mice (Doerflinger et al., 2003
) and Rosa26-EYFP reporter line (Srinivas et al., 2001
) were purchased from the Jackson Laboratory and maintained in C57BL/6 background. These two lines were crossed to obtain Plp-Cre-ERT2
/Rosa26-EYFP (PCE/R) double transgenic mice. In PCE/R mice, Cre-ERT2
fusion protein is expressed in the cytosol under the control of plp promoter. After binding with tamoxifen, Cre recombinase translocates into the nucleus to mediate Cre-loxP recombination, thus eliciting permanent expression of the EYFP reporter gene in PPEPs and their progenies. All animal procedures were performed according to the Institutional Animal Care and Use Committee, UC Davis, and National Institutes of Health guidelines.
Tamoxifen and BrdU or EdU treatments
Tamoxifen (TM) (T5648, Sigma) was dissolved in an ethanol/sunflower seed oil (1:19) mixture at a concentration of 10 mg/ml. At P7, PCE/R pups were injected twice, 6–8 h apart intraperitoneally with tamoxifen 75 μg/g body weight. 5-Bromo-2′-deoxyuridine (BrdU, B5002, Sigma) or 5-ethynyl-2′-deoxyuridine (EdU, A10044, Invitrogen) (Buck et al., 2008
) was prepared in sterile 1 × phosphate-buffered saline (1 × PBS) (pH7.4) at a concentration of 10 mg/ml. Single or multiple intraperitoneal injections of EdU or BrdU (100μg/g body weight) were given at the times indicated in the figures or figure legends.
PCE/R mice were sacrificed at 1 (n = 7), 3 (n = 6), 8 (n = 5), 27 (n = 3), or 53 (n = 3) d after TM injection, i.e. at P8, P10, P15, P30 and adult (P60). After anesthesia with ketamine (150 mg/kg body weight)/xylazine (16 mg/kg body weight), mice were perfused transcardially with 1 × PBS and then with 4% paraformaldehyde (PFA) in PBS. Brain and spinal cord were collected, post fixed in 4% PFA in PBS for 1 h at room temperature (RT), then cryoprotected in 30% sucrose (v/v) prepared in 1 × PBS at 4 °C overnight, then transferred to OCT compound for embedding. Anterior forebrain with lateral ventricle (LV) from Bregma 0 mm- Bregma 1mm, posterior forebrain with hippocampus from Bregma −0.9 mm - Bregma −1.8 mm (Paxinos and Franklin, 2001
) and the lumbar segment were cut transversely on a Leica cryostat (Model: CM3050 S) to prepare 12–14 μm thick sections, which were stored at −80 °C until use. For the purpose of stereological counting, 40 μm thick cryo-sections were cut on a Leica cryostat (Model: CM3050 S), air-dried for 4 h at 37 °C or RT overnight, and stored at −80 °C until use.
Sections were air dried for 30 min at RT, followed by incubation in normal serum blocking solution (dependent on the secondary antibodies used) at RT for at least 30 min (10% normal serum + 0.1 % Triton X 100 in 1 × PBS). When a streptavidin (SA)-biotin detection system was used, the section was treated with a streptavidin-biotin blocking kit (SP-2002, Vector) prior to normal serum blocking. Primary antibodies (see Supplemental Table 1
) diluted in 5% normal serum + 0.1% Triton X-100 in 1 × PBS were incubated at 4 °C overnight (12–20 h) or 37 °C for 3 h, followed by 3 × 20 min washes in PBS + 0.1 % Triton X-100. After incubation with secondary antibodies (see supplemental Table 1
for details) for 2 h at RT, the sections were washed 3 times (20 min each) in PBS + 0.1 % Triton X-100 at RT. For streptavidin-biotin detection system, FITC-, Rhodamine X- or Pacific blue-SA (see supplemental Table 1
), diluted in PBS were incubated for 15 min at RT, followed by 3 × 10 min washes in PBS + 0.1 % Triton X-100 at RT. Finally, Hoechst 33258 was used to label nuclei, and the sections were mounted with Vectashield mounting medium for fluorescence (Vector, H-1000). Because intrinsic fluorescence of the EYFP reporter protein was weak, an antibody against EYFP was used to amplify the EYFP signal.
For BrdU immunostaining, after all the immunostaining steps except Hoechst staining were completed, the sections were postfixed with 2% PFA in 1 × PBS at RT for 15 min, then denatured in 2N HCl at 37 °C for 45 min. After 3 × 5 min washes in PBS, the sections were incubated with BrdU antibody (see supplemental Table 1
) diluted in 5% normal serum + 0.1% Triton X-100 at 4 °C overnight or 37 °C for 3 h. For detection of EdU, an EdU imaging kit (C10084, Invitrogen) was used as per the manufacture’s instructions.
