Phase 1: Generation of Spp1 Knockout AD Mouse Models — Weeks 1-4
Breed Spp1-/- mice (The Jackson Laboratory #004936) with 5xFAD mice (#034840-JAX) to generate Spp1-/-;5xFAD double mutants. Establish four experimental groups (n=12 per group based on power analysis for Cohen's d=1.0): wild-type controls, Spp1-/- only, 5xFAD only, and Spp1-/-;5xFAD double mutants. Confirm genotypes using PCR with primers for Spp1 knockout allele, 5xFAD transgenes (APP and PS1), and appropriate controls. Age mice to 6 months when synaptic loss is established but not maximal in 5xFAD model. Maintain mice under standard housing conditions with 12:12 light/dark cycle.
...Phase 1: Generation of Spp1 Knockout AD Mouse Models — Weeks 1-4
Breed Spp1-/- mice (The Jackson Laboratory #004936) with 5xFAD mice (#034840-JAX) to generate Spp1-/-;5xFAD double mutants. Establish four experimental groups (n=12 per group based on power analysis for Cohen's d=1.0): wild-type controls, Spp1-/- only, 5xFAD only, and Spp1-/-;5xFAD double mutants. Confirm genotypes using PCR with primers for Spp1 knockout allele, 5xFAD transgenes (APP and PS1), and appropriate controls. Age mice to 6 months when synaptic loss is established but not maximal in 5xFAD model. Maintain mice under standard housing conditions with 12:12 light/dark cycle.
Phase 2: Behavioral Assessment of Cognitive Function — Weeks 5-6
Conduct comprehensive behavioral testing battery to assess cognitive function. Perform Y-maze spontaneous alternation test (15-minute sessions) to measure working memory, with >60% alternation considered normal. Execute novel object recognition test with 24-hour retention interval, measuring discrimination index [(time with novel - time with familiar)/(total exploration time)]. Conduct contextual fear conditioning with 24-hour and 7-day memory tests, measuring percentage time freezing. Use Morris water maze with 5-day acquisition phase and probe trial on day 6, recording escape latency and time in target quadrant. Randomize testing order and ensure experimenter blinding to genotypes.
Phase 3: Synaptic Density Analysis by Immunohistochemistry — Week 7
Sacrifice mice by transcardial perfusion with PBS followed by 4% paraformaldehyde. Process brains for immunohistochemistry using 40μm vibratome sections. Perform triple immunofluorescence staining for presynaptic marker synaptophysin (Millipore MAB368, 1:500), postsynaptic marker PSD95 (Millipore MAB1596, 1:300), and neuronal marker MAP2 (Sigma M4403, 1:800). Focus analysis on hippocampal CA1 stratum radiatum and cortical layers II/III where synaptic loss is prominent in 5xFAD mice. Use confocal microscopy (63x oil objective, NA 1.4) to acquire high-resolution z-stacks (0.2μm steps) of dendrite segments. Quantify synapse density as colocalized synaptophysin/PSD95 puncta per μm of MAP2+ dendrite length using ImageJ with synaptic puncta analysis plugins.
Phase 4: Electron Microscopy Ultrastructural Analysis — Week 8
Prepare separate cohort of mice (n=6 per group) for electron microscopy to validate immunofluorescence findings. Perform transcardial perfusion with 2.5% glutaraldehyde in 0.1M phosphate buffer. Process hippocampal CA1 tissue through osmium tetroxide post-fixation, dehydration series, and Epon embedding. Cut 70nm ultrathin sections and stain with uranyl acetate and lead citrate. Image synapses in stratum radiatum using transmission electron microscopy at 15,000x magnification. Quantify synaptic density per μm² neuropil, synaptic vesicle density, and active zone length. Identify synapses by presence of synaptic vesicles, electron-dense postsynaptic density, and synaptic cleft.
Phase 5: Molecular Analysis and Mechanistic Assessment — Week 9
Extract proteins from hippocampal tissue using RIPA buffer with protease inhibitors. Perform Western blot analysis for synaptic proteins: synaptophysin (Millipore MAB368, 1:2000), PSD95 (Millipore MAB1596, 1:1000), and synapsin I (Millipore AB1543P, 1:1000) with β-actin normalization. Additionally, assess microglial activation markers CD68 (Bio-Rad MCA1957, 1:500) and complement protein C1q (Abcam ab182451, 1:1000) to examine potential mechanisms of SPP1-mediated synaptic loss. Perform qPCR analysis for inflammatory cytokines (Il1b, Tnfa, Il6) and synaptic genes (Dlg4, Syn1, Syp) using TaqMan assays with Gapdh normalization.
Phase 6: Statistical Analysis and Data Integration — Week 10
Employ two-way ANOVA with factors of Spp1 genotype (WT vs KO) and AD pathology (control vs 5xFAD) for all primary outcomes. Use Tukey's multiple comparison test for post-hoc analysis. Calculate effect sizes (Cohen's d) for all pairwise comparisons. Perform correlation analysis between behavioral scores and synaptic density measurements. Apply Bonferroni correction for multiple comparisons (adjusted α = 0.0125 for 4 primary outcomes). Use GraphPad Prism for statistical analysis and generate comprehensive correlation matrices between behavioral, morphological, and molecular measures.