(A,B,E,F,H) Survey images of coronal sections of the SEZ (A,B,E), RMS (F), and OB (H) stained by bright-field immunohistochemistry. A small number of cells in the SEZ express Cre (A, low mag; B, high mag). (C,D) Confocal micrographs of single optical slices through cells in the SEZ that are double stained by immunofluorescence for Cre and GFAP (C) or Cre and Nestin (D). Individual channels and orthogonal analysis show that all Cre-expressing cells in the SEZ also express Nestin (C) and GFAP (D). Orthogonal images (ortho) show three-dimensional analysis of individual cells at specific sites marked by intersecting x, y, and z axes. Many cells express the reporter protein β-gal (E) in the neurogenic proliferative regions of the SEZ. Migrating neuroblasts in the RMS (F) and granule neurons in the OB (H) also express β-gal. Box in G indicates the region shown in H. Arrows in A, B, and E indicate representative Cre or β-gal-positive cells in SEZ. SEZ, subependmyal zone; LV, left ventricle; STR, striatum; RMS, rostral migratory stream; OB, olfactory bulb; GCL, Granule Cell Layer; Gl, Glomeruli.

(A) Neurosphere cultures generated from the adult SVZ of PTEN knockout (Mutant) and wild-type (Control) mice were cultured at clonal density (1000 cells/ml), passaged and re-seeded at clonal density every 2 weeks. Clonal neurosphere numbers, representative of the number of stem cells present in the culture, were significantly higher in mutant cultures at all passages (P<0.001). (B) The clonal neurospheres were then dissociated and the total cell number was counted at each passage followed by re-culturing at clonal density. After the first passage, mutant cultures consistently produced higher total cell numbers even after long-term serial clonal passaging whilst control cultures produced decreased cell numbers with time, eventually senescing by passage 24. Data are means ± SEM; n = 4. (C) In addition, the clonal neurospheres from mutant mice maintained their robust neurogenesis even at late clonal passages whilst the ability to generate neurons was dramatically attenuated in control cultures over time. Immunocytochemistry for TUJ1+ cells (green) and the counterstain Hoescht (blue) are shown at passage 4 and 16. n = 4.

Survey images of coronal sections of control and mutant SEZ. (A) Compared with littermate controls, mutant mice showed increased GFAP- and P-S6-positive labeled cells which co-localized in the SEZ. Images and graphs quantifying SEZ volume (B), Ki-67 counts (C), and DCX volume (D) demonstrate increased proliferation in mutant SEZ as compared to control regions. DAPI counter stain is used to visualize nuclei. Data are means ± SEM, n = 5. LV, lateral ventricle; CC, corpus collosum; STR, striatum; SEZ, subependymal zone; DCX, doublecortin.

(A) Quantification of BrdU⁺ cells in RMS revealed an increase in Pten null SEZ-born progenitors migrating in the RMS when compared to control. (B) Representative images of RMS stained for BrdU, P-S6, and their colocalization shows the BrdU⁺ population in mutant RMS is Pten null. (C and D) Survey images of sagittal sections of control and mutant RMS stained with DCX (C) and graph quantifying DCX⁺ cells show an increase in the number of migrating neuroblasts in mutant RMS compared to control (D). Inserts show higher magnification of boxed area. (E) Quantification of the percentage of DCX⁺ cells diverging from RMS pathway shows no difference between control and mutant. DAPI counter stain is used to visualize nuclei. Data are means ± SEM, n = 6. RMS, rostral migratory stream; DCX, doublecortin; BrdU, 5-Bromo-2′-Deoxyuridine.

(A) Enlarged OB of mutant mice. Photo shows a brain hemisphere from control and mutant mouse. Panel on right depicts the progressive increase in the ratio of mutant/control OB weight. n = 40. (B) Histological analysis of OB section from control and mutant mice reveals normal histoarchitecture. (C) GCL volume is increased in mutant mice whereas no significant change is observed in EPL. n = 10. (D) SEZ-born neuroblast migration was analyzed by BrdU pulse-labeling. Mice were injected with 200 mg/kg BrdU peritoneally, and distribution of BrdU-labeled cells was determined 2 weeks after injection. An increased number of BrdU-labeled cells were seen within the GCL. Quantification of BrdU-labeled cells within GCL indicates Pten mutant mice had a significant increase in cell number at 2 weeks post injection. n = 5. (E) Using the TUNEL assay, we observed a significant decrease in the percentage of dying cells in the GCL of mutant animals as compared with control. n = 5. (F) Representative GCL images show enhanced immunoreactivity for NeuN and increased NeuN/P-S6 double positive cells in mutant when compared to a matched control region. Data are means ± SEM. OB, olfactory bulb; GCL, granule cell layer; EPL, external plexiform layer; BrdU, 5-Bromo-2′-Deoxyuridine; DAPI is used as counterstain to visualize nuclei.

Control and mutant mice were subjected to the olfactory habituation test. Pre-training of wild-type control mice to a cotton swab soaked in water habituated mice to the presence of the swab in their cage. This was manifested as a decline in the number of sniffs with subsequent exposures to the swab. During odorant testing, the cotton swab was laced with 50 µM isoamyl acetate or 50 µM hexyl alcohol and introduced on three successive trials. The fact that the control mice sniffed the isoamyl acetate- and hexyl alcohol-laced cotton swabs more times than during the third exposure to the water swab indicated that the animal was able to smell these odorants. (A) Compared to control, mutant mice sniffed the swabs less frequently in the second and third exposures (all three conditions) signifying they habituated to the novel odorant faster than the control. n = 14 (control) and 12 (mutant). (B) Both control and mutant mice lost their ability to detect odorants at 2 weeks post treatment (top), whereas the ability of mutant mice to detect odorants began recovering at 4 weeks post treatment (middle), and their ability to detect odorants was restored to pre-treatment levels by week 6 (bottom). Data are means ± SEM; n = 8.

(A) Quantification of DCX labeling in peri-infarct cortex and SEZ. DCX⁺ cells in control and mutant 7 d after stroke. Right panel shows stereological quantification of DCX⁺ cells in peri-infarct cortex. Scale bars: top, 100 µm; bottom, 25 µm. Data are means ± SEM; n = 6. (B) Representative images of p-S6 staining from both hemispheres of mutant mouse. Left panel shows hemisphere containing peri-infarct cortex which has increased staining for p-S6 in peri-infarct cortex when compared to uninjured cortex in the same animal (right panel).