Overview of behavioral recording. Representative plot of swim distance of larvae exposed to 500 μM 1-methyl-4-phenylpyridinium (MPP+; red) vs. control (black). Inset (top right) shows the swim distance during photomotor assay measured between 1:00 pm and 6:00 pm consisting of five 30-min intervals alternating dark (OFF, gray boxes) and light (ON, white boxes). Arrows indicate the lights-on phases used for analysis. Sleep parameters were evaluated after the transition to darkness from 10:00 pm to 8:00 am the following day.

MPP+ effects on motor activity of zebrafish larvae. Motor activity was evaluated at 6 days post fertilization (dpf) in absence (Control) or presence of 500 μM MPP+ for (A) average velocity (mm/s) during lights-on and; (B) lights-off; (C) distance swam (mm); (D) frequency of movement; (E) cumulative time of movement (s) and (F) movement bout duration (s) were examined during five consecutive light-on phases. Data are presented as means ± SEM from two independent experiments pooled, control group n = 64, MPP+ group n = 61. Normality was tested using the D’Agostino and Pearson omnibus normality test followed by the nonparametric two-tailed Mann–Whitney U test.

MPP+ effects on sleeping pattern of zebrafish larvae. Sleeping pattern was evaluated in absence (Control) or presence of 500 μM MPP+ for (A) sleep fragmentation; (B) sleep ratio; (C) sleep latency (s); (D) swim velocity (mm/s); (E) wake bout duration (s) and (F) sleep bout duration (s). Data are presented as means ± SEM from two independent experiments pooled, control group n = 64, MPP+ group n = 61. Normality was tested using D’Agostino and Pearson omnibus normality test followed by a two-tailed unpaired t-test or nonparametric two-tailed Mann–Whitney U test.

Effects of MPP+ on thigmotaxis. Thigmotaxis was evaluated at 6 dpf in the absence (Control) or presence of 500 μM MPP+ and represented as (A) the fraction of total distance swum in the outer zone of the well and (B) the average distance from the center of the well during lights-on. Data are represented as means ± SEM from four independent experiments pooled, control group n = 45, MPP+ group n = 42. Normality was tested using D’Agostino and Pearson omnibus normality test followed by a two-tailed unpaired t-test or two-tailed Mann–Whitney U test.

Effects of glial-derived neurotrophic factor (GDNF) on motor activity of MPP+-treated zebrafish larvae. Larvae were incubated in the absence (black bars) or presence (white and gray bars) of 500 μM MPP+ and injected with 72.9 ng GDNF (gray bars) at 4 dpf before assessing motor activity at 6 dpf for (A) distance swum (mm); (B) frequency of movement; (C) cumulative time of movement (s) and (D) swim bout duration during five consecutive light-on phases. Data are represented as means ± SEM from a single experiment, control group n = 32, MPP+ group n = 31, GDNF group n = 32. Normality was tested using D’Agostino and Pearson omnibus normality test followed by the two-tailed Kruskal–Wallis test (p-value shown above graph) and Dunn’s post hoc analysis. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, for multiple comparisons.

Effects of GDNF on the sleeping pattern of MPP+-treated zebrafish larvae. Larvae were incubated in the absence (black bars) or presence (white and gray bars) of 500 μM MPP+ and injected with 72.9 ng GDNF (gray bars) at 4 dpf before assessing (A) sleep fragmentation; (B) sleep ratio; (C) sleep latency (s); (D) swim velocity (mm/s); (E) wake bout duration (s) and (F) sleep bout duration (s) at 6 dpf. Data are presented as means ± SEM from a single experiment, control group n = 32, MPP+ group n = 31, GDNF group n = 32. Normality was tested using D’Agostino and Pearson omnibus normality test followed by the two-tailed Kruskal–Wallis test (p-value shown above graph) and Dunn’s post hoc analysis. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, for multiple comparisons.

Effects of 4-phenylbutyrate (PBA) on motor activity of MPP+-treated zebrafish larvae. Larvae in the absence (Control) or presence of 500 μM MPP+ were treated with vehicle (black bars) or 100 μM PBA (white bars). Motor activity was evaluated for (A) distance swum (mm); (B) frequency of movement; (C) cumulative time of movement (s) and (D) movement bout duration (s) during five consecutive light-on phases. Data are presented as means ± SEM from a single experiment, control group n = 32, MPP+ group n = 31, PBA group n = 32, MPP+/PBA group n = 32. Normality was tested using D’Agostino and Pearson omnibus normality test followed by two-way ANOVA and Tukey’s post hoc analysis. P-value for interaction between neurotoxin exposure and drug treatment is shown above the graph. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, for multiple comparisons.

Effects of PBA on the sleeping pattern of MPP+ treated zebrafish larvae. Larvae in the absence (Control) or presence of 500 μM (MPP+) was treated with vehicle (black bars) or 100 μM PBA (white bars). Sleeping pattern were evaluated for (A) sleep fragmentation; (B) sleep ratio; (C) sleep latency (s); (D) swim velocity (mm/s); (E) wake bout duration (s) and (F) sleep bout duration (s). Data are represented as means ± SEM from a single experiment, control group n = 32, MPP+ group n = 31, PBA group n = 32, MPP+/PBA group n = 32. Normality was tested using D’Agostino and Pearson omnibus normality test followed by two-way ANOVA and Tukey’s post hoc analysis. P-value for interaction between neurotoxin exposure and drug treatment is shown above the graph. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, for multiple comparisons.