For immunostaining for stereological counting, a modified protocol was used. Briefly, the 40 μm thick cryo-sections were incubated with blocking solution (10% normal serum + 0.5 % Triton X 100 in 1 × PBS) for 4–5 h at RT. Primary antibodies were diluted in10% normal serum + 0.1 % Triton X 100 and incubated at 4 °C for 48 h. Secondary antibody incubations were at RT for 4 h. Hoechst 33258 was used to label nuclei, and the sections were mounted with Vectashield mounting medium (Vector, H-1000). We ascertained that, after tissue processing and staining procedure, the 40 μm thick sections typically underwent 20% shrinkage, to reach an approximate final thickness of 32 μm, still sufficient for the stereological counting.
vGLUT1 mRNA In situ hybridization
a 30 mer oligonucleotide probe 5′-GCACTGGGAACAAGGGAGGACTTGCATCTT-3′ targeted mouse vesicular glutamate transporter 1 mRNA was synthesized and 5′ labeled with digoxigenin by Integrated DNA Technologies. 12 μm thick sections were air dried at RT for 30 min. Sections were incubated in 0.3 TritonX-100/DEPC-PBS for 5 min, followed by 2 X 5min wash in DEPC-PBS. Sections were then digested in 1 μg/mg protenase K for 10 min at RT, followed by 2 X 5min washes in DEPC-PBS, and incubated for 10 min in acetylating solution (2.33 ml triethanolamine from Sigma + 500 μl acetic anhydride from Sigma + 1 ml HCl, volume up to 200 ml in water), then washed 2 X 5min with DEPC-PBS. Probe was added in hybridization buffer (50% formamide + 0.3 M NaCl + 20 mM Tris-HCl, pH8.0 + 5 mM EDTA + 10 mM NaPO4, pH8.0 + 10% Dextran sulphate + 1X Denhardt’s + 0.5 mg/ml yeast RNA) to a final concentration of 1 μg/ml. The probe was then denatured at 65 °C for 5 min, and immediately cooled on ice. Sections were incubated at 37 °C overnight, then washed in 2 X SSC at 55 °C for 15 min, 1 X SSC, 4 x for 15min at 55 °C, and 1 × SSC at RT for 15min, followed by the immunohistochemistry protocol. We used goat anti-DIG antibody and a rhodamine conjugated-donkey anti-goat secondary antibody to visualize the in situ signal.
Microscopy and stereological quantification
A Nikon Eclipse C1 confocal laser scanning microscope was used to image FITC (488 nm laser line excitation), Rhodamine X (561 nm laser line excitation) and Hoechst 33258 or Pacific blue (404 nm laser line excitation). Optical sections (z = 0.45) were acquired using 20× (numerical aperture, NA, 0.75), 40× (NA, 1.30) or 60× (NA, 1.40) oil objective lens with Nikon EZ-C1 software, version 3.40. The Nikon EZ-C1 3.20 FreeViewer was used to create single channel views, merged views and orthogonal views of the images, and Photoshop CS3 was used to combine the images, which were exported from EZ-C1 3.20 FreeViewer without any manipulation of contrast. We considered two antigens as co-localized only if colocalization extended from the top to bottom of the z-plane images.
For cell counting, 3–4 sections from each brain or spinal cord (3–5 animals for each time-point) were examined. The dorsal cortex area (D-ctx) (depicted in ) refers to the cerebral cortex above the corpus callosum that extends from the midline laterally to the tip of hippocampus CA3, including retrosplenial cortex, parietal association cortex, trunk region and part of barrel field of primary somatosensory cortex. The ventral cortex area (V-ctx) (depicted in ) refers to cerebral cortex located ventrally down from the rhinal fissure, including amygdala and piriform cortex. Fimbria (Fi) and hippocampus (Hip) counting areas were the whole Fi and Hip area respectively.
Figure 1 A subpopulation of early postnatal NG2+ oligodendrocyte progenitor cells is targeted by Cre-mediated recombination. A, Experimental design: two tamoxifen I.P. injections at P7, and analysis at P8. B, Diagram showing anatomical structures of mouse forebrain (more ...)
Stereological unbiased estimates of volumes and cell numbers were obtained by using a computer interfaced with an Olympus BX61 microscope equipped with a motorized stage, running StereoInvestigator software, version 8.24 (MicroBrightField, Williston, VT, USA). The regions of interest were traced at low magnification (4 × with NA, 0.16 for brain, 10 × NA 0.4 for spinal cord), and the counting was conducted at 60 × oil (NA, 1.42). Every 12th section was selected for counting. Counting grids randomly placed by the software were applied onto D-ctx (300 × 300, x and y size, in μm), V-ctx (300 ×300), fimbria (80 × 80), hippocampus (200 × 200) and spinal cord gray matter (200 × 200). The counting frames were set at 100 × 100 ( x and y size, in μm) in D-ctx, V-ctx, hippocampus and spinal cord gray matter, and at 40 × 40 in fimbria. In most instances, 30–40 counting sites were evaluated in one section for the region of interest. The optical dissector and guard zone were set at 15 μm and 5 μm, respectively. Cells immunoreactive for a specific marker were counted only if they overlapped with the Hoechst 33258 nuclear staining. Cell densities were calculated by dividing the total cell number in question by the total volume (mm3) counted. All counting data were expressed as mean ± S.D.