4.1. Voluntary exercise mouse model
All animal experiments were conducted in compliance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, with the approval of Washington University’s Animal Studies Committee. Two month old female mice (C57BL/6) were either housed in standard mouse shoebox cages with 4 mice per cage (control) or in a larger rat shoebox cage with 2 exercise wheels and 3 mice per cage (runner). The mice were allowed to run for 12 days. Use of exercise wheels was visually confirmed. On day 13, mice were injected with the appropriate thymidine analogue. Mouse brains were then harvested 4 hours after injection of EdU or BrdU in all studies except for the survival studies of the EdU positive cells, in which the mouse brains were harvested 30 days after EdU injection as described below.
For the EdU dose-response experiment, five groups of mice received single intraperitoneal injection of EdU (Invitrogen, Carlsbad, CA) at a dose of 10, 20, 50, 100 or 200 mg/kg body weight. Each group had six control mice and six running mice. For comparison of EdU and BrdU staining, two groups of mice received a single intraperitoneal injection of EdU (200 mg/kg) or the equimolar dose of BrdU (Sigma, St. Louis, MO) (243.5 mg/kg). Each group had six control mice and six running mice. For survival studies, six control mice and six running mice received a single injection of EdU (100 mg/kg). The mice continued in the respective experimental conditions for 30 days. The brains were then harvested. For co-localization studies of EdU and BrdU, two independent experiments were performed. In one experiment, one control mouse and one running mouse received a single intraperitoneal injection of EdU (200 mg/kg) and a single injection of BrdU (243.5 mg/kg). In the other experiment, two control mice were treated in the same way.
Mouse brains were harvested 4 hours or 30 days after injection of the thymidine analogue. Mice were euthanized, and brains were removed, snap-frozen and stored at −80°C. The frozen mouse brains were sectioned at 20 μm with a cryostat and mounted onto Fisher Superfrost Plus slides. Every eighth 20 μm coronal section (160 μm apart) throughout the whole hippocampus was collected as one set; a total of eight sets were collected for each brain. One set of sections was randomly chosen for each of the following processes: EdU or/and BrdU staining, quantification of the fluorescence intensity of EdU-labeled cells, or immunohistochemical staining for protein markers, as described below.
4.2. Restraint stress mouse model
Two month old male mice (BL6/SJL) were randomly assigned to restraint stress or control groups (n=6 per group). Mice were subjected to restraint stress in a 50 mL conical tube as previously described (Drew and Hen, 2007
) for two hours daily for 7 days in total. Control mice remained in their home cages. On the 7th
day, all mice were injected i.p. with EdU 100 mg/kg and sacrificed four hours later. Brains were removed, snap-frozen on powdered dry ice, and sectioned using a cryostat as described above.
4.3. EdU staining
EdU staining was conducted using Click-iT™ EdU imaging kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. This protocol is normally intended for use in cell culture, but was adapted for histological staining of brain tissue as follows. Slides containing mounted frozen brain sections were allowed to thaw to room temperature, and then fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) for 15 min. After washing twice with 3% bovine serum albumin (BSA) in PBS the sections were permeablized with 0.5% Triton X-100 in PBS for 20 min. The sections were again washed twice with 3% BSA in PBS and then incubated with a Click-iT™ reaction cocktail containing Click-iT™ reaction buffer, CuSO4, Alexa Fluor® 594 Azide, and reaction buffer additive for 30 min while protected from light. The sections were washed once more with 3% BSA in PBS. For subsequent DNA staining, sections were washed once with PBS and then incubated with 5 μg/mL Hoechst 33342 for 30 min. The slides were then washed twice with PBS and coverslipped with Vectashield mounting media (Vector Laboratories Inc, Burlingame, CA). All steps were carried out at room temperature.
4.4. BrdU immunohistochemistry
Slides containing mounted frozen brain sections were allowed to thaw to room temperature, then were fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) for 15 min and permeablized with 0.5% Triton X-100 in PBS for 20 min. The sections were then incubated with 2 N HCl for 60 min at 37 °C to denature DNA and neutralized with 0.1 M boric acid (pH 8.5) for 30 min. The sections were blocked with 1.5% normal horse serum (Vector Laboratories Inc, Burlingame, CA) in PBS for 1 h and incubated with rat anti-BrdU antibody (Accurate Chemical & Scientific, Westbury, NY) at 1:1000 dilution in the blocking serum overnight at 4 °C. After washing, sections were incubated with FITC-conjugated donkey anti-rat IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for 1 h. The slides were then immediately coverslipped with Vectashield mounting media (Vector Laboratories Inc, Burlingame, CA).
