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 fall....

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): <....

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)