nNOS deficiency-induced cell proliferation depletes the neurogenic reserve.

The consequences of nitric oxide synthase (NOS) gene knockout on proliferation, survival and differentiation of neuronal precursors in the subgranular (SGZ) and subventricular (SVZ) zones were analyzed. Comparative studies were performed in neonatal, adult and old (18-month) wild-type (WT), nNOS, eNOS, and iNOS knockout (KO) mice. Effects on brain cell proliferation were studied by sacrificing animals at 24h after injecting BrdU, while effects on survival and differentiation of dividing brain cells were studied by sacrificing other animals at three weeks after injections and double immunostaining with cell phenotype-specific antibodies. In the neonatal SGZ, cell proliferation was higher than at any other age, with a significantly decreased level in eNOS-KO mice. In the neonatal SVZ, cell proliferation in each of the three NOS-KO strains was significantly lower than in WT. In the adult, in both the SGZ and SVZ, all strains showed lower levels of cell proliferation than in neonates. Thereby, the significant highest cell proliferation was found in the SGZ and SVZ of nNOS-KO mice. In the SGZ and SVZ of old mice, in each strain, BrdU-positive cell counts were further reduced from adult levels, whereby cell proliferation of nNOS-KO mice attained the most massive reduction (in the SGZ almost to zero). In adult animals sacrificed 21 days after BrdU injections, values of BrdU-/NeuN-positive cells in all knockout animals were the same as WT, indicating that the initial cell proliferation changes were not sustained or translated into neuronal differentiation. The effect of nNOS-KO, inducing cell proliferation only temporarily, consists with the concept that neuronal stem cells have a finite proliferation capacity.