Ten week-old male C57BL6 mice (SLC, Shizuoka, Japan) were group-housed under controlled conditions (21 °C room temperature, 12 h light/12 h dark cycle) with food and water available ad libitum. All animal experiments were approved by the Laboratory Animal Care and Use Committee of Keio University School of Medicine.
Donepezil (Aricept) was generously provided by Eisai Co., Ltd, Japan. Mice were intraperitoneally injected with saline (control, n = 5 or 6) or donepezil (0.5, 1 or 2 mg/weight (kg)/day, n = 4 or 5) for 1, 2 or 4 weeks consecutively (see Figs 3–5 for details).
Chronic stress protocol
Mice were separated into three groups. In the restraint group, mice were injected (i.p.) with either ChEI (donepezil 1.0 mg/weight (kg)/day, n = 4) or saline (n = 5) for 1 week, injected twice with bromodeoxyuridine (BrdU, Sigma, St. Louis, MO, USA) (50 mg/kg, i.p.) with a 2-h interval, and then placed in a close-fit cylindrical restrainer (acrylic tube 2.8 cm in diameter with ventilating holes on the top and bottom to avoid elevating body temperature) inside their home cage for 6 h per day for 4 consecutive weeks. To decrease effects on the motor activities and food intakes of the mice, the restraint was performed during the light period, while the mice were inactive. ChEI or saline was injected immediately before each restraint period. Mice in the control group (n = 5) were treated with saline and BrdU for 1 week as described above, and then left undisturbed in their home cages for another 4 weeks. Before the final restraint, blood was sampled for measurement of the plasma corticosterone level using an enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA).
To assess the effects of the donepezil treatments on cell-proliferation in the DG, SVZ, RMS and OB, mice were intraperitoneally injected with BrdU (50 mg/kg) immediately after 2 weeks of donepezil treatment, and allowed to survive for another 24 h before perfusion. To assess the differentiation and survival of the newborn cells, mice were injected with BrdU (50 mg/kg) twice with a 2-h interval at the beginning of the donepezil treatment or restraint stress exposure.
Mice were deeply anesthetized with diethyl ether and transcardially perfused with PBS, followed by 4% paraformaldehyde in 0.1 m phosphate buffer. The brains were removed and postfixed in the same fixative for 24 h at 4 °C. Fifty-micrometer serial sections were coronally cut on a vibratome (VT1000S, Leica, Heidelberg, Germany) and collected in PBS.
For AChR-immunostainings (Fig. 1), sections were incubated with antibodies against muscarinic AChR m1 (m1AChR, rabbit, 1 : 300, Sigma), m2 (m2AChR, rabbit, 1 : 300, Alomone labs, Israel) or m4 (m4AChR, rabbit, 1 : 300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or the β2 subunit of nicotinic AChR (β2nAChR, rabbit, 1 : 100, Santa Cruz) combined with antibodies, against the highly polysialylated neural cell adhesion molecule (PSA-NCAM) or neuronal nuclei (NeuN), at 4 °C overnight. After washing with 0.1% TritonX-100 in PBS (PBS-T), the sections were incubated with biotinylated anti-rabbit IgG antibody (goat, 1 : 500, Jackson) for 2 h, followed by signal amplification with the avidin-biotin-complex (ABC)-system (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) for 1 h, and visualized with rhodamine-labeled thyramide (PerkinElmer, Boston, MA, USA). The sections were then incubated in Alexa Fluor 488-conjugated anti-mouse IgM or IgG antibody (goat, 1 : 500, Molecular Probes) for 2 h. To detect α7nAChR, sections were incubated in rhodamine-conjugated α-bungarotoxin (α-BTX) for 2 h at 37 °C, followed by incubation in antibodies for PSA-NCAM or NeuN overnight at 4 °C. After washing, the sections were incubated with Alexa Fluor 488-conjugated anti-mouse IgM or IgG antibodies (goat, 1 : 500, Molecular Probes) mixed with Hoechst 333442 (Sigma) for 2 h.
