For over 50 years, researchers have preferentially used male mice in experiments, in part due to concern that the hormone cycle in females would cause behavioral variation that could negatively impact results. Over time, this practice has resulted in a poorer understanding of the female brain, likely contributing to the misdiagnosis of mental and neurological conditions in women, as well as the development of drugs that have more side effects for women.
“This bias starts in basic science, but the repercussions are rolled into drug development, and lead to bias in drugs being produced, and how drugs are suited for the different sexes,” said Dana Levy, a research fellow in neurobiology at Harvard Medical School (HMS).
Now, a new study led by Levy has not only turned the long-held, sexist assumption on its head but has demonstrated the opposite—male mice are the ones that exhibit excessive behavioral variation that can harm research results.
“I think this is really powerful evidence that if you’re studying naturalistic, spontaneous exploratory behavior, you should include both sexes in your experiments. 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, professor of neurobiology at HMS, who co-led the study.
For the study, published in Current Biology, 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.
The mice were placed in a 5-gallon bucket for 20 minutes while researchers used a camera to capture their movements and behaviors as they freely explored the space. The researchers then swabbed each female mouse to determine its estrous status before repeating the bucket test with the same individual multiple times.
The team analyzed the videos with MoSeq, an artificial intelligence technology previously developed by the Datta Lab. The technology uses machine learning algorithms to break down a mouse’s movements into around 50 different “syllables,” or components of body language. With MoSeq, the researchers gathered in-depth, high-resolution data about the structure and pattern of mouse behavior during each session.
According to the study results, estrous status had a negligible effect on exploratory behavior in female mice. Instead, patterns of behavior varied widely across individual mice—regardless of gender.
“If you give me any random video from our pile, I can tell you which mouse it is. That’s how individualized the pattern of behavior is,” said Datta. “[This] suggests that in behavioral studies a dominant aspect of variation in the data is the fact that individuals have subtly different life histories.”
That being said, when the team compared the recordings of females with males, they saw that males actually exhibited more behavioral variation within a single mouse and between mice than females did.
“The fact that female behavior is more reliable suggests that including females might actually decrease the overall variability in your data under many circumstances,” said Datta.
Surprised by the amount of individuality they recorded in their results, Datta and Levy are now delving deeper into the neural basis of individuality, examining mouse behavior from birth to death to better understand how patterns of behavior emerge, crystallize and change.
“We want to understand the mechanisms of individuality: how variability between individuals comes about, how it affects behavior, what can alter it, and what brain regions support it,” said Levy.
The researchers are also interested in exploring how internal states beyond hormonal status, such as hunger, thirst, pain and illness, affect exploratory behavior. They hope their current study will open the door for more rigorous, quantitative research on mouse behavior patterns.