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Fig. 2
Transgenic FingR lines reflect endogenous synapses and do not affect synapse number or behavior.
(A) FingR(PSD95)-GFP signal is adjacent to Synapsin immunohistochemistry (top panels) and overlaps PSD-95 immunohistochemistry (bottom panels). Quantification of FingR(PSD95)-GFP signal and endogenous PSD-95 signal demonstrates 95% overlap of PSD-95 immunohistochemistry with GFP signal from FingR (bar graph: median 95% +/− 1%, SEM; n = 6 larvae; p 0.01). Confocal images of sections from immunostained Tg(otpb.A:Gal4); Tg(FingR(PSD95)-GFP) larvae, scale bar 10 μm, 5 μm in inset panels. (B) Ex vivo sparse zebrafish primary neuron cell culture demonstrates co-localization of FingRs and endogenous synaptic proteins. Top row, dissociated neurons from Tg(otpb.A:Gal4); Tg(FingR(PSD95)-GFP) embryo, immunohistochemistry for anti-GFP and anti-PSD95. Bottom row from Tg(HuC:Gal4); Tg(FingR(GPHN)-Cherry) embryo, immunohistochemistry for anti-mCherry and anti-GPHN. Confocal images of slides, scale bar 10 μm, 5 μm in inset. (C–E) Pan-neuronal expression in Tg(FingR(PSD95)-GFP); Tg(HuC:Gal4) larvae. (C) FingR(PSD95)-GFP expression does not affect Synapsin protein expression. Data is double-transgenic larvae compared to Tg(HuC:Gal4) only. Images are confocal z-stacks, dorsal views, rostral to top; dotted circle indicates area of Synapsin puncta quantification for bar graphs. Western blots are for Synapsin in whole larvae, standardized to β-catenin; quantified bar graphs to right. (D) FingR(PSD95)-GFP expression does not affect spontaneous swimming behavior (percent time swimming and number of movements), n = 20 embryos each test; three separate experiments; SEM. (E) FingR(PSD95)-GFP expression does not affect audiomotor startle response, n = 27 (control) and 30 (FingR) embryos each test; three separate experiments, SEM (velocity, body curvature, or latency).