'Let's just study males and keep it simple': How excluding female animals from research held neuroscience back, and could do so again

Actions taken by the Trump administration have spurred changes at America's major science agencies — and if worst comes to worst, these shifts may jeopardize our understanding of how the brain works and how to treat neurological conditions, experts told Live Science.

For years, the National Institutes of Health (NIH) has upheld a policy that requires scientists who work with vertebrate animals to consider "sex as a biological variable." To earn grants, researchers must explain how their study designs account for sex, such that any differences between the sexes will be made apparent. Researchers must provide strong scientific justification to include only one sex in a study.

But recently, The Transmitter reported that the NIH seems to have archived this policy. The NIH has yet to issue an official statement on the matter, but the move followed executive orders issued by the Trump administration that called for overturning "gender ideology" and "radical" diversity, equity and inclusion programs.

In apparently archiving its "sex as a biological variable" policy, the NIH could be signaling a shift away from requiring both males and females in research. And such a shift could be particularly dire for basic neuroscience research.

Related: Is there really a difference between male and female brains? Emerging science is revealing the answer.

Emerging animal research is revealing fundamental differences between male and female brains. These differences manifest in how sex hormones influence brains at the basic level of memory formation and neuronal firing, for example, and evidence suggests these differences may be relevant not just to lab animals, but also to humans. Failing to include both sexes in lab research could lead us to miss fundamental forces that shape the human brain and how drugs affect it.

"If the fundamental mechanisms by which molecules sculpt neurophysiology differ between the sexes, then we need to know that as early as possible in the process," said Catherine Woolley, a professor of neurobiology at the Northwestern University Weinberg College of Arts and Sciences.

A long legacy of bias

Are male and female brains wired differently? The question has beguiled humanity for centuries, but scientists have only recently begun to find answers.

That's partly because, to uncover differences between males and females, you must study both sexes, including in research with lab animals. But scientists haven't done that until relatively recently.

Animal studies enable experiments that would be impossible with humans. Researchers can't slice living people's heads open, pluck out their neurons or implant electrodes with wild abandon.

Human subjects also come with baggage — namely, men and women are raised differently and have different life experiences. These cultural factors shape the brain alongside biological factors, like hormones and chromosomes, making it difficult to separate nature from nurture.

Historically, however, scientists simply ignored the variable of sex by excluding females altogether. This bias was particularly pronounced in neuroscience — one 2009 review found that studies with exclusively male lab animals outnumbered those with females 5.5-to-1.

There was a concern that the reproductive cycle of female lab animals would "mess up the data, make it too variable," said Lise Eliot, a professor of neuroscience at the Rosalind Franklin University of Medicine and Science in Chicago. That idea has been thoroughly debunked, but for a time, the prevailing viewpoint was, "let's just study males and keep it simple," Eliot told Live Science.

Only in recent years has that attitude begun to shift.

Inclusive research breeds discovery

Including female lab animals has led to findings that unseated long-held assumptions about how the brain works.

For instance, Woolley uses lab rats to study how the sex hormone estrogen drives synaptic plasticity, the brain's ability to dynamically strengthen or weaken connections between neurons over time. This process enables the ability to learn and form memories, and it also underlies the brain changes behind psychiatric conditions like addiction.

Woolley has shown that plasticity works differently in males and females.

Connections between two neurons grow stronger via a process called long-term potentiation, which unfolds in two phases: an early phase, which lasts a few hours, and a late phase, which lasts longer. A key enzyme — protein kinase A (PKA) — was thought to participate in only the later, long-lasting phase.

"We've shown that that's true only in males," Woolley said. For females, PKA is crucial for the early phase of long-term potentiation, she and her colleagues revealed.

In another study, the team uncovered a sex-specific difference in the hippocampus, a brain region key for learning and memory. They showed that, exclusively in female rats, a specific type of estrogen makes neurons more likely to trigger a signal, thanks to a "previously unknown" mechanism. This particular estrogen is made in the brains of both males and females, and it doesn't follow the same cycle as estrogen made by the female reproductive system.

In that research, the team was "doing an experiment that has been done before … and our results were different," Woolley told Live Science. "It dawned on me that perhaps the results that we were getting differed from the published literature because we were using female animals." Previous work had included only males.

Many drugs act at the connection points between neurons, so it's important to understand how those connections are formed and maintained, Woolley said. Therefore, findings from these animal studies could help inform future treatments and diagnostics for people, she added.

There are some key differences between rodents and people, including that the human menstrual cycle is about seven times longer than the comparable cycle in rodents. But many studies have probed the similarities and differences in how estrogens act on the rodent brain and the nonhuman primate brain.

Based on these data, Woolley suspects many observations in the rat brain will translate to humans, even if they're not exactly the same.

Related: Faster brain aging tied to X chromosome inherited from Mom

Differences in fear processing

Beyond Woolley's lab, scientists have also uncovered nuances in how male and female mice store fearful memories.

When the researchers blocked signals from part of the amygdala — a key emotion-processing center involved in fear conditioning — it stopped fear memories from being "saved" in male mice, but not in females.

Then, they studied people with a genetic mutation in that same signaling pathway. Males with the mutation had trouble remembering that a cue on a screen would come with a mild electric shock, but the same mutation had no effect on females' memories.

That hints that males and females store fearful memories differently, and that could have implications for post-traumatic stress disorder (PTSD), the authors speculated. For instance, using a drug to block those amygdala signals after a traumatic event could help prevent PTSD in males but likely not in females.

Another study revealed cell-level differences in the nucleus accumbens, the core of the brain's reward system. More excitatory, or "activating," signaling occurs in the nucleus accumbens of female mice and rats, compared with males.

And several methods of damping that excitement in males do not work in females, the team found. That finding could be relevant for better understanding depression and addiction, since signaling in the reward system underlies both conditions, the study authors suggested.

The road ahead

A better understanding of these sex differences could lead to improved psychiatric and neurological treatments tailored to each sex, scientists argue. But the future of this research is uncertain given the changes that seem to be unfolding at America's premier science institutions.

The fate of the NIH's "sex as a biological variable" policy is currently unknown. But if including both sexes in research is no longer prioritized, it could set back the whole field of biomedicine, Woolley emphasized.

"It is very important to understand that the Sex as a Biological Variable policy is not about sex differences research," Woolley told Live Science. "This policy is about all biomedical research and ensuring that the results of taxpayer-funded biomedical research are relevant to everyone: both men and women, both boys and girls."