Assessment of Motor Coordination and Balance in Mice Using the Rotarod, Elevated Bridge, and Footprint Tests

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

In order fully to utilize animal models of disease states, to test experimental therapeutics, and to understand the underlying pathophysiology of neurodegenerative disease, behavioral characterization of the model is essential. Deterioration of normal motor function within a disease state signals the progression of an underlying pathological process, and identifies disease?sensitive time points according to which the onset of therapeutic trials may be scheduled. Deterioration in the performance of motor tasks may also indicate the point when motor deficits begin to compromise our ability to measure other deficits within cognitive and behavioral domains. In acute therapeutic trials, the separation of motor from cognitive or behavioral function may be crucial in determining the functional specificity of the drug effect. If we are to accurately measure motor performance in disease progression or during drug trials, tests of motor function that have been highly optimized with respect to sensitivity must be applied. Since motor coordination and balance are essential to normal motor function, tests that probe these facets are ideal for the purpose. In this chapter, we describe in detail three test protocols that principally measure motor coordination (the rotarod and footprint tests) and balance (the elevated bridge test) in mice. Curr. Protoc. Mouse Biol. 2:37?53 © 2012 by John Wiley & Sons, Inc.

Keywords: rotarod; elevated bridge; footprint; gait; balance; coordination; motor tests; mice

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Introduction
Basic Protocol 1: Measuring Motor Coordination and Balance on the Rotarod
Basic Protocol 2: Measuring Motor Coordination and Balance on the Elevated Bridge
Basic Protocol 3: Gait Analysis Using the Footprint Test
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Measuring Motor Coordination and Balance on the Rotarod

Materials

The appropriate mouse strain and housing facility
70% ethanol spray and absorbent paper towel for the cleaning of the apparatus

A rotarod apparatus, for example the Ugo Basile (http://www.ugobasile.com) rotarod (Fig. ), which typically comprises:
- A horizontal rod of 3 cm diameter and 30 cm length
- A drive motor and pulley system that allows the rod to be turned at multiple speed settings; the simplest versions permit a series of fixed preset speeds, whereas newer apparatus typically includes an accelerating option where the angular velocity increases between preset start and terminal speeds over a fixed time
- Six partitions of the beam to provide segregated areas for 5 individual mice
- A trip switch below each segment for measuring the latency to fall from the beam

NOTE: Rotarod speeds should be designed with the severity of disease in mind. There should be at least one slow speed where the majority of mice are able to remain on the beam. Subsequent speeds should ideally produce a data curve that is representative of the disease or treatment severity.NOTE: In‐house construction of a rotarod apparatus is possible with the beam length, diameter, and mouse capacity designed as required. The commercial rotarods may be fitted with a basic gearing system, which is not required if the investigator wishes to run the beam at a single speed. Gearing can be achieved through the use of different gear wheel sizes (Fig. ). Similarly, an electronic trip switch that is fitted to the commercial apparatus is not necessary; as the manual timing of a mouse falling from the beam will suffice, in this instance a stopwatch would be required.NOTE: The rotating rods in commercial apparatus are typically made of Perspex or other plastic and inscribed with horizontal square cut grooves to aid grip. Unlike rats, mice can effective grip on with their claws, hang on, and rotate with the rotating rod. A simple solution to this problem is to re‐cover the rod with a rough rubber surface, such as provided by gluing on a segment of bicycle inner tube. See Commentary for more details.

Basic Protocol 2: Measuring Motor Coordination and Balance on the Elevated Bridge

Materials

The appropriate mouse strain and housing facility
70% ethanol spray and absorbent paper towel for the cleaning of the apparatus

An elevated bridge apparatus (see Fig. ); our current bridge has the following features:
- An elevated beam of 1 m (but could be shorter) that is inclined at a 17° angle such that the highest point of the beam is 58 cm above the floor; start and stop areas marked on the beam that are 15 cm from the lower end of the beam and 10 cm from the goal box, respectively
- The beam is tapered in width from 1.2 cm at the start point to 0.5 cm at the stop point with a ledge running the length of either side of the beam that is 2 cm below the level of the beam and 1.5 cm wide
- A dark goal box ( 10.5 × 10.5 × 10.5 cm) positioned at one end of the beam
Towels or other soft material to place beneath the beam.
Stopwatch.
Video camera, tripod and video tapes.
Mouse identification numbers for use with video camera
Video viewing facility for scoring the tapes

