Microglia morphology parameters and clustering, in scn1Lab-KD larvae. (A,B) Dorsal views of the optic tectum of 4 dpf Tg[mpeg1:mCherryF] (A₁) and Tg[mpeg1:mCherryF]; scn1Lab-KD (B₂) larvae showing microglial cells, and 3D reconstructions of microglia in Tg[mpeg1:mCherryF] (A₂,A₃) and Tg[mpeg1:mCherryF]; scn1Lab-KD (B₂,B₃) individuals. (C–G) Quantification of microglia sphericity (C), number of processes (D), total process length (E), mean process length (F), and ramification index (G), in 4 dpf Tg[mpeg1:mCherryF]; scn1Lab-KD (N = 11; n = 239) and Tg[mpeg1:mCherryF] larvae (N = 11; n = 228). (H) Sholl analysis of Tg[mpeg1:mCherryF] (black) and Tg[mpeg1:mCherryF]; scn1Lab-KD (blue) microglia branching. (I) Scheme of the head of a zebrafish larva with the area of interest (the periventricular stratum) framed in red. (J) Cluster analysis of microglia populations in 4 dpf Tg[mpeg1:mCherryF]; scn1Lab-KD larvae (N = 11; n = 239) and Tg[mpeg1:mCherryF] sibling controls (N = 11; n = 228), according to sphericity (S), number of processes (NP), total process length (TL), area (A), and volume (V), leading to three cell populations. (K) Repartition of control (white) and scn1Lab-KD (blue) microglia populations in the three clusters defined (“branched”, “amoeboid”, and “intermediate”). All data are represented as the mean ± sem. p-values were determined using Mann–Whitney or unpaired Student t-test depending on the normal distribution of the values. n.s., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bars: 50 µm (A₁,B₁); 10 µm (A₂,A₃,B₂,B₃).

Microglia dynamics in scn1Lab-KD larvae. Confocal time-lapse analysis of microglia dynamics in the brains of 4 dpf not-injected Tg[mpeg1:mCherryF] control (A₁,A₂) and Tg[mpeg1:mCherryF]; scn1Lab-KD larvae (B₁–B₄), and merged images of two-time points highlighting the increased move of microglia cell bodies in scn1Lab-KD larvae (C₂) compared to that observed in WT controls during a 36-min period (C₁) (0′: cyan, 36′: magenta, merge: white). Quantification of microglia cell body displacements (D) and mean displacement speed (E) over a 60-min period, in 4 dpf scn1Lab-KD larvae (N = 6; n = 55) and WT controls (N = 4; n = 46). (F) Microglial cell tracking over a 60-min period, in 4 dpf control (F₁) and scn1Lab-KD (F₂) larva brain. The scale bar is 20 µm in all images. N = number of larvae and n = number of microglial cells. All images were acquired with an SP8 Leica laser scanning confocal equipped with a 40×/water 1.1 objective. All data are represented as the mean ± sem. p-values were determined using the Mann–Whitney test. ****, p < 0.0001.

Cytokine expression in scn1Lab-KD larvae. (A,B) Quantification of the expression of genes encoding pro- (Il1β and Il8) (A), and anti-inflammatory cytokines (Il10, Il4, and Tgfβ3) (B), relative to that of the tbp gene, in the brains of 4 dpf not-injected (white) and scn1Lab-KD (blue) larvae. (C,D) Dorsal view of the periventricular stratum of 4 dpf Tg[mpeg1:mCherryF] (C₁–C₃) and Tg[mpeg1:mCherryF]; scn1Lab-KD larvae (D₁–D₃), showing Il1β-expressing microglial cells (C₂,D₂). Note that while part of “amoeboid” microglial cells only expressed the il1β:GFP transgene, few highly branched microglia also expressed il1β:GFP. (E) Scheme of a zebrafish larval head with the area of interest (the periventricular stratum) framed in red. (F) Quantification of the percentage of microglial cells expressing Il1β:GFP in not-injected (N = 19) and scn1Lab-KD larvae (N = 16). Scale: 10 µm. N = number of larvae analyzed. All images were acquired using a Leica SP8 confocal microscope, equipped with a 20×/water objective with a numerical aperture of 0.75. All graphs represent mean ± sem. The p-values were calculated using a Student’s t-test. n.s., not significant; ****, p < 0.0001.

Microglia depletion increases neuron hyperactivity in scn1Lab-KD larvae. (A) Scheme of the experimental setup. (B) Calcium imaging of Ca⁺⁺ uptake events recorded over a 20-min period in 4 dpf WT (N = 11), scn1Lab-KD (N = 16), microglia-depleted WT (N = 9) and microglia-depleted scn1Lab-KD larvae (N = 9). (C) LFP recording over a 20-min period of neuron activity in 4 dpf WT (N = 5), scn1Lab-KD (N = 5), microglia-depleted WT (N = 5) and microglia-depleted scn1Lab-KD larvae (N = 5). Scale bars of calcium traces: 0.05 ΔF/F₀ and 1 min; and that of LFP traces: 0.5 mV and 1 min. Quantification of the frequency (D₁) and amplitude (D₂) of calcium events with intensity > 0.04 ΔF/F₀, recorded during a 1-h period in 4 dpf not-injected (white) and scn1Lab-KD larvae (blue). (E). Quantification of the frequency (E₁) and amplitude (E₂) of LFP traces with downwards depolarization > 0.3 mV and lasting > 100 ms, recorded during a 1-h period in 4 dpf not injected (white) and scn1Lab-KD larvae (blue). N = number of larvae. All data are represented as the mean ± sem. ****, p < 0.0001: indicates a statistically significant difference between control and scn1Lab-KD larvae. $$, p < 0.01; $$$, p < 0.001; $$$$, p < 0.0001: indicates a statistically significant difference between larvae with or without microglia. p-values were determined using two-way ANOVA with Tukey post-tests.

Microglia ablation increases the locomotor activity of scn1Lab-KD larvae. (A) Scheme of the experimental setup. (B) Traces of the moves of WT (N = 148), scn1Lab-KD (N = 184), microglia-depleted WT (N = 45), and microglia-depleted scn1Lab-KD larvae (N = 28). The swimming speeds of larvae are represented according to the following color codes: black (low speed), green (medium speed), and red (high speed). (C) Quantification of the total distance traveled by 4 dpf WT (white) and scn1Lab-KD larvae (blue) during a 30-min period recording. (D) Quantification of the time spent by 4 dpf WT (white) and scn1Lab-KD larvae (blue) in a motionless position (D₁), or swimming at low (D₂), medium (D₃), and high speed (D₄). N = number of larvae. All graphs represent the average ± sem. The p-values were calculated by a two-way ANOVA followed by a Tukey post-test. ***, p < 0.001; ****, p < 0.0001: indicates a statistical difference between genotypes. $$$, p < 0.001; $$$$, p < 0.0001: indicate significant differences depending on the presence or absence of microglia.