behavior in the stressful environment. Therefore, ABCC10/0 mice kept a normal phenotype under non-stress conditions, but in- tensified anxiety in the light area (stress). These observations indicate the need for further studies that will elucidate the complex behavioral changes in these strains. Deficiency in ABC transporter expression somehow seems to minor effect brain ontogenesis in layer I and layer VI (layers with least cellular density and mostly interconnecting axons and dendrites) according to the altered cortical architecture in ABCB1 and ABCC1 transporter-deficient mice. Whether these changes are primary or secondary due to changes in the cytoarchitecture of other layers remains unclear since the total number of neurons is not changed (Figure 7B). In addition to ontogenetic changes in brain structure, it seems likely that ABC transporters alter the biological milieu within the cells/brain. Steroids, for example, are substrates for ABC transporters [54] that are able to alter anxiogenic functions in specific sets of hippocampal neurons [55]. Neurogenesis may contribute to certain behaviors, such as enabling spatial learning in orientation tasks [56]. Because the analyzed ABC transporter-deficient mice have an FVB/N background with a known retinal degeneration leading to visual deficits within the first 30 days of life [57,58], we were not able to analyze the animals’ abilities in spatial orientation tasks based on visual cues. Regarding performance on the other tasks, the visual impairment should not be of consequence, due to the fact that close-range orientation is based also on tactile stimuli [59]. The mice are able to orient within the narrow corridors of the Y-maze and easily recognize the open field and abyss surrounding the open arms of the plus-maze, as shown in previous studies [60]. In summary, the diverse ABC transporter subfamily members play important roles in physiologic and disease states of the brain. Alteration of their function due to genetic predisposition (e.g. promoter activity), slight structural variations (e.g. amino acid exchanges), or transport activity (e.g. by drugs or metabolites) may lead to early or prolonged effects on stem cell homing and differentiation. ABC transporters thus help to regulate two functions that are important for maintaining the integrity of the brain during aging and neurodegeneration – they export specific agents via the brain’s barriers, and they also maintain the integrity and functionality of the stem cell pool. The large number of ABC transporter-modulating drugs that are used for the treatment of chronic diseases suggests the feasibility of testing such agents as potential modulators of neurodegeneration in acute and chronic disorders. Materials and Methods Animals FVB.129P2-Abcg2tm1Ahs N7 (ABCG20/0), FVB.129P2-Abc- b1atm1BorAbcb1btm1Bor N12 (ABCB10/0), and FVB.129P2-Abc- c1atm1Bor N12 (ABCC10/0) mice were purchased from Taconic Farms (Denmark) [28,61,62]. All mice were housed under a 12 h/ 12 h light/dark cycle at 22uC with free access to food (SNIFF, Germany) and water. All protocols involving the use of animals were approved by the local authorities (LALLF M-V TSD 7221.3- 1.1-035/10 and LALLF M-V TSD 7221.3-1.1-024/11). CCI-Surgery and BrdU injections For each group (FVB controls, ABCB10/0, ABCG20/0, ABCC10/0), 6 mice (3 males, 3 females) were used for controlled cortical impact (CCI)-surgery at the age of 90 days. Animals were anesthetized using a ketamine/xylazine mixture (1.9 mg/ml ketamine; 0.5 mg/ml xylazine) at 20 ml per gram bodyweight. Animals were placed in a stereotactic frame and a midline incision ABC Transporters in Neurogenesis was made to expose the skull. The skull was opened using a drill (ø 2 mm) at the position bregma 22.0 mm anterior and bregma 22.0 mm lateral. The impact device used for the CCI consisted of a 1.2 mm diameter steel cannula with a deformed tip. The impact device was driven 2.2 mm into the brain above the hippocampus. After surgery, the wound was closed using suture clips [38,39]. To analyze the proliferation capacities, a daily dose of 50 mg BrdU per gram body weight was administered intraperitoneally for 7 consecutive days prior sacrifice. Immunofluorescence and immunohistochemistry Immunofluorescence. All animals were killed by cervical dislocation and immediately transcardially perfused with 20 ml of PBS followed by 20 ml of buffered, 4% paraformaldehyde (PFA) as described previously [63]. The brains were extracted and post- fixed in 4% PFA overnight and consecutively immersed in 15% sucrose and 25% sucrose solutions, each step overnight. Whole brains were mounted and frozen in cryo media (OCT Compound, Tissue Tek). We chose a specific range of the hippocampus (bregma 21.5 mm to bregma 22.2 mm) and cut (Leica CM3050 S) serial coronal sections (16 mm) within this 720 mm range from whole brains and separated every third section alternately to one of three containers for the different immunofluorescence staining protocols. The entire number of sections was 45, which yielded 15 sections per group. For each protocol, 6 slices were taken randomly from the respective pool and were stained immunofluorescently. Cells were counted if within the subgranular zone or no more than two cell diameters away [64] in both hemispheres. The spacing of at least two sections between the evaluated sections of each marker group maintains the key consideration of unbiased stereological cell quantification that no cell will occur in more than one section and therefore would not be counted twice. To reveal BrdU labeling, the brain sections were pre-incubated in 1.5 M HCl for 30 min at 37uC to denature the DNA. Then, the slices were washed three times in PBS before blocking for an hour in 5% chicken serum in PBS with 0.5% Triton X-100. The sections were incubated free-floating with the primary antibodies anti-BrdU (1:600; Sigma clone BU-33), anti-Sox2 (1:400; Santa Cruz clone Y-17), anti-DCX (1:400; Santa Cruz clone C-18) and anti-calretinin (1:2000; Santa Cruz clone N-18) overnight in blocking buffer at 4uC in the appropriate combinations of BrdU/ Sox2, BrdU/DCX and BrdU/calretinin. Incubation with fluores- cence-labeled secondary antibodies (1:2000 anti-mouse Cy2; 1:2000 anti-goat Cy3; Dianova, Germany) was performed for 2 hours at room temperature after washing in PBS. The slices were covered with DePex (Serva Electrophoresis, Germany) and visualized using a Zeiss LSM 700 microscope. Cells were counted if they were within the subgranular zone (SGZ) and excluded if they were more than two cell diameters from the SGZ [64]. For quantification of different cell populations, the reviewers remained incognizant of the experimental groups. The labeled cells in the dentate gyrus of each hemisphere were counted in 3 to 6 slices per animal and marker combination. Six animals per group (3 males, 3 females) were analyzed. Immunohistochemistry. Brains were harvested as described above and post-fixed for at least 24 hours. The tissue was paraffin-embedded, and 4 mm-thick coronal sections were cut. Neurons were stained using the NeuN primary antibody (1:1000; Millipore clone MAB377) on a Bond-MaxTM (Leica, Germany) automated staining system and the BondTM Polymer Refine Detection kit (Leica, Germany). PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35613