Stress-induced Changes in Hippocampal Synapses

A separate cohort of restraint stress, multimodal stress and control mice (n=5–9 mice per group) were used to assess the effects of each stress on the number of hippocampal synapses. The postsynaptic density 95 protein (PSD-95), which is localized primarily to dendritic spine heads, was used as a reliable marker for mature synapses.^{28 (link), 29 (link), 30 (link)} Mice were perfused immediately after the stress, and brains were postfixed and processed for immunohistochemistry as described in previous studies.^{31 (link)} Briefly, brains were sectioned coronally (for dorsal hippocampus) or horizontally into 20 μm thick slices. Immunohistochemistry was performed on free-floating sections and mouse anti-PSD-95 antibody (1:2000; Affinity BioReagents, Golden, CO, USA) was used. Antibody binding was visualized with anti-mouse IgG conjugated to Alexa Fluor 568 (1:200, Molecular Probes, Eugene, OR, USA). The restraint experiments and the multimodal stress experiments were run separately and each included a control group. Sections from each stress group and its control group were run concurrently in the same conditions, and analyzed without knowledge of treatment group (blindly). Deconvolution analysis was performed on wide-field three-dimensional images as described previously.^{26 (link),31 (link),32 (link)}