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. 2020 Jun 24;9:e56273. doi: 10.7554/eLife.56273

Figure 5. Dispersion dynamics of various SynGAP isoforms from synaptic spines during LTP.

(A) Live confocal images of hippocampal neurons expressing individual GFP-tagged SynGAP isoforms and mCherry during basal conditions and chemLTP conditions. Yellow arrows indicate dendritic spines that enlarged following chemical LTP treatment. Blue arrowheads mark dendritic spines that did not enlarge after chemical LTP treatment. Scale Bar, 5 μm. (B) Quantification of averaged relative change in the GFP-SynGAP isoform at synaptic spines following chemLTP treatment. Synaptic localization of SynGAP isoforms was determined by calculating the ratio of GFP intensity within dendritic spine heads and dividing by GFP intensity localized to the dendritic shaft at the base of the dendritic spine. Error bars indicate ± SEM. Two-way ANOVA followed by Tukey's post hoc test (SynGAP isoforms F(3,296) = 21.43; p<0.0001, chemLTP F (1,296)=119.9; p<0.0001, Interaction F(3,296) = 6.607; p<0.0001, n = 37–39 spines from 4 independent experiments, ***p<0.001, **p<0.01, *<0.05) was performed. Error bar indicates ± SEM.

Figure 5.

Figure 5—figure supplement 1. shRNA efficiency and rescue SynGAP construct titration assay.

Figure 5—figure supplement 1.

(A) Efficiency of shRNA-SynGAP construct. Hippocampal neurons were transfected with shRNA-SynGAP#5 construct together with GFP (marker for transfected cell) and stained for pan-SynGAP antibody (**p<0.01, 77.3 ± 0.1% reduction of SynGAP expression upon our shRNA-SynGAP#5 construct, n = 4 cells, unpaired T-test (two-tailed), Error bars indicate ± SEM). Scale Bar, 10 μm. (B) Expression levels of our shRNA-resistant SynGAP isoform construct. Hippocampal neurons were transfected with mCherry and GFP-SynGAP isoform construct and stained for SynGAP isoform specific antibodies described in Figure 1 (α1 88.3 ± 0.2%, α2 97.0 ± 0.2%, β 83.5 ± 0.1% for GFP-SynGAP portion, n = 4 cells). Dotted line showed endogenous SynGAP expression levels. Error bars indicate ± SEM. One-way ANOVA followed by Tukey's post hoc test (Transfections F(4,10) = 126.8; p<0.01, n = 3 cells, *p<0.05) was performed. Scale Bar, 10 μm.