ABC Transporters in Neurogenesis Figure 2. ABCC1 deficiency impairs stem cell proliferation and accelerates late neuronal differentiation. The graphs show the newly generated population (BrdU incorporation) of Sox2+ (A), DCX+ (B) and calretinin+ (C) cells in the SGZ of the DG. The number of proliferating Sox2+ NSPCs was significantly decreased in ABCB1- (225%), ABCG2- (228%) and ABCC1- (237%) deficient mice, implicating slower proliferation of NSPCs within the 7 days of BrdU treatment. In contrast, late neuronal differentiation seems to be accelerated due to the significantly greater proportion of BrdU+ postmitotic early granule cells expressing calretinin in animals lacking ABCG2 (+43%) and ABCC1 (+51%) expression. The photomicrographs display expression examples for marker combinations: Sox2/BrdU (D), DCX/BrdU (E) and calretinin/BrdU (F). Arrowheads indicate double-labeled cells. Error bar: SEM; * p,0.05; **p,0.01; Scale bar: 20 mm. doi:10.1371/journal.pone.0035613.g002 neurons in the induced hemisphere of ABCB10/0 and ABCC10/ 0 mice (229% ABCB10/0; 224 ABCC10/0) (Figure 6D). Comparing the different strains, we saw only a slight, non- significant decrease of Sox2+ progenitors in ABC transporter- deficient mice as compared to controls, indicating that the lack of ABCB1 or ABCC1 significantly reduces the number of cells differentiating into a neuronal lineage. The numbers of both DCX+ and calretinin+ cells were significantly lower in ABCB1- and ABCC1-deficient mice, while there was no difference observable in animals lacking the transporter ABCG2. The only parameter that was consistently affected in all three ABC transporter-deficient strains was the proliferation capacity. subgranular zone. Thus, we wanted to determine whether these differences also influence the density of neurons in the neocortex. To assess neuronal area in the cerebral cortex, we established a reviewer- independent method to determine the total area covered by NeuN+ cells in a normalized cortical area. Paraffin-embedded brain sections were cut and immunohistochemically stained for the neuronal marker NeuN. Brain sections were digitized at a resolution of 230 nm/pixel, and the NeuN+ areas were discriminated from unstained cortex [41] to determine the NeuN+ fraction of the cortical area (Figure 7B). Comparison of the neuronal densities did not reveal significant differences between the ABC transporter- deficient strains and genetic background controls. ABC transporter-deficiency does not affect neuronal area in the neocortex The above analyses indicate that ABC transporter-deficiency differentially influences neurogenic function in the hippocampal ABC transporter-deficiency effects cortical cytoarchitecture To assess whether loss of ABC transporter function possibly results in deficits in cortical development, which might be reflected PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35613