1：Experimental Design and Method Selection:

The study combined tau seeding with chronic in vivo two-photon calcium imaging in awake head-fixed mice expressing human tau with the P301S mutation. This allowed for the investigation of functional consequences of NFT formation in intact neuronal circuits and direct comparison of NFT-bearing and NFT-free neurons over the disease course.

2：Sample Selection and Data Sources:

Homozygous transgenic male and female mice expressing human mutant P301S tau (on a C57BL/6J background) and wild-type C57BL/6J mice were used. Mice were injected at 2 months of age with tau preformed fibrils (tau-PFFs) or vehicle, and imaging was performed over up to 50 days post-injection.

3：List of Experimental Equipment and Materials:

Equipment included a stereotaxic frame, dental drill (Schick-Technikmaster C1), two-photon microscope (La Vision Trim Scope), tunable Ti:sapphire lasers (Coherent Chameleon and Mai Tai), cranial windows, AAV1.hSyn1.mRuby2.GSG.P2A.GCaMP6s.WPRE.SV4 virus, tau-PFFs, FSB dye, antibodies (AT8), confocal microscope (LSM 780), and software (MATLAB, ImageJ, Photoshop, GraphPad Prism, STATISTICA). Materials included ketamine-xylazine, dexamethasone, carprofen, cefotaxime, dental acrylic, coverslips, and various chemicals for immunohistochemistry.

4：hSynmRubyGSG.P2A.GCaMP6s.WPRE.SV4 virus, tau-PFFs, FSB dye, antibodies (AT8), confocal microscope (LSM 780), and software (MATLAB, ImageJ, Photoshop, GraphPad Prism, STATISTICA). Materials included ketamine-xylazine, dexamethasone, carprofen, cefotaxime, dental acrylic, coverslips, and various chemicals for immunohistochemistry. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Mice underwent stereotaxic injection of tau-PFFs or vehicle into the frontal cortex, followed by cranial window implantation. After recovery and training for head-fixation, in vivo two-photon calcium imaging was performed in awake mice. FSB was injected to label NFTs in vivo. Imaging sessions recorded spontaneous activity of layer 2/3 neurons, with behavioral state (quiet vs. active) monitored via whisking movement. Data were processed for calcium transient analysis and correlation patterns.

5：Data Analysis Methods:

Calcium transients were detected and analyzed using custom MATLAB code, including image registration, neuropil correction, and transient frequency calculation. Statistical analyses used one-way ANOVA, two-way ANOVA, MANOVA, and Student's t-tests. Correlation coefficients and pattern persistence were evaluated over time.