Mice have long been used in neuroscience research because they provide a flexible model that scientists can control and examine to understand more about the brain. Previously, researchers preferred male mice over female mice because they were concerned that the female hormone cycle would skew the data. However, new Harvard Medical School research suggests this concern may be unjustified.
The researchers examined how mice behaved as they freely explored an open space using a type of mice widely researched in experimental conditions. They discovered that the hormone cycle had little effect on behavior and that behavioral differences between individual female mice were significantly bigger. Moreover, behavioral variations between men and females were significantly higher within and between mice.
According to the research team, the findings highlight the necessity of including both sexes in mouse studies.
Sandeep Robert Datta, professor of neurobiology at the Blavatnik Institute at HMS, who co-led the study with Rebecca Shansky of Northeastern University, said, “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,”
When neuroscientists attempt to understand the human brain better, they frequently turn to the mouse, which Datta refers to as the “standard bearer vertebrate model for learning how the brain functions.”
Because the mouse and human brains share a great deal of structural organization and genetic information, scientists can readily edit the mouse genome to answer specific experimental questions and construct models of human disorders.
Datta said, “Much of what we understand about the relationship between genes and neural circuits, and between neural activity and behavior, comes from basic research in the mouse, and mouse models are likely going to be central tools in our fight against a broad array of neurological and psychological diseases.”
Male mice have been utilized preferentially in trials by researchers for more than 50 years, and nowhere is this practice more prevalent than in neuroscience. In fact, according to a 2011 study, there were more than five times as many single-sex neuroscience studies of male mice than female mice. This practice has resulted in a poor understanding of the female brain over time, likely contributing to the misdiagnosis of mental and neurological conditions in women and the development of drugs with more side effects for women — issues highlighted by Shansky in a 2021 perspective in Nature Neuroscience.
The gap in sex representation that is frequent in animal research has historically been replicated in human subjects research.
Lead author Dana Levy, a research fellow in neurobiology at HMS, said, “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. For example, Levy noted that conditions such as anxiety, depression, and pain are known to manifest differently in female mice and women than in the male mice that are more often used in early-stage drug testing.
Datta said that the reasons for such a long-standing bias in neuroscience are complicated: “Part of it is just plain old sexism, and part of it is conservatism in the sense that people have studied male mice for so long that they don’t want to make a change.”
Female mice are often excluded from research due to the assumption that their behavior is affected by cyclic variations in hormones such as estrogen and progesterone, known as the estrous cycle. According to Datta and Levy, Estrous status is known to affect specific social and sexual behaviors in mice strongly. However, data on its influence in other behavioral contexts are mixed.
The scientist said, “We wanted to measure how much the estrous cycle seemed to influence basic exploration patterns. 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.”
He added, “We assumed, like everybody else, that adding females was just going to complicate our experiments. “And so we said, ‘why not test this.'”
Researchers studied genetically identical males and females from a common strain of lab mice in a circular open field. They placed the mice in a 5-gallon Home Depot bucket for 20 minutes. They used a camera to record their movements and behaviors in 3D. They swabbed each female mouse to determine its estrous status. They repeated the bucket test with the exact individual multiple times.
The researcher said, “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, “For example, Levy noted that conditions such as anxiety, depression, and pain are known to manifest differently in female mice and women than in the male mice that are more often used in early-stage drug testing.”
The scientists used MoSeq, an artificial intelligence technique developed by the lab before, to analyze the structure and pattern of mouse activity during each session. They discovered that whereas the estrous state did not affect exploratory behavior in female mice, behavioral patterns varied far more across female mice than over the estrous cycle.
When the researchers analyzed female and male mice, they discovered something unexpected: Men demonstrated behavioral individuality as well. However, they had more behavioral variety within a single mouse and between mice than females.
The findings make a strong scientific case for using female mice in experiments, as female behavior is more reliable and could decrease the overall variability in data under many circumstances. The study looks at only one mouse strain in one lab setup, so the results can only be generalized to other strains and setups with further testing.
However, the strain and setup are commonly used in neuroscience research. Datta and Levy are interested in exploring how internal states beyond hormonal statuses, such as hunger, thirst, pain, and illness, affect exploratory behavior in mice. They also want to delve deeper into the neural basis of the individuality of mouse behavior that they saw in the study. To this end, the Datta lab examines mouse behavior from birth until death to understand how individualized patterns of behavior emerge and crystallize during development and how they change throughout life. The researchers also hope their work will open the door for more rigorous, quantitative research on whether and how the estrous cycle affects mouse behavior in other contexts, such as completing complex tasks.
Scientist Said, “This is a very interesting example of how assumptions that affect the way that we conduct and design our science are sometimes just assumptions — and it is important to test them because sometimes they’re not true directly,”
Journal Reference:
- Dana Rubi Levy, Sandeep Robert Datta, et al. Mouse spontaneous behavior reflects individual variation rather than an estrous state. Current Biology. DOI: 10.1016/j.cub.2023.02.035