The Brain On Stress

For humans and rats, the same brain regions - the hypothalamus, hippocampus, and amygdala - are engaged under stress. Likewise, the same neuroendocrine glands - the pituitary and the adrenal - are activated. The impacts of adrenal steroid hormones on the adult rat brain had been widely studied, but hardly anything was known about stress and the adolescent brain. Was it possible that stress affected young brains and older brains differently, in ways that researchers and clinicians had overlooked?

His question was simple: Do adolescents and adults undergo a similar neuroendocrine response when stressed? He knew of two other experiments in the previous 20 years that had involved stressing adolescent rats by using foot shock or ether inhalation. Romeo was subjecting his teenage rats to a different kind of stress: restraint. Restraint stress, he says, is both a physical and a psychological stressor, and he knew that it would activate the hypothalamic-pituitary-adrenal (HPA) axis, the part of the neuroendocrine system that regulates stress.

When an animal encounters a stressor, the HPA axis is immediately activated. Cells in the hypothalamus release corticotropin-releasing hormone (CRH), which triggers the pituitary, a pea-sized gland nestled into a bony enclave at the base of the brain, to secrete adrenocorticotropic hormone. ACTH then acts on the adrenal glands to release glucocorticoids: cortisol in humans and corticosterone in rats. Romeo wanted to study this system.

Romeo's plan was to first compare the stress response of young rats with that of adult rats to find out whether stress reactivity changed during puberty. Half of his rats were adults, 77 days old, and half were juveniles, 28 days old and still prepubescent. In his experiment, he would put each rat under stress for a short time, and then measure the levels of stress hormones - corticosterone and ACTH - in its blood.

He would need to record their hormone levels at different stages - before, during, and after the stressor. Using a tiny guillotine from the lab, Romeo began the experiment by decapitating 12 rats - six adults and six juveniles - to collect blood from the neck and heart, in order to capture the pre-stress hormone levels. He removed their brains, testes, and adrenal glands for later testing.

Then Romeo coaxed each of the remaining rats one at a time into a wire mesh restrainer, only slightly bigger than the rat itself. It was lined with thick tape and held shut with a binder clip. The restraint did not hurt the rat but induced stress by holding it perfectly still, somewhat like a miniature straightjacket. Half an hour later, after the rats' stress levels had climbed, he removed them from the restrainers. He immediately decapitated 12 of them - again, six adults and six pups - and collected their blood and organs. He returned the remaining rats to their cages. One group was killed 30 minutes later, another one hour after that.

After the experiment, Romeo set up an immunoassay to measure the hormone levels in the blood samples. It would take several hours for the hormones to bind to their antibodies. So he waited overnight.

Romeo had first begun studying gonadal steroid hormones in puberty when he was a PhD student at Michigan State University. After receiving his degree in 2001, he took a postdoc in Bruce McEwen's neuroendocrinology laboratory at Rockefeller. For the first few years, his research strayed from adolescence as he worked with McEwen on maternal neglect and its influence on the physiology and behavior of animals as they grow into adulthood.

Autoradiograms showing NMDA binding sites. Romeo exposed the hippocampus to 3 H-glutamate plus cold NMDA (top) and 3 H-glutamate alone. Arrows mark the extent of the CA1 strata oriens and radiatum measured.

© 2005 Neuroendocrinology; S Karger AG, Basel

In the meantime, he closely followed the research of adolescent specialists such as Ronald Dahl, Judith Rapoport, and Jay Giedd, and he tracked the newest discoveries. The human brain, which scientists had long believed changed little after the first few years of life, was blooming madly with activity and growth in adolescence. The brains were exhibiting waves of growth so significant that they nearly rival the period of brain growth just before birth.

In the mid-1990s, Giedd, a neuroscientist at the National Institute of Mental Health, realized he was near one of the largest brain banks in the country, the Yakovlev-Haleem Collection, housed at the National Museum of Health and Medicine at Walter Reed Army Medical Center. Giedd was immediately struck by how few adolescent brains were in the collection. Of more than 1,500 brains, only about 14 were from adolescents. "There was this huge hole in the knowledge," he says.

Using tissue samples from the brain bank as well as scans of live brains, he launched into the first large-scale, longitudinal study of human adolescent brains. Giedd found that as puberty begins, the gray matter blooms in a flourish of growth, and then over time begins pruning back, as some neurons selectively sever their connections to other neurons while others die off. It also seemed that the frequently used connections become stronger during this time, and those used less-often are severed at the synapse. The brain was organizing itself. Synapses must have a purpose to survive.

