MOLECULAR, BEHAVIORAL AND ELECTROPHYSIOLOGICAL INVESTIGATION OF ZEBRAFISH EXPOSED TO DIFFERENT CONVULSIVE AGENTS: DOES ZEBRAFISH BECOME CHRONICALLY EPILEPTIC?

RESUMO

INTRODUÇÃO: Zebrafish (Danio rerio) is a well-established animal model for acute seizure investigations [1]. To date, we have no indications that zebrafish can become chronically epileptic through repetitive, prolonged, or single acute chemical stimuli. Previous studies performed by our research group have shown that subconvulsant stimuli are not sufficient to make zebrafish chronically epileptic. In the present study, we sought to investigate whether the immature zebrafish can become epileptic through different stimuli using pentylenetetrazole (PTZ) and pilocarpine, followed by measuring behavioral, molecular, and electrical parameters.

OBJETIVOS: The main aim of this study was to investigate the impact of convulsive doses of PTZ and pilocarpine on the zebrafish immature brain.

MÉTODOS: This study was approved by the Ethics Committee on Animal Use (CEUA) of UNICAMP: #5541-1, #5079-1. Wild-type zebrafish larvae were separated into three main groups (n = 5, each sample comprising a pool of 5 larvae heads), (i) Single Acute Seizure, SAS (ii) Single status epilepticus, SSE (3-hour of seizure), (iii) Daily Acute Seizure, DAS and their respective control groups. The first group consisted of applying convulsant doses of PTZ (15 mM) or Pilocarpine (30 mM) to larvae at 7 days post fertilization (dpf) for 20 minutes. The second group used the aforementioned drug concentrations, but the stimulus was applied for a period of 3 hours, followed by two weeks of resting until 21 dpf. In the third group, animals received the same convulsant concentrations, however, the stimuli were applied daily from 7 dpf to 15 dpf. At the end of the stipulated time for each group, behavioral and molecular analyses were performed. Behavioral analyzes were performed in Danio Vision hardware and Ethovision software. To the molecular study, we investigated cfos, fosb, bdnf, and il1b genes by RT-qPCR. EEG analyzes were implemented for acute stimuli of up to 3 hours with 7 dpf larvae induced to seizures with PTZ or pilocarpine using the Intan RhD2000 System with both, a single electrode and MEA. Statistical analysis was carried out by unpaired t-test and Mann-Whitney.

RESULTADOS: We found that chemical stimuli with PTZ and pilocarpine significantly changed the behavior in zebrafish larvae. Larvae treated with PTZ showed a high average speed and total distance traveled compared to the controls. On the other hand, larvae from SAS group treated with pilocarpine significantly decreased behavioral parameters. Molecular data from the SAS group indicated a significant increase in the cfos expression for PTZ-treated larvae but not for pilocarpine-treated larvae. The PTZ-treated SSE group showed a significant increase of the il1b gene. SSE group treated with pilocarpine showed that cfos, bdnf, and il1b genes were significantly up regulated. Finally, larvae from DAS group showed a significant up-regulation for all genes investigated in both, PTZ and pilocarpine-treated groups. Electrophysiological data were acquired, and differences in amplitude and duration of seizures were detected between groups.

CONCLUSÃO: The present study suggests that even no spontaneous seizures were observed in the zebrafish brain, we found behavioral and molecular repercussions occurring throughout the stimuli, which may be valuable for future studies.

BIBLIOGRAFIA: 1. Baraban SC et al., doi: 10.1016/j.neuroscience.2004.11.031.

PALAVRA-CHAVE: zebrafish; epilepsy; genetic; molecular; immature; brain; eeg