2.1. Zebrafish husbandry and chemical exposure
The fish were maintained at 28°C with a light to dark cycle of 14:10 hours. Egg water was used as the swimming solution with a 60 mg/ml concentration of instant ocean sea salt (Instant Ocean, Blacksburg, USA) and 0.1% methylene blue (Fisher Scientific, Ottawa, Canada). Relevant guidelines were followed to ensure proper care, under the Canadian Council for Animal Care (CCAC) guidelines, Animal Care Committee (ACC) protocol GZ 2020-7 R3, and York University Biosafety Permit PR 02-19.
All experiments were conducted using 6 dpf Tupfel long fin (TL) zebrafish. Fish were divided into several groups to generate sample sizes of 45 fish per condition to study the effects of DA agonists and antagonists presented in Table 1. All exposures were done off-chip, with all larvae in each category exposed to a specific test chemical and washed off at the same time. The concentrations and exposure times presented by Iron et al. [33] were used for each drug and the desired compounds were administered in the swimming media. A total of 15 embryos per well were placed in a 12-well plate, with each well containing 3 ml of the prepared solution.
All chemical compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA) and mixed with deionized (DI) water to produce the desired stock concentration with one exception. Haloperidol was the only drug that required dimethyl sulfoxide (DMSO) as a solvent due to its low solubility. The solutions were then diluted with egg water to reach the desired concentrations ranging between 0.2–50 μM. In contrast, Haloperidol was diluted with 0.4% DMSO to make the stock solution and further diluted with egg water to form the desired concentration.
Table 1. DA agonists and antagonists used in our chemical screening assays along with their concentrations and exposure times which were selected based on the literature[33].