Materials and Reagents

1. Aged C57Bl/6 male mice (24-months-old) which model 56-69 year-old humans (Flurkey et al., 2007)
   Notes:
   
   1. Differences in the behavioral C57Bl/6 mice have been observed compared to other stains (Crabbe et al., 1999). Furthermore, sex differences in behavioral tests have also been widely described in the literature (Kopp et al., 2006).
   2. For testing on the spontaneous alternation y-maze, animals can be maintained on a diet of standard mouse chow and water ad-libitum. The mice were maintained on a regular light cycle.
2. Surface disinfectant, a surface disinfectant to remove odors on the y-maze between animals (e.g., 70% ethanol)
   Note: The literature should be consulted when selecting a disinfectant to determine which solution is ideal for mouse strain being tested (Campagna et al., 2016) and equipment being used.
   

Equipment

1. Y-maze (Custom made or commercially purchased) (Stoelting, catalog number: 60180), the y-maze can be made of gray, white, or black plexiglass with the dimensions 39.5 x 8.5 x 13 cm for mice (Figure 1A)
   Note: The plastic fiber makes it easy to disinfect between animals to remove any odors. The three arms of the maze are interconnected at an angle of 120°. Animals should be habituated to the room for at least 30 min prior to testing, without the y-maze being visible.
2. Video camera
   Notes:
   
   1. Animal behavior can be tracked using a video camera recording, real-time analysis, or automated tracking equipment such as AnyMaze (Stoelting, catalog number: 60000). For video camera recordings, no special frame rate is required. 
   2. We video recorded all animals which facilitated scoring by two individuals blinded to experimental groups. The set-up should be above the y-maze and not interfere with the testing as shown in Figure 1B.
3. Visual cues
   Note: Paper visual cues can be custom made and hung on the walls of testing room. These cues should be uniform in color and examples are shown in Figure 1B.
   
   
   Figure 1. Pictorial representations of spontaneous alternation y-maze apparatus (A) and testing set-up (B)
   

Software

1. Animal tracking program, AnyMaze (Stoelting, catalog number: 60000)
2. Statistical testing, GraphPad Prism (GraphPad Software)
   

Procedure