Dating the Birth of Human Cells—Carbon 14 Runs Rings around Competition


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Dating the Birth of Human Cells—Carbon 14 Runs Rings around Competition

15 July 2005. Analysis of growth rings from pine trees in Sweden shows that the proliferation of atomic tests in the 1950s and 1960s led to an explosion in levels of atmospheric carbon 14. Now, Jonas Frisen and colleagues at the Karolinska Institute in Stockholm have taken advantage of this spike in C14 to devise a method to date the birth of human cells. Because this test can be used retrospectively, unlike many of the current methods used to detect cell proliferation, and because it does not require the ingestion of a radioactive or chemical tracer, the method can be readily applied to both in vivo and postmortem samples of human tissues. In today’s Cell, Frisen and colleagues report how they used the dating method to dismiss the possibility that neurogenesis takes place in the adult human cortex.

Because of its extremely long half-life (over 5,000 years), carbon 14 content has typically been used to date only very old artifacts or fossils. The method has traditionally failed to resolve dates of samples that differ in age by less than a few hundred years—accurate enough perhaps to date the youngest and oldest parts of the most ancient redwood trees, but not to tell how many newborn cells might be present in the human brain. But the almost tenfold increase in atmospheric C14 that peaked around the mid-1960s has been followed by a rapid decline since the nuclear test ban treaties and the cessation of high-yield, above-ground nuclear tests. In fact, C14 is assimilated so rapidly that from about 1963, its half-life in the atmosphere has only been about 11 years. Current atmospheric C14 is about twice the level it was before the 1950s.

First author Kristy Spalding and colleagues capitalized on this relatively rapid decline in C14 to develop their dating method. The authors first established that there is a relationship between the C14 content of DNA and the atmospheric C14 in the local area when that DNA was made. Unlike many other macromolecules in a cell, DNA is chemically stable once laid down, so its C14 levels are not expected to change even if the DNA ages. Indeed, when Spalding and colleagues examined samples that dated prior to or after the proliferation of atmospheric nuclear tests, they found that the C14 content in the DNA correlated with the predicted atmospheric C14 at the time the DNA would have been synthesized. Next, examining samples from single individuals born after the test ban treaty went into effect, the authors found that the C14 content in DNA isolated from the cerebellum, cortex and intestine were, as would be expected, not the same age. Cerebellar DNA was a few years older than the DNA in the cortex, which in turn was about 12 years older than the DNA in the small intestine. This matches the pattern that would be expected when one considers the time taken for development of the human brain and the relatively rapid turnover of epithelial cells in the intestine. The authors estimated that using their method, they can put an age on human cells to within +/- 2 years.

Turning to individual cell types rather than tissues, Spalding and colleagues set about to determine if cortical neurons are as old, or perhaps younger, than the individual. Ever since it was shown that neurogenesis takes place in hippocampus of the adult brain (see ARF related news story), an outstanding question has been whether or not cortical neurons can be replenished in adults. To tackle this question, the authors isolated neuronal and non-neuronal nuclei from cerebral cortex tissue samples. While they found that total DNA C14 levels in these samples are younger than the donor, indicating cell turnover, they found that the cortical neurons are always as old as the individual donor. This was true for people born prior to, during, or after the spike in atmospheric C14.

The authors suggest that previous reports of adult cortical neurogenesis might be technical artifacts. But they also state that their method is only sensitive enough to detect a minimum of 1 percent of newborn cells in a given cell population, so that leaves open the possibility that there is some, albeit a very small amount, of newborn cells in the cortex.

As Paola Arlotta and Jeffrey Macklis from the Harvard Medical School write in an accompanying Cell perspective, historically, methods to label newborn cells, such as the use of tritium, bromodeoxyuridine (BrdU), or other halogenated urides, are toxic and cannot be used in humans, so the strategy developed by Spalding and colleagues “enables a more direct understanding of cell turnover, aging, and lifespan throughout the human body and those of other long-lived animals.”

