Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

Figure 2

Astrocyte contact regulates actin mobile fraction. (a-b) The pictures show two neurons growing over a layer of astrocytes ((a) “Ast high” condition), or in the near-absence/absence of astrocytes ((b) “Ast low” condition). Notice how in the “Ast low” condition a large part of the neuron has no contact with the surrounding astrocytes. Neurons were transfected with GFP-actin (green) and astrocytes were identified by GFAP staining (red). Scale bar: 10 μm, 21 days in vitro. (c) Cumulative distribution of spine head areas comparing “Ast high” (black) versus “Ast low” (red) ( spines “Ast high” and spines “Ast low”) (Kolmogorov-Smirnov test, ns). (d) Frequency distribution of mobile fractions in “Ast low” conditions (red bars) ( spines). Of interest is that the MF was drastically reduced in the “Ast low” condition, as compared to the “Ast high” condition (Mann-Whitney test, ). The mobile fraction values distribution of “Ast high” conditions was included for comparison (black bars). (e and f) In vivo confocal images. (e) To evaluate the presence of direct astrocyte contact with the recorded spine, membranes were stained with the lipophilic dye FM4-64 (4 μM, red). The dendrite shown in (e) “Ast high” condition was lying on top of an astrocytic layer. Longitudinal sections at higher magnification were performed to study spine surroundings (approximately marked as a dashed line), pictures (A) to (C) (overlap, FM4-64 and GFP-actin, resp.). Scale bar: 1 μm. Bottom: detailed pictures of selected frames obtained from the FRAP experiment, prebleach, bleach, and postbleach. Scale bar: 1 μm (). (f) Detailed picture of a dendrite growing in “Ast low” conditions. Scale bar: 1 μm. Notice how FM4-64 staining was only present along the dendrite, but not surrounding the spine. A section of the optical longitudinal acquisition is shown in pictures (A), (B) and (C). Bottom: selected frames obtained from the FRAP experiment, prebleach, bleach, and postbleach. Scale bar: 1 μm (). (g) Normalized fluorescence recovery curve of the depicted spines growing in “Ast high” (black dots) and “Ast low” (red dots). Notice that spines growing in “Ast high” conditions are characterized by a recovery close to 80%, while spines growing in “Ast low” conditions present a recovery close to 50%.

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