4.5. Double labeling of EdU and BrdU, or EdU and other molecular markers
EdU staining and BrdU staining were performed sequentially as described above. Briefly, brain sections were fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) for 15 min and permeablized with 0.5% Triton X-100 in PBS for 20 min. The sections were then incubated with a Click-iT™ reaction cocktail for 30 min for EdU staining. The sections were then incubated in 2 N HCl for 60 min at 37 °C to denature DNA and neutralized with 0.1 M boric acid (pH8.5) for 30 min. The sections were blocked with 1.5% horse normal serum (Vector Laboratories Inc, Burlingame, CA) in PBS for 1 h and incubated with mouse anti-BrdU antibody (Sigma, St. Louis, MO) at 1:200 dilution in the blocking serum overnight at 4 °C. After washing, sections were incubated with FITC-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for 1 h. For immunostaining of neuronal nuclear protein (NeuN), mouse anti-NeuN antibody (Millipore, Billerica, MA) at 1: 50 dilution and FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at 1:100 dilution were used. For immunostaining of glial fibrillary acidic protein (GFAP), rabbit anti-GFAP polyclonal antibody (Abcam Inc, Cambridge, MA) at 1:500 dilution and FITC-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at 1:100 dilution were used. The slides were coverslipped with Vectashield mounting media (Vector Laboratories Inc, Burlingame, CA).
4.6. Microscopy and cell counting
Every eighth 20 μm coronal section throughout the entire hippocampus was analyzed from each animal after immunostaining for EdU and/or BrdU. The positive cells in the SGZ of the DG were manually counted using a 20x/0.75 objective under an epifluorescence microscope (Nikon Eclipse E600, Nikon Instrument Inc., Melville, NY) equipped with a digital camera (Nikon DXM1200F) while focusing down through the tissue. Resulting numbers were multiplied by eight to provide an estimate of the total number of positive cells in the SGZ of the DG for each half brain and were reported as the comparative numbers of EdU positive cells. For counting BrdU positive cells, a B-2E/C FITC filter block containing a 465-495 nm excitation filter and a 515-555 band-pass filter for collecting emission was used. For counting EdU positive cells, a G-2E/C TRITC filter block containing a 528-553 nm excitation filter and a 600-660 band-pass filter for collecting emission was used. The optical path was set to 100% of the binocular eyepiece to provide the maximal fluorescent intensity to visualize the positive cells for cell counting. Control brain sections from animals that were not injected with EdU or BrdU were immunostained as background controls. In the majority of cases, BrdU (or EdU) positive cells displayed significantly stronger fluorescent intensity than the background control and allowed for easy manual recognition of the positive cells. In the cases when the fluorescent intensity was weak, a 40x/0.95 objective was used to carefully examine the fluorescent intensity and the nuclear shape of the staining. The cells which showed nuclear staining and a brighter fluorescence intensity than controls were counted as positive.
4.7. Quantification of the fluorescence intensity in EdU-labeled cells
To study the EdU-dose dependence of the fluorescence intensity in EdU-labeled cells, we used two control mice and two running mice for each EdU dose group (10, 20, 50, 100 or 200 mg/kg). For each mouse two brain sections were chosen so that the sections were from the same region of the DG for all the analyzed mice. About one hundred cells were analyzed for each dose group. The EdU-labeled cells in the sections were imaged with a 20x/0.75 objective under an epifluorescence microscope with the same exposure setting for all the dose groups. The fluorescence intensity of each EdU-labeled cell was quantified using Image J software (National Institute of Health) and averaged for each dose group.
4.8. Statistical analysis
EdU and BrdU positive cell numbers were expressed as the mean ± SEM. The EdU dose response data were analyzed by nonlinear regression using GraFit software, version 5 (Erithacus Software Limited ,UK). The data were fitted by Eq. A
Where N is the EdU positive cell number at each EdU dose, Nmax
is the maximal EdU positive cell number, and D50 is the EdU dose at which the EdU positive cell number equals one-half of Nmax
. The standard errors, which were equal to the squared standard deviation, were calculated for Nmax
and D50 by the GraFit software.
The differences in EdU positive cell numbers between the control and running groups at each EdU dose were analyzed using a two-tailed student’s t-test. The differences in EdU and BrdU positive cell numbers in both the control and running groups were analyzed by a two way ANOVA using PRISM software (GraphPad Software, Inc. San Diego, CA). A p value of < 0.05 was considered significant.