Meanwhile, he noticed that the white matter, or myelin, steadily increases throughout adolescence. Over time, more and more myelin surrounds each of the neurons' stringy axons like a sheath, speeding connections between the cells, and improving function in the brain. That's all happening while teenagers are making choices during high school that can influence the rest of their lives: decisions as simple as music and fashion taste, as life-changing as college choice, or as loaded as whether to use a condom. In other words, events that occur during adolescence can make imprints on the brain, shaping neural circuitry and potentially influencing who a person becomes.

These cellular changes come at a particularly vulnerable time for teenagers. The overwhelming majority of people with schizophrenia start showing symptoms as adolescents. The prevalence of depression, which affects about two percent of children, climbs to seven percent after puberty. Although genetic factors play a large role in the onset of these diseases, some scientists say that chronic stress during adolescence tips the balance, causing someone who would otherwise be mentally sound to have a mental disorder. For all of these reasons, Romeo felt that the effects of stress on the changing brain during adolescence was an area that needed more research.

All of this was on Romeo's mind in May 2003 when he went to a neuroscience retreat at the Mohonk Mountain House, a Victorian castle deep in New York state's Hudson Valley. Rockefeller's Fernando Nottebohm, whose unconventional research on songbirds had unearthed some of the earliest evidence for neurogenesis, was giving a talk that day. He encouraged scientists to approach their research with independence, irreverence, even mischief. "Science is not really about logic," he said in a recent interview. "It's about the things you imagine. If you go with your gut feelings and act like an artist, not like a scientist, you're likely to be more original, and there will be more of you in it." Encouraged and inspired by the speech, Romeo returned to the lab, more motivated than ever. Without telling his boss, he had already started his experiment.

Russell Romeo working at his lab bench.

© Greg Kessler

Romeo is an amiable man, with a short, spiky haircut, fashionable black-rimmed glasses, and an athletic build. He keeps records on every rat, every purchase order, and every experiment neatly filed in a thick black binder. McEwen once called him the most organized scientist ever to work at the lab. Romeo knows that describing his work to any nonscientist requires explaining the basics of neuroscience and the neuroendocrine system, and he takes the time to get it right, drawing diagrams and clarifying questions with detail and precision.

When he arrived at the lab the morning after his experiment and started to check the levels of corticosterone and ACTH in the rats' blood, he remembers the raw data of the hormone levels coming off the printer. To maintain the integrity of the experiment, the data was blinded, so he didn't know which number was associated with which rat. Romeo began matching number to animal and punching them into a computer spreadsheet. "I'm going, holy s***,' as I'm putting them in," he says. "I'm looking at the data and thinking, wow, this really is a robust effect'."

He hit the "analyze" button on the computer, and a set of numbers appeared on the screen. "Lo and behold, I saw this robust, differential response," he says. "The animals had gone through the exact same stimulus, but their response was very, very different."

In all the animals, the corticosterone levels had shot up to about 420 nanograms immediately followed the stressor. After 30 minutes, however, adult levels had dropped down to about 150 ng, while the juveniles' levels remained suspended at 400 ng. By 60 minutes, adult cortisone levels were at 120 ng, while levels for the young rats had dropped to only 260 ng. Two hours later, adult cortisone had returned to basal level, while the juveniles' cortisone levels were still at about 80 ng, far above base level. Both released similar amounts of corticosterone, but the young rats took nearly an hour longer than the adults to recover. ACTH levels were showing a similar pattern.

The juvenile animal was mounting a longer stress response, which meant that the juvenile brain was exposed to stress hormones for a longer period than the adult brain. Romeo thought the physiologic and behavioral implications of the extended response were unknown, but since puberty is a time of increased susceptibility to drug abuse and mental disorders, and since these disorders appear to be exacerbated by stress, this area of research, he concluded, needed further investigation.

After collecting and analyzing the data, Romeo wrote a manuscript. He typed both his own name and McEwen's at the top, printed it out, and paper clipped the pages together. Then, paper in hand, he walked across the hall to his boss's office and knocked on the door. McEwen was at his desk, working. "Here are some results I've collected," Romeo remembers saying. "It's a little far afield from what I've been doing. What do you think?"

As a young postdoctoral researcher, McEwen had made an exciting discovery of his own. For a long time, scientists believed that adrenal steroid hormones circulated in the body but didn't bind to anything above the neck. McEwen discovered that stress hormones also bind to cells in the hippocampus. It was a major discovery for any researcher, especially a young one, and it was published in Nature in 1968 (220:911-2). McEwen's early research played a vital role in the emergence of a new field of science: neuroendocrinology.

