Behavioral Test Questions

The Influence of Animal Age in Behavioral Testing

By September 23, 2017 No Comments

Age has a strong influence on our behavior. Just look around the next time you are at the grocery store: a toddler is flailing on the ground in a temper tantrum; a young girl spins fervently in circles while waiting with her mother in line; an old woman slowly inches her way down the aisles, pausing for long periods to choose each item. While some of these behavioral differences may be due to socialization and the lifetime of governing influences on our behavior, there are also innate biological changes in behavior with aging due to changes in brain function.

Such changes in humans are mirrored in the behavior of our laboratory mice. Many of us have had the experience of opening a cage of weanling mice, only to have one vault from the cage as if on springs. I have spent many an afternoon crawling around on the vivarium floor, trying to retrieve a vital study participant. We’ve also witnessed the aging of our mice; like humans, they become fatter, their activity begins to slow, and, if the timing of our experiments allows, we see their hair begin to gray.

What is the Optimal Testing Age of Mice and Rats?

Laboratory mice and rats have a compressed lifespan in comparison to humans. The lifespan is strain-dependent and averages 1.5 to 3 years in the mouse[1], and 2.5 to 3.5 years in the rat[2]. Rodents are best tested when they reach adulthood and are past the hormonally turbulent period of puberty, but before the onset of age-related cognitive and motor decline.

Mice and rats typically reach sexual maturity by age six to eight weeks[3, 4]. Rats are considered to reach social maturity considerably later, around 12 weeks of age[4]. Thus, at minimum, mice and rats should be 2 and 3 months of age, respectively, when behavioral testing is initiated

How does age affect behavioral testing results?

Declines in reproductive capacity begin to occur around 8 months in mice and between 15 and 20 months in rats. Reproductive senescence leads to changes in the levels of hormones that can influence behavior. As mice enter late life, they may experience age-related hearing loss, memory impairments, and general motor weakness, which can be exacerbated by aspects of behavioral testing such as fasting[5]. For this reason, it is important to avoid testing animals that are too old.

A key study in this regard examined the effect of the progression from young adulthood to middle age on a battery of behavioral tests in C57BL/6J mice[6]. The mice were divided into four age groups: 2–3 months old, 4–5 months old, 6–7 months old, and 8–12-months old. The mice were evaluated in tests of memory, anxiety, depression, fear learning, and motor function. Age was a significant factor in performance in all of the tests. As one might predict, the youngest mice (2-3 months old) were the spryest and outperformed the other groups in tests in which motor function was a primary component, such as the rotarod test. They also had the sharpest memories, outperforming all other groups in the Barnes maze and fear-conditioning test.

Influence of animal age in behavioral testing

In older mice, body weight increased, while grip strength decreased. The activity of older mice in the open field test was reduced. Tests of anxiety showed increased entries into the open arm in the elevated plus maze, but reduced time in the light in the light-dark box in older mice.

In the social interaction test, older mice displayed a reduction in social contacts. Older mice also displayed decreased immobility in the tail suspension test and forced swim tests. Their startle response to sound was decreased with aging, and prepulse inhibition was increased.

The 4-5 and 6-7-month-old mice exhibited detectable differences from the youngest mice in many of the tests. However, the 8-12-month-old mice showed the most pronounced differences in behavior compared to the other groups. In parallel with the reproductive decline that occurs around 8 months of age, these data suggest more pronounced changes in function begin to occur at this age in mice.

Overall, this study shows us that careful attention must be paid to the age of the animals when designing behavioral tests and interpreting behavioral data. Relatively small age differences can have a large impact on results.


  1. Coleman DL et al. Biology of the Laboratory Mouse, 2nd edition. Dover Publications, 1966.
  3. Sengupta P. The Laboratory Rat: Relating Its Age With Human’s. Int J Prev Med. 2013 Jun; 4(6): 624–630.
  4. Mayer C1, Acosta-Martinez M, Dubois SL, Wolfe A, Radovick S, Boehm U, Levine JE. Timing and completion of puberty in female mice depend on estrogen receptor α-signaling in kisspeptin neurons. Proc Natl Acad Sci U S A. 2010 Dec 28; 107(52): 22693–22698.
  5. Kennard JA, Woodruff-Pak DS. Age sensitivity of behavioral tests and brain substrates of normal aging in mice. Front Aging Neurosci. 2011 May 25;3:9.
  6. Shoji H, Takao K, Hattori S, Miyakawa T. Age-related changes in behavior in C57BL/6J mice from young adulthood to middle age. Mol Brain. 2016 Jan 28;9:11.

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