Knowledge Base

Introduction

The Y-maze

The Y-maze is constructed with three arms of equal length that extend from a central platform at a 120° angle. In the basic version, the animal is free to explore the maze arms. In a modified version, it is possible to close off some arms either to keep the animal in a specific arm for a fixed time once it has entered that arm, or to limit exploration to some arms, not others.

The Y-maze is generally used to investigate alternation behavior, where animals are supposed to alternate choices of maze arms on successive opportunities. Irrespective of the precise function of alternation, the animal must remember which arm it had entered on a previous occasion to enable it to alternate its choice on a following trial. Therefore, the use of the Y-maze is based on the assumption that, following some pharmacological manipulation, it is primarily modified memory processes that are reflected in changed alternation rates.

Spontaneous Alternation

Definition

Spontaneous Alternation is a behavioral test for measuring the willingness of rodents to explore new environments. Rodents typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited. Many parts of the brain, including the hippocampus, septum, basal forebrain, and prefrontal cortex, are involved in this task (Conrad et al. 1996).

Classical Procedure

The animal is released in the center of the maze, and allowed to freely explore the three arms (A, B, C). Over the course of multiple arm entries, the subject is expected to show a tendency to enter a less recently visited arm (A > B > C > A > ...). The observer records the number of arm entries and the number of visits to the three arms without a re-visit in between (for example, A > B > C, B > C > A, etc.) in order to calculate the percentage of alternation. An arm entry is scored when all four limbs are within the arm.

Calculation of Alternation Index

Given a zone sequence:

A > B > C > B > ...

An alternation is defined as multiple entries into the three different arms on overlapping triplet sets. In this example the first three visits are an alternation:

A > B > C > B > ...

The next overlapping triplet set, B > C > B, is not an alternation.

The Alternation index is given by the number of alternations divided by the maximum number of possible alternations, that is, the number of alternations that would be obtained if the animal visited the zones in the sequence: A > B > C > A > B > C ...

For a 3-arm maze like the Y-maze, the maximum number of alternations is equal to the total number of visits minus 2.

Alternation index is expressed in percentage:

Alternation index = (Number of alternations / (Total number of zone visits - 2)) x 100

Spontaneous Alternation in EthoVision XT

You can easily calculate the Alternation index using the outputs Alternations and Max Alternations in the Zone alternation dependent variable. For more information, see Zone alternation in the EthoVision XT Help.

Delayed Alternation

The delayed alternation task allows assessing spatial working memory in a T- or Y-maze. The main difference with spontaneous alternation is that performance is evaluated between trials.

Classical Procedure

In the first trial of the test, the animal is placed at the end of the start arm and has to choose between the two other arms that are baited. Once the choice is made, the subject is removed and after a variable delay, is returned to the start arm. In this second trial, the one arm baited is now the opposite arm to which was chosen during the first trial. The animal has to make a different choice than its first one (correct choice) to get the reward.

Delayed Alternation in EthoVision XT

For all tests where maze arms can be considered as targets of a choice, and others non-targets (errors), you can use outputs Target first visits and Total errors in the Target visits and errors dependent variable. For more information, see Target visits and errors in the EthoVision XT Help.

References

• Conrad, C.D., Galea, L.A., Kuroda, Y., McEwen, B.S. (1996). Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment. Behav Neurosci 110: 1321-1334.
• Conrad, C.D., Lupien, S.J., Thanasoulis, L.C., McEwen, B.S. (1997). The effects of type I and type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze. Brain Res. 759, 76-83.
• Hughes, R.N. (2004) The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory. Neurosc. Biobehav. Rev. 28, 497-505.
• Detrait, E., Brohez, C., Hanon, E., De Ryck, M. (2010). Automation of Continuous Spontaneous Alternation to Increase the Throughput for In Vivo Screening of Cognitive Enhancers. Optimization of the Ethovision System for the Y-maze Test in Mice. Proceedings of Measuring Behavior 2010 (Eindhoven, The Netherlands, August 24-27, 2010), 141-144.

EthoVision XT Settings

Create an experiment. For details, see the Setup an Experiment section in the EthoVision XT Help (press F1 in EthoVision XT).

Experiment Settings

Choose Setup > Experiment Settings and configure the following:

• Number of Subjects per Arena: 1.
• Tracked Features: Center-point detection, or center point, nose-point, and tail-base detection.
• If you want to use the Deep Learning technique to track the subject's nose, under Body point detection technique choose Deep learning. Note that in order to use this technique there are additional requirements, including a powerful graphics card. See Deep learning: Requirements and Limitations in the EthoVision XT Help.

Arena Settings

For the Spontaneous Alternation Task

1. Choose Setup > Arena Settings > Open Arena Settings 1.
2. Choose the video or camera image and click Grab.
3. Click 2. Select Shape and Draw Arena. Resize and move the outline of the arena to make it overlap with the Y-maze image.

For details on how to resize and move the arena outline, see Arena Settings in the EthoVision XT Help.

For the Delayed Alternation Task

1. Choose Setup > Arena Settings > New.
2. After calibrating and drawing the outline of the arena, click 3. Select shape and draw zones. Draw the three arm zones.
3. To draw the start box zone and the target zones (that is, the arm ends which will be baited with food), create an additional zone group.

For details on how to work with zone groups and draw and edit shapes, see Arena Settings in the EthoVision XT Help.

Trial Control Settings

In the Trial Control Settings you can define conditions for the start and stop of data acquisition.

Choose Setup > Trial Control Settings > New.

The templates for the Y-maze contain the following Trial Control Settings profiles:

• Default. Tracking starts when the subject's center point is detected for 1 second in the arena. Tracking stops at the end of the video or when you give the stop command.
• Tracking Duration 5 min. Tracking starts when the subject's center point moves from the start box to the center arm. Tracking stops after 5 minutes.

