Kelly Juliana Seibt

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.

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.

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.

MK-801 alters Na + , K + -ATPase activity and oxidative status in zebrafish brain: reversal by antipsychotic drugs

Schizophrenia is a debilitating mental disorder with a global prevalence of 1% and its etiology remains poorly understood. In the current study we investigated the influence of antipsychotic drugs on the effects of MK-801 administration, which is a drug that mimics biochemical changes observed in schizophrenia, on Na+, K+-ATPase activity and some parameters of oxidative stress in zebrafish brain. Our results showed that MK-801 treatment significantly decreased Na+, K+-ATPase activity, and all antipsychotics tested prevented such effects. Acute MK-801 treatment did not alter reactive oxygen/nitrogen species by 2′7′-dichlorofluorscein (H2DCF) oxidation assay, but increased the levels of thiobarbituric acid reactive substances (TBARS), when compared with controls. Some antipsychotics such as sulpiride, olanzapine, and haloperidol prevented the increase of TBARS caused by MK-801. These findings indicate oxidative damage might be a mechanism involved in the decrease of Na+, K+-ATPase activity induced by MK-801. The parameters evaluated in this study had not yet been tested in this animal model using the MK-801, suggesting that zebrafish is an animal model that can contribute for providing information on potential treatments and disease characteristics.

Inhibitory effect of lithium on nucleotide hydrolysis and acetylcholinesterase activity in zebrafish (Danio rerio) brain

Lithium has been used as an effective antimanic drug in humans and it is well known for its effects on neuropsychiatric disorders and neuronal communication. ATP and adenosine are important signaling molecules, and most nerves release ATP as a fast co-transmitter together with classical neurotransmitters such as acetylcholine. In this study, we evaluated the in vitro and in vivo effects of lithium on acetylcholinesterase and ectonucleotidase activities in zebrafish brain. There was a significant inhibition of ADP hydrolysis after in vivo exposure to lithium at 5 and 10 mg/l (27.6% and 29% inhibition, respectively), whereas an inhibitory effect was observed for AMP hydrolysis only at 10 mg/l (30%). Lithium treatment in vivo also significantly decreased the acetylcholinesterase activity at 10 mg/l (21.9%). The mRNA transcript levels of the genes encoding for these enzymes were unchanged after exposure to 5 and 10 mg/l lithium chloride. In order to directly evaluate the action of lithium on enzyme activities, we tested the in vitro effect of lithium at concentrations ranging from 1 to 1000 μM. There were no significant changes in zebrafish brain ectonucleotidase and acetylcholinesterase activities at all concentrations tested in vitro. Our findings show that lithium treatment can alter ectonucleotidase and acetylcholinesterase activities, which may regulate extracellular nucleotide, nucleoside, and acetylcholine levels. These data suggest that cholinergic and purinergic signaling may be targets of the pharmacological effects induced by this compound.

Influence of mercury chloride on adenosine deaminase activity and gene expression in zebrafish (Danio rerio) brain

Mercury is a widespread environmental contaminant that is neurotoxic even at very low concentrations. In this study we investigated the effects of mercury chloride on soluble and membrane adenosine deaminase (ADA) activity and gene expression in zebrafish brain. Inhibition of ADA activity was observed in the soluble fraction at 5-250 microM HgCl(2) (84.6-92.6%, respectively), whereas inhibition occurred at 50-250 microM in membrane fractions (20.9-26%, respectively). We performed in vitro experiments with chelants (EDTA and DTT) to test if these compounds prevented or reversed the inhibition caused by HgCl(2) and found that the inhibition was partially or fully abolished. The effect on ADA activity in soluble and membrane fractions was evaluated after acute (24h) and subchronic (96h) in vivo exposure of zebrafish to 20 microg/l HgCl(2). ADA activity in the soluble fraction was decreased after both acute (24.5%) and subchronic (40.8%) exposures, whereas in brain membranes the enzyme was inhibited only after subchronic exposure (21.9%). Semiquantitative RT-PCR analysis showed that HgCl(2) did not alter ADA gene expression. This study demonstrated that ADA activity was inhibited by mercury and this effect might be related to the neurotoxicity of this heavy metal.