Behavioral Assessment of the Aging Mouse Vestibular System

Summary

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Abstract

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Introduction

Our sense of balance is perhaps one of the most overlooked yet vital components of even the most basic motor activities including walking and turning. Balance is influenced by numerous factors including muscle strength, motor coordination and vestibular function, and it is only in the presence of vestibular neuropathies or during normal aging that the importance of a fully functioning balance system is appreciated. Disturbances to the vestibular system are often associated with experiences of vertigo or dizziness and disequilibrium resulting in an increased risk of falls and subsequent injuries¹. This is particularly critical in older populations where falls are one of the leading causes of injury².

Vestibular function tests are commonly based on the vestibular reflexes, in particular, the vestibulo-ocular (VOR) or the vestibulo-collic reflex (VCR). The VOR and VCR are essential for the stabilization of images on the retina and head position during movements of the head and body respectively. Commonly, VOR measurements require invasive implantation of search coils to measure eye movements or video tracking of eye movement³. This is challenging in mice due to the small nature of the mouse eye and the difficulty in detecting the pupil for video analysis³. As an alternative, the VCR has been used to measure stabilization of the head in response to body movements in mice without the need for invasive surgery⁴. Despite this, few studies focus specifically on how the vestibular system performs as a whole and more importantly how it changes during aging.

To assess overall balance performance simply and noninvasively we modified two commonly used behavioral tests. The rotarod and inclined balance beam tests assess different aspects of motor performance in rodents and in previous studies have been used in a test battery to acquire a complete profile of motor capability. This capability can be affected by disease or genetic modification, and is also sensitive to processes associated with normal development and aging^5-7. Earlier work using the rotarod has shown that motor coordination in mice declines after 3 months of age⁸. In addition, rats show noticeable balance deficits with increasing age on the balance beam test⁹.

This paper describes the use of the rotarod and balance beam tests in conjunction with a vestibular stimulus in order to challenge the vestibular system and characterize the subsequent impact on balance performance in young and older mice. While the simple and noninvasive methods described are not designed as stand-alone measures of peripheral vestibular function, they do provide a useful and simple behavioral measure to compare cellular and subcellular changes at multiple stages of vestibular processing during normal aging in mice.

Protocol

1. Animals

1. Mice (C57/BL6) of ages 1, 9, and 13 months old were obtained from the Animal Resources Centre (Perth, Australia). These mice were housed in standard mouse cages in the Bosch Rodent Facility at the University of Sydney on a 12/12 hr light/dark cycle with access to food and water ad libitum. The procedures outlined below were approved by the University of Sydney Animal Ethics Committee.
2. Bring mouse cages into the testing room prior to each test for 10 min to allow mice to acclimatize to the testing environment.

