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.
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.
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 aging5-7. 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.
1. Animals
2. Rotarod
3. Balance Beam with Vestibular Challenge
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 were considered training (Figure 2), while measurements of TTF that form the plateau were recorded and used for data analysis (Figure 5).
Figure 5 shows that motor performance on the rotarod deteriorates with age. When compared with their 9 month-old counterparts (n = 8), 1 month-old mice (n = 6) were able to stay on the rotarod significantly longer (18.38 ± 4.66 vs. 14.13 ± 2.53 sec; p < 0.05, Student’s t-test). This shows that the rotarod and the test parameters used are sensitive to changes in motor performance with age.
Balance Beam
Figure 6 shows the times to traverse (TTT) the balance beam and cross the finish line before and after the vestibular stimulus for 1 month-old, 9 month-old and 13 month-old mice. In 1 month-old mice (n = 9), the vestibular stimulus had a minimal effect on the time taken for them to traverse the balance with similar TTT before (3.49 ± 0.62 sec) and after (3.81 ± 0.66 sec) the stimulus. In contrast, 9 month-old mice (n = 6) required more time to traverse the balance beam after the vestibular stimulus (4.85 ± 1.67 vs. 8.45 ± 2.59 sec; p <0.05, Student’s t-test). In 13 month-old mice (n = 5) TTT increased following the vestibular stimulus (6.48 ± 2.19 vs. 9.24 ± 4.11 sec) but this was not statistically significant.
To further examine the interaction between age and vestibular stimulus-related changes in TTT we used repeated-measures ANOVA with a Tukey post hoc test. Figure 6 shows that the impact of vestibular stimulation on balance beam performance is significantly greater in 9 month-old (p < 0.01) and 13 month-old (p < 0.001) mice when compared with 1 month-old mice. Together these results indicate that the simple balance beam apparatus can be used in conjunction with the custom built rotator to measure vestibular-related changes in balance performance throughout the murine lifespan.
Figure 1. The rotarod apparatus. (A) The rotarod apparatus has 5 lanes and is able to test a maximum of 5 rodents at any one time. The cylindrical dowels (arrowhead) upon which mice are placed are situated above the metal landing platforms (*) that trigger pressure sensors for data acquisition. (B) A photo of one 1 month-old mouse and one 9 month old mouse sitting on the dowels facing the back of the rotarod in preparation of a test.
Figure 2. Rotarod training curves. An example of one 1 month-old and one 9 month-old mouse and their measurements of time to fall on the rotarod. The measurements of time to fall for both mice increased steadily during trials 1 to 5 and therefore were considered as training trials. Once trials stabilized (performance increases plateaued; dashed line) measurements of time to fall were recorded for data analysis.
Figure 3. The custom built rotator. (A) The custom-built rotator is used to stimulate the vestibular system of mice. This rotator consists of a Dremel (arrowhead) and a rodent running wheel (*). (B) A superior view of the rotator. Mice are placed inside the chamber (arrowhead) at the center of the running wheel.
Figure 4. Balance beam apparatus. (A) The inclined balance beam apparatus has an 80.3 cm long base, with the start of the beam situated 52.5 cm above the ground and the goal box (*) raised 60 cm above the ground. (B) The behavior of mice on the balance beam is recorded by two cameras placed at the lower end of the beam. The videos recorded provide left and right views of the mice as they traverse the beam and are used for later analysis.
Figure 5. Rotarod performance decreases with age. 1 month-old mice (n = 6) were able to stay on the rotating dowels significantly longer than 9 month-old (n = 8) mice (*; p < 0.05). Data are represented as mean ± SD.
Figure 6. The impact of vestibular stimulation on balance beam performance is greater with age. Vestibular stimulation increased the time to traverse in 9 month-old (n = 6) and 13 month-old (n = 5) mice but not in 1 month-old (n = 9) mice. When the interaction between age and vestibular stimulus-related changes in TTT is assessed the impact of vestibular stimulation on balance beam performance is significantly greater in 9 month-old and 13 month-old mice when compared with 1 month-old mice. Data are represented as mean ± SD. *; p < 0.05 **; p < 0.01 ***; p < 0.001.
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 opposite direction (i.e. towards the start line)15. Ultimately, overtraining can lead to underestimates of actual motor capability. It is therefore critical that training curves be assessed prior to analysis.
