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David Strumfels' Comment: I t is often amazing how much good can come from things that are generally (and rightfully) regarded as evil.
Kirsty Spalding was one of the scientists who doubted that assessment. Spalding, once a postdoc at the Karolinska Institute in Stockholm, Sweden and now a professor there, knew there were tantalizing hints that the adult hippocampus—a seahorse-shaped region deep in the brain that is important for memory and learning—could regenerate neurons. But without knowing exactly when each neuron was created, scientists couldn’t say with any certainty that this was true.
Spalding and her postdoc advisor Jonas Frisén had a hunch that a pulse of radioactive carbon created by above-ground nuclear tests during the Cold War could help solve the riddle. “A geopolitical phenomenon—this Cold War bomb testing—has, in a way, put a date stamp on everything and everybody,” Spalding says. The bomb pulse has been declining since the 1963 above-ground test ban treaty, creating a sort of clock they could exploit. By determining how many radioactive carbon atoms a cell contained, Spalding and Frisén hoped they could calculate its birthdate. To test theory, they needed neurons—a lot of them.
Aboveground nuclear bomb tests, like this French test over Fangataufa Atoll, created a pulse of radioactive carbon in the atmosphere.
Spalding’s curiosity eventually leading her to a slaughterhouse on the outskirts of Stockholm. Standing outside the low, gray industrial building, she watched as horses went in one side and, about 15 minutes later, a worker appeared on the other end, holding a head, neurons and all. “It was precisely as revolting as it sounds,” she says. Spalding would then spend hours chipping away to extract the necessary cells, a grisly procedure that was just the first in a decade-long stretch of hurdles she had to surmount. “Had we known how difficult it was going to be, we never would have stuck with it,” says physicist Bruce Buchholz, one of Spalding’s co-authors and an expert on bomb pulse dating at the Lawrence Livermore National Laboratory outside San Francisco.But Spalding persevered, and her hard work eventually paid off. Last June, she published a paper in which she conclusively stated that adult human did indeed build new neurons in their brains. The human hippocampus, Spalding and Frisén discovered, was continually creating small numbers of neurons. They and their co-authors had solved one of neuroscience’s longstanding mysteries.
In the years leading up to that, Spalding and Frisén pioneered a new field of research, using the Cold War bomb pulse to answer a number of questions about human physiology, including neuron formation and lipid cycling. “It’s an amazingly powerful tool, whether you want to look at a fat cell or a brain cell,” Spalding says. Their work has been so fruitful that it could provide them with a lifetime worth of projects. But she and her collaborators can’t waste any time. By 2050, Frisén and Spalding estimate, the bomb pulse will have completely dissipated.
Bombs Away
The premise of bomb pulse dating is fairly straightforward. Most aboveground nuclear bomb testing happened between 1955 and 1963, and those detonations released untold numbers of neutrons into the atmosphere. These slammed into nitrogen atoms, causing their nuclei to eject a proton. What was once a seven-proton nitrogen became a six-proton carbon. But unlike most carbon atoms, which have six protons six and neutrons, this radioactive carbon, known as 14C, retained nitrogen’s two extra neutrons, a difference in atomic mass that is small, but measurable. Normally, only a tiny fraction of the world’s carbon is 14C, so little that scientists measure it in parts per trillion. The bomb pulse doubled this amount. While 14C concentrations are still low even after the bomb pulse, the difference is obvious to scientists who know what they’re looking for.To measure the small amounts of 14C, scientists use a technique called mass spectrometry, which sorts atoms by weight. When looking for carbon isotopes, the instrument strips carbon atoms of some of their electrons and launches them into a magnetic field, which alters each atom’s trajectory. Inertia causes heavier atoms, like 14C, to take a wider path than lighter ones. By measuring how many carbon atoms travel along the various paths, scientists can determine how much 14C is in a sample.
Atmospheric 14C spiked in 1955 and rapidly dropped off after the Limited Test Ban Treaty was signed in 1963, which banned all aboveground detonations. Without nuclear explosions producing new 14C, existing 14C began to mix with other carbon sinks, diluting its concentration in the air.
Radioactive 14C is incorporated into all living things: by plants that use carbon dioxide for photosynthesis, by the animals that eat the plants, by the animals that eat other animals that have eaten plants. To our bodies, one type of carbon is as good as any other, so 14C is used when building our cells, our proteins, and our DNA.
A cell’s DNA reflects the amount of 14C in the atmosphere at the time it was made. (For plants, that’s shortly after they fixed CO2 containing 14C; for animals, it’s when they ate a plant or animal containing 14C.) Scientists measure the amount of 14C in DNA because, while other molecules are frequently refreshed throughout a cell’s life, DNA remains constant. By determining the age of the DNA, researchers can determine when, exactly, a cell was created.
