography-based method to analyze activity of GABA transporters in central nervous system.

The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation medium. The evaluation of the GAT kinetics returned values of Km = 382.5 ± 32.2 µM and Vmax = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na+- and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.

Euterpe oleracea fruit (Açai)-enriched diet suppresses the development of experimental cerebral malaria induced by Plasmodium berghei (ANKA) infection.

BACKGROUND: Cerebral malaria is one of the most severe complications attributed to protozoal infection by Plasmodium falciparum, gaining prominence in children mortality rates in endemic areas. This condition has a complex pathogenesis associated with behavioral, cognitive and motor sequels in humans and current antimalarial therapies have shown little effect in those aspects. Natural products with antioxidant and anti-inflammatory properties have become a valuable alternative therapeutic option in the treatment of distinct conditions. In this context, this study investigated the neuroprotective effect of Euterpe oleracea (açai) enriched diet during the development of experimental cerebral malaria induced by the inoculation of Swiss albino mice with Plasmodium berghei ANKA strain. METHODS: After Plasmodium infection, animals were maintained on a feeding with Euterpe oleracea enriched ration and parameters such as survival curve, parasitemia and body weight were routinely monitored. The present study has also evaluated the effect of açai-enriched diet on the blood-brain barrier leakage, histological alterations and neurocognitive impairments in mice developing cerebral malaria. RESULTS: Our results demonstrate that between 7th-19th day post infection the survival rate of the group treated with açai enriched ration was higher when compared with Plasmodium-infected mice in which 100% of mice died until the 11th days post-infection, demonstrating that açai diet has a protective effect on the survival of infected treated animals. The same was observed in the brain vascular extravasation, where Evans blue dye assays showed significantly less dye extravasation in the brains of Plasmodium-infected mice treated with açai enriched ration, demonstrating more preserved blood-brain barrier integrity. Açai-enriched diet also attenuate the histopathological alterations elicited by Plasmodium berghei infection. We also showed a decrease of the neurological impairments arising from the exposure of cerebral parenchyma in the group treated with açai diet, ameliorating motor and neuropsychiatric changes, analyzed through the SHIRPA protocol. CONCLUSION: With these results, we conclude that the treatment with açai enriched ration decreased the mortality of infected animals, as well as protected the blood-brain barrier and the neurocognitive deficits in Plasmodium-infected animals.

Oxidative Stress Mediates Anxiety-Like Behavior Induced by High Caffeine Intake in Zebrafish: Protective Effect of Alpha-Tocopherol.

Anxiety is a common symptom associated with high caffeine intake. Although the neurochemical mechanisms of caffeine-induced anxiety remain unclear, there are some evidences suggesting participation of oxidative stress. Based on these evidences, the current study is aimed at evaluating the possible protective effect of alpha-tocopherol (TPH) against anxiety-like behavior induced by caffeine (CAF) in zebrafish. Adult animals were treated with CAF (100 mg/kg) or TPH (1 mg/kg)+CAF before behavioral and biochemical evaluations. Oxidative stress in the zebrafish brain was evaluated by a lipid peroxidation assay, and anxiety-like behavior was monitored using light/dark preference and novel tank diving test. Caffeine treatment evoked significant elevation of brain MDA levels in the zebrafish brain, and TPH treatment prevented this increase. Caffeine treatment also induced anxiety-like behavior, while this effect was not observed in the TPH+CAF group. Taken together, the current study suggests that TPH treatment is able to inhibit oxidative stress and anxiety-like behavior evoked by caffeine.

Adenosine A1 receptors modulate the Na+-Hypertonicity induced glutamate release in hypothalamic glial cells.

Glutamate release in response to a hypertonic stimulus is a well described phenomenon in the hypothalamus. Evidence suggests that hypothalamic glial cells release glutamate into the extracellular environment in hypertonic conditions. In the current study, we described autocrine regulation of adenosine on glutamate release induced by Na+hypertonicity in hypothalamic glial cell cultures. We showed that glial cells cultured from the cerebral cortex did not release glutamate or adenosine under hypertonic conditions. The findings suggest that the hypothalamus has specialized glial cells, which are responsive to osmotic variations. Stimulation or inhibition of adenosine A1 receptors modulates extracellular glutamate levels in hypothalamic glial cell cultures under hypertonic stimulation. Our results extend previous observations regarding the role of glial cells in the control of hypothalamic physiology. They further demonstrate for the first time that hypothalamic glial cells regulate Na+-hypertonicity-induced glutamate release by activation of adenosine A1 receptors via adenosine release.

Ascorbic acid prevents chloroquine-induced toxicity in inner glial cells.

Ototoxicity is a collateral effect of prolonged treatment with chloroquine which is a widely utilized as an anti-lupus and anti-malarial drug. Glial cells of inner ear are responsible for maintenance of neuronal cells homeostasis in auditory system. In the current study we have evaluated chloroquine-induced toxicity and protective effect of ascorbic acid treatment on Schwann glial cell cultures of inner ear. Glial cells were cultured from organ of Corti of mice cochlear structure. Purity of Schwann glial cell was confirmed by S100 protein staining. Cell viability was evaluated in control and cultures treated with different concentrations of chloroquine. Glutamate uptake and ROS production were measured by HPLC and DCFH-DA probe fluorescence, respectively. Results have shown that chloroquine treatment evoked concentration and time -dependent toxicity (LC50 = 70 µM) as well as significant decrease on glutamate uptake and high production of ROS in glial cell cultures. Co-treatment with ascorbic acid has prevented both chloroquine-induced ROS production and chloroquine toxicity on glial cell cultures. This pre-clinical study is the first one to demonstrate chloroquine-induced ROS production by glial cells of inner ear as well as the protective effect exerted by ascorbic acid on these cells.

Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.

Serotonin (5-HT) is a neurotransmitter that is involved in many behavioral functions, including the organization of defense, and its putative pathological correlate, anxiety and stress disorders. Recently, behavioral tests for anxiety have been proposed in zebrafish. Exposure to the novel tank test or to the light/dark test increased extracellular fluid 5-HT content in the brain; anxiety-like behavior correlated positively with 5-HT content in the novel tank test, while the correlation was negative in the light/dark test. Acute treatment with a low dose of fluoxetine was anxiolytic in the geotaxis test and anxiogenic in the scototaxis test, while treatment with a higher dose produced a hyperlocomotor effect in both tasks. Buspirone and WAY 100635 were anxiolytic in both tests, while SB 224289 was anxiolytic in the geotaxis and slightly anxiogenic in the scototaxis test. Serotonin depletion with pCPA was anxiogenic in the geotaxis and anxiolytic in scototaxis. These results underline the differential sensitivity of these tasks to assess serotonergic agents; alternatively, serotonin might regulate zebrafish behavior differently in the novel tank test and in the light/dark test.