Mice have long been used in neuroscience research because they provide a flexible model that scientists can monitor and examine to understand more about the brain. Previously, researchers preferred male mice to female mice because they were concerned that the female hormone cycle would skew the data. However, new research from Harvard Medical School suggests that these concerns may be unfounded.

The researchers studied how mice behaved while freely exploring an open space using a type of mouse that has been extensively studied in experimental conditions. They found that the hormone cycle had little effect on behavior and that the behavioral differences between individual female mice were significantly greater. In addition, behavioral variations between males and females were significantly higher within and between mice.

According to the research team, the findings highlight the need to include both sexes in mouse studies.

Sandeep Robert Datta, a professor of neurobiology at HMS’s Blavatnik Institute, who co-led the study with Northeastern University’s Rebecca Shansky, said: “I think this is really strong evidence that if you’re studying naturalistic, spontaneously exploratory behavior, you should include both sexes in your experiments — and it leads to the argument that in this setting if you can only choose one sex to work on, then you should actually be working with women,”

When neuroscientists try to better understand the human brain, they often turn to the mouse, which Datta refers to as the “vertebrate standard-bearer model for learning how the brain functions.”

Because the mouse and human brain share much structural organization and genetic information, scientists can easily edit the mouse genome to answer specific experimental questions and construct models of human disorders.

data 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 to become central tools in our battle against a wide variety of neurological and mental illnesses. .”

Male mice have been the preferred use of researchers in experiments for over 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 same-sex neuroscience studies in male mice than in female mice. Over time, this practice has led to a poor understanding of the female brain, which likely contributed to the misdiagnosis of mental and neurological disorders in women and the development of drugs with more side effects for women – issues Shansky points out brought in a 2021 perspective in Nature Neuroscience.

The gender representation gap that often appears in animal research has historically been replicated in human research.

Lead author Dana Levy, a research fellow in neurobiology at HMS, said: “This bias starts in basic science, but the repercussions feed into drug development and lead to biases in drug manufacturing and how drugs are appropriate for the different genders. For example, Levy noted that conditions such as anxiety, depression and pain are known to manifest differently in female and female mice than in male mice, which are more commonly used in early-stage drug testing.

Datta said the reasons for such a longstanding 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 been studying male mice for so long they don’t want to change anything.”

Female mice are often excluded from research because of the assumption that their behavior is influenced 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 strongly influence specific social and sexual behavior in mice. However, data on its influence in other behavioral contexts are mixed.

The scientist said: “We wanted to measure to what extent the estrous cycle appeared to affect fundamental patterns of exploration. Our question was whether these ongoing changes in the hormonal state of the mouse affect other neural circuits in a way that is confusing to researchers.”

He added, “We assumed, like everyone else, that adding females would just complicate our experiments. And so we said, ‘why don’t you test this.'”

Researchers studied genetically identical males and females of a common strain of laboratory 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 behavior in 3D. They swabbed each female mouse to determine estrous status. They repeated the bucket test several times with the exact person.

The researcher said, “This bias starts in basic science, but the repercussions feed into drug development and lead to biases in drug manufacturing and how drugs are appropriate for the different genders.” For example, Levy noted that conditions such as anxiety, depression, and pain known to manifest differently in female mice and females than in male mice which are more commonly used in early stage drug testing.”

The scientists used MoSeq, an artificial intelligence technique previously developed by the lab, to analyze the structure and pattern of mouse activity during each session. They found that while estrous state did not affect exploratory behavior in female mice, patterns of behavior varied much more between female mice than during the estrous cycle.

When the researchers analyzed female and male mice, they discovered something unexpected: males also showed behavioral individuality. However, they had more behavioral variation within a single mouse and between mice than females.

The findings make a strong scientific case for using female mice in experiments because female behavior is more reliable and could reduce overall variability in data under many conditions. 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 arrangement are often used in neuroscience research. Datta and Levy are interested in exploring how internal states beyond hormonal states, such as hunger, thirst, pain and disease, influence exploratory behavior in mice. They also want to dig deeper into the neural basis of the individuality of mouse behaviors they saw in the study. To that end, the Datta lab examines mouse behavior from birth to 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 to more rigorous, quantitative research into whether and how the estrous cycle influences mice’s behavior in other contexts, such as completing complex tasks.

scientist said, “This is a really interesting example of how assumptions that affect the way we conduct and design our science are sometimes just assumptions — and it’s important to test them because sometimes they aren’t directly true,”

Magazine reference:

  1. Dana Rubi Levy, Sandeep Robert Datta, et al. Spontaneous behavior of mice reflects individual variation rather than an estrous state. Current Biology. DOI: 10.1016/j.cub.2023.02.035