DFP exposure induced major morphological changes of microglial cells. (A) Scheme of the experimental set up. Live 5 dpf Tg[mpeg1:mCherryF] larvae were exposed to either 1% DMSO (control) or 15 µM DFP for 6 h and then imaged with a confocal microscope. The region of interest is framed in red. (B–C) Representative microglial cells in control (B) and DFP-treated larvae. (C) Scale bar: 50 µm. (B′,B″) Magnification of the white-circled microglia from a control larva (B′), and corresponding 3D reconstruction (B″). (C′,C″) Magnification of the white-circled microglia from a DFP-treated larva (C′), and corresponding 3D reconstruction (C″). Scale bar: 10 µm. (D–I) Changes in microglia morphological parameters: sphericity (Sp) (scaled from 0, fully disordered morphology, to 1, perfect sphericity) (D), surface area (S) (E), volume (V) (F), mean branch number (NB) (G), total branch length (TL) (H), and mean branch length (ML) (I) in control (DMSO) (N = 13 embryos, n = 327 cells) and DFP-treated larvae (DFP) (N = 14 larvae, n = 294 cells). (J) Sholl analysis of microglia branch complexity in control (black) and DFP-exposed larvae (blue). Error bars on all graphs represent the standard error of the mean (SEM). Statistics: ***, p < 0.001; n.s., not significant. (K) Clustering of microglial cell populations in control (DMSO) and DFP-exposed larvae (DFP), using five of the previously described morphological parameters (Sp, NB, TL, ML, and S) (see Materials and Methods). Each column corresponds to a single microglial cell, and each parameter is scaled from black (‘resting’ state) to red (‘activated’ state); black dotted lines separate the different microglial populations (ramified, transitional, and amoeboid).

Dynamics of microglia remodelling in DFP-exposed embryos. Selected snapshot views of live imaging of microglia showing the dynamics of microglia morphological changes in a living 5 dpf Tg[mpeg1:mCherryF] larva, before (0 h) and at different time points of a 6 h exposure to 15 µM DFP. Coloured arrowheads indicate individual microglial cells. Scale bar represents 20 µm.

DFP exposure induced massive expression of pro-inflammatory cytokines (A–C). Expression levels of transcripts encoding cytokines Il1β (A), Il8 (B), and Il4 (C) relative to that of reference tbp transcripts, in RNAs from control (DMSO) and DFP-exposed larvae (DFP) at different exposure times. In each condition, N = 8 samples, n = 7 larvae/sample. Error bars on all graphs represent the standard error of the mean (SEM). Statistics: *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

DFP exposure induced massive brain inflammation. Expression levels of transcripts encoding cytokines Il1β, Il8, and Il4, relative to that of reference tbp transcripts, in dissected brain RNAs from larvae exposed for 6 h to 15 µM DFP or DMSO (in each condition, N = 5 samples, n = 10 brains/sample). Error bars on all graphs represent the standard error of the mean (SEM). Statistics: *, p < 0.05; **, p < 0.01; n.s., not significant.

DFP exposure rapidly induced massive neuronal activation. Expression levels of fosab RNA relative to that of tbp RNA transcripts, from control (DMSO) and DFP-exposed (DFP) larvae, at different time points of exposure (in each condition, N = 8 samples, n = 7 larvae/sample). Error bars on all graphs represent the standard error of the mean (SEM). Statistics: **, p < 0.01; ***, p < 0.001.

Microglia are key players in DFP-induced inflammation. Expression levels of transcripts encoding cytokines Il1β, Il8, Il4, and Tnfα relative to that of reference tbp transcripts, from control larvae and pU.1 morphants exposed for 6 h to either 1% DMSO (DMSO) or 15 µM DFP (DFP) (in each condition, N = 7–8 samples, n = 7 larvae/sample). Error bars on all graphs represent the standard error of the mean (SEM). Only statistically significant differences between samples are shown: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Microglial depletion markedly reduced DFP-induced neuronal hyperactivation. Expression levels of fosab RNAs relative to that of reference tbp transcripts, from control larvae and pU.1 morphants exposed for 6 h to either 1% DMSO (DMSO) or 15 µM DFP (DFP) (in each condition, N = 7–8 samples, n = 7 larvae/sample). Error bars on all graphs represent the standard error of the mean (SEM). Statistics: **, p < 0.01; ***, p < 0.001.