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. 2023 Jul 28;18(7):e0288904.
doi: 10.1371/journal.pone.0288904. eCollection 2023.

A comparative study to optimize experimental conditions of pentylenetetrazol and pilocarpine-induced epilepsy in zebrafish larvae

David Szep  1   2 , Bianka Dittrich  1   2 , Aniko Gorbe  1   2 , Jozsef L Szentpeteri  1 , Nour Aly  2 , Meng Jin  3 , Ferenc Budan  1   2 , Attila Sik  1   2   4
Affiliations

A comparative study to optimize experimental conditions of pentylenetetrazol and pilocarpine-induced epilepsy in zebrafish larvae

David Szep et al. PLoS One. .

Abstract

A common way to investigate epilepsy and the effect of antiepileptic pharmaceuticals is to analyze the movement patterns of zebrafish larvae treated with different convulsants like pentylenetetrazol (PTZ), pilocarpine, etc. Many articles have been written on this topic, but the research methods and exact settings are not sufficiently defined in most. Here we designed and executed a series of experiments to optimize and standardize the zebrafish epilepsy model. We found that during the light and the dark trials, the zebrafish larvae moved significantly more in the light, independent of the treatment, both in PTZ and pilocarpine-treated and the control groups. As expected, zebrafish larvae treated with convulsants moved significantly more than the ones in the control group, although this difference was higher between the individuals treated with PTZ than pilocarpine. When examining the optimal observation time, we divided the half-hour period into 5-minute time intervals, and between these, the first 5 minutes were found to be the most different from the others. There were fewer significant differences in the total movement of larvae between the other time intervals. We also performed a linear regression analysis with the cumulative values of the distance moved during the time intervals that fit the straight line. In conclusion, we recommend 30 minutes of drug pretreatment followed by a 10-minute test in light conditions with a 5-minute accommodation time. Our result paves the way toward improved experimental designs using zebrafish to develop novel pharmaceutical approaches to treat epilepsy.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flowchart of the research design.
Fig 2
Fig 2. The difference between the means of moved distances during light and dark trials (Mann-Whitney U test).
Fig 3
Fig 3. The means of moved distances between the treated groups during the light and dark trials.
Fig 4
Fig 4. The difference in the means of total distance moved between the PTZ, pilocarpine treated and control groups (Mann-Whitney U test).
Fig 5
Fig 5. Means of the total distance moved by the differently treated and control groups in 5-minute time intervals (Ncontrol = 184, NPTZ = 154, Npilocarpine = 177).
Fig 6
Fig 6. Mean of total distance moved by the differently treated and control groups during the 9 different trials.
Fig 7
Fig 7. Cumulative means of distance moved by PTZ, pilocarpine treated and control zebrafish groups.
Ncontrol = 184, NPTZ = 154, Npilocarpine = 177.
Fig 8
Fig 8. The regression lines calculated for PTZ, pilocarpine treated and control groups.
See this image and copyright information in PMC

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