Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Y-maze
• Procedure: Balance beam test
• Procedure: Inclined screen test
• Procedure: Grip strength test
• Procedure: Elevated plus maze
• Procedure: Contextual fear conditioning
• Procedure: Blood glucose
• Procedure: Beta-amyloid levels
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Body weight	Scales	Body weight
2	Y-maze test	Y-maze apparatus	Spontaneous alternation, distance traveled, number of arm entries
3	Balance beam test	Narrow beam	Time to cross beam
4	Incline screen test	Screen at 45° angle	Time to right body
5	Grip strength test	Grip strength meter	Grip strength
6	Elevated Plus Maze	Elevated Plus Maze apparatus	Distance traveled, number of arm entries
7	Contextual fear conditioning	Fear conditioning chamber	Time spent freezing during training and memory sessions
8	Blood samples collected and glucose levels determined	Glucose meter	Blood glucose levels
9	Brains harvested and beta-amyloid levels determined	Spectrophotometer, Biotek plate reader	Beta-amyloid levels

Equipment, software and supplies

• Y-maze
  • Clear acrylic arms 2" wide x 12" long x 2" high
  • Placed on table in dimly lit room
  • Spatial cues displayed on walls around the table
• Software: ANY-maze (Stoelting Co., IL, USA)
• Balance beam
  • 3 ft long x 0.5" wide
  • Elevated above a table 20.75"
  • "Safe" platforms on either side of the beam
• Inclined screen
  • Wire-mesh grid (holes 1 cm x 1 cm)
  • 45° angle
• Grip strength meter (Coulbourn Instruments, PA, USA)
• Elevated plus maze
• Fear conditioning chamber
• Glucose meter and strips
• Dissection kit
• Sonicator
• NanoDrop 2000 UV-Vis Spectrophotometer (ThermoScientific, USA)
• Beta-amyloid ELISA (Wako Chemicals (Cat #298-92410), Richmond VA, USA)
• Biotek plate reader (BioTek, USA)

Reagents and solutions

• Phosphate buffered saline (PBS)
• Triton X-100
• TissueLyser II System (QIAGEN)
• Guanidine hydrochloride (GuHCl)

Procedure: Y-maze

1. Mice are habituated to transport and to the testing room for 3 days prior to testing.
2. Mice are placed in a randomized start arm and video tracking software is used to monitor arm entries.
   • An arm entry is called when the mouse's entire body (including hindpaws) enter the arm.
   • The sequence and total number of arms entered is recorded.
   • Percentage of successful alternations is calculated as follows: number of alternations/maximum possible alternations (total number of arms entered - 2) x 100.
3. For each mouse that is measured longitudinally, the age at which the animal becomes impaired, or performed below chance levels (50%), is recorded and used as the "age at onset".

Procedure: Balance beam test

1. Mice are place in the center of the beam.
2. Time taken for mouse to cross to a safe platform is measured.
3. Three-minute maximum time limit.
4. Repeated in triplicate and scores averaged.

Procedure: Inclined screen test

1. Mice are placed face down on the wire mesh grid at 45° angle.
2. Time taken for mouse to right itself (negative geotaxis) is recorded.
3. Three-minute maximum time limit.
4. Repeated in triplicate and scores averaged.

Procedure: Grip strength test

1. Grip strength is measured with a standard grip strength meter.
2. Repeated in triplicate and scores averaged.

Procedure: Elevated plus maze

1. Mice are placed in the center of the maze and allowed to explore for 6 min.
2. Video tracking software is used to track the mouse and calculate the time spent in open versus closed arms of the maze as well as the number of arm entries into either open or closed arms, the total number of arm entries, and the total distance traveled.

Procedure: Contextual fear conditioning

1. Mice are habituated for 3 days to transport and to the testing room.
2. Mice are trained on a standard contextual fear conditioning paradigm.
   • Training consists of a 180 s baseline period followed by 4 mild foot shocks (1s, 0.9 mA), separated by 115 +/- 20 s.
   • A 40 s interval following each foot shock is defined as the post-shock interval.
   • The percentage of time spent freezing during the final post-shock interval is used as an index of contextual fear acquisition.
3. Twenty-four hours later, contextual fear memory is tested by returning the mouse to the testing chamber for 10 min.
   • The percentage of time spent freezing during the testing trial was measured using FreezeFrame software and used as an index of contextual fear memory.

Procedure: Blood glucose

1. Mice are bled through the tail vein, collecting a small drop of blood on a testing strip.
2. Strips are read using a glucose meter.
3. Note: tests are run between 12-2 pm to approximate fasting blood glucose as mice typically eat less during the light cycle.

Procedure: Beta-amyloid levels

1. Mice are euthanized and brains harvested and hemisected.
2. Beta-amyloid 1-42 (A-beta42) levels are quantified from sections of the temporal cortex.
3. Tissue is homogenized in 1X PBS + 1% Triton X-100 using the TissueLyser II system and sonicated 2x10x on low power.
4. Protein concentration is determined using a spectrophotometer.
5. Brain homogenates (10 mg/mL) are extracted in a final concentration of 5M GuHCl overnight at 4°C.
6. Samples are then diluted appropriately and run in duplicate on a A-beta42-specific sandwich colorimetric ELISA according to the manufacturer's protocol.
7. Optical densities at 450 nm are read on a plate reader and A-beta42 concentration is determined by comparison with A-beta42 standard curves.
8. Only readings in the linear range of the standard curve are included in analysis.
9. Duplicates are averaged to determine the concentration of A-beta42 in each sample.
10. A-beta42 concentrations are normalized to total protein concentration (ng/mg).

Data collected by investigator

• Body weight (2-16 mo)
• Y-maze (2-16 mo)
  • Percentage spontaneous alternation
  • Age at onset of working memory deficits (age at which spontaneous alternation percentage dropped below 50%)
  • Distance traveled
  • Number of arm entries
  • Percentage of alternating arm returns
• Balance beam: mean time to cross beam across 3 trials (6, 14 mo)
• Inclined screen: mean time to right body across 3 trials (6, 14 mo)
• Grip strength across 3 trials (6, 14 mo)
• Sensorimotor composite score (beam, screen, grip), derived by Z-scores (6, 14 mo)
• Elevated plus maze (6, 14 mo)
  • Distance traveled
  • Number of arm entries
  • Percentage of entries into open arms
  • Percentage of time spent in open arms
• Contextual fear conditioning (6, 14 mo)
  • Percentage of time spent freezing during training period on day 1 for baseline and after each shock (4 shocks)
  • Change in percentage of time spent freezing from 1st post shock to 4th post shock (slope)
  • Percentage of time spent freezing during memory period on day 2 for 10 min (data collected in 2-min bins)
• Blood glucose levels (6, 14 mo)
• Temporal cortex amyloid levels (6, 14 mo)