Zebrafish Maintenance Conditions Affect Behavioral and Biochemical Biomarkers: A Possible Interfering Factor on the Research Results.

Over the years, scientific research with fish models has grown at a rapid pace, and issues such as animal welfare are becoming increasingly important in various areas of animal husbandry and experimentation. Here, we evaluated whether Danio rerio behavior is affected by long-term maintenance (75 days) in an enriched environment or a chronic stress (CS) situation. In addition, we evaluated some biochemical parameters related to redox status. We concluded that long-term maintenance of zebrafish in enriched environment might induce an anxiety-like behavior pattern when these fish are faced with an acute subsequent stressor. These anxiety results, the increased school cohesion, and the absence of oxidative damage allow us to hypothesize that the fish maintained in environmental enrichment (EE) situation is more reactive, showing a strong protective reaction to the stress. From an applicable perspective, we show that both too much stress and too little stress are not ideal for zebrafish stocks. In CS situations, fish can habituate and might not respond optimally to test conditions. In opposite, the low stress promoted by environmental enrichment also renders the fish incapable of dealing with occasional stressors optimally, because now even normal conditions appear stressful to them and may elicit fear behaviors they normally would not exhibit.

Pyriproxyfen Exposure Impairs Cognitive Parameters and Alters Cortisol Levels in Zebrafish.

Pyriproxyfen is one of the most used larvicides and insecticides; it acts as an analog of juvenile insect hormone (a growth regulator). It is highly toxic during all stages of mosquito development, suppresses metamorphosis, and interferes in insect reproduction and proliferation. Pyriproxyfen and its main metabolite have been shown to affect brain development in rodents. This compound is employed mainly to eliminate outbreaks of the genus Aedes, even in potable water. Despite the increasing number of toxicological studies about larvicides and insecticides-with an indication of continuous use-there have been few studies about the effects of pyriproxyfen in non-target species such as fish. This study evaluated the effects of pyriproxyfen on behavioral, cognitive, and endocrine parameters in zebrafish. We exposed adult zebrafish to different pyriproxyfen (Pestanal®) concentrations (0.125, 0.675, and 1.75 mg/l) for 96 h. We analyzed behavioral parameters, memory, cortisol levels, and gene expression of glucocorticoid receptor (gr) and corticotrophin-releasing factor (crf) after pyriproxyfen exposure. This exposure did not alter locomotion (distance or mean speed), anxiety-like behavior (latency to enter to the top zone of the tank or time in the top zone of the tank), and social or aggressive behavior. However, there was impaired inhibitory avoidance memory at all tested pyriproxyfen concentrations. Cortisol levels were reduced in exposed groups when compared to control or vehicle. However, gr and crf gene expression in pyriproxyfen-treated animals were unaltered when compared to control or vehicle groups. Taken together, these findings indicate that pyriproxyfen may induce cognitive impairment and altered cortisol levels in zebrafish, a non-target species.

Novel object recognition and object location tasks in zebrafish: Influence of habituation and NMDA receptor antagonism.

This study aims to establish a protocol for evaluating the object recognition memory and object location tasks in zebrafish. We evaluated novel the object recognition memory and analyzed the exploration time of the objects during training and testing. Zebrafish explored more the new object in comparison to the familiar object (61% of exploration time during test session). We also tested the object location task and measured the exploration time of each object in the familiar and novel object location. There was a preference to explore the object in the novel location (63% of exploration time during test session). The effect of the non-competitive NMDA receptor antagonist MK-801 was investigated on the object recognition and object location memory. Control (water only) and treated animals (5 µM MK-801) presented a significant preference in exploring the familiar object in comparison to the new object (66 and 68% of exploration time, respectively, during test session); however, 10 µM MK-801-treated animals did not show differences in the exploration time of the objects. In the object location task, the animals treated with the 5 or 10 µM MK-801 did not show a preference for the familiar or novel location whereas the control group had a higher preference in exploring the object in the familiar location (64% of exploration time during test session). Considering the different responses of the control group between original task and in the regimen treatment, we evaluated the impact of habituation on cortisol levels of animals in three different protocols: (1) habituated at the experiment apparatus for 3 days (C1 condition), (2) habituated at the experiment apparatus for 3 days plus treatment tank exposure at fourth day (C2 condition), (3) habituated at the treatment tank and experiment apparatus for 3 days and exposed to treatment tank again at fourth day (C3 condition). The results showed higher levels of cortisol in animals submitted to C2 and C3 conditions compared to animals submitted to C1. When introduced to an acute stressor during C1 condition, we observed an increase in the cortisol levels and an absence of preference for the objects in comparison to control group, which had a preference for novel object and novel location. Fluoxetine treatment induced a decrease in cortisol levels and an absence of preference for the objects in C2 and C3 conditions in comparison to control group, which had a preference for familiar object. However, fluoxetine treatment induced a preference to the novel location in C2 and C3 conditions in comparison to control group, which had a preference for familiar location. These results indicate that treatment tank exposure induced a different performance in object recognition and object location memory due to stress responses. Therefore, these tasks are prone to evaluate memory in physiological and pathological conditions, but its use is limited due to sensitivity to stress caused by manipulation.