2. Rotarod

1. Set up the rotarod apparatus (Figure 1A):
   1. Install the dowels in each lane of the rotarod.
      Note: In this instance rat dowels (70 mm in diameter) are used instead of mouse dowels (32 mm in diameter) to discourage the mice from clinging to the dowel and performing “passive rotations”¹⁰.
   2. Position the magnetic landing platforms on the wire situated at the bottom of each lane of the rotarod making sure that they are not tilting to touch the floor of the rotarod and are placed as close as possible to the magnetic right wall of each lane without touching.
      Note: During rotarod testing mice are required to walk in a forward direction to stay on the rotating and accelerating dowels. When a mouse is no longer able to stay on the dowel, they fall and displace the landing platform that subsequently activates a magnetic sensor. The time taken to fall from the rotating dowel, the dowel rpm at time of falling and the distance traveled is automatically calculated for each mouse and recorded on the display screen at the front of the rotarod.
   3. Slide 2 clear plastic panels into the front of each rotarod lane with the shorter panels at the bottom and the longer panels above.
   4. Input the test parameters using the keypad located at the front of the rotarod. Follow steps 2.1.4.1 to 2.1.4.6 for the accelerating rotarod test parameters and steps 2.1.4.7 to 2.1.4.12 for the fixed speed rotarod test parameters.
      1. Set the maximum duration of the test to 60 sec.
      2. Set the number of lanes to be used (or the number of mice to be tested).
      3. Set the starting speed of the test to 5 rpm.
      4. Set the top speed of the test to 44 rpm.
      5. Set the ramp speed of the test to 60 sec.
      6. Set the size of the dowels chosen and the direction of rotation to rat dowels rotating in a forward direction.
      7. Set the maximum duration of the test to 240 sec.
      8. Set the number of lanes to be used to 1 as the mice are tested individually.
      9. Set the starting speed of the test to 15 rpm.
      10. Set the top speed of the test to 15 rpm.
      11. Set the ramp speed of the test to 0 sec.
      12. Set the size of the dowel chosen and the direction of rotation to a rat dowel rotating in a forward direction.
          Note: The settings above may be altered to suit the needs of different experiments.
   5. Place a camera in front of the rotarod for the fixed speed rotarod test, so that the behavior of the mouse during trials can be recorded and the videos used for later analysis to determine the duration of time the mice were able to stay on the rotarod.
2. Follow steps 2.2.1 to 2.2.4 for the accelerating rotarod test:
   1. Place one mouse on each stationary dowel for 5 min to allow mice to acclimatize to the rotarod.
   2. Gently nudge the mice to face the back of the rotarod and start the rotarod test when all subjects are facing in this direction (see Figure 1B).
   3. Return all mice to their cages when they have fallen from the rotating dowels and leave them to rest for 10 min with access to food and water.
   4. Repeat steps 2.2.1 to 2.2.3 to complete a total of 8 trials making sure to clean the dowels, lanes, and landing platforms of the rotarod for urine and feces, and move the landing platforms back to its starting position betwen each trial.
      Note: The first 3-5 trials are used as training trials to allow the mice to familiarize themselves with the task. The time to fall, distance walked and end rpm of the dowel at the time of the fall for each subsequent trial is recorded for later analysis (Figure 2).
3. Follow steps 2.3.1 to 2.3.8 for the fixed speed rotarod test:
   1. Place one mouse on a dowel for 5 min to allow it to acclimatize to the rotarod. Return the mouse back to its cage.
   2. Start video recording on the camera and press start on the rotarod. Then place the mouse on the rotating dowel ensuring it faces the back of the rotarod.
   3. Stop video recording on the camera when the mouse falls from the rotating dowels, and return the mouse to its cages for 10 min with access to food and water.
   4. Repeat steps 2.3.2 and 2.3.3 until a total of 8 trials is acquired making sure to clean the dowels, lanes, and landing platforms of the rotarod for feces and urine, and move the landing platforms back to its starting position between each trial.
   5. Switch on the custom built rotator at 3 Hz for 20 sec to allow the mice to become familiar with the sound. Stop the rotator after 20 sec by switching off the drill and place hands on either side of the running wheel to stop it from continuing to spin past the initial 20 sec.
      Note: The rotator itself consists of a rodent running wheel secured to a drill (Figure 3A). At the center of the running wheel is a small chamber with a mesh lid where the mouse is placed (Figure 3B). The rotator spins in a counter-clockwise direction about the vertical axis. The magnitude of the stimulus is in line with previous studies that show rotary stimulations ranging from 0.2 to 3 Hz are sufficient to generate VOR and VCR responses^4,11,12.
   6. Place the mouse inside the chamber at the center of the rotator and replace the lid.
   7. Switch on the rotator at its lowest setting of 3 Hz for 20 sec. Start the rotarod and begin video recording on the camera during this time in preparation for the upcoming trial. Switch off the drill at the end of the 20 sec and place hands on either side of the running wheel to stop it from spinning. Retest the mouse on the rotarod immediately after by transferring it as quickly as possible to the rotating dowel.
   8. Stop video recording on the camera when the mouse falls from the dowel and return the mouse to its cage.
4. Clean the clear plastic panels with a mild detergent/water mix and the cylindrical dowels, lanes and metal landing platforms of the rotarod with 70% ethanol when all mice have been tested.

