Figure 4
Cyfip1 deficiency affects F-actin dynamics in spines, occludes structural responses to cLTD and increases surface AMPA receptor mobility. Cyfip1+/− and WT hippocampal neurons were transfected with actinGFP or LifeactGFP at 17–20DIV and subjected to FRAP live imaging 2 days later. Spines were imaged for 100 s and bleaching occurred after the first 20 s. (a) Representative images over time in seconds of actinGFP fluorescence recovery in WT and Cyfip1+/− spines. The red circles highlight the bleached spine. Scale bar, 2 μm. Quantification of GFP fluorescence intensity within the spine head region of WT compared with Cyfip1+/− neurons transfected with actinGFP (b) or LifeactGFP (d) shows Cyfip1+/− spines recover to a greater extent than WT (data points represent an average of 25–33 movies, *P<0.05). Data are fitted with single exponentials (coloured lines). The mobile fraction, quantified as the final amount of recovered fluorescence presented as a percentage of the total bleached fluorescence, is significantly increased in Cyfip1+/− neurons for both actinGFP (c) (n=29–33, *P<0.05) and LifeactGFP (e) (n=24–29, *P<0.05). 21DIV WT and Cyfip1+/− hippocampal neurons were transfected with actinGFP and treated with either 20 μM NMDA+20 μM glycine (NMDA) or saline (SAL). (f) Representative images of spines following each treatment. Scale bar, 5 μm. Spine volume is significantly decreased in WT neurons following NMDA treatment compared with saline (n=8000–13 000 spines per condition, ****P<0.0001). However, Cyfip1+/− spines have basally low volumes and fail to remodel in response to NMDA (n=12 000–14 000 spines per condition, ****P<0.0001) (g). Spine volume is significantly decreased in WT NMDA-treated neurons compared with saline treatment whereas Cyfip1+/− saline- and NMDA-treated neurons show no leftward shift (n=8000–14 000 spines, ****P<0.0001, one-way analysis of variance (ANOVA)) (h). GluA2SEP-containing receptors were labelled with quantum dots (QDs) and live imaged. (i) Representative QD trajectories are shown for WT and Cyfip1+/− neurons, arrowheads indicate GluA2SEP-containing synapses and green dotted line indicates synapse area. Scale bar, 2 μm. Quantification of receptor diffusion inside (j) and outside (k) synaptic clusters revealed that GluA2SEP-containing receptors are more mobile in synaptic clusters of Cyfip1+/− neurons compared with WT (n=140–160 QD tracks, **P<0.01). cLTD, chemical long-term depression.