NOTE: Typically, elevated bridge apparatuses are made in‐house and consequently come in a wide variety of shapes and sizes. The one described here is our latest model, based on the original design of Schallert (Schallert et al., ) that has been used extensively on mouse models of neurodegenerative disease. It features a single fixed beam of graduated width rather than the commonly used changeable beam system. Some apparatus designs have a goal box used as a sanctuary that the mice can attain if they run the full length of the beam. Some beam designs have a beam set at an incline. The incline on our current beam is set at 17° from horizontal (plane, or parallel with respect to the ground) with the goal box placed at the highest end, where the beam reaches its narrowest point. The incline can increase the sensitivity of the task, but also seems to encourage the mice to run the beam.NOTE: Two areas of the beam have been designated the “start” and “stop” areas. Essentially, these are simply demarcation lines positioned at either end of the beam designed to allow the operator to start and stop the timing of the animal when running the beam. The start area is the area where the mouse is placed at the onset of the trial and where it will orient itself prior to running the beam. The stop area is the area of the beam where the mouse slows down prior to entering the goal box. The use of these areas permits a more accurate assessment of the traverse speed, as they exclude preparation and goal‐box entering times.NOTE: The use of a ledge on the bridge (Schallert et al., ) permits the accurate assessment of the number of foot slips that the mouse makes as it traverses the beam while allowing the mouse to complete the task. Hind‐limb foot slips are the most sensitive measure of disability in the mouse lines that we have tested (see Fig. ).

Basic Protocol 3: Gait Analysis Using the Footprint Test

Materials

The appropriate mouse strain and housing facility
70% ethanol spray and absorbent paper towels for the cleaning of the apparatus

A corridor runway, 60 cm in length and 5 to 10 cm wide, with side walls high enough to prevent the mice from escaping (Fig. A; see note below)
A dark goal box to place at one end of the corridor
Two colors of non‐toxic, water‐based paint (children's poster paint is ideal)
Small dishes to decant the paint into
Paint brush
Strips of paper cut to fit the corridor floor. The paper should be stiff and slightly absorbent to provide good paint marking without smearing
A 30‐cm ruler and pencil
Stopwatch

NOTE: Corridors are usually constructed in house. In our laboratory, we use a 60‐cm‐long corridor for mice (Brooks et al., ). This allows multiple gait cycles to be assessed, reducing variability and increasing the reliability in the scoring. However, the length of the corridor can be varied depending on the requirements/preferences of the experimenters. The width of the corridor can also vary. A narrow corridor of 5 cm is preferable, as it prevents the mouse from meandering as it runs towards the goal box; this improves the quality of the data. Mice also frequently change direction, which will prevent consistent gait cycles from being collected, and a narrow corridor reduces the likelihood of this, while encouraging the mice to head straight to the opposite end of the corridor. If a dark goal box or tunnel is put at the far end of the corridor, it will provide further incentive for the mice.NOTE: A variety of digital apparatuses are available that use treadmill technology and which produce precise, high‐definition images of the mice while running. This approach is highly sensitive, with the main drawback being the expense (see Commentary).

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Figures

Figure 1. A commercial rotarod apparatus (A ) with digital interface (B ). Note that the newer models have electronic gearing with an older gearing system depicted in (C ).

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Figure 2. Progressive decline in rotarod performance in the HdhQ150 Huntington's disease mouse model, over 4 to 16 months of age.

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Figure 3. The elevated bridge apparatus set up for the photograph without the video recording system (A ). The goal box and “start” and/or “stop” marks are clearly visible in white on each picture. Note the ledge on the beam (B ), which makes foot slips (C ) easier to record.

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Figure 4. A typical data set from the R6/1 Huntington's disease mouse model on the elevated bridge apparatus. Measures of turn (A ) and crossing (B ) latencies, along with fore‐limb (C ) and hind‐limb (D ) foot slips all indicate progressive impairment in the transgenic mice.

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Figure 5. (A ) Narrow corridor apparatus used for gait analysis. (B ) Mouse leaving painted footprints, using blue paint on the two forepaws and red paint on the two hind paws. (C ) Illustration of the measurements taken to collect stride length, fore and hindpaw base, and right and left overlap.

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Figure 6. Sample data from Q150 Huntington's disease mouse model on stride and gait analysis test. (A ) Stride length of transgenic and wild‐type mice for hind‐paw and fore‐paw strides. (B ) Base width hind‐paw and forepaw steps of transgenic and wild‐type mice.

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Literature Cited

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