Figure 5. Neurogenesis is induced by controlled cortical impact (CCI) trauma. The photomicrographs show the SGZ of the DG after CCI (LEFT: Control hemispheres; RIGHT: hemispheres after CCI). Shown are the marker combinations Sox2/BrdU (A), DCX/BrdU (B) and calretinin/BrdU (C). Arrowheads indicate double labeled cells. Scale bar: 20 mm. doi:10.1371/journal.pone.0035613.g005 their overall activity and changes in spontaneous alternation behavior using the Y-maze. We did not detect significant effects of transporter deficiency on alternation behavior, but the activity of ABCB10/0 or ABCC10/0 mice was significantly reduced as detected by the shortened running distance (Figure 8A) and reduced number of arm entries (Figure 8B). Specifically, ABCB1 deficiency led to a 13% decrease in the running distance and a 16% decrease in arm entries, and these effects were even more pronounced in ABCC1-deficient mice (running activity 217%, arm entries 223%). The elevated plus maze was used to assess anxiety levels in the mice. The basic principle of this maze is that the exploratory drive is equal for each arm, while the anxiety-driven aversion to enclosed arms is lower than to open arms, resulting in an increased exploration of the enclosed arms [47]. Analyzing the time spent in the open arms revealed a significant decrease in ABCB10/0 or ABCG20/0 mice (244% ABCB10/0; 231% ABCG20/0) (Fig- ure 9A), while the decline did not reach statistical significance in ABCC10/0 mice. All transporter deficient mice remained longer in the enclosed compartments of the maze (+43% ABCB10/0, +24% ABCG20/0, +32% ABCC10/0) (Figure 9B). Regarding the running velocity in the open compartment, we could not find any significant alterations (Figure 9C). In contrast, a significant reduction in running speed within the enclosed compartments was measured in ABCB10/0 and ABCC10/0 mice (223% ABCB10/0; 214% ABCC10/0), but not in ABCG20/0 mice (Figure 9D). The light/dark box experiments allowed us to assess the overall exploratory behavior as in an open-field test (running distance, rearing, transitions) as well as anxiety like behavior (light ABC Transporters in Neurogenesis avoidance, differing activity under light and dark). Comparing the distances travelled by mice within the open compartment of the maze, no significant alterations were observed with regard to ABC transporter-deficiency (Figure 10A). However, mice deficient for ABCB1 showed increased activity within the dark compart- ment of the maze (Figure 10B), as indicated by increased running distance (+64%). This finding is consistent with the significantly increased proportion of time spent in the dark compartment compared to controls (Figure 10C). A lower number of transitions between the two compartments showed impairments in explor- atory behavior of both ABCB1 and ABCC1 transporter-deficient animals. The number of transitions was significantly decreased by 59% (ABCB10/0) and 49% (ABCC10/0) compared to controls (Figure 10D). Rearing activity was differentially altered within the different maze compartments. While ABCC1-deficient mice showed significantly decreased rearing activity in the open-field compartment (267%; Figure 10E) and no difference in the dark compartment (Figure 10F), mice lacking ABCB1 did not show alterations in rearing activity in the open field (Figure 10E), but increased activity in the dark compartment (+71%; Figure 10F). Discussion ABC transporter function has been proposed to influence hematopoietic and neuronal stem cell proliferation and differen- tiation in vitro [31]. Here, we demonstrate for the first time that the loss of ABC transporter function also leads to specific impairments in neurogenesis and neurofunctional changes in vivo. We show that the deficiency of the ABC transporter ABCB1 significantly affects stem cell homeostasis in vivo, while these effects do not appear to be based upon cell-intrinsic effects due to unaffected NSPC differentiation in vitro. Furthermore, we were able to determine that the lack of ABCC1, a transporter not yet recognized in the setting of NSPC functions, leads to a significant decline in the neurogenic differentiation of NSPCs during disease states such as traumatic brain injury. With regard to cognitive functions, we found that ABC transporter status influences the levels of overall activity and anxiety. Recent in vitro studies revealed ABCB1 and ABCG2 expression in proliferating neuronal progenitors. The expression disappeared during differentiation, thus suggesting a role of the transporters in maintaining the cells in a specified state [29,30,48]. With this in mind, we sought to find quantitative impairments in the neuronal stem and progenitor cell pool. However, ABC transporter- deficiency did not significantly alter the Sox2+ NSPC pool. Rather, the absence of ABCB1 impaired the differentiation of NSPCs into the neuronal lineage, as detected by significantly decreased amounts of DCX+ and calretinin+ cells. Although ABCG2 was found to be expressed in NSPCs [30], in our analysis, its deficiency only slightly altered neurogenic functions in ABCG2- deficient mice in vivo. Analyzing the BrdU+ fraction of Sox2+ cells, we found impaired proliferation in the ABC transporter-deficient mice. For ABCG20/0 and ABCC10/0 mice, the BrdU+ fraction of calretinin+ cells increased significantly. Therefore, it is likely that NSPCs lacking one of the analyzed ABC transporters experience a higher turnover because they are not able to stabilize their stem cell character, thus supporting the view that ABC transporters are involved in NSPC homeostasis. The impairment of neurogenic differentiation in ABCC1 transporter-deficient mice was disclosed using an induction model in which the brain was stressed by acute traumatic injury. Thus, disease states demanding increased regeneration potential help to uncover functions of ABC transporters beyond their known roles in normal cell physiology. Indeed, such disease-related functions PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35613