Ts65Dn and euploid B6EiC3 animals were generated by backcrossing Ts65Dn females to C57BL/6JEi X C3H/HeSnJ F1 hybrid (B6EiC3) males. The parental generation was obtained from the Jackson Laboratory (Bar Harbor, ME). Quantitative PCR (qPCR) genotyping was performed on genomic DNA extracted from tail tips7
double heterozygote animals in C57BL/6J background37
were obtained from David Anderson’s laboratory and bred in our animal facility. All experiments involving animals were performed in accordance with institutional and federal guidelines. Mutant mice were genotyped using RT-PCR as described previously37
. Ts65Dn females were crossed with Olig1/2+/−
heterozygote males to generate Ts65DnOlig1/2+/+/−
animals and the mutants were genotyped using both qPCR and PCR.
Ts65Dn and euploid littermates at postnatal day (P) 8, P15 and P30 were anesthetized by Forane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) and perfused intracardially with 1M phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA). The perfused brains of the postnatal animals as well as the fresh embryonic brains were removed and fixed in PFA overnight at 4°C followed by 30% sucrose. All samples were embedded in Tissue-Tek OCT compound (Sakura Finetek Inc., Torrance, CA), frozen on dry ice and sectioned (20 μm) by Microm HE505E cryostat (ThermoFisher Scientific, Kalamazoo, MI). All measurements and analyses were done at the level of sensorimotor cortex.
All immunohistochemical reactions were performed on 20μm frozen brain sections from P8, P15 and P30 pups unless otherwise stated. The antibodies used were rabbit anti-calretinin, calbindin, parvalbumin (1:1000, Chemicon, Temecula, CA), Olig2 (1:500, Abcam, Cambridge, MA), Ki67 (1:500, Vector Laboratories, Burlingame, CA), Phospho-histone3 (PH3, 1:250, Upstate, Lake Placid, NY) and Tbr1 (1:2000, a gift from Dr. Robert Hevner, University of Washington, Seattle, WA), rat anti- somatostatin (1:250, Chemicon) and BrdU (1:100, Abcam) and mouse anti- β-tubulin III (TUJ1, 1:500, Covance, Berkeley, CA) and parvalbumin (!:500, Sigma). The secondary antibodies used were AlexaFluor 488, AlexaFluor 546 conjugated or biotinylated goat anti-rabbit, donkey anti-rat, or goat anti-mouse (1:200, Invitrogen, Carlsbad, CA). All frozen sections were mounted with Vectashield (Vector Laboratories) and paraffin sections were mounted with permount (Fisher Scientific).
Dioxygenin-labeled RNA in situ hybridization
RNA in situ hybridization was performed on E13.5 and E14.5 paraffin sections and E15.5 frozen sections for Dlx2, Dlx5, Nkx2-1, Lhx6 and somatostatin probes. Sections were postfixed in 4% PFA for 10 min followed by 2 × 5 min rinses in PBS. Proteinase K (Invitrogen) digestion (20 μg/mL in PBS) was carried out for 6 min followed by 1 × 5 min rinse in PBS, refixing for 5 min in 4% PFA and another PBS rinse. The sections were acetylated for 10 min (2.2 g triethanolamine hydrochloride, 540 μL of 10 N NaOH, 300 μL of acetic anhydride in 60 mL of molecular grade water) prior to 3 × 5 min rinses in PBS. RNA probes, prepared at a dilution of 2 μL/mL of hybridization solution (50% formamide, 10% dextran sulfate, 1% 100× Denhart’s, 250 μg/mL yeast tRNA, 0.3 M NaCl, 20 mM Tris–HCl, pH8, 5 mM ethylene diaminetetraacetic acid [EDTA], 10 mM NaPO4, 1% sarcosyl in diethylpyrocarbonate-treated H2O, were incubated at 80 °C for 2 min. Thereafter, 250 μL of the probe mix was applied to each slide, coverslipped with Hybri-slips (Sigma) and placed in a sealed box humidified with 50% formamide and H2O and incubated at 55 °C overnight. The next day, the Hybri-slips were floated off by placing the slides in 5× saline-sodium citrate buffer, prior to a 30-min high stringency wash in prewarmed 50% formamide, 2× SSC at 65 °C. Next, the sections were rinsed in 3 × 10 min rinses in RNase