Brain zinc chelation by diethyldithiocarbamate increased the behavioral and mitochondrial damages in zebrafish subjected to hypoxia.

The increase in brain levels of chelatable zinc (Zn) in dysfunctions involving oxygen deprivation has stimulated the treatment with Zn chelators, such as diethyldithiocarbamate (DEDTC). However, DEDTC is a redox-active compound and it should be better evaluated during hypoxia. We use the hypoxia model in zebrafish to evaluate DEDTC effects. The exploratory behavior, chelatable Zn content, activities of mitochondrial dehydrogenases, reactive species levels (nitric oxide, superoxide anion, hydroxyl radical scavenger capacity) and cellular antioxidants (sulfhydryl, superoxide dismutase) of zebrafish brain were assessed after recovery, with or without 0.2 mM DEDTC. The increased brain levels of chelatable Zn induced by hypoxia were mitigated by DEDTC. However, the novel tank task indicated that DEDTC did further enhance the exploratory deficit caused by hypoxia. Furthermore, these behavioral impairments caused by DEDTC were more associated with a negative action on mitochondrial activity and brain oxidative balance. Thus, due to apparent pro-oxidant action of DEDTC, our data do not support its use for neuroprotection in neuropathologies involving oxygen deprivation.

Topographical analysis of reactive zinc in the central nervous system of adult zebrafish (Danio rerio).

Reactive zinc (Zn) is crucial for neuronal signaling and is largely distributed within presynaptic vesicles of some axon terminals of distinct vertebrates. However, the distribution of reactive Zn throughout the central nervous system (CNS) is not fully explored. We performed a topographical study of CNS structures containing reactive Zn in the adult zebrafish (Danio rerio). Slices of CNS from zebrafish were stained by Neo-Timm and/or cresyl violet. The Zn specificity of Neo-Timm was evaluated with Zn chelants, N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), sodium diethyldithiocarbamate (DEDTC), Zn sulfide washing solution, and hydrochloric acid (HCl). Unfixed slices were also immersed in the fluorescent Zn probe (zinpyr-1). Yellow-to-brown-to-black granules were revealed by Neo-Timm in the zebrafish CNS. Telencephalon exhibited slightly stained regions, while rhombencephalic structures showed high levels of staining. Although stained granules were found on the cell bodies, rhombencephalic structures showed a neuropil staining profile. The TPEN produced a mild reduction in Neo-Timm staining, while HCl and mainly DEDTC abolished the staining, indicating a large Zn content. This result was also confirmed by the application of a Zn probe. The present topographical study revealed reactive Zn throughout the CNS in adult zebrafish that should be considered in future investigation of Zn in the brain on a larger scale.

Evaluation of spontaneous recovery of behavioral and brain injury profiles in zebrafish after hypoxia.

Cerebral hypoxia-ischemia can lead to motor and sensory impairments which can be dependent on the extent of infarcted regions. Since a better understanding of the neurochemical mechanisms involved in this injury is needed, the use of zebrafish as a cerebral hypoxia model has become quite promising because it could improve the knowledge about hypoxia-ischemia. In the current study, we aimed to investigate the spontaneous recovery of brain and behavioral impairments induced by hypoxia in adult zebrafish. Brain injury levels were analyzed by spectrophotometric measurement of mitochondrial dehydrogenase activity by staining with 2,3,5-triphenyltetrazolium chloride, and behavioral profiles were assessed by the open tank test. The induction of hypoxia substantially decreased mitochondrial activity in the brain and impaired behavior. The spontaneous recovery of fish subjected to hypoxia was assessed after 1, 3, 6, 24, and 48h under normoxia. The quantification of brain injury levels showed a significant increase until 24h after hypoxia, but after 48h this effect was completely reversed. Regarding behavioral parameters, we verified that locomotor activity and vertical exploration were impaired by hypoxia and these effects were reversed after 3h under normoxia. Taken together, these results show that zebrafish exhibited transient cerebral and behavioral impairments when submitted to hypoxia, and 1h under normoxic conditions was insufficient to reverse both effects. Therefore, our data help to elucidate the time window of spontaneous recovery in zebrafish after hypoxia and also the behavioral phenotypes involved in this phenomenon.

Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.

Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol.

Taurine (TAU) is an amino sulfonic acid that plays protective roles against neurochemical impairments induced by ethanol (EtOH). Mounting evidence shows the applicability of zebrafish for evaluating locomotor parameters and anxiety-like behavioral phenotypes after EtOH exposure in a large scale manner. In this study, we assess the effects of TAU pretreatment on the behavior of zebrafish in the open tank after acute 1% EtOH (v/v) exposure (20 and 60 min of duration) and on brain alcohol contents. The exposure for 20 min exerted significant anxiolytic effects, which were prevented by 42, 150, and 400 mg/L TAU. Conversely, the 60-min condition induced depressant/sedative effects, in which the changes on vertical activity were associated to modifications on the exploratory profile. Although all TAU concentrations kept locomotor parameters at basal levels, 150 mg/L TAU, did not prevent the impairment on vertical activity of EtOH[60]. Despite the higher brain EtOH content detected in the 60-min exposure, 42, 150, and 400 mg/L TAU attenuated the increase of alcohol content in EtOH[60] group. In conclusion, our data suggest that both protocols of acute EtOH exposure induce significant changes in the spatio-temporal behavior of zebrafish and that TAU may exert a preventive role by antagonizing the effects induced by EtOH possibly due to its neuromodulatory role and also by decreasing brain EtOH levels. The hormetic dose-response of TAU on vertical exploration suggests a complex interaction between TAU and EtOH in the central nervous system.

Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.

The open tank paradigm, also known as novel tank diving test, is a protocol used to evaluate the zebrafish behavior. Several characteristics have been described for this species, including scototaxis, which is the natural preference for dark environments in detriment of bright ones. However, there is no evidence regarding the influence of "natural stimuli" in zebrafish subjected to novelty-based paradigms. In this report, we evaluated the spatio-temporal exploratory activity of the short-fin zebrafish phenotype in the open tank after a short-period confinement into dark/bright environments. A total of 44 animals were individually confined during a 10-min single session into one of three environments: black-painted, white-painted, and transparent cylinders (dark, bright, and transparent groups). Fish were further subjected to the novel tank test and their exploratory profile was recorded during a 15-min trial. The results demonstrated that zebrafish increased their vertical exploratory activity during the first 6-min, where the bright group spent more time and travelled a higher distance in the top area. Interestingly, all behavioral parameters measured for the dark group were similar to the transparent one. These data were confirmed by automated analysis of track and occupancy plots and also demonstrated that zebrafish display a classical homebase formation in the bottom area of the tank. A detailed spatio-temporal study of zebrafish exploratory behavior and the construction of representative ethograms showed that the experimental groups presented significant differences in the first 3-min vs. last 3-min of test. Although the main factors involved in these behavioral responses still remain ambiguous and require further investigation, the current report describes an alternative methodological approach for assessing the zebrafish behavior after a forced exposure to different environments. Additionally, the analysis of ethologically-relevant patterns across time could be a potential phenotyping tool to evaluate the zebrafish exploratory profile in the open tank task.

Neuropsychiatric evaluation in subjects chronically exposed to organophosphate pesticides.

Long-term exposure to low levels of organophosphate pesticides (OP) may produce neuropsychiatric symptoms. We performed clinical, neuropsychiatric, and laboratory evaluations of 37 workers involved in family agriculture of tobacco from southern Brazil who had been exposed to OP for 3 months, and in 25 of these workers, after 3 months without exposure to OP. Plasma acetylcholinesterase activity levels of all subjects were within the normal range (3.2 to 9.0 U/l) and were not different between on- and off-exposure periods (4.7 +/- 0.9 and 4.5 +/- 1.1 U/l, respectively). Clinically significant extrapyramidal symptoms were present in 12 of 25 subjects, which is unexpected in such a population. There was a significant reduction of extrapyramidal symptoms after 3 months without exposure to OP, but 10 subjects still had significant parkinsonism. Mini-mental and word span scores were within the expected range for this population and were not influenced by exposure to OP. Eighteen of the 37 subjects (48%) had current psychiatric diagnoses in the first interview (13 with generalized anxiety disorder and 8 with major depression). Among the 25 subjects who completed both evaluations, the total number of current psychiatric diagnoses, after 3 months without using OP, dropped from 24 to 13 and the number of affected individuals with any psychiatric diagnosis dropped from 11 to 7. In conclusion, this study reinforces the need for parameters other than acetylcholinesterase activity to monitor for chronic consequences of chronic low-dose OP exposure, and it suggests that subjects have not only transient motor and psychiatric consequences while exposed, but may also develop enduring extrapyramidal symptoms.

Glutamate uptake in cultured astrocytes depends on age: a study about the effect of guanosine and the sensitivity to oxidative stress induced by H(2)O(2).

Relatively few studies have been conducted to investigate the relationship between glutamate and development and/or aging. Rat cortical astrocyte cultures were used as a model to investigate glutamate uptake during development. The immunocontent of the markers glial fibrillary acidic protein (GFAP) and S100B increased, while basal secretion of S100B decreased, in astrocytes from 10 to 40 days in vitro (DIV). Basal glutamate uptake increased with age. Exposure to hydrogen peroxide decreased glutamate uptake more potently at 40 than 10 DIV. Moreover, 40 DIV astrocytes showed earlier loss of integrity (at 6 h) than 10 DIV astrocytes (at 24 h) after H(2)O(2) exposure. Addition of guanosine stimulated glutamate uptake only in 10 DIV astrocytes. The present work shows that mature astrocytes in culture present some neurochemical alterations also observed in astrocytes of aged animals. These results can contribute to the understanding of some consequences of the excitotoxicity and oxidative stress during brain aging.

Time-related increase in mitochondrial superoxide production, biomolecule damage and antioxidant enzyme activities in cortical astrocyte cultures.

According to the free radical theory of aging, biological senescence processes develop from a general failure to maintain organism's homeostasis, probably due to oxidative stress. The brain is particularly susceptible to oxidative damage, and astrocytes are chiefly responsible for its antioxidant defense. Here we evaluated and compared the enzymatic antioxidant activities, mitochondrial superoxide production, and oxidative damage in biomolecule in cortex astrocytes from newborn Wistar rats maintained for 10-13 or 40-47 days in culture. We show that, besides an increase in antioxidant enzyme activities in matured astrocyte cultures, there was an increase in lipoperoxidation and in protein oxidation, probably due to an increase in mitochondrial electron transport chain superoxide production. This could indicate that the increasing in defense mechanisms was not sufficient to avoid oxidative biomolecule damage during maturation.

ATP diphosphohydrolases: an overview

This review aimed at discussing the protocols used to characterize and to distinguish ATP diphosphohydrolases from other enzymes which can promote the degradation of ATP and ADP, since there is a confusion about the identily of this enzyme and ATPases.