Hippocampal slices that received TBS, together with paired control slices from the same mice, were collected at specified post-stimulation time points to evaluate dendritic spine levels of GTP-bound (activated) Rac1 or phosphorylated (p) PAK (Ser141), respectively. Specifically, double-immunolabeling for pPAK and the postsynaptic scaffold protein PSD95 or for Rac1-GTP and cofilin was performed (Chen et al., 2007 (link)). Cofilin was used as a spine marker in combination with localization of Rac1-GTP because the antisera are raised in different species and our work has shown that cofilin is highly localized within hippocampal dendritic spines (Chen et al., 2007 (link)). For experiments evaluating basal levels of PAK, adult mouse brains were fast-frozen in 2-methyl butane (-45°C) and cryostat sectioned on the coronal plane at 20 μm. The slide-mounted tissue was fixed in -20°C methanol for 15 min and processed for dual immunohistochemical localization of PAK3 and PSD95. Primary antisera used included mouse anti-PSD95 (1:1000; #1-054 Affinity BioReagents/Thermo Fisher Scientific, Rockford, IL), rabbit anti-cofilin (1:250; #ACFL02, Cytoskeleton, Denver, CO), mouse anti-Rac1-GTP (1:1000, #26903 NewEast Biosciences, Malvern, PA), rabbit anti-phospho-PAK1,2,3 Ser141 (1:100; #44-940G, Invitrogen), and rabbit anti-PAK3 (1:500; #06-902, Millipore, Billerica, MA). Alexa488 anti-mouse IgG and Alexa594 anti-rabbit IgG (Invitrogen) were used for visualization.
In all cases a sample field of 136 × 105 × 3 μm (42,840 μm3) was photographed with a 63× objective (1.4 NA) and a CCD camera (Orca ER; Hamamatsu Photonics, Bridgewater, NJ). For LTP experiments, the sample field was placed between the two stimulating electrodes. For analysis of whole brain, sections through mid-septotemporal hippocampus were similarly evaluated. In all instances, digital Z-stacks (0.2 μm steps; 3 μm thick) were collected and processed for iterative deconvolution (Volocity 4.1 Restorative Deconvolution, Improvision, Walthem, MA). Automated in-house systems were then used to count single- or double-labeled puncta within the size range of dendritic spines. Three-dimensional (3D) analyses of spine immunofluorescent labeling in field CA1 str. radiatum were performed using a multiple intensity threshold series protocol as described (Rex et al., 2009 (link); Chen et al., 2010 (link); see Suppl. Fig. 1). Briefly, image Z-planes were normalized to a target background intensity (30% of maximum) and iteratively binarized at regular intensity thresholds (4% steps ranging from 39-78% of maximum) using exclusion criteria for object size and ellipticity, followed by dilation and erosion filtering. Repeated observations were binned and analyzed to assess object boundaries and discriminate neighboring objects. This process accurately identifies both faintly and densely labeled elements. Finally, analyses were reconstructed in 3D to calculate label volume and position. Multiple labels in the same image Z-stack were analyzed independently; immunolabeling for the two antigens (spine marker and the target protein) were considered colocalized if any overlap was detected between their respective boundaries. Counts of single-labeled and double-labeled elements from each section were then averaged to obtain a value for each slice or brain. Values in text and figures are group means ± sem. Significance was determined by ANOVA and individual comparisons by Tukey's HSD post-hoc test.