Another critical step within the rotarod protocol is ensuring that the mice face the correct direction (opposite direction to rotation) prior to starting each trial. Mice facing the wrong direction when the dowels start to rotate have difficulty maintaining balance on the dowel and consequently fall early, potentially overestimating the impact of the test. Further, in the balance beam test, it is important to transfer the mice as quickly as possible from the rotator to the balance beam as recovery from the vestibular challenge begins immediately. This can mean that disequilibrium caused by the rotator and subsequent reduction in performance can be underestimated.
Modifications and Troubleshooting
Modifications can be made to both the rotarod and the balance beam test in order to alter the sensitivity of the tests. The rotarod test can easily be modified to change the level of difficulty of the motor task necessary to detect balance and motor deficits. This is can be achieved by manipulating the speed at which the dowels rotate during the test, and also whether or not these rotations accelerate over the duration of the test. For the balance beam test different beam widths can be used to adjust the sensitivity of the test, with smaller beam widths engendering a higher level of difficulty. Beams with rectangular cross-sections can also be used, although in a previous study using this approach, it was shown that mice were able to grip onto the sides of the beam which lead to aberrant measurements of time to traverse15. In both rotarod and balance beam tests, mice can be challenged with the vestibular stimulus and retested on the apparatus up to 3 times. However, it should be noted that mice are often reluctant to complete the task after undergoing the first trial with the vestibular stimulus.
Limitations
Measurements of balance and motor ability can be affected by the size and weight of individual mice being tested14. This means that there is a possibility that the impact of age on motor performance can be augmented by the effects of gravity and center of mass. Indeed, mice with relatively higher body mass have been shown to perform worse on the rotarod test16. The application of the vestibular rotator however minimizes the extent to which balance performance is confounded by weight, and facilitates the attribution of balance performance to the impact of aging on the vestibular system.
Significance of the Technique with respect to Existing Methods and Alternative Methods
There have been few studies that directly investigate aging in the vestibular system of any species. Commonly these studies have used the VOR to assess vestibular function and have shown that VOR function is preserved up until 60 weeks of age with only small changes after adulthood is reached17,18. In addition VOR tests typically require a degree of invasiveness to attach the recording coils to the animal’s cornea, and often require a recovery period3. Due to the small size of the mouse eye the most commonly used alternative, video eye tracking, is also difficult to achieve. Together these difficulties have limited the number of VOR studies in the murine model.
The methods described in this paper employ apparatus commonly used to assess motor coordination and balance. In addition, these methods have been used to investigate the changes that occur during development and aging and those due to genetic modifications5,7,19,20. As motor coordination and balance has been shown to decline after 3 months of age, the additional use of a simple vestibular stimulus in this paper facilitates the investigation of the vestibular system in an aging murine model without the use of more difficult and invasive techniques outlined above8. This information can then be used to correlate behavior with underlying cellular and subcellular changes that occur in the vestibular system with age.
Future Application or Directions after Mastering this Technique
Although the methods described here do not quantify the level of disequilibrium experienced by the animals post-rotation, further application of a vestibular stimulus can be modified to include a scoring system based on the presence of symptoms including urination, defecation and tremors21. Other ways of quantifying the amount of disequilibrium experienced by mice include measuring saccharin and Kaolin uptake as shown previously11,21. Ultimately, the ability to score the vestibular-related effect of aging in an individual mouse allows for investigation on correlations between balance performance and cellular/subcellular processes using subsequent electrophysiological, molecular and two-photon microscopy techniques22.
The authors have nothing to disclose.
The authors would like to acknowledge The Garnett Passe and Rodney Williams Memorial Foundation and the Bosch Institute Animal Behavioural Facility.
Rotarod | IITC Life Science Inc. | #755 | "Rat dowels" = 70 mm diameter. Do not allow ethanol contact perspex. |
iPhone | Apple | Can use any type of camera (e.g. Logitech webcam described above). 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: 1m 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 centre of this block, forming a tunnel which runs perpendicular to the walking beam. | Brace all joins with small steel brackets. | ||
Adjustible metal ring (13 mm wide) | Pass this through the aperture in the block, pass the starting end of the balance beam through this ring and tighten until the beam is firmly in place. | ||
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 | ||
Electrical tape | Fix webcam to roof. | ||
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) |