Figure 5

Larval activity. (a) Untreated 5 day-old zebrafish larvae show substantial variability in activity over time. (b) Similar variability is observed in larvae treated with 1 µl/ml DMSO, a solvent or vehicle that is commonly used in high-throughput screens. (c) Larval activity is stable over time when averaging a row of 12 larvae. (d) Day-to-day variability is limited when imaging four rows of 12 larvae, i.e. separate experiments carried out on different days do not display significant differences in activity (chi-squared test, n = 48 larvae per experiment). (e) Treatment with tricaine, a fish anesthetic. Larvae displayed reduced activity, as compared to the untreated controls, in response to a broad range of tricaine concentrations. (f) Treatment with diazepam (Valium), a positive allosteric modulator of the GABA receptor. Treatment with 80 µM diazepam induced a complete loss of activity. At lower diazepam concentrations (50–70 µM), larvae displayed elevated activity as compared to the DMSO-treated controls (C1). A second set of three experiments with three experimental groups per experiment confirmed the loss of activity after treatment with 80 µM diazepam and showed an increase in activity after treatment with 200 µM flumazenil, a diazepam antidote, as compared to the DMSO-treated controls (C2). Note: all measurements were made after a 1-hour acclimation period in the imaging system. In panel d-f, larval activity was averaged during a 10-minute period immediately following the 1 hour acclimation period. An asterisk or double-asterisk indicates a significant difference of a treatment group as compared to the corresponding control (*p < 0.05/n, **p < 0.01/n), using a chi-squared test with the conservative Bonferroni correction for multiple comparisons (n = 6 in panel e, n = 6 and 2 in panel f). Error bars indicate the standard error of the mean. Activity in ‘lmpi’ = larval movement per interval, using a 6 second interval.