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup®. This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup®-treated adult zebrafish demonstrated a significant impairment in memory. Both glyphosate and Roundup® reduced aggressive behavior. Our data suggest that there are small differences between the effects induced by glyphosate and Roundup®, altering morphological and behavioral parameters in zebrafish, suggesting common mechanisms of toxicity and cellular response.

Inhibition of protein synthesis or mTOR in the basolateral amygdala blocks retrieval-induced memory strengthening.

Fear memory retrieval can lead to either reconsolidation (accompanied or not by strengthening of the memory trace) or extinction. Here, we show that non-reinforced retrieval of inhibitory avoidance (IA) conditioning can induce memory strengthening assessed in a subsequent retention test trial. Infusion of the protein synthesis inhibitor cycloheximide or the mTOR inhibitor rapamycin into the rat basolateral complex of the amygdala (BLA) after a reactivation (retrieval) session impaired retrieval-induced strengthening. Intra-BLA infusion of the mRNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) after retrieval had no effect. These findings provide the first evidence suggesting that non-reinforced IA retrieval can lead to memory strengthening through a mechanism dependent on protein synthesis and mTOR activity in the BLA.

Administration of the phosphodiesterase type 4 inhibitor rolipram into the amygdala at a specific time interval after learning increases recognition memory persistence.

Here we show that administration of the phosphodiesterase type 4 (PDE4) inhibitor rolipram into the basolateral complex of the amygdala (BLA) at a specific time interval after training enhances memory consolidation and induces memory persistence for novel object recognition (NOR) in rats. Intra-BLA infusion of rolipram immediately, 1.5 h, or 6 h after training had no effect on retention tested at 1, 7, and 14 d later. However, rolipram infused 3 h post-training promoted memory persistence for up to at least 14 d. The findings suggest that PDE4 inhibition in the BLA can enhance long-term memory formation when induced specifically 3 h after learning.

Impairment of object recognition memory by rapamycin inhibition of mTOR in the amygdala or hippocampus around the time of learning or reactivation.

The role of the basolateral complex of the amygdala (BLA) in recognition memory remains poorly understood. The mammalian target of rapamycin (mTOR) in the BLA and other brain areas has been implicated in synaptic plasticity and memory. We have recently shown that mTOR signaling in both the BLA and the dorsal hippocampus (DH) is required for formation and reconsolidation of inhibitory avoidance, a fear-motivated memory task. Here we examined the effects of infusions of the mTOR inhibitor rapamycin into the BLA before or after either training or reactivation on retention of novel object recognition (NOR) memory in rats, and compared the effects with those obtained using intra-DH infusions. Male Wistar rats received bilateral infusions of vehicle or rapamycin into the BLA or DH before or after NOR training or reactivation. Rapamycin impaired NOR retention tested 24h after training when given either before or immediately after training into the BLA or DH. Rapamycin also impaired retention measured 24h after reactivation when infused before reactivation into the BLA or DH, or immediately after reactivation into the BLA, but not when given 6h after reactivation into either the BLA or DH. The results suggest that mTOR signaling in the BLA and DH is involved in NOR memory formation and stabilization.

Inhibition of mTOR by rapamycin in the amygdala or hippocampus impairs formation and reconsolidation of inhibitory avoidance memory.

Mammalian target of rapamycin (mTOR), a central regulator of protein synthesis in neurons, has been implicated in synaptic plasticity and memory. Here we show that mTOR inhibition by rapamycin in the basolateral amygdala (BLA) or dorsal hippocampus (DH) impairs both formation and reconsolidation of memory for inhibitory avoidance (IA) in rats. Male Wistar rats received bilateral infusions of vehicle or rapamycin into the BLA or DH before or after IA training or retrieval. Memory retention was tested at different time points after drug infusion. Rapamycin impaired long-term IA retention when given before or immediately after training or retrieval into the BLA. When infused into the DH, rapamycin produced memory impairment when given before training or immediately after retrieval. The impairing effects of post-retrieval rapamycin required memory retrieval and were not reversed by a reminder shock. The results provide the first evidence that mTOR in the BLA and DH might play a role in IA memory reconsolidation.