Detection Settings

Choose Setup > Detection Settings.

Check in the Video Section that the sample rate is set to:

• For rats: 5 samples/second.
• For mice: 12.5-15 samples/second.

When tracking the nose-point, center-point, and tail-base point of the rodents, always choose the maximum sample rate.

If you selected the Deep Learning technique to track the subject's nose, under Method next to Deep learning click Define and select a box around the subject. Make sure that the box includes the subject's nose. Try not to include objects like walls, especially if they are the same color as the subject. For details, see the EthoVision XT Help.

Trial List

Independent Variables

If you perform a Y-maze test where one of the arms is closed off or provided with food, it is always a good idea to create independent variables that mark the type of trial, which you can use to select a subset of trials for answering a specific question. For example, create the independent variable Correct choice with possible values Yes and No, depending on whether the subject chose the arm not visited in the previous trial. At the end of each trial, enter the value for that subject. You can use this variable to filter the trials based on the value of Correct choice.

Acquiring Data

Continuous Spontaneous Alternation

1. Release the subject in the center of the Y-maze.
2. Start the trial.
3. Let the subject explore the maze for the time required (typically 5-6 minutes). Then stop the trial.
4. Put the subject back in its home cage. Proceed with the next subject.

Forced-choice Alternation

In this variant, the subject is forced to explore one of the two arms in the first trial.

1. Close one of the arms of the maze. Place the subject into the start arm, and allow it to explore the maze for 15 minutes.
2. After a fixed inter-trial interval, open the door in the blocked arm. Return the subject to the maze by placing it in the start arm. The subject is allowed to explore freely all three arms of the maze for 5 minutes. The Y-maze is often rotated between the two trials.

Two-trial Spontaneous Alternation

In this task, the subject is confined for a fixed time in the T- or Y-maze arm chosen in the first trial. This allows manipulation of the time that the animal has experience with one of the arms.

1. In the first trial, let the subject access one of the arms after having left the start arm. Once an arm has been entered, close off that arm by lowering a guillotine door behind the subject. Leave the subject there for a fixed time (this is called the intra-trial interval).
2. Remove the subject and, after an inter-trial interval, let it choose the arm in a second trial. This two-trial sequence is repeated a number of times.

Delayed Alternation

This task is usually designed with a T- or Y-maze where one arm is the starting location and the other arms are baited.

1. Release the subject in the start arm. Once the subject enters one of the other two arms, close the door for that arm (trial 1).
2. After a predetermined inter-trial interval, release the subject and let it freely choose one of the two arms (trial 2).

If the subject chooses the alternate arm, this is scored as alternation. This two-trial procedure is repeated several times.

Data Analysis

Analysis Profiles

Choose Analysis > Analysis Profile.

The template experiment contains the following analysis profiles:

• Distance, Time and Movement. Calculates the total Distance moved (a measure of activity of the animal), the mean Velocity, and the total time spent in the two states of Movement (moving and not moving). Group means with their standard errors are also calculated.
• Zone alternation. Includes the variable In zone (to calculate the frequency, total time, and latency to first visit for each zone) and Zone alternation (to calculate the number of alternations and the maximum number of alternations for that set of zone visits).

Alternation Behavior

Spontaneous Alternation

1. Choose Analysis > Results > Statistics and Charts.
2. Select the Analysis profile that contains the Zone Alternation variable.
3. Locate the column Zone Alternation in the table.
4. To calculate the Alternation index, divide Alternations by Max Alternations and multiply by 100.

To change the Zone alternation definition, open the Analysis profile Zone Alternation and double-click the variable Zone alternation. Here you can specify which statistics you want. You can also enter a Zone exit threshold to remove false re-entries in a zone from the statistics, due to small movements of the body point near the zone borders.

For more information, see Zone alternation in the EthoVision XT Help.

Delayed Alternation

For delayed alternation tests, Zone alternation is not the correct dependent variable. To analyze correct and incorrect choices, use Target visits and errors to compare performance between trials. Because the target arm changes between trials (for trial 2, the target is the arm which was not visited in the previous trial), you must define multiple instances of Target visits and errors.

Whether an arm can be defined as "target" depends on the outcome of the previous trial. If the subject went to the Left arm in trial x, then for trial x+1 the target should be the Right arm, and a revisit to the Left arm should be scored as an error.

To configure this:

1. In the Trial List (Setup > Trial List), mark each trial with a variable which states which arm was the correct choice. By definition, the first trial of each subject has no "correct" choice, as the values for subsequent trials depend on what the animal does in that trial.
2. In the Data Profile, filter the trials based on this variable. Create two Data profiles: one for "Right arm correct" and one for "Left arm correct".
3. Create two corresponding Analysis profiles:
   • Analysis profile: Target visits and errors for when the target (correct choice) was the Left arm. The Right arm is a non-target; visits to that arm are scored as errors.
   • Analysis profile: Target visits and errors for when the target (correct choice) was the Right arm.

When analyzing the data, make sure to combine each Data profile with the correct Analysis profile.

Visualizing Data

Choose Analysis > Results > Integrated Visualization.

The first plot shows the variable In zone defined in the Analysis profile, where you can see which arms were visited during the trial.

The second plot shows Zone alternation. The subject visits first the zone marked in blue, then the one in red, and when the subject enters the green-marked zone, an alternation is completed. This is scored as Alternations (blue vertical segment). At every visit except the last two in a trial, Max Alternations is also scored (purple vertical segment), marking the moment a possible alternation could have occurred.

Source: EthoVision XT 17.5 Application Manual - The Y-maze Test