Slice preparation and electrophysiology
Standard techniques approved by the University of California, Los Angeles (UCLA) Institutional Animal Care and Use Committee were used to prepare 400-μm-thick slices from hippocampi obtained from 2- to 3-month-old C57BL/6 mice. For some experiments, “mini-slices” containing just the hippocampal CA1 region were prepared by removing the dentate gyrus, CA3 region, and subiculum from freshly cut slices. In all experiments, slices were maintained at 30°C in an interface-type chamber (Fine Science Tools, Foster City, CA) and continuously perfused with an oxygenated (95% O2/5% CO2) artificial CSF (ACSF) consisting of 124 mm NaCl, 4.4 mm KCl, 25 mm Na2HCO3, 1 mm NaH2PO4, 1.2 mm MgSO4, 2 mm CaCl2, and 10 mm glucose. Slices were allowed to recover for at least 2 h before the start of an experiment. Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells were activated using a bipolar, nichrome wire electrode placed in stratum radiatum of the CA1 region of the slice, and the resulting synaptic potentials were recorded using an ACSF-filled glass micro-electrode (5–10 MΩ) placed in stratum radiatum. Single pulses of presynaptic fiber stimulation were delivered at 0.02 Hz using a stimulation intensity that evoked field EPSPs that were 50% of the maximum amplitude that could be evoked using strong stimulation intensities. To examine the effects of synaptic stimulation on GluR1 phosphorylation at T840, we used CA1 mini-slices maintained in interface-slice chambers and used larger bipolar stimulation electrodes fabricated from 66-μm-diameter, Formvar-coated nichrome wire (A-M Systems, Carlsborg, WA). The tip separation of the stimulation electrode was adjusted such that the electrode spanned the width of stratum radiatum and the stimulating electrode was placed at one end of the slice while an extracellular recording electrode was placed in stratum radiatum at the opposite end of the slice.
Slices were prepared and maintained using techniques identical with those used for electrophysiological recordings. In general, slices obtained from the same animal were placed into up to four separate chambers (three slices per chamber). One chamber was exposed to ACSF alone to provide control, untreated tissue while the remaining chambers were treated with various pharmacological reagents. This allowed us to use a within-subjects design and, by pooling multiple slices per condition, provided sufficient amounts of protein for several immunoblots. Thus, different blots could be used to measure phospho- and total GluR1 levels from the same samples. Pharmacological treatments and tissue homogenization were performed using previously described methods (Delgado and O’Dell, 2005
). Synaptoneurosomes were prepared using a previously described protocol (Ho et al., 2004
). Proteins (20 μ
g/lane) were resolved on 12% SDS-PAGE gels, transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membranes, and incubated overnight with primary antibodies. After a 2–4 h incubation with HRP-conjugated secondary antibodies (1:2000), immunoreactive bands were visualized using enhanced chemiluminesence (Immun-Star; Bio-Rad, Hercules, CA). Image acquisition and analysis were done using a cooled CCD camera and the Quantity One software package from Bio-Rad. To control for potential variations in loading, all blots were reprobed with anti-tubulin or anti-actin antibodies and the optical density values for each band of interest were normalized to the density values obtained for these loading controls in the same lane.
The rabbit polyclonal antibody against phospho-T840 GluR1 was made by Abcam (Cambridge, UK), using a synthetic peptide (conjugated to KLH) derived from the C terminus of mouse GluR1 phosphorylated at T840. The antibody was subsequently immunogen affinity purified. Phosphospecific antibodies to GluR1 phosphorylated at S845 and S831 (both used at 1:1000) as well as total GluR1 (1:2000) were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibodies to actin (1:2000) and βIII tubulin (1:20,000) were obtained from Upstate Biotechnology and Sigma (St. Louis, MO), respectively.
Hippocampal slices were maintained at 30°C in submerged-slice type chambers and perfused (at 1–2 ml/min) with oxygenated (95% O2/5% CO2) ACSF. Slices were allowed to recover for at least 3 h and then homogenized in 20 mm 4-morpholinepropanesulfonic acid (MOPS), pH 7.4, 2 mm EDTA, 5 mm EGTA, 1% Triton X-100, 0.5% DOC (sodium deoxycholate), 30 mm NaF, 40 mm β-glycerophosphate, 20 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and Roche (Basel, Switzerland) Complete protease inhibitor mixture. Protein concentrations were determined by Micro BCA Protein Assay kit (Pierce, Rockford, IL) and 500 μg of total protein was immunoprecipitated overnight at 4°C with 2 μg of anti-GluR1 (Chemicon, Temecula, CA). After a 2 h incubation with protein G-Sepharose, samples were washed three times with 20 mm MOPS, pH 7.4, 1% Triton X-100, and 1 mm sodium orthovanadate. Proteins were then separated by SDS-PAGE and procedures described above were used for Western blotting.
