What impact does early maternal separation have on the brain? And will the answer affect the way societies care for abandoned children?
By Sally West Johnson '72
Illustrations by Chris Buzelli
In the aftermath of the fall of Romanian dictator Nicolae Ceaucescu in 1989, the world came face to face with a grim spectacle: thousands upon thousands of children locked away in orphanages, warehoused in steel cribs, deprived of love, affection, even simple human touch. It was a problem unknown in degree, if not in kind.
Most of us understood intuitively that these children had suffered both physically and emotionally, but just how damaged were these children? Could anything be done to help them? Questions that might have been of academic interest to the scientific community before took on new urgency.
The imperative to find answers is why David Parfitt and Dana Helmreich, both of the Middlebury College biology department, plan to spend the next few years of their careers proving that babies separated from their mothers at birth have a harder time as adults. The husband-and-wife team have joined a worldwide effort to produce the kind of data-based proof that governments and scientific institutions require to formulate public policy or dole out money for social-service programs—namely that babies separated from their mothers develop abnormally high levels of a stress hormone in the brain, and that those excessive hormone levels have a lasting impact on animal behavior.
"All indicators are that the accumulation of chronic stress hormones can have damaging effects on the brain," explains Parfitt, an assistant professor of biology who joined the Middlebury College faculty in 1999. Together, he and Helmreich, a research scholar who holds a Ph.D. in neuroscience, run a research laboratory in John M. McCardell, Jr., Bicentennial Hall, in which they study the behavior of live mice and analyze blood and tissue samples from dead mice to assess the effect of stress in infancy. In time, they hope to bring their research findings to bear on humans, particularly on children raised in institutional settings.
"We're looking at behavioral inhibitions and developmental delay as a function of hormone secretion," explains Parfitt. "We also want to know whether some degree of replacement of maternal behavior can alleviate behavioral problems in adulthood."
The most intriguing sight in Parfitt's office on the third floor of Bi Hall is the huge dry-erase board that takes up much of one wall. Lists, made of letter-number combinations, in green, blue, and red marker, designate which mice were born into which litter and when. The lists are further sorted by the light cycles to which the mice have been subjected, the amount of time they've been separated from their mothers, and the collection times of their blood samples—after they have been subjected to new stresses as adults. Beside the lists, noted in purple marker, are exclamatory comments such as "Breeding and separation complete!!" and "Yahoo!!!! Blood sampling done!"—the kind of thrill that must be unique to scientists.
Then there are the personal touches: a photo and a ticket stub from a 1994 Michigan vs. Notre Dame football game (Parfitt got his doctorate at the University of Michigan), a blue-framed candy dispenser that looks like a wall hanging but actually dispenses M&M's to the couple's two young daughters and to the deserving students who assist them (many are seniors working on their theses). Parfitt's lab next door, where all biochemical and neuroanatomical analyses of mouse blood and brain tissue take place, has its own idiosyncrasies—a blue brain that radiates purple brain waves is the resident good luck charm.
Parfitt and Helmreich have spent much of their working lives in the lab. Indeed, they met in a research lab at the University of Pittsburgh, where she was studying the effect of stress on the brain and he was studying how the neural circuitry of the brain controls behavior. Their current research evolved out of a chance meeting with Dr. Judy Cameron while attending the annual meeting of the Society for Neuroscience, in New Orleans, in 2000. Cameron had been a mentor to both of them in Pittsburgh. At that time, she had joined the Research Network on Early Experience and Brain Development, a project funded by the MacArthur Foundation for the express purpose of bringing scientific information to bear on those who decide public policy for the care of orphans in a country such as Romania.
A specialist in non-human primates, Cameron was looking at the effects of certain hormone levels on rhesus monkeys and trying to equate the animals' responses to stress to the behavior of depressed human adolescents. Other scientists were working with other animals, but nobody was working with mice. She proposed that Parfitt and Helmreich tackle mouse research, and they agreed.
Mice, says Parfitt, have an innate advantage over other populations of research animals because they are inbred, unlike rats, which are outbred (interbred with unrelated individuals). Thus, discrete mouse populations are genetically similar, allowing researchers to study the impact of environmental factors without having to factor in the messy complications of significant genetic variability.
To begin, the researchers had to create the stress that would elevate the hormone levels in the pups. Parfitt started by raising three groups of mice: three distinct family lines of mothers and their juvenile male offspring. After birth, each litter was assigned to one of three groups: (1) mothers and pups never removed from the home cage, (2) mother and pups removed from the home cage for 10 minutes a day and separated, and (3) mother and pups removed from the home cage for three hours a day and separated. The separations began one day after birth and continued for 10 days before the initial experiment ended. Then the pups were left to grow to adulthood, which takes about 60 days.
