Discovery of novel hippocampal neurogenic agents by using an in vivo stable isotope labeling technique

Neurogenesis occurs in discrete regions of adult mammalian brain, including the subgranular zone of the hippocampus. Hippocampal neurogenesis is enhanced by different classes of antidepressants, but screening for neurogenic actions of novel antidepressants has been inefficient because of limitations of 5-bromo-2'-deoxyuridine labeling techniques. We describe an efficient in vivo method for measuring hippocampal neurogenesis involving incorporation of the stable isotope, H-2, into genomic DNA during labeling with (H2O)-H-2 ( heavy water). Male rodents received 8 to 10% (H2O)-H-2 in drinking water; DNA was isolated from hippocampal progenitor cells or neurons. Label incorporation into progenitor cells of Swiss-Webster mice revealed subpopulation kinetics: 16% divided with t(1/2) of 2.7 weeks; the remainder did not divide over 1 year. Progenitor cell proliferation rates in mice were strain-dependent. Chronic antidepressant treatment for 3 weeks, with (H2O)-H-2 administered during the final week, increased progenitor cell proliferation across all the strains tested. Fluoxetine treatment increased 2H incorporation into DNA of gradient-enriched neurons or flow-sorted neuronal nuclei 4 weeks after (H2O)-H-2 labeling, representing the survival and differentiation of newly divided cells into neurons. By screening 11 approved drugs for effects on progenitor cell proliferation, we detected previously unrecognized, dose-dependent enhancement of hippocampal progenitor cell proliferation by two statins and the anticonvulsant topiramate. We also confirmed stimulatory activity of other anticonvulsants and showed inhibition of progenitor cell proliferation by isotretinoin and prednisolone. In conclusion, stable isotope labeling is an efficient, high-throughput in vivo method for measuring hippocampal progenitor cell proliferation that can be used to screen for novel neurogenic drugs.