Figure 3.

Effects of theta-burst stimulation on the number of spines with high concentrations of pCofilin. a, Laser confocal micrograph shows immunolabeling for total cofilin in the proximal CA1 str. radiatum. Scale bar, 5 μm. b, Micrographs showing immunostaining for pCofilin, PSD95, and the merged image of the two for the same field of CA1 str. radiatum. The pCofilin immunoreactivity (red) was localized to discrete structures that fell within the size range expected for dendritic spines. These were much less numerous than the comparable profiles (a) labeled by antisera against total cofilin. Comparison of the three panels in b shows that a subpopulation of the numerous PSD95-IR structures (green) also contained pCofilin immunoreactivity (evident as yellow puncta in the right Merge panel) and that the majority of pCofilin-IR structures were associated with concentrations of PSD95 immunoreactivity. Scale bar, 5 μm. c, Enlargement of the structure indicated by the arrowhead in b illustrates the spatial relationship of areas occupied by pCofilin (red) and PSD95 (green) immunoreactivities and the extent to which the two overlap (yellow at bottom). Scale bar, 0.25 μm. d, Three-dimensional reconstruction of immunolabeling for pCofilin (red) and PSD95 (green) structures after wide-field microscopic acquisition at 0.2 μm z-steps followed by restorative deconvolution: the adjacent but distinct localizations of pCofilin and PSD95 immunoreactivities are evident. The images are successive 90° rotations (from top to bottom). Scale bar, 0.5 μm. e, Slices were collected 30 s, 2–7 min, or 15–30 min after the delivery of a single train of TBS to the Schaffer-commissural projections and processed for pCofilin immunostaining. Controls (cont; open bar) received baseline stimulation. The marked increase in pCofilin-IR puncta at 2–7 min was highly significant (**p = 0.008, Tukey's HSD) relative to controls and 30 s post-TBS groups (n = 8 for cont and 2–7 min, n = 9 for 0.5 and 15–30 min). These results provide a close replication of those obtained with pPAK. f, Spine counts were combined within the various groups (control, 30 s post-TBS, etc.) from the separate pPAK and pCofilin experiments. Z-scores were calculated using the means and SDs for the control groups in each experiment; thus, each slice value was expressed as the difference from the mean of the control slices divided by the SD for the control slices. The planned comparisons of 2 and 7 min versus control was highly significant [ANOVA, p = 0.003, **p = 0.003, and ***p = 0.0009 for 2 (n = 7) and 7 (n = 6) min, respectively].