Abstract

Methodological differences may explain discrepancies in adult mammalian neurogenesis in the brain outside the widely accepted neurogenic regions, i.e. hippocampus and olfactory bulb/subventricular zone. Here, we describe a method to dissolve and administer bromodeoxyuridine (BrdU) at high concentrations (150mg/mL) into the adult mouse brain to demonstrate neuronal incorporation of this thymidine analogue in CNS regions with a low rate of neurogenesis. The dosage and duration of BrdU appear critical, since exposure to low doses did not result in a robust label using this marker for proliferation [Zhao et al., 2003. Proc Natl Acad Sci USA 100; 7925]. Mice (five per cage in an enriched environment) received BrdU in the right cerebral ventricle from a subcutaneous osmotic pump (0.9mg/day, 3 weeks, infused at 0.25microL/h). To avoid labelling of cells with a fast turnover, the mice were allowed to survive 3 weeks after ending the BrdU delivery. Midbrain sections were processed for tyrosine hydroxylase (TH) immunohistochemistry, post-fixed with 4% paraformaldehyde, denaturated with 2N HCl and 0.025% pepsin, followed by immunolabelling of nuclear BrdU in single-stranded DNA. Double-labelled cells were analysed in a confocal laser microscope and showed segmented nuclear BrdU-label surrounded by TH-immunoreactive cytoplasm, never displaying apoptotic morphological features. Nigral neuronal proliferation was confirmed using another marker [(3)H]thymidine, delivered via an intraperitoneal osmotic pump. The protocol was well tolerated by the mice and not found to be toxic for the region studied, i.e. did not alter the total number of nigral neurons identified with TH/cresyl violet immunohistochemistry.