Effect of mangiferin, a naturally occurring glucoxylxanthone, on fear memory in rats.

Mangiferin (1,3,6,7-tetrahydroxy-2-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] -xanthen-9-one, CAS 4773-96-0), a naturally occurring glucosylxanthone, is widely distributed in higher plants and a constituent of folk medicine. In the present study the effect of systemic administration of mangiferin on behavioural outcomes of neurological function in normal rats was investigated. A single intraperitoneal injection of mangiferin (10, 50 and 100 mg/kg body weight) immediately post-training produced an impairment of long-term memory for aversive training and a reduced freezing in a dose independent manner, when given immediately post-training. The administration of mangiferin 6 h post-training did not affect fear memory. The results indicate that mangiferin might induce deficits of emotionally motivated memory.

Posttraining systemic administration of the histone deacetylase inhibitor sodium butyrate ameliorates aging-related memory decline in rats.

Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) ameliorated an aging-associated deficit in object recognition memory in rats when the injection was given immediately, but not 6h after training. NaB had no effect in younger rats with normal memory retention. The results indicate that HDACis can ameliorate aging-related memory impairments by influencing the early consolidation phase of memory formation.

Possible enhancement of long-term fear conditioning performance by cisplatin administration in rats / Possível facilitação do desempenho em condicionamento aversivo por administração de cisplatina em ratos

Neurocognitive deficits associated with chemotherapy represent an increasing concern, and the development of animal models to investigate chemotherapy-induced alterations in memory is warranted. Aims: to examine the effects of systemic injection of cisplatin on formation of fear-motivated memory in rats. Methods: male Wistar rats were given an intraperitoneal (i.p.) injection of saline or cisplatin followed by inhibitory avoidance (IA) training. Memory retention was tested 1 and 7 days after training. Control experiments using an open field were carried out to confirm the specificity of the cisplatin-induced alteration in IA performance. Results: cisplatin induced a unexpected enhancement of IA performance measured 7 days after drug injection and training. Control experiments suggested that the effect could not be attributed to sensorimotor alterations or toxic effects. Discussion: the findings are discussed in the light of previous preclinical evidence that cancer chemotherapy can, under some conditions, lead to memory enhancement

É crescente a preocupação com disfunções cognitivas associadas ao uso de quimioterapia para tratamento de câncer. É necessário o desenvolvimento de modelos experimentais que permitam avaliar alterações na memória induzidas por antineoplásicos. Objetivos: avaliar os efeitos da administração sistêmica de cisplatina sobre a formação de memória motivada por medo em ratos. Métodos: ratos Wistar machos receberam uma injeção intraperitoneal (i.p.) de solução salina (controles) ou cisplatina antes de uma sessão de treino em esquiva inibitória (EI). A retenção da memória de EI foi avaliada em testes realizados 1 e 7 dias depois do treino. Experimentos controle em um campo aberto foram usados para confirmar a especificidade das alterações induzidas por cisplatina no desempenho em EI. Resultados: a administração de cisplatina levou a um inesperado aumento do desempenho de EI medido 7 dias após o treino. Os experimentos controle indicam que esse efeito não deve estar relacionado à toxicidade ou alterações em funções sensoriais e motoras. Discussão: os resultados são discutidos em relação a estudos prévios que indicam que, em algumas condições, quimioterápicos antineoplásicos podem levar a uma facilitação da memória

Mangiferin, a naturally occurring glucoxilxanthone improves long-term object recognition memory in rats.

Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a xanthone widely distributed in higher plants showing antioxidative, antiviral, anticancer, antidiabetic, immunomodulatory, hepatoprotective, and analgesic effects. In the present study, we have investigated the effects of systemic administration of mangiferin on behavioral outcomes of neurological function in normal rats. A single intraperitoneal injection of mangiferin (10, 50, or 100mg/kg body weight) enhanced novel object recognition (NOR) memory when given immediately post-training. The administration of mangiferin 6h post-training did not affect NOR memory. There were no significant differences between groups in the total time exploring both objects, indicating that mangiferin did not affect locomotion or motivation. Mangiferin stimulated cell proliferation and induced a significant increase in the supernatant levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-alpha in vitro in human U138-MG glioblastoma cells. The results indicate that mangiferin enhances recognition memory through a mechanism that might involve an increase in neurotrophin and cytokine levels.

Systemic administration of doxorubicin impairs aversively motivated memory in rats.