For choline acetyltransferase (ChAT) immunohistochemistry (Fig. 2), sections were pretreated in cold (−20 °C) methanol for 6 min, and then boiled in 0.01 m citric acid (pH 6.0) for 30 s. Following treatment with 1% H2O2 for 1 h, the sections were preincubated in TNB blocking reagent (PerkinElmer) for 2 h at room temperature, and incubated overnight in anti-ChAT antibody (goat, 1 : 100, Chemicon International, Temacula, CA, USA) mixed with anti-PSA-NCAM antibody (mouse, 1: 2000, kindly provided by Dr Tatsunori Seki) (Seki & Arai 1993) at 4 °C. After washing with PBS-T, sections were incubated with biotinylated anti-goat IgG (donkey, 1 : 500, Jackson Immuno Research Laboratories, West Grove, PA, USA) for 2 h. The ChAT signal was amplified using an ABC-system (Vector Laboratories) and visualized with fluorescein-labeled thyramide (TSA Fluorescence Systems, PerkinElmer). These sections were then incubated with Alexa Fluor 568-conjugated anti-mouse IgM or anti-mouse IgG antibody (goat, 1 : 500, Molecular Probes, the Netherlands), and Hoechst 33342 (Sigma) to stain the nuclei for 2 h at room temperature.
For BrdU staining (Figs 3–5), a series of floating sections, with 250-µm intervals, from each animal were incubated in 2 N HCl for 30 min at 60 °C, and then incubated with anti-BrdU antibody (mouse, 1 : 200, Becton Dickinson, Franklin Lakes NJ, USA) overnight at 4 °C. After washing with PBS-T, the sections were incubated in biotinylated anti-mouse IgG antibody (1 : 500, Jackson). The signal was visualized using an ABC Elite kit (Vector Laboratories) and diaminobenzidine (DAB).
For double-labeling with BrdU and NeuN (Figs 6 and 7), following the incubation in HCl, sections were incubated in antibodies for BrdU (rat, 1 : 200, Abcam Ltd, Cambridge, UK) and NeuN (mouse, 1 : 200, Chemicon) overnight at 4 °C, then incubated in biotinylated anti-rat IgG antibody (donkey, 1 : 500, Jackson) and finally Alexa Fluor 488-conjugated anti-mouse IgG antibody (goat, 1 : 500, Molecular Probes) for 2 h at room temperature. The BrdU signal was amplified with an ABC system, and visualized by rhodamine-labeled thyramide (PerkinElmer).
For Ki67 staining (Figs 5 and 7), sections were incubated in anti-Ki67 antibody (rabbit, 1 : 800, Novocastra, Newcastle, UK) overnight at 4 °C, and then in Alexa Fluor 488-conjugated anti-rabbit antibody (goat, 1 : 500, Molecular Probes) for 2 h.
Image processing and quantification
All the slides were coded before quantitative analysis, and the code was not broken until the analysis was complete. Cell counting was consistently performed by the same investigator (NK) blind to the group identification of each section. BrdU-labeled cells in the GCL and SGZ in the DG were counted under 400× magnification (Axioplan 2, Carl Zeiss, Germany). Cell numbers were counted using six or seven 50 µm-thick sections (250 µm apart) per animal. Therefore, the total number of cells in the DG was calculated by multipling the number of cells counted in these sections by six. For the SVZ, RMS and OB, absolute cell numbers were counted and presented. BrdU-labeled cells and Ki67 positive cells in the DG (SGZ and GCL), SVZ, RMS and OB were counted at 400× magnification under a microscope (Axioplan 2, Carl Zeiss, Germany). Pyknotic cells were identified and counted using DG and OB sections stained with Cresyl violet. The areas of the GCL and SGZ in each section were measured using Photoshop (Adobe) to calculate the density of pyknotic cells In the DG. BrdU-labeled cells in the DG and five randomly chosen areas scanned in the OB were captured by confocal laser microscopy (Carl Zeiss, LSM510, C-apochromat 40×/1.20 W objective and C-apochromat 63x/1.20 W objective lenses) to confirm double-labeling with NeuN. Cells were considered double-labeled if colabeling was seen throughout the extent of the nucleus on 1 µm optical sections. Co-localizations of AChR signals with neuronal markers (PSA-NCAM and NeuN) were analyzed on 1.0 µm optical sections, and contacts of ChAT-positive cholinergic fibers on to PSA-NCAM-positive immature neurons were analyzed on 0.4 µm optical sections using confocal laser microscopy (LSM510; Carl Zeiss).
The obtained data were expressed as mean ± SE. Differences between means were determined by one-way anova, followed by the Bonferroni post hoc multiple comparison test. Differences were regarded as statistically significant when P < 0.05.