There is one small caveat, however. Because atmospheric C14 levels are falling relatively quickly, the method will decrease in sensitivity with time, so the period during which it will be useful is limited and people born around the time of the nuclear tests will remain the most suited for study. Nevertheless, as Spalding and colleagues point out, there are plenty of tissue banks with archived material which will always be useful.—Tom Fagan.

References:
Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, Frisen J. Retrospective birth dating of cells in humans. Cell. 2005;122:133-143. Abstract

Arlotta P, Macklis JD. Archeo-cell biology: Carbon dating is not just for pots and dinosaurs. Cell. 2005;122:4-6. Abstract

	Comments on News and Primary Papers

	Comment by: Elizabeth Gould
	Submitted 20 July 2005 \| Permalink	Posted 20 July 2005
	About 40 years ago, Joseph Altman and his colleagues used the 3H-thymidine autoradiographic method to birthdate cells in the brains of adult rats and cats, and reported evidence for neurogenesis in the olfactory bulb, hippocampus, and neocortex (1-5). These findings were corroborated and extended by Michael Kaplan and his colleagues by combining 3H-thymidine autoradiography with electron microscopy (16-19). Despite this rather large body of work, the findings were not well-received by the neuroscience community, and adult neurogenesis in the mammalian brain remained a matter of debate (17,27, 28). In the late 1990s, the use of a newer technique, bromodeoxyuridine (BrdU) labeling, in combination with confocal microscopy, led to new evidence in support of this phenomenon and the acceptance of adult neurogenesis in the olfactory bulb and the hippocampus (11,13,14,24). These two areas have been named “neurogenic” because they are now widely believed to support adult neurogenesis. However, similar evidence has been reported for “non-neurogenic” brain regions, including the neocortex,... Read more About 40 years ago, Joseph Altman and his colleagues used the 3H-thymidine autoradiographic method to birthdate cells in the brains of adult rats and cats, and reported evidence for neurogenesis in the olfactory bulb, hippocampus, and neocortex (1-5). These findings were corroborated and extended by Michael Kaplan and his colleagues by combining 3H-thymidine autoradiography with electron microscopy (16-19). Despite this rather large body of work, the findings were not well-received by the neuroscience community, and adult neurogenesis in the mammalian brain remained a matter of debate (17,27, 28). In the late 1990s, the use of a newer technique, bromodeoxyuridine (BrdU) labeling, in combination with confocal microscopy, led to new evidence in support of this phenomenon and the acceptance of adult neurogenesis in the olfactory bulb and the hippocampus (11,13,14,24). These two areas have been named “neurogenic” because they are now widely believed to support adult neurogenesis. However, similar evidence has been reported for “non-neurogenic” brain regions, including the neocortex, striatum, amygdala, hypothalamus, and substantia nigra (6,7,9,12,30,32). One of the most disputed claims is that of adult neurogenesis in the neocortex. We and others have reported positive evidence for adult-born neurons in the neocortex of rat and monkey (7,9,13,15). However, others have not corroborated these findings (20,22,23), or have found evidence for adult neurogenesis only under conditions of damage (21,25,31). Methodological flaws have been proposed as explanations for putative false positive and false negative data on this subject (13,26). The development and application of new methods is clearly needed to resolve the debate about adult neurogenesis in neocortex and other “non-neurogenic” brain regions. The recent publication in Cell by Spalding and colleagues (29) reported the use of a novel and highly innovative method to search for adult neurogenesis in human postmortem brain tissue. This method was designed with the following facts in mind: 1) Levels of atmospheric C14 were very high during the time of intensive nuclear weapons testing (in the mid 1950s to early 1960s); and 2) carbon