Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat

Abstract

Bromodeoxyuridine (BrdU) is broadly used in neuroscience to study embryonic development and adult neurogenesis. The potential toxicity of this halogenated pyrimidine analogue is frequently neglected. In this study, we administered BrdU in small doses by the progressively delayed cumulative labeling method to immunocytochemically tag different cerebellar cell types with antibodies to specific markers and BrdU in the same section. The well-known structure of the cerebellum made it possible to ascertain several toxic effects of the treatment. Time-pregnant rats were given five or six injections of 5 or 6 mg of BrdU ( approximately 12-20 mg/kg) at 8-hour intervals over 2 successive days between day 11 and 21 of pregnancy (E11-E12 to E20-E21), and the adult progeny was processed by immunocytochemistry. We demonstrate that this treatment effectively labeled distinct cerebellar cell populations but produced striking defects in the proliferation, migration, and settling of the Purkinje cells; reduced the size of the cerebellar cortex and nuclei; produced defects in the patterning of foliation; and also affected litter size, body weight, and mortality of the offspring. The observed toxic effects were consistent within individual treatment groups but varied between different treatment groups. Treatment with BrdU at the peak of neurogenesis of cerebellar projection neurons (E14) produced the most severe malformations. We observed no overt effects on the timing of neurogenesis for cerebellar neurons and glia across experimental groups. In conclusion, BrdU is a useful tool to study neural development, but its cytotoxicity represents a serious pitfall particularly when multiple doses are used to label cells.

Different Types of Cerebellar GABAergic Interneurons Originate from a Common Pool of Multipotent Progenitor Cells

“…Together with previous birthdating studies (Altman and Bayer, 1997;Sekerková et al, 2004a;Yamanaka et al, 2004), our present analyses indicate that cerebellar GABAergic interneurons are generated between E13 and P15. Within this time window, the different phenotypes are produced during overlapping periods, according to an inside-out sequence: first in the deep nuclei, then in the granular layer, and, finally, in the molecular layer.…”

“…Although birthdating studies indicate that all nuclear neurons are generated at the beginning of cerebellar neurogenesis [i.e., between E13 and E15 in the rat Bayer, 1978, 1997;Sekerková et al, 2004a)], other reports suggest that local inter- 9c 14 0 0 0 14 7c 62 0 0 0 62 10c 525 1 0 0 526 2c 492 3 0 0 495 11c 299 0 0 1 300 3c 513 0 0 0 513 1c 279 0 0 0 279 6c 417 0 0 0 417 4c 439 0 0 0 439 5c 101 0 0 0 101 8c 582 0 0 0 582 tot 2239 1 0 1 2241 tot 1484 3 0 0 neurons have a different origin than projection neurons (Hoshino et al, 2005;Wang et al, 2005;Fink et al, 2006) and may be also generated at later stages (Maricich and Herrup, 1999). Therefore, as a preliminary step for our study, we reexamined deep nuclei neurogenesis to define the time window in which local interneurons are born.…”

Cell Proliferation in the Forebrain and Midbrain of the Adult Bullfrog, <i>Rana catesbeiana</i>

“…A higher dose was thus employed to maximize the amount of label at long survival times. Although BrdU has toxic and mutagenic effects when administered to pregnant or developing mammals [Kolb et al, 1999;Sekerková et al, 2004], no such effects have been observed in adult mammals administered doses as high as 600 mg/kg [Cameron and McKay, 2001]. We observed no obvious toxic effects of or behavioral abnormalities resulting from the high doses of BrdU administered to our ex-perimental animals.…”

Programmed Cell Death of Adult-Generated Hippocampal Neurons Is Mediated by the Proapoptotic Gene Bax

“…Some animals received a single BrdU injection of 200 g/gm body weight. This dose of BrdU is reported to more completely label DNA synthesizing cells in situ (Cameron and McKay, 2001), although high concentrations of BrdU have been reported to be toxic (Sekerkova et al, 2004). Using this protocol, we again obtained no apparent difference in the number of BrdU ϩ cells between WT and Bax-KO mice (3150 cells in WT mice, and 2870 cells in Bax-KO mice; n ϭ 1 in each group).…”