Primary cortical neuron culture
Cortices from E18 rat embryos were digested with papain (0.5 mg/ml in EBSS, 37°C for 20 min), washed and gently triturated by passing the tissue through a Pasteur pipette with a fire-polished tip. Neurons were counted and plated onto poly-L-lysine (Sigma, 0.5mg/ml) pre-coated 60 mm Petri dishes (Becton Dickinson, Bedford, MA) at 4×106/dish for biochemistry or dishes containing five coverslips (0.3×106 per 60 mm dish) for immunostainings. To ensure high-quality cell adhesion and growth, coverslips were first incubated in nitric acid overnight and thoroughly washed with 4 changes of large amounts of water every 2 hours. Dried coverslips were then baked at 200°C for 2 hours, coated with poly-L-lysine (Sigma, 0.5mg/ml) overnight and washed again before being incubated in plating medium for cell plating. The plating medium is MEM containing 10% fetal bovine serum (FBS), 5% horse serum (HS), 31 mg cystine and 1% P/S/G. 24 hrs after plating, the culture medium was completely replaced with feeding medium (Neurobasal medium supplemented with 1% HS, 2% B-27 and 1% P/S/G). Thereafter, neurons were fed twice a week with 2 ml feeding medium/dish until use. Since glia are important in neuron growth and synaptogenesis, glia cells were inhibited by adding FDU at DIV 5, but not completely eliminated from the culture.
Western blot analysis of protein abundance
2 wk-old cultured cortical neurons in 60 mm dishes (4×106/dish) were incubated with ouabain (50 μM) in culture medium for 1 hr or as indicated. For calcium-free or sodium replacement experiments, cells were incubated with ACSF containing 1 mM EGTA and 0 calcium, or sodium being replaced with NMDG or LiCl, supplemented with ouabain 10 min later for 1 hr. After treatment cells were rinsed with ACSF, harvested in ice-cold lysis buffer (PBS supplemented with 1% triton X-100, 0.5% deoxycholate, 0.1% SDS and 1:300 protease inhibitor cocktail containing AEBSF, Aprotinin, Bedysyin, E-64, Leupeptin and Pepstatin A, Sigma), and rotated at 4°C for 1 hr. Following centrifugation of the lysates at 14,000 rpm for 15 min, supernatants were boiled with 4x sample buffer followed by SDS-PAGE separation and immunoprobing with antibodies against the C-terminus of GluR1 (anti-GluR1Ct, 1:100), GluR2/3 (1:500), NR1 (1:500), PSD-95 (1:800), GRIP (1:500) and NSF (1:1000). The blot was developed using enhanced chemiluminescence detection methods (Amersham). Films were scanned and the optical intensities of the protein bands were quantified using Image-J NIH software.
Isolation of cell-surface AMPARs
Cultured cortical neurons were incubated with antibodies against the extracelluar N-terminal domain of AMPAR GluR1 subunits (anti-GluR1Nt from rabbit, 1:100) for 5 min in culture medium at 37°C. Antibodies against the intracellular C-terminal of GluR1 (GluR1Ct, polyclonal, 1:100) and non-specific rabbit IgG (1:100) were also added to another two dishes, respectively, as controls. All antibodies and IgG have been adjusted to the same stock concentration of 0.5mg/ml. After incubation neurons were washed three times with cold ACSF on ice to remove free antibodies. Cells were homogenized in lysis buffer, to which protein A beads (50 μl in each lysate of 500 μl) were added for 2 h on rotation at 4°C. The final yields were subjected to SDS-PAGE gels and probed with anti-GluR1Ct antibody. In some dishes, surface AMPARs were isolated by surface biotinylation. Following ouabain treatment, culture dishes were placed on ice to stop receptor trafficking, washed with cold ACSF and incubated with biotin (1 mg/ml) for 30 min to label surface proteins. The surface AMPARs were purified by Neutroavidin beads (Pierce) as described previously (Man et al., 2007
GST pulldown assays
Solubilized hippocampal extracts (800 μg of protein) were incubated with glutathione-Sepharose beads (Amersham Biosciences) bound to the indicated GST-fusion proteins (20 μg) at 4°C overnight with shaking. Beads were then washed three times with 500 μl of PBS containing 0.1% Triton X-100 before the bound proteins were eluted with glutathione elution buffer. Elutes were incubated in sample buffer and subjected to 10% SDS-PAGE.
