Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization
Spines can be divided into two categories, according to their fluorescence recovery time. (a) Frequency distribution of tau values in “Ast high” condition (black bars) ( spines). Distribution was adjusted to a two-Gaussian distribution, with two peak values of and seconds ( test, ) (gray line). Insert depicts two representative recovery curves for each category. Only the initial 30 seconds are displayed. Curves were normalized and scaled to the same initial time. (b) Frequency distribution of tau values in the “Ast low” condition (red bars) ( spines). Distribution was adjusted to a two-Gaussian distribution, with two estimates peak values of and seconds ( test, ) (gray line). Black bars show the frequency distribution of tau values in “Ast high” condition, plotted for comparison. (c) The mobile fraction of each “Ast high” spine was plotted against its recovery time value (dotted line, one phase exponential association). Note that spines with higher mobile fractions present slow recovery times, and vice versa. (d) Mobile fraction values of “Ast high” spines were plotted against their spine head area (black dots) ( spines). Of interest, spines with a larger head area show higher mobile fractions, and vice versa. Blue dots represent spines from hippocampal culture slices ( spines). Spines from hippocampal slices show high mobile fractions and larger spine head areas. (e) Left axis: spine head areas were grouped (0.2 μm2 intervals) and their mean average areas were plotted against their average recovery time ( spines, mean ± SEM). The value distribution was adjusted to a linear regression (slope: ). Right axis: same relation employing spines transfected with monomeric GFP (slope: ) ( spines). Both graphs show a lineal relation between bleach area and fluorescence recovery constant, although with a tenfold difference in scale. (f) Right picture. (A) Example of two pyramidal hippocampal neurons expressing GFP-actin from an organotypic slice culture. Scale bar: 20 μm. (B to D) Sequential frames of a FRAP experiment. Scale bar: 2 μm. Average mobile fraction was estimated to be ( spines). Left graph: frequency distribution of tau values obtained from hippocampal slices ( spines, blue bars). Notice how slice spines fall mostly into a single distribution. Spine MF values from cultures and slices were not statistically different (Mann-Whitney test, ns).
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