Knowledge Base

The RotaRod Test

Introduction

The rotarod test is widely used to evaluate drug effects on motor coordination in rodents. The principle of this test is that rats or mice are first trained to walk on a rod rotating at a certain speed. Once the animals have learned this, the effect of a test-compound on their motor performance is evaluated. Animals experiencing impaired motor coordination are unable to cope with the rotating rod and will drop off when the rotation speed exceeds their motor coordination capacity. The more disturbed the animals are, the sooner they fall off the rod.

The use of rotating rods to measure balance in rodents has been described since at least the 1950s (Dunham and Miya, 1957; Kinnard and Carr, 1957). Jones and Roberts (1968) introduced a significant modification of the basic design with an accelerating rod.

The rotarod is a horizontal cylinder that rotates along its long axis. It is situated above the cage floor, high enough to make sure rodents do not jump off the rod and low enough to avoid them being injured when they fall off.

The speed of the rotarod is mechanically driven and can be constant or accelerated during the test.

The test consists of a training phase in which the rats or mice learn to walk on the rod at a certain speed. Then three test trials are carried out in which a drug is tested. A commonly measured parameter is the time until falling off.

With EthoVision XT the Rotarod test can be automated. A camera is placed above the Rotarod, which gives a top-view image of the setup. The rat or mouse that stays on the rod is present in the center of the camera image. When the animal falls off, or clings onto the rod and turns round on it, it moves out of the center of the image. This can be automatically quantified with various parameters in EthoVision XT.

References

• Dunham, N.W. and Miya, T.S. (1957). A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Am. Pharmac. Assoc. Sci. Ed., 46, 208-209.
• Jones, B.J. and Roberts, D.J. (1968). The quantitative measurement of motor inco-ordination in naive mice using an accelerating rotarod. J. Pharm. Pharmacol., 20, 302-304.
• Kinnard, W.J. and Carr, C.J. (1957). A preliminary procedure for the evaluation of central nervous system depressants. J. Pharmacol. Exp. Ther., 121, 354-361.
• Carter, R.J., Morton, A.J., and Dunnett, S.B. (2001). Motor Coordination and Balance in Rodents. In Current Protocols in Neuroscience (2001) 8.12.1-8.12.14.
• Mahieu, M., Willems, R., Hoekstra, L., Ver Donck, L. (2012). Automated Detection of Aberrant Behaviour of Mice on the Rotarod: Use of EthoVision XT. Proceedings of Measuring Behavior 2012 (Utrecht, The Netherlands, August 28-31, 2012) 431-433.

Physical Setup

In most use cases, a video camera is positioned above the rod to record video. For systems with multiple rods, it is best to point one camera towards each rod. You can mix camera images directly with EthoVision XT or a video mixer.

To detect the falling off the rod automatically, place the camera in front of the rotarod. You can have EthoVision XT detect the fall off event when the animal disappears from the arena, or a zone drawn around the rod.

The Rotarod Test in EthoVision XT

Create an experiment. For details, see Chapter 1 of this manual, or in EthoVision XT press F1 and see Setup an Experiment in the EthoVision XT Help.

Experiment Settings

Select the number of cylinders as Number of Arenas.

Arena Settings

1. Choose Setup > Arena Settings > Open Arena Settings.
2. After calibrating, click Select Shape and Draw Arena. The arena covers the region around the rod. Make sure that the animal is always within the arena boundary.

Zones

1. Click Select Shape and Draw Zones.

To quantify the position of the subject's center point relative to the center of the rod, define a zone (Reference zone) at known distance from the rotation axis of the rod. For example, draw a line (Reference line) at the right margin of the arena, and define a zone.

You can then analyze the distance of the subject's center point from the border of this zone. If you know that the border of the zone is for instance at 10 cm from the rod axis (Reference distance), then you can back-calculate the relative distance of the subject-center from the axis. The values of Distance to zone (border) diminished by the Reference distance will be either positive (when the subject moves forward on the rod) or negative (when it moves backward).

To measure the reference distance exactly, place a ruler on top of the rod, and refresh the background image. Then draw the Reference line at a specific distance from the rod.

Note: Do not define the Reference zone in the middle of the rod. If you do that, distance from the zone border will always be positive, no matter what the position of the subject is.

Zones for Turnaround Behavior Detection

To automate turnaround behavior (mice grip themselves to the rod and turn around without falling off), you can define zones in front and at the back of the rod.

Divide the arena in three main zones: Middle (the rod area), the front and the back areas just outside of the rod.

It is then possible to detect turnaround behavior with the following sequence in the Zone transition variable: Middle (mouse in normal position) > Back > Front (the mouse reappears in view) > Middle.

Detection Settings

1. Click Advanced.
2. Under Subject Contour, select one or more pixels for the first Erosion and Dilation filter.
3. Increase the first Erosion until the subject's tail is not detected anymore.
4. Then increase Dilation until the entire body is detected.

Acquiring Data

Training Phase

This phase lasts typically three days. Each subject is placed on the rotarod for a maximum of 1 minute. The trial is repeated a few times each day, with 5 to 10 minutes inter-trial interval.

Testing Phase

Each subject is placed on the rotarod for a maximum of 1 minute. The trial is repeated two times with 5 to 10 minutes inter-trial interval.

Scoring Behaviors Manually

If you want to score when the animal falls off the rod, define the behavior Falling off under Manual Scoring Settings. After you have started the trial, watch the subjects in the video and in the Manual Scoring tab at the bottom of the screen, click the button for the arena (subject) that applies.

Data Analysis

Choose Analysis > Analysis Profile > New.

Mouse Position

In the Analysis profile, choose Distance to zone and select the Reference zone.

Turnaround Behavior

In the Analysis profile, click the button next to Zone transition. Select the following sequence: Middle > Back > Front > Middle. The zones must be defined in the Arena Settings.

Latency to Fall

If the camera is placed in front of the rotarod apparatus, you can measure the latency to fall by using the In zone variable. This calculates the time that the animal was detected in the arena (that is, until it fell off the rod).

The Y-maze Test

Introduction

The Y-maze is constructed with three arms of equal length that extend from a central platform at a 120 degree 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 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 in 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.

---

Source: EthoVision XT 19 - Application Manual