Katiucia Marques Capiotti

Unpredictable chronic stress model in zebrafish (Danio rerio): Behavioral and physiological responses

Zebrafish (Danio rerio) have emerged as a promising model organism to study development, toxicology, pharmacology, and neuroscience, among other areas. Despite the increasing number of studies using zebrafish, behavioral studies with this species are still elementary when compared to rodents. The aim of this study was to develop a model of unpredictable chronic stress (UCS) in zebrafish. We evaluated the effects of UCS protocol during 7 or 14 days on behavioral and physiological parameters. The effects of stress were evaluated in relation to anxiety and exploratory behavior, memory, expression of corticotrophin-releasing factor (CRF) and glucocorticoid receptor (GR), and cortisol levels. As expected, UCS protocol increased the anxiety levels, impaired cognitive function, and increased CRF while decreased GR expression. Moreover, zebrafish submitted to 7 or 14 days of UCS protocol presented increased cortisol levels. The protocol developed here is a complementary model for studying the neurobiology and the effects of chronic stress in behavioral and physiological parameters. In addition, this protocol is less time consuming than standard rodent models commonly used to study chronic stress. These results confirm UCS in zebrafish as an adequate model to preclinical studies of stress, although further studies are warranted to determine its predictive validity.

Quercetin and rutin prevent scopolamine-induced memory impairment in zebrafish

Demographic aging gives rise to a growing population with age-associated behavioral and cognitive deficits that may be associated at least partially to the increasing prevalence of neurodegenerative disorders, such as Alzheimers disease (AD). In this disease, it has been observed a decrease in the cholinergic system, which is crucial to memory formation. Scopolamine-induced amnesic effect, through the disruption of the cholinergic neurotransmission, is one of the approaches used to investigate the mechanisms involved in cognitive impairment observed in AD. The aim of our study was to investigate the potential protective role of quercetin and rutin against scopolamine-induced inhibitory avoidance memory deficits in zebrafish. Scopolamine (200 μM dissolved in the tank water for 1h) given pre-training hindered memory formation while both quercetin and rutin pretreatments (50mg/kg, single injection, i.p.) prevented the scopolamine-induced amnesia. None of the compounds affected zebrafish general locomotor activity. Together, these results contribute to the increase of the knowledge about plant compounds applicability as medicines to prevent and treat neurodegenerative diseases, like Alzheimers disease.

Zebrafish neurotransmitter systems as potential pharmacological and toxicological targets

Recent advances in neurobiology have emphasized the study of brain structure and function and its association with numerous pathological and toxicological events. Neurotransmitters are substances that relay, amplify, and modulate electrical signals between neurons and other cells. Neurotransmitter signaling mediates rapid intercellular communication by interacting with cell surface receptors, activating second messenger systems and regulating the activity of ion channels. Changes in the functional balance of neurotransmitters have been implicated in the failure of central nervous system function. In addition, abnormalities in neurotransmitter production or functioning can be induced by several toxicological compounds, many of which are found in the environment. The zebrafish has been increasingly used as an animal model for biomedical research, primarily due to its genetic tractability and ease of maintenance. These features make this species a versatile tool for pre-clinical drug discovery and toxicological investigations. Here, we present a review regarding the role of different excitatory and inhibitory neurotransmitter systems in zebrafish, such as dopaminergic, serotoninergic, cholinergic, purinergic, histaminergic, nitrergic, glutamatergic, glycinergic, and GABAergic systems, and emphasizing their features as pharmacological and toxicological targets. The increase in the global knowledge of neurotransmitter systems in zebrafish and the elucidation of their pharmacological and toxicological aspects may lead to new strategies and appropriate research priorities to offer insights for biomedical and environmental research.

Antipsychotic drugs prevent the motor hyperactivity induced by psychotomimetic MK-801 in zebrafish (Danio rerio)