atoms in the DNA of cells do not turn over. Thus, the relative content of C14 in a population of neurons, particularly those generated during or immediately after the weapons testing, can be used to birthdate the cells. This method can be used for examining cell populations, but not individual cells, because C14 incorporation into DNA is a rare event, even at the time when C14 atmospheric levels were very high. Nonetheless, for large homogeneous populations of cells, an average age can be estimated. In tissues comprising multiple cell types, cells can be sorted and C14 levels can be assayed in subpopulations. Using this approach, the average age of cells in two regions of the adult human brain autopsy tissue—the cerebellum and occipital cortex—were estimated. The authors found that cells in the occipital cortex were younger than cells in the cerebellum, but when neurons and non-neurons were separated, the occipital cortex neurons were the oldest, almost as old as the individual. Thus, the findings support the view that cortical neurons are generated during development. The authors leave open the possibility that adult neurogenesis occurs in brain regions that were not examined in the study. While it might appear that these findings rule out the possibility of adult neurogenesis, at least in the occipital cortex of humans, there are some important points to consider. The first is whether or not this method is sufficiently sensitive to detect adult neurogenesis in any brain region. This can be tested by examining an area where this phenomenon is well-established and occurs at a relatively high rate. For the human brain, there is only one such area—the hippocampus. In 1998, Eriksson and colleagues reported evidence for adult neurogenesis in the hippocampus of adult human cancer victims, using BrdU labeling (11). Spalding and colleagues (29) did not examine the hippocampus in the present study, so this issue will require further experimentation. The second question is whether a substantially lower rate of adult neurogenesis could be detected with this method. Studies reporting new neurons in putatively “non-neurogenic” brain regions indicate that the rates of neuronal addition are very low relative to those in the hippocampus (8,9,15,32). Since carbon dating does not allow for the birthdating of individual cells, a relatively large proportion of adult-born neurons would be necessary to detect an average age difference. The authors suggest that detectability limits are about 1 percent for their method (29), so one out of 100 neurons must be produced in adulthood to find adult neurogenesis in a given region. If adult-generated neurons survived for long periods of time, accumulating over decades, then the 1 percent detectability limit may be sufficient. However, studies in animals report that adult-generated neurons have a relatively short lifespan and many new neurons appear to die shortly after their production (10,15). This would preclude the ability to detect an accumulation of adult neurogenesis over a very long period of time with a method that cannot examine individual cells. In summary, the paper by Spalding and colleagues (29) presents a creative and novel approach to investigating the age of neurons in the brains of humans born around the time of nuclear bomb testing. Further characterization and refinement of this method will undoubtedly provide useful information about brain development and function. In the meantime, we can conclude that most, if not all, neurons in the occipital cortex of humans are generated during development. The possibility for adult neurogenesis in this area remains only if its rate is exceedingly low and the new neurons do not have a lengthy lifespan. References: 1. Altman J, Das GD. (1965a) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124: 319-335. Abstract 2. Altman J, Das GD. (1965b) Post-natal origin of microneurones in the rat brain. Nature. 207:953-956. Abstract 3. Altman J. (1962) Are new neurons formed in the brains of adult mammals? Science. 