buffer (0.5 M NaCl, 10 mM Tris–HCl, pH 7.5, 5 mM EDTA), followed by RNaseA (Roche, Indianapolis, IN) treatment (20 μg/mL in RNase buffer) for 30 min and one 15 min rinse in RNase buffer, all at 37 °C. The high stringency washes were repeated twice for 20 min each at 65 °C, followed by a 15 min rinse in 2× SSC, then 0.1× SSC, both at 37 °C and a PBT (PBS + 0.1% Tween 20; Sigma) rinse for 15 min at room temperature. The sections were blocked with 10% goat serum in PBT for 1 h at room temperature, prior to a 3-h incubation with an alkaline phosphatase-coupled anti-DIG antibody (1:5000 in 1% goat serum in PBT; Roche) in a humidified chamber at room temperature. Then, the sections were rinsed extensively in PBT at room temperature (4 × 15 min rinses) and then underwent 2 × 10 min rinses in freshly prepared NTMT buffer (100 mM NaCl, 100 mM Tris–HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween 20). The sections were then placed in a light-protected humidified chamber with approximately 400 μL of BM-purple AP substrate (Roche) containing ~0.25 mg/mL levamisol (Sigma) until satisfactory staining was achieved, typically overnight. Finally, the sections were rinsed twice in PBS, coverslipped using Crystal mount aqueous mounting media (Sigma) and photographed immediately.
In vivo neurogenesis assay
Pregnant Ts65Dn females at E13.5 were injected once with BrdU (50 μg per gm body weight, i.p.) and killed 24 hrs (n=3) after injection. Twenty micron coronal sections of the frozen heads were stained for either BrdU and TUJ1 or BrdU and Ki67. Images with Z-stacks (1 μm steps) were collected on a confocal microscope (Zeiss LSM 510 Meta). In the images with BrdU/TUJ1 double staining, BrdU+ and BrdU+/TUJ1+ cells were counted in the SVZ of ganglionic eminences in the ventral forebrain. We considered the BrdU+/TUJ1+ labeling to represent the neurons which had exited the cell cycle just after the BrdU injection. Similarly, in the images with BrdU/Ki67 double staining, BrdU+ and BrdU+/Ki67− cells were counted in the VZ and SVZ of the medial ganglionic eminence. The BrdU+/Ki67− cells represent the cells exiting the cell cycle just after the BrdU injection.
Cell cycle analysis
A cumulative BrdU labeling protocol was followed to determine (i) the growth fraction (i.e. the proportion of cells that comprise the proliferating population), (ii) Tc, the length of the cell cycle and (iii) the time to reach the growth fraction (Tc-Ts)7
. Pregnant Ts65Dn females at E13.5 or E14.5 were injected with BrdU (50μg per gm body weight, i.p.) every 2 hrs with survival times of 2, 6, 10, 14, 18 and 24 hrs (n=3–6 for each time point per genotype). The last injection was given 2 hrs before sacrifice. The labeling index (LI), or fraction of cells labeled with BrdU, was determined at each time point using immunohistochemistry. All labeling experiments were started at 7 AM.
Western blot analysis
For Western blot analysis, calbindin protein from the cortex of P15 pups was obtained by tissue homogenization in RIPA buffer (50mM Tris-HCl pH 7.4, 0.9% NaCl, 1% NP-40, and 0.25% sodium deoxycholate). Protein concentration was determined according to manufacturer’s instruction using BCA Protein Assay Kit (Pierce, Rockford, IL). Twenty micrograms of protein homogenate were loaded per well for electrophoresis after which the proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% milk for 1 hour at room temperature. The blots were incubated overnight at 4°C with rabbit anti- calbindin (1:5000, Chemicon) and mouse anti-β-actin (1:20,000, Chemicon). After washing, blots were incubated for one hour with HRP-conjugated secondary antibodies (1:10,000, Chemicon). Blots were developed by chemiluminescence using SuperSignal Kit (Pierce). Quantification of band densitometry was performed using Scion Image software.
