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Figure 4. Newly synthesized FUS is required for visual experience-dependent structural plasticity.(A) Protocol to test the effect of MO-mediated CPP knockdown on VE-dependent structural plasticity. Tecta were co-electroporated with MOs and GFP-expression plasmid 2 days before imaging. GFP-expressing tectal neurons were imaged in vivo before and after 4 hr in dark followed by 4 hr of VE. Images of a control neuron are shown. Dendritic arbors of individual neurons were reconstructed and total dendritic branch length (TDBL) was compared across imaging time-points. (B) A schematic of different regulatory steps of gene and protein expression, including nuclear transcription, RNA splicing, and cytosolic translation. (C–C’’) Plots of VE-dependent growth rates (changes in TDBL over 4 hr) in dark and VE in tecta electroporated with control MO (C), FUS MO (C’), or NONO MO (C’’). Gray lines connect data points for individual neurons and black lines are average growth rates in dark and VE. Neurons treated with control MO increase growth rate with VE compared to dark. FUS MO blocked the normal increase in growth rate in response to VE. VE-dependent structural plasticity was unaffected by NONO MO. Control MO: n = 14 cells; FUS MO: n = 9 cells; NONO MO: n = 10 cells. (D–D’’) The impaired experience-dependent structural plasticity seen with FUS knockdown (D’) was rescued by expression of exogenous FUS (D’’). Control MO: n = 9 cells; FUS MO: n = 11 cells; FUS MO + FUS: n = 10 cells. (E, F) Validation of FUS knockdown and overexpression (OE). Normalized mRNA or protein expression of fus/FUS in tecta electroporated with FUS MO (E) or FUS expression construct (F), compared to controls. (E) Left: Representative gels of fus-a and control rps13 transcripts from tecta electroporated with control or FUS MO. Right: fus-a expression normalized to rps13 from tecta treated with control or FUS MO. FUS knockdown significantly reduced fus-a (0.78 ± 0.08, p=0.0302), n = 4 independent experiments. (F) Left. Representative blots of FUS and β-tubulin expression from tecta electroporated with control or FUS expression construct. Right: FUS expression normalized to β-tubulin from tecta treated with control or FUS expression constructs. FUS expression construct significantly increased FUS protein. FUS-OE: 1.4 ± 0.13, p=0.0172; n = 5 independent experiments. (G) Left: Representative gels of gria1 and gria2 and control rps13 transcripts from tecta electroporated with control MO or FUS MO. Right: plots of gria1 and gria2 expression normalized to rps13 from tecta treated with control or FUS MO. Fus MO significantly decreased gria1 (0.68 ± 0.12, p=0.0365) but not gria2 (0.79 ± 0.18, p=0.1642); n = 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001, two-tailed paired Student’s t test for comparisons between two matched pairs (C–D) and one-tailed Student’s t test for comparisons of two independent groups (E–G). Error bars represent ±SEM (E–G). Image published in: Liu HH et al. (2018) © 2018, Liu et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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