The Case for Including Female Mice in Neuroscience Research Just Got Stronger

New research from Harvard Medical School challenges a long-held assumption that hormones have a broad effect on behavior in female mice, making them less suitable for research  and suggests that for many experiments, the concern may not be justified. The study results, published in Current Biology, reveal that female mice, despite ongoing hormonal fluctuations, exhibit exploratory behavior that is more stable than that of their male peers.

Using a strain of mice commonly studied in lab settings, the researchers analyzed how the animals behaved as they freely explored an open space. They found that the hormone cycle had a negligible effect on behavior and that differences in behavior between individual female mice were much greater. Moreover, differences in behavior were even greater for males than for females, both within and between mice. 

“I think this is really powerful evidence that if you’re studying naturalistic, spontaneous exploratory behavior, you should include both sexes in your experiments—and it leads to the argument that in this setting,  if you can only pick one sex to work on, you should actually be working on females,” said Sandeep Robert Datta, who co-led the study.

Perhaps the biggest reason for excluding female mice, Datta said, stems from a widespread assumption that their behavior is broadly affected by cyclic variations in hormones such as estrogen and progesterone—the rodent version of a menstrual cycle, known as the estrous cycle. According to Datta, estrous status is known to have a strong effect on certain social and sexual behaviors in mice. However, data on the influence of estrous status in other behavioral contexts have been mixed.

“We wanted to measure how much the estrous cycle seemed to influence basic patterns of exploration,” Datta said. “Our question was whether these ongoing changes in the hormonal state of the mouse affect other neural circuits in a way that’s confusing for researchers.” “We assumed, like everybody else, that adding females was just going to complicate our experiments,” lead author Dana Levy added, “And so we said, ‘why not test this.’”

The researchers studied genetically identical males and females from a common strain of lab mouse in a circular open field—a standard lab setup for behavioral neuroscience experiments. In practice, the test involved placing a mouse in a 5-gallon bucket for 20 minutes and using a camera to record the mouse’s movements and behaviors in 3D as it freely explored the space. The researchers swabbed each female mouse to determine its estrous status and repeated the bucket test with the same individual multiple times.

The team analyzed the videos with MoSeq, an artificial intelligence technology previously developed by the lab. The technology uses machine learning algorithms to break down a mouse’s movements into around 50 different “syllables,” or components of body language: short, single motions such as rearing up, pausing, stepping, or turning. With MoSeq, the researchers gathered in-depth, high-resolution data about the structure and pattern of mouse behavior during each session.

The researchers found that estrous status had very little effect on exploratory behavior in female mice. Instead, patterns of behavior tended to vary much more across female mice than they did throughout the estrous cycle.

When the researchers compared female and male mice, they found something that surprised them: Males also exhibited individuality of behavior, but they had more behavioral variation within a single mouse and between mice than females.

“People have been making this assumption that we can use male mice to reliably make comparisons within and across experiments, but our data suggest that female mice are more stable in terms of behavior despite the fact that they have the estrous cycle,” Datta said. Based on their findings, researchers in the Datta lab have already switched from male mice to mixed groups or female mice in their other experiments that involve circular, open-field testing.