Abstracts

Rationale: Animal models play an important role to understand the physiopathology and treatment of human diseases, including epilepsies. Zebrafish has many advantages as an experimental model such as genetic homology to humans, external fertilization and development, transparency, and small size, which simplify genetic manipulations and drug screening.  In 2005, the zebrafish was characterized as a model for acute seizures, since this animal is susceptible to the pentylenetetrazole (PTZ) (Baraban, et al., 2005). Kindling is a classical model for development of spontaneous seizures and offers a great opportunity to investigate the epileptogenic process. The present study aimed to explore the development of kindling in the zebrafish by analyzing seizure behavior and cfos mRNA levels after application of subconvulsant doses of PTZ. Methods: Wild-type adult zebrafish were separated in kindling (KG) and control (CG) groups. Animals from KG were individually exposed to a subconvulsant dose of PTZ (7.5mM) for 2 minutes repeated daily for up to 60 days (5 days per week). Animals from CG were handled for 2 min/day, 5 days/week, but in PTZ-free water. The animals were sacrificed at 5th, 15th, 30th and 60th days and their brains were collected for total RNA extraction (n=5 per time point/group). Reverse transcriptase quantitative-PCR amplifications were carried out in triplicates with cfos and ef1α as endogenous control using TaqManTM System. The relative quantification (RQ) was calculated by the equation RQ=2 –ΔΔCT. Data are represented as mean ± Standard Error of Mean (SEM). Each time point was analyzed by Mann-Whitney test and significance was considered when p≤0.05. Results: During 60 days, no induced or spontaneous seizure-like behavior was achieved in the animals exposed daily to PTZ; only a slightly increased swim activity was observed. RT-qPCR showed an up-regulation of the cfos mRNA levels in the KG at 5th, 30th and 60th day compared to their respectively CG. The mean ± SEM of cfos gene and the p value obtained for comparisons between CG and SG were the following: CG05 1.49± 0.28 vs. KG05 5.13 ± 1.60 (p=0.004); CG15 0.43± 0.23 vs. KG15 0.29 ± 0.06 (p=0.34); CG30 0.96± 0.12 vs. KG30 0.98 ± 0.14 (p=0.05); CG60 0.64± 0.2 vs. KG60 1.14 ± 0.2 (p=0.008). Conclusions: So far there is no chronic model of epilepsy described in the literature for zebrafish. Such a model would be of high impact. While we have not yet established a chronic model, our results show a significant difference in cfos mRNA levels between the KG and CG groups. This indicates that neurons were more prone to stimulation in the kindling group. Reference: Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience. 2005;131(3):759-68. Funding: FAPESP #14/15640-8, CEPID-BRAINN- FAPESP #13/07559-3