The Great Debate
Neuroscience dogma had long dictated that the adult human brain did not create any new neurons. The only time neuron numbers could increase was thought to be during fetal development and early childhood. Once the peak number was reached—usually around age four—it was all downhill. But by the late 1960s and early 1970s, rodent studies led some experts like Fred Gage, a neuroscientist at the Salk Institute in La Jolla, California, to question this notion.“We found stem cells in the hippocampus of adult mice and rats that could create new neurons,” Gage says. It was groundbreaking work, but at the time not everyone was convinced of its importance. “In order for this to have significance, we needed to know whether this occurred in humans.”
To identify dividing cells in mice and rats, Gage had been using a molecule known as bromodeoxyuridine, or BrdU. BrdU is a synthetic nucleoside that can be incorporated into newly synthesized DNA in place of the standard nucleoside thymidine, the T in ATCG. Instead of thymidine’s standard methyl group, BrdU has a bromine atom, a small molecular difference which researchers can detect. If a cell divides shortly after BrdU is injected into the body, the DNA of the new cells will contain BrdU.
DNA in new cells in a rat hippocampus is labeled here in green.
At that time, in the mid-1990s, BrdU was mainly used in humans as a cancer diagnostic agent to determine which parts of a tumor were rapidly dividing. Gage saw this as an opportunity to test his hypothesis, so he obtained the brains of recently deceased cancer patients who had been treated with BrdU, checking if there were neurons in the hippocampus that contained the chemical. There were. “This was the first evidence that there were dividing stem cells in an adult brain, and that these cells could give rise to new neurons in the hippocampus,” Gage says. He published his data in a 1997 paper in Nature Neuroscience.Although Gage felt he had settled the debate, not all scientists agreed. Some disputed his results because they came from cancer patients, whose bodies contained cells that weren’t behaving normally. “The reception was pretty rocky in the early days,” Gage says. “It was hard to accept that this could happen in mice and rats, not to mention people.”
Across the Atlantic, in his Stockholm lab, Frisén read Gage’s paper with interest. He, too, had believed that humans could grow new neurons, but never had the evidence to back up his hunch. BrdU was potentially toxic and carcinogenic—no research safety committee was going to allow its use on healthy subjects.
A rat hippocampus as seen through an ultra-widefield high-speed multiphoton laser microscope
Frisén initially turned to radiocarbon dating, which archaeologists use to determine the age of ancient artifacts. At best, however, the technique could only date something to within one hundred years—not nearly sensitive enough for what he needed. In his literature search, however, he stumbled across a variation on the technique that also used 14C. No one had used it to answer major questions in biology, though. “It was a high-risk project,” Frisén says. “I talked to all of the postdocs in my lab, and Kirsty was the only person willing to take on this research.”Hopes and Isotopes
One of the first significant biological applications of bomb pulse dating came in forensic science back in the early 1990s. Detectives in Vienna had found two wealthy, elderly sisters dead in an apartment in 1992, and they called on University of Vienna physicist Walter Kutschera to help to solve the mystery. To disperse the women’s substantial estates and life insurance payouts, police needed to know which had died first or if they died at the same time. The detectives had heard about bomb pulse dating, and Kutchera was a resident expert. Could he help, the police asked? Kutschera agreed. “We focused on the cells that renewed quickly, like skin and hair. If you measure something that is renewed very regularly, you can determine when that person died,” he says.Working with forensic experts, Kutschera found that the newest cells in one of the sisters dated from 1988. Sister B, however, had cells that were born in 1989. The detectives ultimately concluded that Sister A had died at home while Sister B continued to live in the same apartment as her sister’s dead body. One or two years later, Sister B also died. Since Sister B was the last alive, the detectives could disperse the sisters’ estates according to Sister B’s wishes.
Other forensic teams soon began taking advantage of bomb pulse dating. Detectives could determine date of birth and death for John and Jane Does, helping them put a name to a body. It would occasionally solve the case; other times it would give distraught relatives an answer to what had happened to their missing loved one.
The forensics technique was also used to identify victims the deadly Indian Ocean tsunami in 2004. A large number of Swedish tourists had been in the tsunami’s path, and although officials knew who had gone missing, they were having trouble matching names with the bodies. The Swedish government asked Frisén and Spalding if they could use their expertise in bomb pulse dating to make the identifications. The experience emphasized for Frisén and Spalding how useful the technique could be. “This work actually spurred us on to keep going with some of our biological research,” Frisén says.