Subsequent research by McEwen and others has shown that when an adult animal is under chronic stress, glucocorticoids can attack nerve cells in the hippocampus, causing dendrites to shrivel and cells to shrink. Stress can also halt neurogenesis. In the amygdala, the brain's emotional center, the opposite occurs: More dendrites sprout forth from the neurons when an animal is chronically stressed.

Russell Romeo (left) and Bruce McEwen looking at a sketch of the rat brain that they use when making comparisons.

© Greg Kessler

Scientists believe these changes may help to explain the behavior a person shows months after climbing out of a car wreck, surviving an assault, or losing a family member. Shrinkage of cells in the hippocampus is associated with depression and memory loss. The growth of cells in the amygdala has been linked to overwhelming emotions, and in turn, anxiety disorders. These changes in the hippocampus are usually reversible. Once the stress is relieved, memory and mood improve. But in adults, changes in the amygdala - emotional changes - don't always change back.

"The basic idea that has come from the kind of studies that I and Robert Sapolsky and others have done on animal models, is that the human brain does change volumetrically and functionally in some of these stress-related disorders," McEwen says. Chronic stress is reshaping the brain in sometimes permanent, sometimes harmful ways. The amygdala, the hippocampus, and the prefrontal cortex are three brain areas that undergo major changes during puberty.

Romeo had found in his first experiment that juveniles were exposed to stress hormones longer than the adults. "All things being equal, they're going to be experiencing higher levels for a longer period of time," he recalls thinking. "This might be why stress can be so damaging to an individual during adolescence."

When McEwen looked at Romeo's manuscript on that day in September, he was pleased not only with Romeo's findings, but also his chutzpah. "Most of the good things that have happened in this laboratory have been from people taking the initiative and introducing something that was their own, not just following the line of the laboratory," McEwen would say long afterward. "The things that have been really creative in this lab have been serendipitous."

With McEwen's blessing, Romeo launched into a second experiment. In his first, he had examined the hormonal stress response between adults and juveniles by looking at an acute, one-time stressor and found that hormonally, juveniles take longer to recover. In the second, he wanted to test the difference between the pups and the adult rats under chronic stress. This was an important step, because scientists knew that chronic stress was linked to depression and other mental disorders in adults. Knowing how chronic stress affected the adolescent brain could potentially shed light onto treatment for these disorders.

This time, Romeo stressed the rats - 36 young and 36 old - for thirty minutes each day for seven days. His questions: How do young rats and adults compare in their reactions to long-term stress? If they've experienced the stressor before, will they react to it differently when it repeats?

In the adult rats, hormone levels shot up on the first day of restraint but then dropped each ensuing day, as if they were growing accustomed to the stress. In the juveniles, on the other hand, corticosterone levels shot up even higher after the first day of stress and didn't drop until the stressor was removed. But, once it was removed, the juveniles showed a much faster return to baseline.

The pattern was confusing, but it was obvious again that the adult and young rats were responding in very different ways. "Now we've got a whole added level of complexity," he remembers thinking. "From the [beginning] they're different, then you throw in experience, and it's even more different."

Using triple-label immunofluorescence staining, he also found that among the stressed juvenile animals, a larger number of CRH neurons in the paraventricular nucleus of the hypothalamus were expressing Fos, which meant that more gene transcription was occurring in those cells. At least part of the difference in stress reactivity is associated with different levels of activity in these cells, he concluded. It was hard to say exactly what this meant, but it narrowed down the search, providing a candidate for future investigations.

Chronic stress during adolescence, Romeo found, also led to changes in the behavior of his rats. When he subjected rats to stress throughout the course of puberty, they lost weight, had elevated levels of corticosterone, and showed symptoms of depression, like learned helplessness. This was not the case for adults subjected to stress. These data, he says, indicate that animals are particularly sensitive to stress during adolescence, but it will take more investigation to determine exactly what is changing during puberty to account for the difference in stress reactivity.

Romeo's next line of research, however, will be to investigate neurotransmitter systems, with a particular focus on norepinephrine and serotonin. He will investigate how adolescent stress affects the serotonergic and noradrenergic pathways in the brain. "We know that dysfunction of these pathways in adulthood can lead to psychological disorders, but know relatively little how challenges during puberty may affect their immediate or long-term functioning," Romeo wrote in a recent e-mail.

Plasma corticosterone concentrations in prepubertal and adult males exposed to a single 30-minute session of restraint or a daily 30-minute session of restraint for one week. A single explanation for the results is still elusive, but Romeo showed in this experiment that young and adult rats were responding in very different ways.