3. Balance Beam with Vestibular Challenge

1. Set up the balance beam apparatus as seen in Figure 4A.
   Note: The balance beam apparatus was adapted from an apparatus described in Carter et al. (2001)¹³. For this test, mice walk from the lower end of the beam, which is 52.5 cm above the ground, to a darkened goal box (13 x 22 cm, with a 5 x 6 cm doorway) situated 60 cm above the ground (Figure 4A). Mice naturally seek out the darkness and protection of the goal box in favor of the exposed beam and are further encouraged to traverse the beam by the slight incline which exploits their natural escape mechanism to run in an upwards direction¹⁴. The beam itself is 1 m in length and has a circular cross section with a diameter of 14 mm. A tailored range of beam diameters can be used which allows the experimenter to adjust the sensitivity of the test or accommodate larger subjects. At the lower end of the balance beam a white line indicates the starting line. Another line has been drawn 60 cm from the start line at the higher end of the beam to indicate the finish line (Figure 4A).
   1. Position 2 cameras, one on either side of the balance beam, at the lower end of the balance beam (Figure 4B).
      Note: These cameras should be angled to capture the entire length of the balance beam and ensure that start and finish lines marked on the balance beam are clearly visible. These cameras will be used to video record the behavior of mice as they traverse the balance beam, with resulting videos being used for later analysis.
   2. Line the floor of the goal box with paper towel, to enable easy cleaning of urine and feces after testing each mouse, and place the housing dome from the subjects home cage inside the goal box.
   3. Place adequate foam or other cushioning material underneath the raised beam to protect any subjects that fall from the apparatus. Mice that fall will be picked up immediately by the experimenter and placed inside the goal box to rest.
2. Place one mouse in the goal box for 2 min so that it becomes familiar with this environment. Cover the opening to the goal box with a gloved hand for 5 sec if the mouse tries to walk onto the beam during this time to discourage this behavior.
3. Train the mouse by placing it on the beam just outside the opening to the goal box and allowing it to walk into the goal box. Continue to train the mouse by placing it on the beam progressively further away from the goal box until the mouse is able to walk from the start line to the goal box with no assistance and minimal hesitation. Leave the mouse to rest in the goal box for 1 min after each run.
4. Begin testing the mouse when training is complete. 
   1. Start video recording on cameras.
   2. Place the mouse at the start line of the beam and wait while it traverses the beam in the direction of the goal box.
   3. Stop video recording on cameras when the mouse reaches the box.
   4. Leave the mouse to rest in the goal box for 1 min. Remove any urine or feces that may have been deposited during the trial while waiting.
   5. Repeat steps 3.4.1 to 3.4.4 until a total of 5 trials have been completed.
5. Switch on the custom built rotator at 3 Hz for 20 sec (like in the fixed speed rotarod test) to allow the mice to become familiar with the sound. Stop the rotator after 20 sec by switching off the drill and place hands on either side of the running wheel to stop it from spinning.
6. Place the mouse inside the chamber at the center of the rotator and replace the lid.
7. Switch on the rotator at its lowest setting of 3 Hz for 20 sec. Start video recording on the cameras during this time, in preparation for the upcoming trial. Switch off the drill at the end of the 20 sec and place hands on either side of the running wheel to stop it from continuing to spin past the initial 20 sec. Transfer the mouse to the start of the balance beam as quickly as possible and wait while the mouse traverses the beam to the goal box.
8. Stop video recording on cameras when the mouse reaches the goal box and return the mouse to its cage.
9. Clean the balance beam apparatus with 70% ethanol and change the paper towel in the goal box after each mouse has been tested.

Results

Rotarod

The motor performance of mice was described as the Time To Fall (TTF) recorded for each mouse over 8 trials. Using these measurements of TTF, training curves for each mouse can be plotted. Figure 2 shows examples of the motor performance of one 1 month-old mouse and one 9 month-old mouse over the course of 8 trials. These training curves show an increase in TTF during the first 3-5 trials followed by a subsequent plateau. Measurements of TTF recorded before the plateau we...

Discussion

Critical Steps within the Protocol

Previous work has shown that it is easy to overtrain mice on both the rotarod and balance beam apparatus and as a consequence, the acquisition of accurate measurements can be challenging¹⁵. For example, overtraining on the rotarod can lead to mice intentionally jumping off the dowels during both the acclimatization and trial periods, while overtraining on the balance beam can lead to more frequent stopping (exploratory behavior) and travelling in the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rotarod	IITC Life Science Inc.	#755	"Rat dowels" = 70 mm diameter. Do not allow ethanol to contact perspex.
iPhone	Apple		Can use any type of camera. Velcro fixed to the back surface for attachment to the the 3D articulated arm.
3D articulated arm	Fisso/Baitella	Classic 3300-28	Any type of stable vertical stand would be adequate. Velcro is fixed to the apical end of the arm for iPhone attachment.
Wooden walking beam: 1 m long strip of smooth wood with a circular cross-section of 14 mm diameter			A range of diameters and cross section shapes can be used to suit experimental parameters
Wooden goal box (130 x 140 x 220 mm) made from 11 mm thick boards
Support stand made of 41 x 41 mm beams: 2 vertical beams 525 and 590 mm from ground at the start and goal ends respectively; 803 mm horizontal beam that runs along the ground directly under the walking beam; two 20 mm long beams act as "feet", joining the horizontal and vertical beams at each end; a 21 x 21 x 36 mm block hewn at the apical end of the "starting" vertical beam; a 13 x 13 mm aperture cut out of the center of this block, forming a tunnel which runs perpendicular to the walking beam.			Brace all joins with small steel brackets.
Black paint (water based)	Handycan	Acrylic Matt Black	2-3 coats for all wooden surfaces of the balance beam apparatus
Clear finish	Wattle Estapol	Polyurethane Matt	Single coat for all beams. Double coat for all other surfaces of the balance beam apparatus
Foam, packaging material			To cushion any falls from the balance beam
70% Ethanol, paper towels			Clean beam and goal box between each animal.
Gauze pads/paper towels			To line the floor of the goal box
Mouse house (from home cage)