Rotarod test protocol for mice

The mouse walks on a horizontal rod that turns about its long axis, and it must keep walking forward to stay upright.¹ The rotarod is widely used to measure motor coordination, and it is especially sensitive to problems in the cerebellum.³ In models of neurological disease, Brooks and Dunnett note, motor tests give a good read-out of neurological function.⁴

Rustay and colleagues found that rotarod performance changes with the protocol and the test conditions, so no single set of settings captures every difference between mice.⁵ They advise pairing the rotarod with other motor tests.⁵

The typical accelerating test, the kind the steps below follow, is designed to measure peak performance and is not tuned to detect motor learning.³ Shiotsuki and colleagues found a learning deficit in Parkin-deficient mice that the typical test had missed. To get a steep learning curve, with fast gains from trial to trial, they turned a large drum slowly.³

At a set speed, some poorly coordinated mice fall off at the start. For the mice that stay on, the test soon measures endurance.¹ The scores then fall into two clusters, a bimodal spread that may rule out parametric tests.¹

On an accelerating rod the scores are more likely to follow a normal distribution, especially when the rod speeds up only after the mouse has stayed on for 10 seconds.¹ Set the acceleration so fatigue plays no real part. Deacon notes that a mouse is unlikely to tire much in 2 to 3 minutes on the rod.¹ At 20 rpm a minute from a start of 4 rpm, the rod reaches 40 rpm in under 2 minutes.¹

Some commercial rods speed up too slowly to pick out poor coordination from poor endurance.¹ Latency to fall can differ between labs that use the same brand of rod. Bohlen and colleagues point out that a rod may not accelerate at the rate set on it.² Their paper gives a simple way to measure the rate and set it.² Measure your own rod’s acceleration before the study.

The depth of the ridges matters. Too little grip makes the test much harder, and too much lets the mouse hold on and be carried around the rod, called passive rotation.¹ Move the flanges closer for young mice that turn to face the other way on the rod.¹

Keep the rod high enough, because mice drop or jump off a rod set too low.¹ Deacon reports no injury from a fall in hundreds of mice tested on apparatus 30 to 50 cm high, with a soft pad at the base.¹

These steps follow Deacon’s accelerating protocol.¹

1. Set the rod

   Set a start speed of 4 rpm and an acceleration of 20 rpm a minute.¹

2. Place the mouse facing away from the turn

   Hold the mouse by the tail and set it on the turning rod facing away from the direction of rotation, so it has to walk forward to stay upright.¹

3. Start the acceleration after 10 seconds

   Let the rod turn at 4 rpm for 10 seconds, then start the acceleration. If the mouse is not facing forward at 10 seconds, wait until it is.¹

4. Record the fall

   Note the speed at which the mouse falls off.¹ If your rod reports the time on the rod instead, record that, and say which you used.

5. Repeat early falls

   If the mouse falls in the first 10 seconds, note the time and place it again, for up to three tries in all.¹ Do not record a fall in the first 5 seconds caused by poor placing.¹

6. Clean the rod

   Clean and sterilize the equipment between mice, as Deacon advises.¹

Deacon scores the mean speed at fall across the tries. Averaging offsets the extra practice a mouse gets from its early falls.¹ An early fall counts as the 4 rpm start speed. A mouse that falls early once, then stays on until the rod reaches 12 rpm, scores 8 rpm, the mean of 4 and 12.¹ A mouse that falls in the first 10 seconds on all three tries scores 4 rpm.¹

Mice get better with practice, so decide whether practice is part of what you measure.^1,3 To measure peak performance, give every mouse the same practice before the scored trials. For motor learning, score every trial from the first, on a slow rod that gives a steep learning curve.³

Cage enrichment can give practice too. Cardboard tunnels are cylinders that can wobble about their long axis, so mice that balance on them practice the skill the rotarod tests.¹ Keep enrichment the same across groups, and consider enrichment other than cardboard tunnels.

Strain and sex shift the scores. Deacon reports that 129S2/Sv mice did worse than C57BL/6 mice on the rotarod, and notes that male mice seem less well coordinated than females.¹ Balance strain, sex and age across your groups.

• Some mice stop walking and grip the rod, so it carries them around in passive rotation. Most fall off in the end, and Deacon suggests also noting the speed at the first such rotation.¹ A wider rod or shallower ridges may help.¹
• Mice jump off early. Check the rod height, because mice drop or jump from a rod set too low.¹
• Young mice turn to face the other way. Move the flanges closer together.¹
• Scores fall into two clusters. Switch from a fixed speed to an accelerating rod.¹
• Your scores differ from another lab’s. Measure your rod’s real acceleration, and report the rod diameter, surface and protocol.²