Semi quantitative RT-PCR
Total RNAs were prepared using Trizol (Invitrogen) from the medial ganglionic eminence of euploid and Ts65Dn brain at E14.5. cDNA was synthesized from 2 μg of total RNA using ThermoScript RT-PCR system (Invitrogen). Twenty nanograms of the cDNA were used with primers for Olig1 and Olig2 (target gene) and β-actin (control gene) for PCR by SYBR-Green method. The reaction was carried out at 95°C for 5 min, followed by 30 cycles of 95°C for 15 sec, 58°C for 15 sec and 72°C for 15 sec and finally 72°C for 10 min on an iCycler (BioRad, Hercules, CA) with a 96 well format. Relative quantification was calculated by Gene Expression Analysis for iCycler (BioRad).
Mice, 2–3 weeks old, were anesthetized, decapitated and the brain was rapidly removed and placed in ice cold (~4°C) cutting artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 124, KCl 3, CaCl2 2, NaH2PO4 1.25, MgSO4 5, NaHCO3 26, d-glucose 10, bubbled with a mixture of 95% O2/5% CO2. Parasagittal slices, 400-μm thick, were cut on a Lancer Vibratome (Vibratome series 1000; Vibratome, St. Louis, Missouri USA) and transferred to a warmed (~37°C) solution of 50% slicing ACSF and 50% recording ACSF (the same composition as cutting ACSF, but MgSO4 at 1 instead of 5 mM) bubbled with a mixture of 95% O2/5% CO2. After 20 minutes they were transferred to room temperature (~21°C) O2/CO2 bubbled recording ACSF where they were maintained for at least 1 hour before recording. Slices were placed in a recording chamber containing bubbled recording ACSF on the stage of an upright Zeiss FS-1 microscope (Carl Zeiss Microimaging Inc., Thornwood, NY). Using a Photonics IR camera, CA1 pyramidal neurons were identified and a whole-cell patch-clamp configuration was obtained with a borosilicate patch pipette of resistance 3–5 M containing (in mM): K-gluconate 130, KCl 15, HEPES 5, EGTA 1, Mg-ATP 4, Na-GTP 0.3 with pH adjusted to ~7.3 with KOH. Recordings were performed in voltage-clamp configuration and data acquired by way of an Axopatch 200A or 200B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 5 kHz (8-pole Bessel filter, NPI, ALA Scientific Instruments, Inc., Westbury, NY, USA), and recorded on a personal computer using Clampex acquisition software (Molecular Devices). After perfusion of 20 μM CNQX and 50 μM D-APV (to block AMPA/Kainate and NMDA receptor mediated currents) sIPSC were recorded from neurons held at −70 mV under a gap free protocol and later visually inspected and detected offline using MiniAnalysis 6.0 (Synaptosoft, Decatur GA.). sIPSC were eliminated by 20 μM bicuculline (specific GABAA receptor antagonist) in both diploid (n=4) and Ts65Dn (n=5) slices indicating that events were GABAA mediated. IPSC frequency probability distributions were generated from instantaneous event frequencies pooled from all cells for each condition (euploid, n=32; Ts65DnOlig1/2+/+/−, n=27; Ts65Dn, n=34). mIPSC were also recorded at −70 mV in perfusate of 1 μM TTX (to block action potentials and thus sIPSCs), 20 μM CNQX and 50 μM D-APV and analyzed as described above for sIPSCs.
All fluorescent images were taken on a LSM510 Meta confocal microscope (Carl Zeiss Inc., Germany). Immunolabeled cells in each cortical layer (viz. layers II–III, IV, V and VI) were counted every tenth sections of 20 μm each (every 200 μm) in a 500μm width x 150μm height frame in 15 μm thick z-stack images (1μm steps) using Volocity (Improvision, Lexington, MA) and LSM 510 softwares packages. The layers of the neocortex were discriminated using the layer markers, Brn1 (layer I–III), RORβ (layer IV), ER81 (layer V) and Foxp2 (layer VI). The VZ and SVZ abventricular boundary in the MGE was estimated at 100 μm. The bright field images were taken on a BX40 Olympus microscope (Olympus America Inc., Center Valley, PA) and immunolabeled cells were counted using NIH ImageJ software.
Data are presented as mean ± S.D. unless otherwise stated. Comparisons of mean differences between groups were made by unpaired two-tailed Student’s t-test unless otherwise stated. A probability level of p<0.05 was considered to be statistically significant.