Fat Chances
Spalding’s work on the tsunami victims bolstered her confidence that 14C dating could, in fact, accurately date not just individuals, but specific populations of cells in the body. Fortunately, she had already teamed up with Buchholz, the bomb pulse expert at Lawrence Livermore, to fine tune her technique. She then cast her net wide, dating intestinal, skeletal, brain, and blood cells, publishing the first results in July 2005 in Cell. Some cells, such as those in the cerebellum, were just three years younger than the person itself. Blood cells, on the other hand, were very young, confirming existing data about their rapid turnover. In the middle were intestinal cells, which Spalding found had an average lifespan of about 11 years.These results were crucial, Buchholz notes, because it showed the method would work in humans. “Before this study, it seemed bomb pulse dating was more of a curiosity question. People would mostly just say ‘Oh, yeah. We see it in people.’ It wasn’t used as a tool to do biology until Kirsty and Jonas just took this idea and ran with it,” Buchholz says.
To Spalding, those first experiments were satisfying, but merely a step toward her goal. Once she proved the tool worked, she set out to use it. Her first target was fat. “It has very important endocrine functions,” says biologist Philipp Scherer from the University of Texas Southwestern Medical Center in Dallas. “Adipose tissue communicates with other organs using the hormones it creates. As you go from lean to obese, the fingerprint of these hormones changes. The bad ones go up and the good ones go down, so it’s important to understand the basic physiology of these cells.”
Spalding wanted to know whether fat cells were replaced more or less quickly in obese individuals compared with their leaner counterparts. This, she felt, might help explain not only why it’s so hard for many people to lose weight and keep it off, but also some of the health complications that occur in people with excess body fat. Spalding found that the age of cells in both obese and lean people were the same: around 10 years. Her calculations showed that, each year, around 8% of an adult’s fat cells die and are replaced by new cells, a rate that didn’t vary according to body mass index. But the story was different in children: obese kids added new fat cells much more quickly than their old cells died.
Spalding also dated the fats inside adipose cells. She found that throughout a fat cell’s lifetime, lipids were replaced six times, on average. The individuals with the oldest lipids—and thus the slowest turnover rate—were more likely to be insulin resistant. The finding could pave the way for treatments that target the rate of lipid turnover to improve the health of obese individuals, even if it didn’t result in weight loss. “This technique is elegant and useful,” Scherer says. “Targeting fat cell turnover is where the field will have to go in order to get ahold of the obesity and Type 2 diabetes death spiral.”
Results at Last
After years of trial and error, tweaks, and fine tuning, Spalding was finally ready to test her and Frisén’s theory that human brains generated new neurons. The barriers, though, were still significant. Spalding first had to separate hippocampal neurons from the non-neuronal support cells that surrounded them. This took more than a year. She also had to isolate enough of their DNA—typically, she found only one 14C atom for every twelve to fourteen cells. Most detection methods required a full milligram, but Spalding realized she could only isolate a few micrograms of material. She spent a year and a half optimizing her DNA extraction technique, yet it still wasn’t enough.Frustrated, Spalding turned to another brain region that had been hotly debated in neurogenesis circles—the olfactory bulb. For many mammals, smell is the dominant sense, and scientists had discovered newly formed cells in this region in mature animals. In humans, the olfactory bulb is bigger than the hippocampus, and it contains more cells and more DNA. Unfortunately, her results didn’t mirror findings in animals. Humans, she published in Neuron, did not create new cells in the olfactory bulb.
That wasn’t the end, though. Not long after Spalding published the Neuron study, she received word that a group of scientists at Uppsala University in Sweden had developed a new technique, a more sensitive one that could measure her tiny hippocampal samples. Here was new hope, though Spalding was still skeptical. “With each step, I thought I had it, I thought ‘This is great! I’m finally there,’ ” she says, “and then there would be another problem.”
This time, however, was the exception. The determined scientist had finally cleared her last hurdle. The results were conclusive.
Using 55 brains of individuals between the ages of 19 and 92, Spalding separated the hippocampal neurons and isolated their DNA. Buchholz then took the DNA and assayed their 14C ratio. Their results showed that the average hippocampal neuron was only 20-30 years old, even in people who were more than 90 years old. Each day, the researchers estimated, the human brain creates 700 new hippocampal neurons. To the research community, the results were clear: neurogenesis occurs in humans, even long into adulthood. “We found a lot more hippocampal neurogenesis than anyone really expected,” Frisén says.
After years of work and apparent dead ends, Spalding and Frisén finally had their answer. For some, that would be the end of it. But with the bomb pulse expected to dissipate by 2050, their real work is only just beginning.
In the meantime, though, they can revel in overturning one of neuroscience’s long-held tenets. “It presents a whole new picture on our view of the brain,” Gage says of Spalding and Frisén’s research. “It’s not this static structure that just declines with age. Now we see the brain very differently.”
