Knowledge Base

Introduction

the rotarod test

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. Figure 12.1 Rotarod for mice. 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.

EthoVision XT and the Rotarod test

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. Figure 12.2 Camera view.

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

Choose Setup > Arena Settings > Open Arena Settings 1.

After calibrating, click 2. 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

Click 3. 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), than 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 (see Data analysis on page 249).

detection settings

Click Advanced. Under Subject Contour, select one or more pixels for

the first Erosion and Dilation filter. Increase the first Erosion until the

subject's tail is not detected anymore. 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. See also acquiring tracks on page 21.

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 (see page 245).

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 (page 246).

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 calculated the time that the animal was detected in the arena (that is, until it fell off the rod). See also calculate the statistics on page 26.

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Source: EthoVision XT 17.5 Application Manual, The RotaRod Test