Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth
Fig 1
TEA application affects dendritic spine size and actin dynamics at CA3 hippocampal pyramidal neurons.
A. Maximum projection of a stack of multiple optical sections, showing a part of the apical dendrite of a CA3 pyramidal cell expressing fCherry. Representative images of dendritic spines before (15 min) and after (30–50 min) TEA application. The orange arrowheads point to spines exhibiting a substantial increase in their head size, the blue arrowhead indicates a spine remaining stable over time upon 10min TEA treatment. Scale bar, 2μm. B. Quantification showing the change of spine head diameter at different time points before (15min, grey area) and after (30–50 min, grey area) 10min TEA treatment. Error bars represent SEM. (40min after TEA stimulation spine head increase of 16.52% ± 2.2%, p = 0,0012; n = 5 independent experiments / 139 spines of 5 CA3 pyramidal cells). C. Maximum projection before bleaching (on the left) showing the F-actin accumulation in the spine head, (pseudocolor encodes for fluorescence intensity). Scale bar, 2μm. Time series (on the right) showing the fluorescence recovery after photobleaching (FRAP) of eGFP-actin at a single spine. Time point of bleaching at 0 sec (pseudocolor encodes for fluorescence intensity). Scale bar, 2μm. D. Fluorescence recovery curve for eGFP-actin at single spines after photobleaching performed before (n = 16) and after (n = 19) TEA treatment (25mM TEA for 10min). The fluorescence intensity (eGFP-actin) of a single spine is blotted against the time. (plateau level at 110sec after belching before TEA 0.774 ± 0.034 vs. after TEA 0.88 ± 0.017; p = 0.0056).