Time course of surface and total AMPAR reduction
Cortical neurons were cultured in 6 well plates (1×106/well) in order to synchronize the procedures among different treatments. The same amount of ouabain (50 μM) was added to individual wells at different time points so that all wells with varied lengths of ouabain incubation were ready for surface labeling and lysis simultaneously. After treatment, plates were washed, incubated with anti-GluR1N (1:100) for 10 min at 37°C and washed with ACSF on ice to remove the remaining antibody. Following cell lysis, a small portion of lysate was taken to examine total AMPAR levels, and the remainder of the same lysate was incubated with protein A Sepharose beads to isolate surface AMPARs. Time courses of the reduction in both surface-localized and total AMPARs were drawn from the optical intensity of western blots.
Neurons were plated at low density (0.3× 106 per dish with 5 coverslips) to reduce the overlap among neurons so that single dendrites could be readily selected and analyzed. Following 1 hr incubation with ouabain in culture medium, neurons were washed with ACSF, fixed with 4% paraformaldehyde plus 4% sucrose (10 min) and permeabilized with 0.3% Triton X-100 (10 min) on ice. After 1 hr blocking with 10% normal goat serum, cells were incubated sequentially with antibodies against PSD-95 (mouse, 1:500) and NKA α1 subunits (rabbit, 1:300), or GluR2Ct (mouse, 1:100) and NKA α1 (rabbit, 1:300) for 2 h each at room temperature. Cells were then washed and incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 555 red anti-mouse, 1:700, and/or Alexa Fluor 488 green anti-rabbit, 1:700) for 1 hr for visualization.
Image collection on immunolabeled neurons
Immunostained coverslips were mounted onto slides using ProlongGold anti-fade reagent (Invitrogen, Molecular Probe) and kept in the dark for more than 4 hrs before imaging. Using 63x oil immersion objective (NA 1.4), a DIC snap was first taken for morphology purposes. The exposure time for fluorescence signal was set automatically by the software and adjusted manually so that the signal intensity was within the full dynamic range. Either the glow scale look-up table or the histogram was used to monitor the saturation level. Once the parameters were set they were fixed and used for all the imaging in an experiment. For double staining, dual filters were used for PSD-95 or GluR2 (red) and NKA α1 subunits (green). Two channels were imaged sequentially and a merged image was formed automatically by the Zeiss imaging software. Some immunostainings were also imaged by confocal microscopy.
2-3 d after transfection of GFP-GluR1 or PH-GluR1 (1 μg per coverslip) with Lipofectamine 2000, cortical neurons were transferred to an imaging chamber with temperature controlled at 37°C. Receptors were visualized with excitation at 488 nm and imaged every 15 min. Ouabain (50 μM) was added directly to the chamber containing 1 ml ACSF buffered with 10 mM HEPES. Some cells were pretreated with MG-132 for 30 min in culture medium then transferred for imaging in the presence of MG-132. Cells were checked before each imaging to ascertain there was no shift in focus. Fluorescence intensity at the soma was measured at different time points using Image-J and normalized to the starting value.
mEPSC whole-cell patch clamp recording
A coverslip of 14-16 d-old cortical neurons was transferred to the recording chamber with extracellular solution containing (in mM) 140 NaCl, 3 KCl, 1.5 MgCl2, 2.5 CaCl2, 11 glucose and 10 HEPES (pH 7.4), which was supplemented with TTX (1 μM) to block action potentials, APV (50 μM) to block NMDARs and bicuculline (20 μM) to block GABAA receptor-mediated IPSCs. Whole-cell voltage-clamp recordings were made with patch pipettes filled with an intracellular solution containing (in mM) 100 Cs-methanesulfonate, 10 CsCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314 and 10 Na-phosphocreatine (pH 7.4), with the membrane potential clamped at -70 mV. Recordings started 10 min after establishing whole-cell configuration to ensure equilibration between the pipette solution and the cytosol. Following 10 min recording of basal mEPSC, ouabain was added to the bath solution and recording continued for 30-40 min in the presence of ouabain. To examine long-term effects of ouabain treatment, coverslips were incubated with ouabain for 1 hr, then transferred to ACSF immediately for recording (referred to as 1 hr) or to normal culture medium to recover for varied periods of time until being recorded.
ALL values are expressed as Mean ± SEM, and analyzed using a Student's t test for comparison between two groups. Statistical significance is defined as p < 0.05.