Digitization and analyses of NeuN-stained brain slices Brain slices were digitized using a MIRAX Midi slide scanner (Zeiss Microsystems, Germany). The scanned brain slices were processed using a semi-automatic software macro programmed within the AxioVision software package (Zeiss Microsystems, Germany) to separate the stained neurons from the rest of the cortical tissue. Specific regions of interest (ROI) were defined in the cortex and used for further digital processing and area normalization [41]. Furthermore, the NeuN stained sections were used to determine the size of the cortical layers with the AxioVision software package (Zeiss Microsystems, Germany). Therefore, we measured the thickness of layer I, layer II-IV (combined), layer V and layer VI according to Manuel et al. [42]. On each brain section two measurements were performed approximately in the lateral parietal association cortex and the barrel field of the primary somatosensory cortex. The average of both measurements built the value for the according slice. The average value for a distinct animal was calculated of the values of its two associated hemispheres. For the analyses of cortical cytoarchitecture 5–7 animals were analyzed for each strain. Primary NSPC culture Brains were extracted under sterile conditions from male animals aged between 50 and 80 days. Harvested brains from 4 to 6 animals were transferred into ice-cold aCSF-media [65] to cut 2 mm thick coronal slices 6–8 mm behind the olfactory bulbs of each brain. The tissue around the lateral ventricle was harvested from the slices and was enzymatically digested for 10 min at 37uC using a Trypsin/EDTA solution (GIBCO, Invitrogen Inc.). After additional mechanical dissociation, cells were seeded in neuro- sphere culture media (NeuroCultH, STEMCELL Technologies Inc.) supplemented with EGF (20 ng/mL) and FGF (10 ng/mL) (Peprotech Inc., USA) for 7 days until passaging after supplier’s instructions. Cell lines were passaged at least four times before use in proliferation or differentiation assays [66]. Proliferation assay. For measuring proliferation capacity, cells (56104 cells/cm2) were seeded for 24 h on Matrigel-coated coverslips in 24-well plates under proliferation conditions. BrdU (10 mM) was administered 1 h prior to fixation with paraformaldehyde (4% in PBS). Labeling of incorporated BrdU and cell nuclei using DAPI was performed according to the description in the immunofluorescence section. Stained coverslips were mounted in DePex (Serva Electrophoresis, Germany) on glass slides and cells were visualized using a Zeiss laser scanning microscope LSM 700. The relative proportion of BrdU+ cell nuclei against the overall number of DAPI+ nuclei was used to compare the proliferation capacity. The number of independent experiments was n$11 for each strain. Differentiation assay. Cells were seeded on Matrigel-coated coverslips (16105 cells/cm2) and were allowed to differentiate under withdrawal of growth factors in differentiation media (NeuroCultH Differentiation kit, STEMCELL Technologies Inc.). After 3 days, cells were fixed for 10 minutes with paraformaldehyde. The coverslips were incubated with a primary antibody against Tuj1 (1:500, Covance) in blocking buffer at 4uC. Incubation with fluorescence-labeled secondary antibody (1:2000 anti-mouse Cy3; Dianova, Germany) was performed for 2 hours at room temperature after washing in PBS. The coverslips were mounted in DePex on glass slides and were visualized using a Zeiss LSM 700 microscope. The proportion of Tuj1+ cells against the whole number of DAPI+ nuclei was used to compare the proportion of newly differentiated neurons [53]. The number of independent experiments was n$12 for each strain. ABC Transporters in Neurogenesis ABC transporter expression analysis using TaqManH assays. Briefly, RNA was extracted from proliferating neurosphere cultures at day three after passage using the Trizol method. RNA was stored for later analysis at 280Cu in elution buffer. 10 pg RNA were directly used for combined reverse transcription (One-Step RT-PCR, Applied Biosystems) and realtime quantitative PCR (TaqManH assays, Applied Biosystems) in a single tube using a Rotorgene 6000 (Qiagen) according to the supplier’s instructions. Multiplex reactions were performed by simultaneously quantifying the FAM-labeled internal control (GAPDH) and the VIC-labeled test sequence (ABC transporters). Relative expression levels were calculated using the DDCT method [67]. The following TaqManH assays were purchased from Applied Biosystems: ABCB1a (cat# Mm00440761_m1), ABCB1b (cat# Mm00440736_m1). ABCG2 (cat# Mm00496364_m1), ABCC1 (cat# Mm00456156_m1) and GAPDH (cat# Mm99999915_g1). The number of analyzed NSPC RNA samples is n = 3 for each group. Statistics For quantification of different cell populations in stained brain slices or cell culture, the reviewers remained incognizant of the experimental groups. For all quantifications, statistical significance (p#0.05) was determined using unpaired t-tests with Welch’s correction (Graph Pad Prism 5). Behavioral tests Y-maze. Mice were tested in random order in a symmetrical Y-shaped maze (TSE Systems). The dimensions of each arm were 325 mm (length), 85 mm (width) and 150 mm (height). Mice were put into the middle of the maze and were allowed to move freely throughout the maze. Their movement path was recorded with a camera during the test duration of 5 min and analyzed with the VideoMot software package (TSE Systems). The following parameters were assessed: running distance, number of arm entries, and the percentage of alternation [68]. At least 10 animals per group were investigated. Elevated Plus-Maze. Mice were tested randomly in an elevated Plus-maze (TSE Systems). The dimensions of the arms were: length 300 mm and width 50 mm; the maze was elevated 50 cm above the ground. Two opposing arms were either open or enclosed by a wall of 150 mm height. To start, the mice were placed in the center of the maze and allowed to move freely throughout the entire maze. The path was recorded with a camera during the test duration of 5 min and analyzed with the VideoMot software package (TSE Systems). The following parameters were assessed: number of closed arm entries, open arm entries, the mouse’s speed in each compartment, the time spent in each of the arms, and the time spent in the central area [69]. At least 9 animals per group were investigated. Light/Dark box test. Mice were tested randomly in a Light/ Dark-Box (TSE Systems). The maze setup consisted of a box (500 mm6500 mm6400 mm) that is divided into a darkened (1/ 3rd) and an illuminated compartment (2/3rd, illuminated at ,400 Lux) connected by a door (60 mm660 mm). The mice were set in the middle of the illuminated compartment facing away from the door and were allowed to move freely within the two compartments of the maze for 5 min. The course of the animal’s movement was recorded using an infrared camera and infrared light transilluminating the whole maze from below. The software package VideoMot (TSE Systems) was used to analyze parameters such as duration of stay per compartment, distance travelled per compartment, transitions between compartments, and number of PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e35613