Analysis of regional and global neuronal network activity following chronic exposure to PFOS. (A) Illustrative representation of a larval zebrafish brain with anatomical regions outlined: forebrain (FB), optic tectum (OT), cerebellum (Ce), hindbrain (HB), and whole brain (WB). (B) Illustrative representation of neuron-driven CaMPARI: neurons with low intracellular calcium (low Ca2+) remain green following 1-min exposure to 405-nm light, whereas neurons with high intracellular calcium (high Ca2+) photoconvert to red. (C) Confocal micrograph of a 3-dpf larvae expressing neuron-specific CaMPARI [Tg(elavl1:CaMPARI)] following 1 min photoconversion. (D) Generated high-intensity look-up-table (LUT) heat map of the red, photoconverted channel in (C) depicting high-activity neurons. Low-to-high intracellular calcium is depicted by a blue–red–white spectrum. Confocal micrographs of active neurons at 3-dpf in (E) control and (F) 7μM PFOS- and (G) 28μM PFOS-exposed larvae following 1-min photoconversion with 405-nm light. Neuronal activity can be quantified by determining the ratio of fluorescent intensity in the red vs. green channels (FRed/FGreen). (H) Quantification of the regional and global (i.e., WB) neuronal activity as normalized FRed/FGreen ratios of 3-dpf control or 7μM PFOS-treated larvae. (I) Quantification of the regional and global neuronal activity of 3-dpf control or 28μM PFOS-treated larvae. Confocal micrographs of active neurons at 5 dpf in (J) control and (K) 7μM PFOS- and (L) 14μM PFOS-exposed larvae following 1-min photoconversion with 405-nm light. (M) Quantification of the regional and global neuronal activity of 5-dpf control or 7μM PFOS-treated larvae. (I) Quantification of the regional and global neuronal activity of 5-dpf control or 14μM PFOS-treated larvae. Confocal micrographs at 10× magnification. n=21–23 fish per group. **p<0.01; ***p<0.001. 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: Ca2+, calcium ion; CaMPARI, calcium-modulated photoactivatable ratiometric integrator; dpf, days postfertilization; PFOS, perfluorooctane sulfonate.
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