Redrawn from: Ann NY Acad Sci, 1094:202-14, 2006

What Romeo does know is that stress, often linked to depressive and anxiety disorders, affects the adolescent human brain differently than that of an adult. And, he says that treatment should vary accordingly. The lumping together of age groups is a problem, he says. "Psychological dysfunction in adolescence is a very different beast from psychological dysfunction in adults, just like it's very different when you have psychological dysfunction in an aged person versus a young adult." In other words, he says, adults and teenagers may require different drugs and different treatment plans. He says he hopes that his research will help advance treatment designed specifically for teenagers.

Romeo found that for adolescent rats, stress was also linked to signs of depression. By stressing adolescent rats and then observing them in an open maze and a forced swim test, Romeo has found that they are particularly sensitive to stressors, more sensitive than adults. The adolescent rats that were isolated or restrained in Romeo's experiments lost more weight, struggled less in the swim test and showed less movement in the open maze. Rats that he restrained as adolescents were also more prone to showing depressive tendencies later in life.

Elaine Walker, a developmental psychologist and neuroscientist at Emory University, says Romeo's research is important in the context of psychopathologies that emerge during adolescence. Stress is linked to virtually all physical and mental disorders, she says, adding that steroid hormones are believed to have both activational and organizational effects on the human brain. Walker also says that adolescence is a uniquely important time for intervention: "Preventive intervention in adolescence may be most effective because it is a developmental stage when we can most easily identify those who are vulnerable and potentially change the course of development."

A snapshot of Romeo is pasted onto the door of one of Mc-Ewen's labs. He is caught mid-step and face tightened into a grimace, as he sprints the final stretch of the 2006 Berlin marathon, the Brandenburg Gate framing the distance behind him. Romeo had expected the race to end at the famous German monument, but he was wrong. It turned out he had another 400 meters to go. It was a long 400 meters. After the marathon, his coworker, Beth Waters, downloaded the photo from the Internet and stuck it on the door. His face just looked so funny, she says, joking, "It's my only way to roast Dr. Romeo until he retires."

Behind the door, days are long. Lab workers, some with wrinkled clothes and unshaven faces, are hunched over their benches, eyes fixed on their work. In the lab, plastic gloves fill a small trashcan. Finger-picking melodies from a classical guitar compilation album, one of the 15 CDs on rotation, mingle with the sharp hum of a cryostat chamber. It's 3 p.m. on a Friday in mid-January, and everyone is hoping to be finished in time for Happy Hour at the university bar.

On this particular day, Romeo and Erik Bloss, a lab technician, are competing for the title of most mundane job. Romeo is pipetting blood into test tubes, preparing a radioimmunoassay to measure hormone levels in the blood plasma.

Bloss is slicing a rat brain in the cryostat, and mounting brain tissue onto electrically charged microscope slides. The brain, not much larger than Bloss' thumbnail, is white, flesh-colored, and entirely at his mercy. His gloved right hand wheels the brain 20 microns forward on its platform; his left hand pulls a lever to cut a slice of brain tissue. He is wheeling through the olfactory bulb, a protruding section of the forebrain that contains odor receptors. The olfactory bulb is not particularly important for the work he's doing. He's on his way to the paraventricular nucleus.

As they work, Romeo and Waters chat about Flamenco dancing in Miami's Little Cuba, the sleeping patterns of Waters' young daughter, and filter paper. Discussion alternates with long periods of silence, broken only by a sigh or a stretch. "This is mind numbing," Romeo says, and rolls his neck.

While at times studying the adolescent brain can be full of excitement and intrigue, the day-to-day work is a slog, requiring the same patience and endurance it took Romeo to get from the Brandenburg gate to that faraway finish line. But then that's science, a constant seesaw between the thrill and grind of discovery. Not unlike adolescence.

Romeo has accepted a tenure-track assistant professor position in Barnard College's psychology department. Starting this month, he'll have his own lab, with his own students cleaning out cages and researching adolescent stress. Over the past four years, Romeo has had eight papers and two reviews published on stress and adolescence. He has been honored with two young investigator awards for his work, one from The Society for Behavioral Neuroendocrinology in Pittsburgh, and the other from the Conference on Hormones and Brain Function in Turino, Italy.

Romeo's research now is two-pronged. He will focus on the maturation of the HPA axis during and after puberty so he can pinpoint the source of adolescent sensitivity to stress. He will also continue to study the physiologic and behavioral effects of stress on adolescent development. "That is where I really see the big thrust of my research," he says. "I'll be looking at how stress during adolescence influences the individual now and in the future."

Wedged between a computer monitor and a microscope containing tissue samples of rat hippocampus, Romeo explains that the questions that have caught and harnessed his interest are never ending. "Every question you set out to address begs five or six or 10 other questions." He leans back in his rolling desk chair and shrugs cheerfully. "Job security," he says. "You gotta love it."