LY294002, PD98059, rapamycin and roscovitine were purchased from Calbiochem (La Jolla, CA). Fluo-4 AM was purchased from Invitrogen (Eugene, OR). 4-Aminopyridine (4-AP), tetrodotoxin (TTX), picrotoxin (PTX) 4′,6-diamidino-2-phenylindole (DAPI), K252a were purchased from Sigma-Aldrich (St. Louis, MO). BDNF was purchased from R&D Systems (Minneapolis, MN).
Antibodies were used at the following dilutions: polyclonal rabbit anti-phospho-Ser727 STAT3 antibody, anti-phospho-Tyr705 STAT3 antibody, anti-STAT3 antibody, anti-p53 antibody and anti-Bax antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1:500; anti-Erk1/2 antibody, anti-phospho-Erk1/2 antibody, anti-phospho-Ser-743 Akt antibody, cleaved caspase3 (c-cas3) antibody, and monoclonal mouse anti-STAT3 antibody (Cell Signaling Technology, Danvers, MA), 1: 500; monoclonal mouse anti-cytochrome c antibody and NeuN antibody (BD Biosciences Pharmingen, San Diego, CA), 1:1000; monoclonal mouse anti-β-actin antibody, anti-MAP2 antibody (Sigma-Aldrich), 1:10000 and 1:1000, respectively. Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for function blocking experiments.
Dissociated primary hippocampal culture
Culture was prepared as described previously (Murase and McKay, 2006
). Hippocampi from embryonic day 18 (E18) Sprague Dawley rat embryos of either sex were used for both astrocyte (plated at a density of 80,000 cells/ml) and neuron (density: 200,000 cells/ml) cultures. Astrocytes were cultured in Neurobasal (Invitrogen) with 5% fetal bovine serum (FBS) in 5% CO2
at 37°C for 14 days. Medium was changed completely twice weekly. Neurons were plated on confluent astrocyte beds and cultured in Neurobasal and B27 in 5% CO2
at 37°C. Half of the medium was changed every 2 days. Experiments were performed using 14 days in vitro (DIV14) neurons.
Cultures were incubated with 2 μM fluo4-AM for 15 min. Images were obtained with a BX51W1 microscope (Olympus America, Center Valley, PA) equipped with a 10x objective lens. The cultures were imaged in Hepes buffered saline (HBS: 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM Hepes-NaOH (pH 7.4), 290 mOsm) at 37°C using a CCD camera (QImaging, Blaine, WA) at 20 Hz for 40 sec. Typically, 20 neurons in one experiment were used for analyses.
Rat hippocampal culture was incubated with lysis buffer (150 mM NaCl, 1% NP-40, 50 μM Tris-HCl, pH8.0) containing a protease inhibitor cocktail (Roche) for 20 min on ice (60 μl per one 24-well culture dish). Lysate was centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was pre-absorbed with 10% (v/v) of protein A Sepharose beads (Pierce, Rockford, IL) for 1 hr, and incubated with 3 μg/ml of anti-STAT3 antibody for 2 hr followed by incubation with 10 % (v/v) of protein A Sepharose beads for 1 hr at 4°C. The beads were then rinsed 3 times with lysis buffer with protease inhibitor at 4°C prior to elution with an equal volume of 1XSDS loading buffer by boiling for 5 min.
Samples from dissociated culture were collected with 1XSDS loading buffer (60 μl per one 24-well culture dish). Hippocampi from wild-type C57BL/6 mice and Kv4.2−/− mice were homogenized in 300% (v/w) lysis buffer with protease inhibitor on ice. The homogenates were diluted with 2XSDS loading buffer. The samples were boiled for 5 min, then applied to a 4–10% gradient SDS gel (BioRad, Hercules, CA). The proteins were transferred to a nitrocellulose membrane. The membranes were blocked with 4% skim milk in phosphate buffered saline (PBS) for 30 min. Incubation with antibodies was performed in the blocking solution. Membranes were washed with Tris-buffered saline with 0.05% Tween 20. The proteins were visualized with SuperSignal West Pico System (Pierce), and detected and analyzed with a BioChemi System (UVP BioImaging Systems, Upland, CA). Mean±SEM are plotted.
Cultures were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked with PBS containing 5% normal goat serum (NGS, Vector Laboratories, CA). Primary and secondary antibodies were diluted with the blocking solution. Samples were incubated for 2 hr with antibodies.
For the in vivo injection analyses, C57BL/6 and Kv4.2−/− mice were perfused with 4% PFA two days after the injection. Consecutive coronal slices of 50 μm thickness were made by a Leica VT100S vibrating microtome (Leica, Allendale, NJ) and were immunostained with a neuronal marker, NeuN, and the apoptotic marker, c-cas3. Slices were compared with respect to distance from the injection site. Four consecutive slices per animal and three animals per condition were combined for the analyses. The analysis was done blind with respect to the content of the injections.
Neurons were visualized by immunostaining against neuron specific microtubule associated protein 2 (MAP2; (Izant and McIntosh, 1980
)). Fluorescent images were taken with a Zeiss confocal microscope (LSM-510) equipped with 10x lens or a 25x lens. Z-stacked images from eight sections (1 μm intervals) were used for the analyses. All experiments were repeated in at least 3 independent culture preparations. Image analyses were done using ImageJ. Images were taken from 5 fields; one from the center of the coverslip, and two vertically and two horizontally 400–3000 μm from the center. Because the densities of neurons were higher in the rim of coverslips than in other regions, we avoided sampling the edge of coverslips. Each coverslip was defined as an individual culture. Numbers represent mean±SEM. All analyses were done blind.
Transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were transfected with 1.6 μg/ml of pEGFPC1 vector (Clontech, Mountain View, CA), and/or 8 μg/ml activated Akt1/pUSE vector (Millipore, Bedford, MA), or 30 pM rat STAT3 siRNA (Santa Cruz) or wild-type and mutant STAT3 IRES EGFP/pMX plasmids (gift from Dr. Y. Gotoh, Tokyo, Japan) in OPTI-MEM (Invitrogen) for 30 min, then the medium was replaced with NeuroBasal Medium. Transfection was performed 4 days prior to the experiments.
Reverse transcription (RT)-PCR
Hippocampi from C57BL/6 and Kv4.2−/− mice were homogenized in 300% (v/w) lysis buffer on ice. Rat hippocampal culture was incubated with lysis buffer with protease inhibitor cocktail for 20 min on ice (60 μl per one 24-well culture dish). RNA was isolated from the homogenates using TriPure Isolation Reagent (Roche, Welwyn Garden City, UK). RT-PCR was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Using 5 μg of total RNA, first-strand cDNA synthesis reaction by reverse transcriptase was done using Oligo(dT)12–18 as primers. PCR was performed using Taq polymerase (Roche). The sequences of the primers are the following: 5′-CCACACTTTCTACAATGAGC-3′ and 5′-CCGTCAGGATCTTCATGAGG-3′ for rat β-actin, 5′-CTACTAAGGTCGTGAGACGCTGCC-3′ and 5′-TCAGCATACAGGTTTCCTTCCACC-3′ for rat p53, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for mouse GAPDH, 5′-GATGGTGATGGCCTGGCTCC-3′ and 5′-GGTCGGCGGTTCATGCCCCC-3′ for mouse p53. Conditions for PCR reactions are: 44 cycles of 95°C (15 sec), 60°C (20 sec), 72°C (15 sec) for rat p53; and 35 cycles of 95°C (30 sec), 62°C (30 sec), 72°C (30 sec) for rat β-actin, mouse p53 and GAPDH. The PCR products were separated in 2% agarose gel.
Chromatin immunoprecipitation (ChIP)
Chromatin immuno-precipitation assays were performed as described by Ballas et al. (Ballas et al., 2001
). C57BL/6 and Kv4.2−/−
mice were perfused with 4% PFA. Hippocampi were homogenized with cell lysis buffer (CLB; 5 mM Hepes pH 8, 85 mM KCl, and 0.5% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) using a glass tissue grinder on ice. The homogenate was centrifuged at 3000 rpm for 2 min at 4°C, and the pellet was resuspended in CLB with PMSF and centrifuged at 3000 rpm for 2 min at 4°C two times. The pellet was then resuspended in nuclear lysis buffer (NLB; 50 mM Tris-HCl pH8, 10 mM EDTA, 1% SDS) with 1 mM PMSF and was sonicated to yield 100 bp to 1000 bp DNA on ice, and was centrifuged at 12000 rpm for 15 min at at 4°C. The nuclear lysate was pre-absorbed with recombinant protein G agarose (rProtein G agarose; Life Technologies, Grand Island, NY) pre-incubated with 200 μg/ml yeast tRNA and 200 μg/ml salmon sperm (Invitrogen) for 1 hr at 4°C. The chromatin suspension was diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl), then immuno-precipitated with 5 μg/ml of monoclonal mouse anti-STAT3 overnight at 4°C. The chromatin suspension was incubated with rProtein G agarose pre-treated with 3% BSA and yeast tRNA and salmon sperm for 4 hr at 4°C. Agarose beads were washed with series of solutions as following at room temperature: ChIP dilution buffer, dialysis buffer (2 mM EDTA, 50 mM Tris-HCl pH 8, 0.2% sarkosyl), TSE-500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 500 mM NaCl), LiCl detergent (100 mM Tris pH 8, 500 mM LiCl, 1% Triton X-100, 1% deoxycholic acid), and TE (10 mM Tris-HCl pH 8, 1 mM EDTA). To change the solution, the beads were centrifuged at 3000 rpm for 1 min and the supernatant was aspirated. The samples were eluted from the beads with 300 μl of elution buffer (50 mM NaHCO3
, 1% SDS). Samples were incubated overnight at 65°C to reverse PFA cross links following the addition of 20 μl of 5 M NaCl. DNA was then purified from the eluted samples using Qiagen PCR purification kit (Qiagen, Valencia CA). PCR was performed to analyze the STAT3 binding site in the p53 promoter using the following DNA primers: 5′-GGGCCCGTFTTGGTTCATCC-3′ and 5′-CCGCGAGACTCCTGGCACAA-3′. Conditions for PCR reactions were: 30 cycles of 94°C (30 sec), 60°C (30 sec), 72°C (1 min). The PCR products were separated in 1.5% agarose gel.
In vivo injection
The C57BL/6 and Kv4.2KO mice (8 to 9 weeks old) of either sex were anesthetized by ketamine/xylazine cocktail (3.33ml/kg) via intraperitoneal injection, before the surgery. The delivery of reagents to CA1 was done using the following stereotaxic coordinates from bregma: anteroposterior, 2.4 mm; mediolateral, ±2.28 mm; ventrodorsal, 1.6 mm. Reagents (0.5 μl) were delivered at a rate of 0.12 μl/min using a Hamilton needle and syringe attached with a microsyringe pump controller (World Precision Instruments (Sarasota, FL). Anti-TrkB (1.0 mg/ml) and vehicle (PBS) alone were injected, one to each side of the brain. The incision was closed using a polyglycolic acid suture (CP Medical, Portland, OR). Animals were allowed to recover at 37°C for 1–2 hr.
Statistical significance between two groups was determined with a two-tailed paired Student’s t test. For multiple groups, statistical comparisons were made by ANOVA followed by individual group tests with the Bonferroni correction made for multiple comparisons.