Short-term exposure to the simple polyphenolic compound gallic acid induces neuronal hyperactivity in zebrafish larvae

Zebrafish locomotor test

Larval locomotion in zebrafish was investigated at 72 hpf using the automated analyzer DanioVision^TM Observation Chamber (Noldus Information Technology). Larvae were individually exposed to 50 µg/ml GA dissolved in E3 medium. The activity was measured at three time points: 30, 60, and 180 min. The activity measured in treated and control larvae included: 1) total distance covered by each larva (total distance, in mm), 2) total time larvae spent moving (movement, in seconds), 3) mean velocity of each larva across movement measures (velocity, in mm/sec), 4) total number of rotations completed by each larva during the experiment (one rotation was counted every time larvae turned more than 180° left or right with respect to its own body axis, counted as the frequency of left and right rotations).

Cadmium treatment of zebrafish larvae

Two batches of wild-type larvae were exposed for 3 h (from 69 to 72 hpf) to 50 and 100 µM CdCl₂·H₂O in an E3 medium, respectively. Half of the 50 µM and half of the 100 µM treated larvae underwent the exposition to 50 µg/ml GA. The four obtained subgroups were: i. Cd 50 µM only treated larva; ii. Cd 100 µM only treated; iii. Cd 50 µM exposed to GA larvae; and iv. 100 µM exposed to GA larvae. All the groups underwent the behavioral observations reported above. As control groups were used: one group (Control E3) consisting of larvae in E3 medium only, and a second control with larvae exposed to 50 µg/ml GA for 30 minutes only. Larval locomotion was evaluated in a 96-well plate using the automated analyzer DanioVision^TM Observation Chamber (Noldus Information Technology). Each experiment was performed in triplicate.

Neurotransmitter analyses

Neur_data report the final concentration expressed as ng of neurotransmitter per larva.