135:1127-1128. Abstract 4. Altman J. (1963) Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat Rec. 145:573-91. Abstract 5. Altman J. (1969) Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol. 137:433-457. Abstract 6. Bedard A, Cossette M, Levesque M, Parent A. (2002) Proliferating cells can differentiate into neurons in the striatum of normal adult monkey. Neurosci Lett. 328:213-216. Abstract 7. Bernier PJ, Bedard A, Vinet J, Levesque M, Parent A. (2002) Newly generated neurons in the amygdala and adjoining cortex of adult primates. Proc Natl Acad Sci U S A. 99:11464-11469. Abstract 8. Cameron HA, McKay RD. (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol. 435:406-417. Abstract 9. Dayer AG, Cleaver KM, Abouantoun T, Cameron HA. (2005) New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors. J Cell Biol. 168:415-427. Abstract 10. Dayer AG, Ford AA, Cleaver KM, Yassaee M, Cameron HA (2003) Short-term and long-term survival of new neurons in the rat dentate gyrus. J Comp Neurol 460: 563-572. Abstract 11. Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH. (1998) Neurogenesis in the adult human hippocampus. Nat Med. 4:1313-1317. Abstract 12. Fowler CD, Johnson F, Wang Z (2005) Estrogen regulation of cell proliferation and distribution of estrogen receptor-alpha in the brains of adult female prairie and meadow voles. J Comp Neurol 489: 166-179. Abstract 13. Gould E, Gross CG (2002) Adult neurogenesis: some progress and problems. J Neurosci 22:619-623. Abstract 14. Gould E, Reeves AJ, Fallah M, Tanapat P, Gross CG, Fuchs E (1999b) Hippocampal neurogenesis in adult Old World primates. Proc Natl Acad Sci U S A 96:5263-5267. Abstract 15. Gould E, Vail N, Wagers M, Gross CG (2001) Adult-generated hippocampal and neocortical neurons in macaques have a transient existence. Proc Natl Acad Sci U S A. 98:10910-7. Abstract 16. Kaplan MS, Hinds JW (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science 197: 1092-1094. Abstract 17. Kaplan MS (2001) Environmental complexity stimulates visual cortex neurogenesis: death of a dogma and a research career. Trends Neurosci 24: 617-620. Abstract 18. Kaplan MS, Bell DH (1984) Mitotic neuroblasts in the 9-day old and 11- month old rodent hippocampus. J Neurosci 4: 1429-1441. Abstract 19. Kaplan MS (1981) Neurogenesis in the 3-month-old rat visual cortex J Comp Neurol 195: 323-338. Abstract 20. Kodama M, Fujioka T, Duman RS. (2004) Chronic olanzapine or fluoxetine administration increases cell proliferation in hippocampus and prefrontal cortex of adult rat.Biol Psychiatry. 56:570-580. Abstract 21. Kokaia Z, Lindvall O. (2003) Neurogenesis after ischaemic brain insults. Curr Opin Neurobiol. 13:127-32. Abstract 22. Koketsu D, Mikami A, Miyamoto Y, Hisatsune T (2003) Nonrenewal of neurons in the cerebral cortex of adult macaque monkeys. J Neurosci 23: 937-942. Abstract 23. Kornack DR, Rakic P (2001) Cell proliferation without neurogenesis in the adult primate neocortex. Science 294: 2127-2130. Abstract 24. Kuhn HG, Dickinson-Anson H, Gage FH. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci. 16:2027-33. Abstract 25. Magavi SS, Leavitt BR, Macklis JD. (2000) Induction of neurogenesis in the neocortex of adult mice. Nature. 405:951-955. Abstract 26. Rakic P (2002) Adult neurogenesis in mammals: an identity crisis. J Neurosci 22: 614-618. Abstract 27. Rakic P (1985) Limits of neurogenesis in primates. Science 227: 1054-1056. Abstract 28. Rakic P (1985) DNA synthesis and cell division in the adult primate brain. Ann NY Acad Sci 457: 193-211. Abstract 29. Spalding K, Bhardwaj RD, Buchholz BA, Druid H, Frisen J. (2005) Retrospective birth dating of cells in humans. Cell. 122:133-143. Abstract 30. Xu Y, Tamamaki N, Noda T, Kimura K, Itokazu Y, Matsumoto N, Dezawa M, Ide C. (2005) Neurogenesis in the ependymal layer of the adult rat 3rd ventricle. Exp Neurol. 192:251-264. Abstract 31. Yoshimi K, Ren YR, Seki T, Yamada M, Ooizumi H, Onodera M, Saito Y, Murayama S, Okano H, Mizuno Y, Mochizuki H (2005) Possibility for neurogenesis in substantia nigra of parkinsonian brain. Ann Neurol 58: 31-40. Abstract 32. Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci 100: 7925-7930. Abstract View all comments by Elizabeth Gould

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