Glutamate N-methyl-d-aspartate (NMDA) receptor antagonists, such as dizocilpine (MK-801), elicit schizophrenia-like symptoms in humans and a behavioral syndrome in rodents, characterized by hyperlocomotion and stereotyped actions, which is antagonized by antipsychotic drugs. Animal models of schizophrenia have been established and used for the development of new antipsychotic drugs. In this work we characterized the behavioral effects of MK-801 and investigated the effect of typical and atypical antipsychotic treatments on locomotor activity as well on the hyperlocomotion induced by MK-801 in zebrafish. MK-801 (20 microM) increased the locomotor behavior as measured by the number of line crossings, distance traveled, and the mean speed in the tank test after 15, 30, and 60 min of exposure. All tested antipsychotics counteracted MK-801-induced hyperactivity on all parameters analyzed and at doses that, given alone, had no effect on spontaneous locomotor activity. The results suggest a similar profile between typical and atypical antipsychotics in the reversal of locomotor disorders induced by MK-801. Moreover, an anxiolytic effect was verified at 30 and 60 min of MK-801 exposure, which was not reversed by antipsychotics tested in this work. In addition, olanzapine, which alone caused an anxiolytic response, when given with MK-801 potentiated the latters effect on anxiety. In this work we demonstrated the value of the zebrafish, a simple to use animal model, in developing some behavioral features observed in schizophrenia, which may indicate a new approach for drug screening.

The role of CRH in behavioral responses to acute restraint stress in zebrafish.

In teleosts, changes in swimming, exploring, general locomotor activity, and anxious state can be a response to stress mediated by the corticotropin-releasing hormone system activation and its effects on glucocorticoid levels. Zebrafish has been widely used to study neuropharmacology and has become a promising animal model to investigate neurobehavioral mechanisms of stress. In this report the animals were submitted to acute restraint stress for different time lengths (15, 60 and 90 min) for further evaluation of behavioral patterns, whole-body cortisol content, and corticotropin-releasing hormone expression. The results demonstrated an increase in the locomotor activity and an alteration in the swimming pattern during a 5-min trial after the acute restraint stress. Interestingly, all groups of fish tested in the novel tank test exhibited signs of anxiety as evaluated by the time spent in the bottom of the tank. Whole-body cortisol content showed a positive correlation with increased behavioral indices of locomotion in zebrafish whereas molecular analysis of corticotropin-releasing hormone showed a late reduction of mRNA expression (90 min). Altogether, we present a model of acute restraint stress in zebrafish, confirmed by elevated cortisol content, as a valid and reliable model to study the biochemical basis of stress behavior, which seems to be accompanied by a negative feedback of corticotropin-release hormone mRNA expression.

Early exposure to caffeine affects gene expression of adenosine receptors, DARPP-32 and BDNF without affecting sensibility and morphology of developing zebrafish (Danio rerio)

Adenosine receptors are the most important biochemical targets of caffeine, a common trimethylxanthine found in food and beverages. Adenosine plays modulatory action during the development through adenosine receptors and their intracellular pathways activation. In this study, we aimed to evaluate if caffeine gave to zebrafish in the very first steps of development is able to affect its direct targets, through the adenosine receptors mRNA expression evaluation, and latter indirect targets, through evaluation of the pattern of dopamine and cAMP-regulated phosphoprotein and brain-derived neurotrophic factor (BDNF) mRNA expression. Here, we demonstrate that zebrafish express adenosine receptor subtypes (A1, A2A1, A2A2 and A2B) since 24h post-fertilization (hpf) and that caffeine exposure is able to affect the expression of these receptors. Caffeine exposure from 1 hpf is able to increase A1 expression at 72-96 hpf and A2A1 expression at 72 hpf. No alterations occurred in A2A2 and A2B expression after caffeine treatment. DARPP-32, a phosphoprotein involved in adenosine intracellular pathway is also expressed since 24 hpf and early exposure to caffeine increased DARPP-32 expression at 168 hpf. We also evaluate the expression of BDNF as one of the targets of adenosine intracellular pathway activation. BDNF was also expressed since 24 hpf and caffeine treatment increased its expression at 48 and 72 hpf. No morphological alterations induced by caffeine treatment were registered by the check of general body features and total body length. Assessment of tactile sensibility also demonstrated no alterations by caffeine treatment. Altogether, these results suggest that caffeine is able to affect expression of its cellular targets since early phases of development in zebrafish without affect visible features. The up-regulation of direct and indirect targets of caffeine presents as a compensatory mechanism of maintenance of adenosinergic modulation during the developmental phase.