Once the mice had reached adulthood, the experiment continued. Does a mouse, having been subjected to stress as an infant, exhibit more signs of fearfulness and anxiety when stressed as an adult? And does the mother's stress response to separation spill over to the baby, exacerbating the situation for both mother and child?
To help answer those questions, Parfitt built two mazes: an elevated plus maze and an elevated zero maze. As the names suggest, both are elevated off the ground. One has a circular runway, and the other is shaped like a cross. Both mazes had enclosed sections that afforded the mice a feeling of security, but to see what was going on, they had to take a chance and emerge from hiding.
"We wanted to measure the time it takes for the mouse to emerge from the tube," says Parfitt. "We were setting up a conflict between its natural curiosity and its fearfulness. Interestingly, what we found was that the animals that had been separated from their mothers for 10 minutes a day emerged from the tube far more quickly than either the maternal-separated animals or the never-separated animals. A little bit of stress seemed to cause less fearfulness and more boldness than either a lot of stress or no stress at all."
Question number two involved hard science: Did mice subjected to stress as adults have elevated levels of the stress hormone corticosterone in their brains, and if so could the levels be quantified?
To stress the adult mice, the researchers subjected some to very loud noises and drove remote-controlled cars through the cage of others. Some of the mice were killed at regular intervals (up to 90 minutes after they were subjected to stress), their blood was drawn, and their brains were immediately wrapped in foil and frozen to minus 80 degrees. Blood samples were checked for evidence of elevated hormone levels, and whisper-thin brain tissues were mounted on slides and tested for hormone secretions from the hypothalamus.
In the end, of course, all this work is not about mice but about understanding children and the toll taken when they are separated from their mothers at an early age. For several years now, Middlebury has been part of a multifaceted, multifunded effort to improve the lives of the Romanian orphans who were institutionalized in that impoverished former communist state. Outside of this effort, foster care does not exist in Romania, and the researchers hope to show that this option is a viable form of care and preferable to institutionalization,the historically preferred form of care in Romania.
The research also is aimed at determining what, if any, measures can be taken to remedy the damage done after the fact: at what point does intervention no longer have an effect? Is this a literal race against the clock?
One key player in this project has been the Humana Foundation, the philanthropic arm of the health-care giant, Humana Inc. Betty Ashbury Jones, M.A. '86, the wife of the foundation's CEO, is a member of Middlebury College's Board of Trustees, and it is through her efforts that Middlebury and Yale students have made site visits to Romanian orphanages in recent years.
In January 2003, for example, a group of Middlebury students, working under Parfitt, participated in the Bucharest Early Intervention Program as part of their January term study. The students reported back on subjects as diverse as prenatal nutrition, the causes and prevention of head banging in young children, and the role of touch in enriching the lives of children in orphanages. Their efforts were summarized in a report entitled, "From Neurons to Tutova: A Collection of Papers for the Benefit of the Children of Dr. Nicolaescu Hospital."
"We spent a week on campus teaching them what we know about early brain development before we headed out," Parfitt recalls. "The goal of going to Romania was to put the research in context. We wanted them to apply what they learned in the classroom to the human condition."
Many of the 13 students who took part in that January term class have gone on to further study in the field of neuroscience. Kate Stamper, a June graduate, is spending the summer at Vanderbilt University engaged in brain research. Sarah Bunnell '04, another course participant, returned to Romania this past January to implement a study she designed: saliva samples collected from institutionalized children are tested for excessive hormone secretions.
Much of that work has been supported by the MacArthur Foundation, funder of the aforementioned research network, which has posed several questions in the project synopsis that strike at the heart of Parfitt's research:
"What are the brain changes that occur during the first few years of life that may influence the specific behavioral outcomes observed in children raised in orphanages?
"How do the experiences a child has during this time period influence the development of these brain changes?
"To what degree can early abnormalities be remediated by more favorable care-giving environments?
"Is there a specific time period during which intervention is most effective? Is there a time period after which intervention is less likely to be effective?"
"All the indicators are that the accumulation of chronic stress hormones can have damaging effects on the brain," explains Parfitt. "We've studied Romanian children adopted by Canadian families who continue to have learning deficits and hormonal secretions eight years later.
"But with mice, we've seen that the replacement of certain normal maternal behaviors, like licking and grooming the pups with a paintbrush dipped in warm water, seems to alleviate the behavioral problems in adulthood. We've also seen that environmental enrichment, introducing toys and playthings into the mice's environment, can help reverse the effects.
"The $64 million question is this: Can we do something to reverse the effects after the fact? It seems clear to us that the first fix for children should be environmental enhancement. What we're trying to do here is to put science at the service of public policy to help officials make intelligent public-policy decisions."
Sally West Johnson '72 is a writer in Middlebury and a frequent contributor to the magazine.