Effects of tricaine and gradual cooling on the heartrate. (A) Schematic overview of the experimental setup. Fish were agarose-mounted in glass-bottom petri dishes which were modified to allow liquid to flow through only a narrow channel. The dishes had two connectors on each side in order to apply the liquids, and a sticker to ensure consistent placement of the thermal probe. During gradual cooling experiments, hot and cold E3 were dynamically applied via a peristaltic pump into a dish, where they mixed to achieve the desired temperature under control of a temperature feedback loop (see Methods). In tricaine experiments, the peristaltic pump applied tricaine at the standard concentration (168 mg/L), which was then washed out with drug-free E3. Hot and cold E3 beakers also contained 1% v/v 1,2-propanediol. (B) The experimental protocol consisted of 6 min baseline recording, 7 min tricaine or gradual cooling application, and 50 min of recovery. (C) The heartrates of larvae exposed to a standard tricaine concentration (168 mg/L), gradual cooling (11°C) and control fish. Average heart rates are shown, the SEM is shown as shaded envelope. Bars above time points indicate significant differences vs. time-matched control larvae (Tukey's HSD test, for clarity exact p-values are not shown, all p < 0.05, n = 7–8).
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