Wild type zebrafish (Danio rerio, ABC strain) were obtained from the University of Oregon, Zebrafish International Resource Center (Eugene, OR, USA) and maintained at the Northwest Fisheries Science Center in a self-contained re-circulating aquaculture ZMOD system (Marine Biotech Inc.). The facility is operated according to standard procedures for zebrafish husbandry and research (Westerfield 1995). Fish were maintained at 26°C, kept on a 14:10 h light-dark cycle, and fed daily with purified ground salmon feed and brine shrimp. Fish were not fed on the morning of each exposure experiment.
Biomaterial provider
University of Washington
Treatment protocol
To quantify DA-induced neurobehavioral excitotoxicity in zebrafish, adult fish were exposed to DA via intracoelomic (IC) injection at several doses. For injection, fish were netted individually, placed into Ziplock bags and injected through the bag at a point midway between the pectoral fins and the anus. Contents (10 ml) were slowly injected into the IC cavity. IC injections consisted of a vehicle control (phosphate buffered saline (PBS) only) or DA dissolved in PBS. Eight exposure doses (0.21 ± 0.01, 0.25 ± 0.03, 0.32 ± 0.04, 0.47 ± 0.07, 0.82 ± 0.09, 1.2 ± 0.15, 1.6 ± 0.1, and 3.2 ± 0.5 µg DA/g body weight; n = 4 fish per treatment) were used. After injection, four fish from each treatment were placed in 1-liter clear plastic tanks and observed for 6 h. Behavioral toxicity was quantified by the presence or absence of circle or spiral swimming. Time to the onset of excitotoxicity and the number of fish affected were recorded for each treatment. Percentages of fish affected at each dose were used to generate a dose-response curve and an EC50 for behavioral toxicity using Graphpad Prism® software (San Diego, CA). All DA exposure doses were confirmed by HPLC-UV analysis (see HPLC verification of DA exposure doses below). All animal studies were carried out under approved IACUC protocols at the University of Washington.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from individual snap frozen zebrafish brains using a standard TRIzol procedure with the inclusion of 1 µl RNAse inhibitor/sample.
Label
biotin
Label protocol
Total RNA (2 ug) was labeled and hybridized to the Zebrafish Genome Arrays and hybridized according to manufacurer’s instructions.
Hybridization protocol
The standard hybridization protocol was used as recommended by Affymterix.
Scan protocol
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000.
Description
Wild type zebrafish (Danio rerio, ABC strain) were obtained from the University of Oregon, Zebrafish International Resource Center (Eugene, OR, USA) and maintained at the Northwest Fisheries Science Center in a self-contained re-circulating aquaculture ZMOD system (Marine Biotech Inc.). The facility is operated according to standard procedures for zebrafish husbandry and research (Westerfield 1995). Fish were maintained at 26°C, kept on a 14:10 h light-dark cycle, and fed daily with purified ground salmon feed and brine shrimp. Fish were not fed on the morning of each exposure experiment.
