Figure 2.

Figure 2.

Optogenetic modulation of PFOS-exposed microglia. (A) Schematic of halorhodopsin: Optogenetic modulation of microglia electrical state is achieved via photostimulation of the light-gated chloride pump, halorhodopsin (eNpHR3.0). eNpHR3.0 is most responsive to 589-nm wavelength light. (B) eNpHR3.0 was driven under a pan-macrophage promoter [Tg(mpeg1:GalFF;UAS:eNpHR3.0-mCherry)] to achieve optogenetic control of microglia in zebrafish larvae. (C) Experimental paradigm: At 72 hpf, injured or uninjured zebrafish were stimulated for 4 h with 589-nm light in the enclosed Noldus DanioVision Behavior Unit. (D) Confocal micrograph of 3-dpf zebrafish brain exposed to 28μM PFOS. Magenta cells are unstimulated halorhodopsin+ microglia. (E) Pseudocolored (D) in black and white. (E′–E″) 3× magnification of microglia from boxes in (E). Arrows point to projections emanating from the microglia cell bodies. (F) Dorsal view of a representative 3-dpf control-treated larval brain at 4 hpi without stimulation. Injury site marked with magenta dot and area of responding microglia shaded in magenta. (G) Dorsal view of a representative 3-dpf 28μM PFOS-treated larval brain at 4 hpi without stimulation. (H) Confocal micrograph of 3-dpf 28μM PFOS-treated larval brain following stimulation with 589-nm light. Magenta cells are stimulated halorhodopsin+ microglia. (I) Pseudocolored (H) in black and white. (I′–I″) 3× magnification of microglia from boxes in (I). Arrows point to projections emanating from the microglia cell bodies. (J) Dorsal view of a representative 3-dpf control-treated larval brain at 4 hpi following 589-nm light stimulation. (K) Dorsal view of a representative 3-dpf 28μM PFOS-treated larval brain at 4 hpi following 589-nm light stimulation. (L) Quantification of microglia cell area of 3-dpf 28μM PFOS-treated larvae with and without light stimulation. (M) Quantification of microglia cell perimeter of 3-dpf 28μM PFOS-treated larvae with and without light stimulation. (N) Perimeter-to-area ratio of individual microglia of 3-dpf 28μM PFOS-treated larvae with and without light stimulation. n=40–42 cells per group from three independent experiments. (O) Quantification of the area of microglia response around the injury site at 4 hpi. n=9–15 fish per group. Confocal micrographs at 40× magnification (D–E″ and H–I″) or 20× magnification (F,G,J,K). *p<0.05; **p<0.01; ****p<0.0001. Error bars represent standard deviation. Box plot limits represent 25th to 75th percentile, with the midline representing the median. See Excel Table S1 for additional statistical details. Note: dpf, days post fertilization; hpf, hours postfertilization; ns, not significant; PFOS, perfluorooctane sulfonate.