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].