Antipsychotic drugs reverse MK-801-induced cognitive and social interaction deficits in zebrafish (Danio rerio)

Schizophrenia is a severe mental illness characterized by positive and negative symptoms and cognitive deficits. Reduction of glutamatergic neurotransmission by NMDA receptor antagonists mimics symptoms of schizophrenia. Modeling social interaction and cognitive impairment in animals can be of great benefit in the effort to develop novel treatments for negative and cognitive symptoms of schizophrenia. Studies have demonstrated that these behavioral changes are, in some cases, sensitive to remediation by antipsychotic drugs. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs and to discover new pharmacological targets. In the current study we investigated the ability of antipsychotic drugs to reverse schizophrenia-like symptoms produced by the NMDA receptor antagonist MK-801. Results showed that MK-801 (5μM) given pre-training hindered memory formation while both atypical antipsychotics sulpiride (250μM) and olanzapine (50μM) improved MK-801-induced amnesia. The same change was observed in the social interaction task, where atypical antipsychotics reversed the MK-801-induced social interaction deficit whereas the typical antipsychotic haloperidol (9μM) was ineffective to reverse those behavioral deficits. Therefore, MK-801-treated zebrafish showed some behavioral features observed in schizophrenia, such as cognitive and social interaction deficits, which were reverted by current available atypical drugs.

Persistent impaired glucose metabolism in a zebrafish hyperglycemia model

Diabetes mellitus (DM) affects over 10% of the worlds population. Hyperglycemia is the main feature for the diagnosis of this disease. The zebrafish (Danio rerio) is an established model organism for the study of various metabolic diseases. In this paper, hyperglycemic zebrafish, when immersed in a 111 mM glucose solution for 14 days, developed increased glycation of proteins from the eyes, decreased mRNA levels of insulin receptors in the muscle, and a reversion of high blood glucose level after treatment with anti-diabetic drugs (glimepiride and metformin) even after 7 days of glucose withdrawal. Additionally, hyperglycemic zebrafish developed an impaired response to exogenous insulin, which was recovered after 7 days of glucose withdrawal. These data suggest that the exposure of adult zebrafish to high glucose concentration is able to induce persistent metabolic changes probably underlined by a hyperinsulinemic state and impaired peripheral glucose metabolism.

PTZ-induced seizures inhibit adenosine deamination in adult zebrafish brain membranes.

Adenosine exerts neuromodulatory functions with mostly inhibitory effects, being considered an endogenous anticonvulsant. The hydrolysis of ATP by ectonucleotidases is an important source of adenosine, and adenosine deaminase (ADA) contributes to the regulation of this nucleoside concentration through its deamination. In this study, we tested the effect of pentylenetetrazole (PTZ)-induced seizures on ectonucleotidase and ADA activities in adult zebrafish brain. Our results have demonstrated that PTZ treatments did not alter ectonucleotidase and ADA activities in membranes and soluble fraction, respectively. However, ecto-ADA activity was significantly decreased in brain membranes of animals exposed to 5mM and 15 mM PTZ treatments (22.4% and 29.5%, respectively) when compared to the control group. Semiquantitative RT-PCR analysis did not show significant changes after the PTZ exposure on ADA gene expression. The decreased adenosine deamination observed in this study suggests a modulation of extracellular adenosine levels during PTZ-induced seizures in zebrafish.

Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).

Diabetes mellitus, which causes hyperglycemia, affects the central nervous system and can impairs cognitive functions, such as memory. The aim of this study was to investigate the effects of hyperglycemia on memory as well as on the activity of acethylcholinesterase. Hyperglycemia was induced in adult zebrafish by immersion in glucose 111mM by 14 days. The animals were divided in 4 groups: control, glucose-treated, glucose-washout 7-days and glucose-washout 14-days. We evaluated the performance in inhibitory avoidance task and locomotor activity. We also determined acethylcholinesterase activity and gene expression from whole brain. In order to counteract the effect of hyperglycemia underlined by effects on acethylcholinesterase activity, we treated the animals with galantamine (0.05ng/g), an inhibitor of this enzyme. Also we evaluated the gene expression of insulin receptor and glucose transporter from zebrafish brain. The hyperglycemia promoted memory deficit in adult zebrafish, which can be explained by increased AChE activity. The ache mRNA levels from zebrafish brain were decrease in 111mM glucose group and returned to normal levels after 7 days of glucose withdrawal. Insulin receptors (insra-1, insra-2, insrb-1 and insrb-2) and glut-3 mRNA levels were not significantly changed. Our results also demonstrated that galantamine was able to reverse the memory deficit caused by hyperglycemia, demonstrating that these effects involve modulation of AChE activity. These data suggest that the memory impairment induced by hyperglycemia is underlined by the cholinergic dysfunction caused by the mechanisms involving the control of acetylcholinesterase function and gene expression.