Validation of the Zebrafish Pentylenetetrazol Seizure Model: Locomotor versus Electrographic Responses to Antiepileptic Drugs

Abstract

Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABAA antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid first-pass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.

Depdc5 knockdown causes mTOR‐dependent motor hyperactivity in zebrafish

“…Physiological recordings in the Depdc5 knockdown zebrafish model showed an enhanced spontaneous activity in the optic tectum, similar to previous reports of zebrafish epilepsy models 21, 23, 28. This electrical activity recorded at 4–6 dpf lacks, however, the characteristics of epileptiform activity that could be classified as typical ictal and interictal phases.…”

“…4B). Previous genetic models of epilepsy have shown increased sensitivity to PTZ application 20, 21, 23, 28, 31. While both control and Depdc5 knockdown embryos showed significantly increased spontaneous basal activity in response to PTZ, we did not detect a difference in their respective sensitivity to the drug.…”

“…These measures are generally obtained at larval stages 3–7 dpf upon evoked or spontaneous swimming episodes 20, 21, 28, 39. Here we establish a novel measure of epilepsy‐related hyperactivity at 28 hpf in the chorion, characterized by increased frequency of triggering stereotyped movements, such as coils and twitches (as defined in the text).…”

“…We compared the spontaneous activity between controls and Depdc5 knockdown zebrafish larvae from baseline recordings of 60 min duration. To verify that the preparation was intact and able to generate high‐frequency neuronal discharges resembling the epileptiform activity, we then applied 20 mmol/L PTZ to chemically induce epileptiform‐like electrographic activity 20, 28. To account for the variability in electrode placement and individual animals, we eliminated datasets that had outlying spontaneous activity (baseline activity outside two standard deviations of the mean) or where the application of PTZ had no effect on the electrographic activity.…”

What New Modeling Approaches Will Help Us Identify Promising Drug Treatments?

“…A likely explanation is that overnight exposure to these AEDs was either toxic or sedative to developing zebrafi sh, as both possibilities would appear as suppressed locomotion in motion-based tracking assay. More recently, Afrikanova et al [ 1 ] revisited this overnight exposure-PTZ challenge assay and evaluated a similar list of 13 AEDs using a combination of locomotion tracking followed by electrophysiology on agar-immobilized larvae. These latter studies aligned most closely with the original PTZ fi ndings, identifying valproate and diazepam, while also showing that ethosuximide altered burst frequency but not amplitude.…”