🧫 Experiment Protocol
Falsificationproposed
SUMMARY
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- Test MCU overexpression specifically in layer II neurons in healthy vs
## Background and Rationale
This falsification experiment investigates the role of mitochondrial calcium uniporter (MCU) overexpression specifically in layer II cortical neurons during neurodegeneration. MCU dysfunction represents a critical convergence point in Alzheimer's disease pathophysiology, where dysregulated mitochondrial calcium homeostasis contributes to synaptic dysfunction and neuronal death. Layer II ne
METHODOLOGY NOTES
**Phase 1: Animal Preparation and Genotyping (Weeks 1-2)**
• Obtain 8-week-old wild-type C57BL/6J mice (n=40) and APP/PS1 AD model mice (n=40)
• Perform genotyping via PCR to confirm AD transgene status
• Acclimate animals to housing conditions for 1 week
• Randomly assign to experimental groups: WT+Control AAV (n=20), WT+MCU-AAV (n=20), AD+Control AAV (n=20), AD+MCU-AAV (n=20)
**Phase 2: Stereotaxic AAV Injection (Week 3)**
• Anesthetize mice with isoflurane (2-3%)
• Position in stereotaxic frame and inject AAV9-CaMKII-MCU or AAV9-CaMKII-GFP (control) into entorhinal cortex layer II
• Coordinates: AP -4.16mm, ML ±4.2mm, DV -2.8mm from bregma
• Inject 1μL viral suspension (1×10^12 vg/mL) at 0.1μL/min using 33-gauge needle
• Allow 2-week recovery for viral expression
**Phase 3: Calcium Imaging Preparation (Week 5)**
• Inject Fura-2 AM (2μM) or Rhod-2 AM (5μM) calcium indicators via tail vein
• Prepare acute brain slices (300μm thickness) containing entorhinal cortex
• Identify layer I