3.8. Zebrafish Pentylenetetrazole Seizure Model

The maximum tolerated concentration (MTC) of samples and compounds was determined as described before [7] prior to further experiments and used as the highest test concentration. In case no MTC was reached, the highest soluble concentration or 200 µg/mL was used as the highest test concentration.

For screening purposes, no MTC was determined, instead behavioral analysis was followed by visual evaluation of the larvae under a light microscope to assess toxicity of treatment. Overall morphology, heartbeat, and touch response were investigated. MNP samples were scored as being normal or toxic. When embryos showed normal morphology, heartbeat, and touch response the treatment was considered to be normal. In case of an abnormal morphology and/or absence of touch response or heartbeat, a treatment was considered to be toxic.

The locomotion of zebrafish larvae treated with either VHC (1% DMSO) or sample/compound (1% DMSO) for 2 h was investigated by automated behavioral recording at 7 dpf, as described before [7]. In case of the positive control valproate, an 18-h incubation time was used as described in the study of Afrikanova and colleagues [32]. Larval behavior was depicted as mean actinteg units per 5 min during the 30-min recording period and over consecutive time intervals. Data are expressed as mean ±SEM for single experiments and for independent experiments of which the means or raw data are pooled.

In the first secondary screen three replicate wells were used per sample (100 µg/mL) tested and each experimental plate contained 12 internal control wells. In the second secondary screen 6 replicate wells were used per sample and concentration tested (100, 33, and 11 µg/mL), again 12 internal control wells were used per experimental plate.

Non-invasive LFP recordings were measured from the midbrain (optic tectum) of 7-dpf zebrafish larvae pre-incubated with VHC only, PTZ only, compound and VHC, or compound and PTZ. Larvae were incubated for approximately 2 h with VHC (1% DMSO) or test compound (1% DMSO) in a 100 µL volume (in case of the positive control valproate, an 18-h incubation time was used as described in the study of Afrikanova and colleagues [32]), whereafter, an equal volume of VHC (embryo medium) or 40 mM PTZ (20 mM working concentration) was added for 15 min prior to recording. These steps occurred at 28 °C, while further manipulation and electrophysiological recordings occurred at room temperature (21 °C) and were performed as described before [7]. Each recording lasted 600 s. Manual analysis was completed by quantification of the number and duration of epileptiform-like events with Clampfit 10.2 software (Molecular Devices Corporation, San Jose, CA, USA). An electrical discharge was considered epileptiform if it was a polyspiking event comprising at least 3 spikes with a minimum amplitude of three times the baseline amplitude and a duration of at least 100 ms. Data are expressed as mean ±SD.