In this two-part series on the thymidine analog BrdU, we tell the story of how BrdU revolutionized the field of neurogenesis as well as its recently identified limitations as a lineage tracer for neurogenesis studies.
Up until the mid-1960s, scientists believed that the generation of functional neurons from neural precursors (neurogenesis) only occurred during the embryonic and peri-natal stages in mammals. Essentially, it was thought that loss of neurons was an irreversible process in the adult brain. Altman and Das (1965) provided the first evidence of adult neurogenesis in post-natal rats using autoradiographic analysis of [3H] thymidine incorporation into DNA during the S phase of cell growth. However, it was still unclear whether new neurons were produced in the brains of adult humans.
By the 1990s, scientists had begun studying DNA replication using non-autoradiography techniques. Most predominant was the use of bromodeoxyuridine (BrdU), an artificial nucleoside that substitutes for thymidine during DNA replication. Although it is a thymidine analog, BrdU is a halopyrimidine with a different molecular structure than thymidine (see image below). It allows faster detection of DNA synthesis compared to use of [3H] thymidine. In addition, unlike labeled thymidine, it can easily be detected by immunohistochemical methods, which also allow the design of multiplex IHC experiments by simultaneously staining for neuronal stem cell markers such as Nestin and SOX2 (Gratzner 1982).
In clinical settings, BrdU has been used to monitor cancer progression (Waldman et al. 1988). To evaluate tumor growth, BrdU is injected into a patient’s bloodstream. A biopsy of the tumor is then taken and an anti-BrdU antibody is used to detect cells that have incorporated BrdU into their DNA. The ratio of BrdU positive cells to total tumor cells gives an indication of the rate of cell growth.
Neuroscientist Professor Fred Gage of the Salk’s Research Institute later adapted this application of BrdU to neuroscience by hypothesizing that BrdU could be used to birthdate individual cells; he reasoned that the chemical would be incorporated into new but not old brain cells (Eriksson et al. 1998). He further speculated that if BrdU was incorporated into the tumor after injection into the bloodstream, the chemical was also likely incorporated into brain cells. To test their hypothesis Prof. Gage and his research team acquired post-mortem brain samples from cancer patients administered BrdU and set out to answer the question of whether neurogenesis occurs in adult human brains.
Using immunofluorescent labeling of BrdU and the neuronal markers NeuN, calbindin and neuron specific enolase (NSE), Prof. Gage and his team determined that new neurons were generated from dividing progenitor cells in the dentate gyrus of adult humans (Eriksson et al. 1998). They also demonstrated that the human hippocampus retains its ability to generate neurons throughout life (Eriksson et al. 1998).
These findings overturned the long standing dogma that neurogenesis did not occur in adults. The neurogenesis field subsequently took off, and BrdU is now routinely used in neurogenesis studies as a lineage tracer. As a result, BrdU has contributed significantly to our understanding of the timing and pattern of neurogenesis. It is currently the research tool of choice for neurogenesis studies; however, recent studies indicate off-target effects that should be carefully controlled for in your experimental design (Lehner et al. 2011).
Stay tuned to our next blog post where we discuss the “dark side of BrdU” and how you can carefully control your BrdU experiments for accurate data analysis (Lehner et al. 2011).
In the meantime, check out our mouse anti-BrdU antibody (clone Bu20a), which has been used in several neuroscience publications.
In addition to its wide application in neuroscience, the antibody is also suitable for monitoring BrdU incorporation by flow cytometric analysis.
In the image below, we analyzed human BrdU-labeled lymphoma cells using our mouse anti-BrdU antibody, clone Bu20a (MCA2483). The image shows cell cycle profiles, BrdU positive cells are in the S-phase of the cell cycle. For more information about studying the incorporation of BrdU by flow cytometry, check out the respective chapter in our Flow Cytometry Basics Guide.
Don't forget to view the next post in this two-part series, which describes how to control your BrdU experiments.