There is growing clinical evidence of cognitive impairment in cancer patients treated with chemotherapy, especially in women treated with drug combinations for breast cancer. Clinical studies have a difficult task of defining which drugs individually are responsible for the cognitive changes and published papers evaluating single agents in experimental models are scanty. In the present study we have investigated the effect of single escalating doses of doxorubicin (DOX) on memory for inhibitory avoidance conditioning (IA) in rats. The doses used were comparable to those applied in the clinic. When given systemically before training, higher doses of DOX impaired IA memory retention measured 24h and 7days, but not 3h after training. DOX did not affect IA retention when given either before or after training in a multiple-trial IA training protocol. Control experiments showed that DOX produced a decrease in exploratory behavior assessed by the number of rearings performed during exploration of an open field. The results indicate that a single systemic administration of DOX might impair long-term aversive learning.

Alterations in oxidative markers in the cerebellum and peripheral organs in MPS I mice.

Mucopolysaccharidosis type I is a lysosomal storage disease with alterations in several organs. Little is known about the pathways that lead to the pathology. Evidences point oxidative stress on lysosomal storage diseases and mucopolysaccharidosis type I. The aim of the present study was to evaluate oxidative biomarkers on mucopolysaccharidosis type I mice model. We evaluated antioxidant enzymatic activity, protein damage and lipid peroxidation in the forebrain, cerebellum, heart, lung, diaphragm, liver, kidney and spleen. Superoxide dismutase activity was increased on cerebellum, lung, diaphragm, liver and kidney of mucopolysaccharidosis type I mice. Catalase activity was increased on cerebellum, spleen and lung. There was no alteration on glutathione peroxidase activity on any of the analyzed organs. Mucopolysaccharidosis type I mice showed increased carbonyl groups on cerebellum, heart and spleen. There was a decrease of thiobarbituric acid-reactive substances on the cerebellum of mucopolysaccharidosis type I mice. The results indicate a oxidative imbalance in this model. As lysosomes are very susceptible to oxidative damage, leading inclusive to cellular death, and lysosomal storage diseases present several alterations on this organelles, this finding can help to elucidate the cellular damage pathways on mucopolysaccharidosis type I.

An organic selenium compound attenuates apomorphine-induced stereotypy in mice.

Selenium compounds display neuroprotective activities mediated at least in part by their antioxidant actions. Oxidative damage has been implicated in psychiatric disorders including schizophrenia and bipolar disorder, and an alteration in expression of selenium-binding protein-1 (SELENBP-1) has been recently reported in both the blood and brain of schizophrenic patients. In the present study we examined the effects of the organic selenium compound 3'3-ditrifluoromethyldiphenyl diselenide [(F3CPhSe)2] on apomorphine-induced stereotypy in mice, an animal model of psychosis. Systemic administration of (F3CPhSe)2 at the highest dose used (25.0 micromol/kg in a 10.0 ml/kg injection volume) significantly reduced apomorphine-induced stereotyped behaviors. A series of control experiments showed that the same dose of (F3CPhSe)2 did not affect open-field behavior, habituation, or aversively motivated memory. The results indicate that organic selenium compounds should be further investigated as agents with possible antipsychotic properties.

Long-term memory for aversive training is impaired in Idua(-/-) mice, a genetic model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease that leads to neurodegeneration and neurological deficits, among other pathological and clinical consequences. The aim of the present study was to evaluate neurobehavioral parameters in a genetic mouse model of mucopolysaccharidosis type I (MPS I). During exploration of an open field, adult MPS I (Idua(-/-)) mice showed normal locomotion and anxiety but reduced number of rearings. Idua(-/-) mice performed normally in a novel object recognition memory task and showed normal short-term retention of inhibitory avoidance training. By contrast, long-term retention of inhibitory avoidance was impaired in Idua(-/-) mice. The deficit in inhibitory avoidance memory could not be attributed to reduced footshock reactivity. The results indicate that Idua(-/-) mice present deficits in long-term memory for aversive training and reduced exploratory behavior.

NMDA receptors mediate consolidation of contextual memory in the hippocampus after context preexposure.

Male Wistar rats received bilateral infusions of vehicle (VEH) or aminophosphonopentanoic acid (AP5), an N-metil-D-aspartate (NMDA) receptor antagonist, into the dorsal hippocampus immediately after inhibitory avoidance (IA) training. Intrahippocampal infusion of AP5 blocked 24 h IA retention. In the second experiment, animals were preexposed to the IA training context 24 h prior to training and received an infusion of either VEH or AP5 immediately after the preexposure trial and a second infusion of VEH or AP5 immediately after IA training. AP5 did not affect retention in animals preexposed to the IA box and given VEH after preexposure, but blocked retention when given after both preexposure and training. AP5 impaired retention in rats preexposed to an environment distinct from the IA box. These results suggest that NMDA receptors in the dorsal hippocampus mediate the formation of a contextual representation of the task environment.