Effects of ethanol exposure from 1-9dpf on larval development A.

<p>size at 9dpf measured using eLaborant software. <b>B</b> dry weight at 9dpf. <b>C</b> body weight at 6 months. There were no differences between groups. Values are means ± SE, 3 samples were used per group, with 20 (A) or 25 (B) animals per sample.</p

Stress-reactivity measured by whole-body cortisol concentration of zebrafish 6-month-old adults.

<p>Animals were either flash frozen or stressed, either by air exposure at a specific time or acute exposure to ethanol (1%), followed by freezing 6 minutes later. Zebrafish adults showed increased cortisol concentration when air exposed for 30s, but not after 60s exposure. Means ± SE, 3 batches (on different days) of 3 samples of 10 animals were used. <i>P</i><0.01, ANOVA <0.01).</p

Stress-reactivity measured by whole-body cortisol concentration of zebrafish 6-month-old adults.

<p>Treated animals were developmentally exposed to ethanol from 1dpf-9dpf with 20mM or 50mM ethanol concentrations. At 6 months animals were either flash frozen immediately or air exposed and frozen 6 minutes later. Zebrafish adults showed decreased cortisol content with increasing ethanol concentration exposure during development. Means ± SE, 9 batches of animals per group. *<i>P</i><0.05 **<i>P</i><0.01 “t” test and ANOVA.</p

Assessment of embryonic tissue ethanol concentration compared to waterbath ethanol concentration following acute 24-hour ethanol exposure of zebrafish embryos.

<p><b>A</b> Alcohol was assayed in whole embryos developmentally exposed to an ethanol waterbath concentration of 100mM and 20mM for a period of 24 hours, using 1-24hpf, 24-48hpf and 48-72hpf periods. <b>B</b> Tissue ethanol content after exposure to 100mM ethanol during the whole period 1-9dpf. Values are means ± SE, 3 samples collected on different days were used for each time period, 25 animals per sample. Values for tissue ethanol in the absence of added ambient ethanol were less than 1% of those illustrated, and not significantly different from zero.</p

Stress-reactivity in 9dpf larvae measured by whole-body cortisol concentration.

<p>Experimental animals were developmentally treated with ethanol from 1dpf-9dpf with 20mM and 50mM ethanol concentrations. At 9dpf animals were either flash frozen immediately or air exposed then frozen. Air stressed animals showed decreased tissue cortisol concentrations with increasing ethanol concentration exposure during development. Means ± SE, 6 batches of animals per group, *<i>P</i>< 0.05, **<i>P</i><0.01, by ANOVA and “t” test.</p

Zebrafish performance on 2-choice discrimination.

<p>A) Fish were trained in a glass tank (a), within a light-and-sound attenuating box. The divider was raised at the start of each trial, allowing the fish access to the discriminanda. Food reinforcement (artemia suspended in aquarium water) was delivered via a custom-made device (b; adapted, with permission, from 17). B) Percentage of correct responses as a function of set-size (error bars represent standard error). C) Approach latency response times as a function of set-size (error bars represent standard error). Accuracy and response time were unaffected by discrimination set-size, suggestive of parallel search. D) Speed accuracy trade-off function. There was no correlation between accuracy (<i>y</i> axis) and response latency (<i>x</i> axis), suggesting that fish did not trade-off speed for accuracy here, further suggesting parallel processing was occurring during discrimination performance in the zebrafish (<i>r</i> = −0.06).</p

Stress-reactivity measured by novel tank diving in 6-month old adult zebrafish.

<p><b>A)</b> Time course of average time spent per minute at the bottom of the tank, <b>B)</b> overall average time spent per minute at the bottom of the tank each minute, <b>C)</b> mean distance travelled per minute during novel tank diving. <b>D)</b> Effect of diazepam on zebrafish stress-reactivity assessed by novel tank diving. A,B. Zebrafish that had been developmentally exposed to ethanol showed reduced bottom dwelling (<i>F</i> 2, 682 = 3.47, <i>P</i><0.05). There were no significant differences in distance travelled (C). D. Diazepam also significantly reduced time spent by adults at the bottom of the tanks compared to controls (<i>F</i> 1, 408 = 5.45, <i>P</i><0.001). Post hoc t-test, *** <i>P</i><0.001.</p

Stress-reactivity measured by scototaxis in 6-month old adult zebrafish A) Time course of average time spent at the bright side of the apparatus and B) overall average time spent at the bright side of the apparatus.

<p>Adult zebrafish that had been developmentally exposed to ethanol spent more time on the bright side of the tank (<i>F</i> 2, 31 = 3.85, <i>P</i><0.05). Post hoc t-test, ** <i>P</i><0.01, * <i>P</i><0.05.</p

Stress-reactivity of 6-month old zebrafish as assessed by thigmotaxis.

<p><b>A)</b> Time course of average time spent per minute at the edge of the apparatus, <b>B)</b> overall average time spent per minute at the edge of the apparatus, <b>C)</b> distance travelled during thigmotaxis, <b>D)</b> Effect of diazepam on adult zebrafish stress-reactivity assessed by thigmotaxis. <b>A,B</b>) Adult zebrafish that had been experimentally exposed to ethanol spent decreased time at the edge of the tank, (<i>F</i> 2, 127 = 3.09, <i>P</i><0.05). There were no significant differences in distance travelled (C). Adults acutely exposed to diazepam exhibited reduced time spent at the edges of the tanks compared to controls (<i>F</i> 1, 4.98 = 5.44, <i>P</i><0.001) (D). Post-hoc t-test, * <i>P</i><0.05.</p

Tank used for novel tank-diving test.

<p>Fish were netted, placed in the tank and allowed to explore for five minutes. They were filmed during the exploration, and the amount of time spent in the top, middle and bottom of the tank was recorded, as well as the distance swum and velocity.</p