Materials and Reagents

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.
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

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.
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.
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

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

Procedure

Note: Institutional animal ethics protocol approval is required for carrying the experiment described in this protocol.

Habituate mice to handling
A minimum of one to two weeks prior to testing on the y-maze, experimenters should remove animals from cages and handle them (Crawley, 2007). Each week, the animals should be handled daily for 5 out of the 7 days. Each mouse should be handled for at least 5 min.
On the day of y-maze, habituate testing animals to the testing room 30 min prior to beginning behavioral testing (Rutkowsky et al., 2018). Animals should not be able to see the y-maze prior to testing.
Conduct behavioral testing at the same time each day. We tested animals in the afternoon during their light cycle. It is important to remain consistent in testing time parameters.
If possible, the lights in the testing room can be dimmed to increase exploration of the y-maze during testing. There should be indirect and homogenous illumination (150-200 Lux) in the center of the y-maze.
Place each mouse, naive to the maze, at the same end of one arm and allow each mouse to move freely through the maze during an 8-min session (Sarter et al., 1988; Maurice et al., 1994; Holcomb et al., 1998; Li et al., 2010; Lee et al., 2017; de Sousa et al., 2018). Video record the whole process. Record the number of arm entries by two individuals blinded to the experimental groups. If animals are being tested over multiple days, then the orientation of the y-maze should be the same each day.
Notes:
1. If there are loud noises (e.g., construction) during testing, a white noise machine can be used. Animals should be habituated to the white noise prior to testing. In our study, we did not use any white noise during testing.
2. Modifications can be made to the above testing protocol, these modifications are summarized in Table 1–Changes to testing time and paradigm (Mihalick et al., 2003; Võikar et al., 2004; Tchantchou et al., 2005; Barakat et al., 2018) as well as the number of trials per animal (Mihalick et al., 2003). Other modifications include object recognition (Romberg et al., 2013; Ibi et al., 2018) and the two-trial y-maze, which assess short-term memory (Dellu et al., 2000). and the two-trial y-maze, which assess short-term memory (Dellu et al., 2000). In the first trial, one arm was blocked, allowing the mouse to explore only two arms of the maze for 5 min. In the second trial, after a 60 min inter-trial interval, the block to the third arm was removed and the mouse was able to explore all three arms for a period of 5 min. The amount of time spent in the novel arm was calculated within the first minute.
After each trial, the y-maze should be cleaned, disinfected, and dried prior to placing the next mouse. Alternatively, saw dust or bedding can be added to the y-maze and mixed between animals (Ma et al., 2007).

Table 1. Summary of changes that can be made to spontaneous alternation y-maze testing protocol and apparatus

Data analysis

The three main outputs from the spontaneous alteration y-maze analysis are the number of alternations and entries, as well as percent alternations. The number of alternations can be calculated based on the sequence of arm entries. An alternation is defined as successive entries into 3 arms, on overlapping triplet sets (Figure 2). For example, entries into arms 1, 3, and 2 is considered an alternation. Whereas entries into arms 1, 2, and 1 would not be considered an alternation. Alternations can be used to measure short-term spatial memory in mice (Sarter et al., 1988). An arm entry is completed when the hind paws of the mouse had been completely placed in the arm (Ohno et al., 2004). The number of entries per arm is a measurement of activity and locomotion during the testing session and is also be used to calculate the percent alternations. The percentage of alternation can be calculated as the ratio of actual to alternations and it can be calculated by using the formula below:

% alternations = (total number of alternations)/(number of arms entered) × 100%

Figure 2. Representative image depicting an alternation. Alternations are consecutive entries into each arm of the y-maze without any repeats. For each alternation, there are three steps. Steps of alternation outlined in (A) Step one entry into Arm 1; (B) Step two entry into Arm 3; and in (C) Step three entry into Arm 2.

Lack of motivation in older mice may be a problem. In that case water (Lyon et al., 2011) or food (Spence and Lippitt, 1946) motivated y-maze tests can be used. In terms of excluding mice, if a mouse continues to jump out of the maze or not move during testing, their behavior may make them an outlier. Experimenters should carefully review data before making the decision to remove mice. Therefore, we recommend that all trials be video recorded. We advise experimenter not to make premature conclusion concerning memory deficits until enough parametric research has been conducted and alternative explanations have been assessed, please refer to Table 2 for suggestions.

Table 2. Troubleshooting guide for spontaneous alternation y-maze task in rodents

Statistical analysis of the data including the number of alternations and entries, as well as percent alternations can be conducted using GraphPad Prism 6.0 or any other statistical program. Depending on the number of groups, unpaired t-tests or analysis of variance (ANOVAs) can be used.
In Figure 3, we have provided a sample analysis of aged male C57Bl/6 mice deficient in methylenetetrahydrofolate reductase (MTHFR). All animals performed on behavioral task, there were no non-performers. We conducted statistical analysis using GraphPad Prism 6.0. Un-paired t-tests were used to compare genotype groups. In all analysis, a P < 0.05 was considered significant. All data are presented as mean ± standard error of the mean (SEM). Mthfr^+/- mice made less alterations compared to wild-type controls [Figure 3A, t(17) = 2.6, P < 0.05]. It appeared that the Mthfr^+/-mice made fewer total arm entries, but this was not statistically different [Figure 3B, t(17) = 1.1, P > 0.05]. The reduced number of arm entries is a marker for reduced activity. When the number of arm entries was factored into the alternations, there was still a reduction in the percent alternations of Mthfr^+/- mice [Figure 3C, t(17) = 2.39, P ≤ 0.05].

Figure 3. The impact of methylenetetrahydrofolate reductase (MTHFR)-deficiency on the spontaneous alternation y-maze in aged Mthfr^+/+andMthfr^+/- male mice. The number of alternations (A), entries (B), and percent alternations (C). Mean ± SEM of 8-9 mice per group. * indicates P < 0.05, unpaired t-test.

Conclusion: Using this spontaneous alternation y-maze we were able to measure short-term spatial memory in aged animals. In mice, spatial recognition memory can also be assessed by the T-maze, radial arm maze and Barnes maze tasks, but all of these tasks require extensive training compared to the spontaneous y-maze (Crawley, 2007). Furthermore, dietary restriction is required for the T-maze and radial arm maze, this may be challenging for aged mice. The spontaneous y-maze measures spatial recognition memory at a rudimentary level using the natural exploratory behavior of rodents. This type of memory is consistently evaluated in aging studies and is therefore a valuable test. The y-maze does not require significant output resources or training of animals, so assessing aged animals is not difficult. With the increase in the aging population (United Nations Department of Economic and Social Affairs, 2013) and prevalence of age-related diseases (Bach et al., 2011) there is an urgent need to understand disease processes associated to aging and therapeutic development. The spontaneous alternation y-maze provides researchers with data on memory as well as exploratory activity of animals in a short time frame. Furthermore, the standard y-maze is flexible and can be adapted to measure spatial and object recognition, as well as novelty exploration using the 2-trial y-maze.

Acknowledgments

This research was supported by Fonds de la recherché en santé Québec, Council of Ontario Universities Postdoctoral Women’s Health Scholars Fellowship and Natural Sciences and Engineering Research Council of Canada fellowship (N.M.J.).

Competing interests

No conflicts.

Ethics

Mice were bred and maintained in the Department of Neuroscience, Faculty of Science, Carleton University. All experiments performed in this study were in accordance with the Canadian Council on Animal Care (CCAC). Animals were maintained on a diet of standard mouse chow and water ad-libitum.

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