Schematic representation of the experimental setup for the pharmacological zebrafish model. (A) timeline of the experiments, depicting the timepoints for each step illustrated in (B), (C) and (D). (B) Three days post-fertilization (dpf) larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 were injected in the duct of Cuvier with 1 nL of 0.25, 0.5 or 1 mM KA, thus obtaining a final dose of 0.2, 0.4 and 0.9 mg/Kg respectively, or vehicle control (PBS). At 4 and 5 dpf, confocal images (C) were taken from the brains and the number of engulfed and un-engulfed apoptotic nuclei and csf1r-expressing mCherry+ positive cells were quantified in 131 µ (1’, dark orange) or 182 µm (1’ + 2’, light orange) z-stacks, as represented in diagram of the brain of the larva. KA-microinjected larvae at 5 dpf were also incubated with 8 mM PTZ (D), and seizure activity was evaluated with the locomotor assay and tectal field potential recordings. In the locomotor assay, larvae were video-tracked in 96-well plates for the quantification of the distance moved. In the tectal field potential recordings, the electroencephalographic activity in the optic tectum was recorded for 10 min and quantified the number and cumulative duration of seizure-like events.

Visualization and quantification of csf1r-expressing mCherry positive cells. Images of the brain of 4 and 5 days post-fertilization (dpf) larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 previously injected with KA or vehicle control (PBS) at 3 dpf. (A), (B), and (C) are dorsal views (131 µm z-stack images) of 4 dpf larvae and (G), (H) and (I) are dorsal views (182 µm z-stack images) of 5 dpf larvae injected at 3 dpf with vehicle control (Ctrl), KA 0.4 mg/Kg, or KA 0.9 mg/Kg, respectively, showing csf1r-expressing cells labeled in red and un-engulfed and engulfed apoptotic nuclei labeled with acridine orange (AO). (D), (E), and (F) are a zoom-in of a region of interest (white square) from (A), (B) and (C), respectively, and (J), (K) and (L) are a zoom-in of a region of interest (white square) from (G), (H) and (I), respectively highlighting mCherry+ cells with microglial phenotype: cell soma (*) and ramifications (arrows). Scale bars represent 50 µm. (M) Total number of mCherry+ cells at 4 and 5 dpf. Values for injected larvae with vehicle control, KA 0.4 mg/Kg or KA 0.9 mg/Kg are plotted as mean ± SD. Data for total number of mCherry+ cells was pooled from two independent experiments. Statistical differences were determined using two-way ANOVA with Dunnett’s and Šidák’s multiple comparison tests. ****P < 0.0001, *P < 0.05 when compared to the corresponding vehicle control; ££P < 0.01, when compared with the adjacent sample.

Visualization and quantification of un-engulfed and engulfed apoptotic nuclei. Images of the brain of 4 and 5 days post-fertilization (dpf) larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 previously injected with KA or vehicle control (PBS) at 3 dpf. (A) and (B) are dorsal views (131 µm z-stack images) of 4 dpf larvae and (D) and (E) are dorsal views (182 µm z-stack images) of 5 dpf larvae injected at 3 dpf with vehicle control (Ctrl) or KA 0.9 mg/Kg, respectively, showing csf1r-expressing cells labeled in red, as well as un-engulfed apoptotic nuclei displayed as blue spots and engulfed apoptotic nuclei displayed as yellow spots obtained from the AO-labelled nuclei by the software Imaris. (C) and (F) are a zoom-in of a region of interest (white square) from (B) and (E), respectively, highlighting un-engulfed apoptotic nuclei (displayed in blue [white arrowheads]) and engulfed apoptotic nuclei (dots inside mCherry+ cells shown in yellow [white arrows]). Scale bars represent 30 µm. Respectively, (G and H) show a comparison of the total numbers of un-engulfed and engulfed apoptotic nuclei, normalized to vehicle control at 4 and 5dpf. (I) displays the phagocytic index of mCherry+ cells and (J) its phagocytic load. Values for injected larvae with vehicle control, KA 0.4 mg/Kg or KA 0.9 mg/Kg are plotted as mean ± SD. Data for total number of un-engulfed and engulfed AO-labelled nuclei at 4 dpf was pooled from two independent experiments, and at 5 dpf from one experiment. Statistical differences were determined using two-way ANOVA with Dunnett’s and Šidák’s multiple comparison tests. * ** *P < 0.0001, * **P < 0.001, * *P < 0.01, *P < 0.05 when compared to the corresponding vehicle control, and ££££P < 0.0001 and ££P < 0.01, when compared with the adjacent sample.

