Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization
Mobile fraction values do not correlate with culture age or dendrite localization. (a) (A) An example of hippocampal neurons (growing in the presence of astrocytes) transfected with GFP-actin. Scale bar: 20 μm. Pictures on the right (B to E) are higher magnification images showing prebleach (B), bleach (C), postbleach (D), and late phase of recovery (E) (60 s). Scale bar: 1 μm. (b) Normalized fluorescence recovery curve of (a), showing the two fractions of fluorescence recovery: stable and mobile fractions ( spines, black circles). An example of the recovery in the presence of Cytochalasin D (CytD) (5 μM) in the extracellular solution ( spines, blue circles). Top insert: comparison of the initial phase of GFP-actin (black line) and monomeric GFP (red line) recovery curves. Note the similarities between both initial phases in the first 1–3 ms. (c) Graph frequency distribution of mobile fractions from neurons 20 DIV growing in the presence of astrocytes. Mean average was ( neurons, independent cultures, and spines). (d) Neuronal structure drawing indicating the localization of the recorded spines. Note the variability in MF values along neuronal dendrites. (e) Mobile fraction values were averaged according to their dendrite type (primary, secondary, or tertiary), and the same value was calculated at days 18, 20, and 22 in vitro ( neurons, independent cultures, mean ± SEM). As the graph shows, no differences were found for culture age or dendrite localization (two-way ANOVA with Tukey’s multiple comparison test, ns). (f) Average mean of mobile fraction values from ten individual neurons was compared with the overall population of MF mean. No statistical differences were found among neurons or when each neuron was independently compared to the whole population. Only values from 20 DIV were used in this analysis (one-way ANOVA, ns) ( neurons, independent cultures, mean ± SEM).