2.1. Zebrafish Husbandry and Chemical Exposure
This study used zebrafish larvae of the Tupfel long fin (TL) strain and the panx1a-/- line described previously[40]. Larvae were raised in egg water (prepared from Instant Ocean, Blacksburg, USA) supplemented with 0.1% methylene blue (M291-110 Fisher Scientific, USA). Larvae were maintained in a controlled environment at a temperature of 28°C with a 14:10 hour light to dark cycle. Adult TL and Panx1a-/- zebrafish were bred, housed, and raised based on our recent protocol[41].
6-OHDA (Sigma-Aldrich, Oakville, ON, Canada) was used at 250µM based on previous studies[20], [33], [42]. A total of 15 embryos per well were placed in a 12-well plate and 6-OHDA was administered every day starting at 2dpf for either 72hrs (testing at 5dpf) or 120hrs (testing at 7dpf). Considering the light sensitivity of 6-OHDA, the multi-well plates were covered with aluminum foil to avoid light exposure. To keep conditions consistent the untreated control groups were shielded from light in parallel with the test group.
The instructions and specifications outlined in the Canadian Council for Animal Care (CCAC), ACC protocol GZ 2020-7 R3 and York University biosafety permit PR 02-19 were diligently followed.