Visualization and quantification of csf1r-expressing mCherry positive cells, un-engulfed and engulfed apoptotic nuclei in 5 dpf didys552 larvae. (A) and (B) are dorsal views (82 µm z-stack images) of a 5 days post-fertilization (dpf) sibling control (Sib Ctrl) or homozygous didys552 larva, respectively, and outcrossed to Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149. Images show csf1r-expressing cells labeled in red and engulfed and un-engulfed apoptotic nuclei labeled with acridine orange (AO). (C) Number of mCherry+ cells in the brain. (D) Phagocytic load of mCherry+ cells. (E and F) Respectively, number of un-engulfed and engulfed apoptotic nuclei and normalized to sibling controls (Sib Ctrl). Values for homozygous didys552, or sibling controls are plotted as mean ± SD. Data was pooled from two independent experiments. Statistical differences were determined using unpaired t test for (C) and Mann-Whitney test for (D). * P < 0.05, **P < 0.01, when compared with sibling controls. Scale bars represent 50 µm.

Locomotor activity of zebrafish larvae exposed to PTZ. (A) Total distance moved (mm) over 30 min and (B) distance moved per five minutes intervals before (- PTZ) and after (+PTZ) the addition of 8 mM PTZ in 5 days post-fertilization (dpf) larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 previously injected at 3 dpf with KA 0.9 mg/Kg, KA 0.4 mg/Kg, KA 0.2 mg/Kg or vehicle control (PBS). The ROUT (robust regression and outlier removal) method (with Q = 1) to identify outliers was first applied to all data from (A), and one outlier was identified and removed from the KA 0.9 mg/Kg (-PTZ) group and for the subsequent analysis in (B). Values are plotted as mean ± SD (n = 15 larvae for all groups, except for KA 0.9 mg/Kg with n = 14). Statistical differences were determined using two-way ANOVA with Dunnett’s multiple comparison test. #P < 0.05 when compared with vehicle control. *P < 0.05, **P < 0.01, ****P < 0.0001 when compared with control + PTZ.

Electroencephalographic activity in the brain of zebrafish larvae exposed to PTZ. (A) and (B) are illustrations of a 10-minute local field potential recording (LFP) from a 5 days post-fertilization (dpf) larva of the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 incubated with 8 mM PTZ for 45 min and previously injected at 3 dpf with vehicle control (PBS) (Control + PTZ) (A) or 0.9 mg/Kg KA (KA 0.9 mg/Kg + PTZ) (B). (C) and (D) Zoom-in of a seizure-like event from (A, asterisk) and (B, asterisk), respectively. (E) Percentage of larvae with and without seizures. Controls incubated with vehicle (veh) were included. (F) Total number and (G) cumulative duration of seizure-like events within 10-minute recordings for the Control + PTZ and KA 0.9 mg/Kg + PTZ groups. The ROUT (robust regression and outlier removal) method (with Q = 1) to identify outliers was first applied to all data from (F), and one outlier was identified and removed from the Control + PTZ group and for the subsequent analysis of cumulative seizure duration (G). Values are plotted as median with 25–75 percentiles boxed, bars show ranges from 25 percentile minus 1.5 inter-quartile distance (IQR) to 75 percentile plus 1.5 times IQR (Tukey box plot). (F) and (G) n = 21 for Control + PTZ and n = 23 for KA 0.9 mg/Kg + PTZ. Statistical differences were determined using the Mann-Whitney test with *P < 0.05.