Expression of wild-type and T840A GluR1
Mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, TX) was performed according to the manufacturer’s instructions to produce the mutant GluR1 T840A with the following forward and reverse primers: 5′-GCCATACGGACATCGGCCCTCCCCCGG-3′ and 5′-CCGGGGGAGGGCCGATGTCCGTATGGC-3′. The fidelity of the mutagenesis reaction was verified by DNA sequencing. Transfection of HEK 293T cells was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were plated on 6 cm dishes and transfected with 16 μg of DNA and 40 μl of Lipofectamine at ~70–75% confluence. To enhance GluR1 phosphorylation at T840, the protein phosphatase 1 and 2A inhibitor Cantharidin (10 μm; Tocris, Ellisville, MO) was added 24 h after transfection, and cells were incubated for 1 h at 37°C. Cells were then washed three times with ice-cold PBS and lysed in 600 μl of RIPA buffer [50 mm Tris, pH 7.5, 150 mm NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS plus Complete protease inhibitor mixture (Roche) and phosphatase inhibitor mixtures 1 and 2 (Sigma)] on ice for 30 min. GluR1 was then immunoprecipitated (at 4°C) by first preclearing lysates for 3 h with normal rabbit serum. Each lysate was then incubated overnight with 6 μl of anti-GluR1 antibody (Upstate Biotechnology). Immune complexes were then bound to protein A-Sepharose beads (Zymed, South San Francisco, CA) during a 1 h incubation. The beads were then washed four times in wash buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, 10% glycerol), and proteins were eluted in Laemmli buffer.
Cell culture and immunocytochemistry
At UCLA, primary cultures of hippocampal neurons were grown on Matrigel (Becton Dickinson Labware, Bedford, MA)-coated coverslips in MEM media (Invitrogen) containing 5% FBS (HyClone, Logan, UT), 2% B-27 supplement (Invitrogen), 2 mm Glutamax (Invitrogen), 24 mg/ml insulin (Sigma), 0.1 mg/ml transferrin (Calbiochem, La Jolla, CA), 28 mm glucose, and 4 mm AraC (Sigma). After 14–21 d in vitro, cultures were fixed with 4% paraformaldehyde for 10 min and cells were permeabilized for 5 min with 0.1% Triton X-100. After quenching free aldehyde groups with 50 mm NH4Cl for 10–15 min, nonspecific antibody binding was blocked by incubation in 10% goat serum for 30 min. Cells were then incubated with primary antibodies in 10% goat serum for 1–3 h at room temperature or overnight at 4°C. Primary antibodies were detected by incubation with 1:2000 Alexa Fluor 488- or 546-conjugated secondary antibodies in 10% goat serum for 1 h at room temperature. Confocal fluorescence images were obtained using a Zeiss (Oberkochen, Germany) Pascal scanning laser microscope using 63× (1.2 numerical aperture) water immersion objective.
At The Sanger Institute, cultures grown on polylysine/laminin-coated coverslips for 14–24 d in vitro were fixed with ice-cold methanol for 7 min, blocked in PBS containing 3% BSA and 0.2% Triton X-100 for 1 h (blocking solution was also used in both antibody steps); primary antibodies were applied together for 1 h at room temperature. Secondary antibodies were applied together for 20 min at room temperature. Coverslips were mounted with Prolong Anti-Fade Gold (Invitrogen). Primary antibodies used were as follows: mouse anti-GluR1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho T840 GluR1, and chicken antimicrotubule-associated protein 2 (MAP2) (Abcam, Cambridge, UK). Secondary antibodies used were as follows: chicken IgY specific-Cy2 and mouse IgG specific-Rhodamine (Abcam, Cambridge, UK), and rabbit IgG specific-Alexa Fluor 633 (Invitrogen). All images were taken on a Zeiss 510 META confocal microscope using a 63× Plan-apochromat objective.
Peptide array phosphorylation
The 15-aa-long peptides that encompassed the GluR1 threonine 840 (NEAIRTST
LPRNSGA) were synthesized on cellulose membranes in a parallel manner using SPOT technology (Reineke et al., 2001
) and covalently immobilized to glass slides. Each peptide was present in triplicate and a negative control peptide for the phosphorylation site was included, replacing threonine with valine (NEAIRTSV
LPRNSGA). As a positive control of kinase activity, consensus phosphorylation peptides were attached. These peptide sequences were included in a broader screen of synaptic phosphorylation sites to be reported elsewhere. Peptide arrays were sealed with Gene-Frame incubation chambers (Abgene House, Surrey, UK), and the chambers were filled with 330 μ
l of kinase buffer, 20 mm
MOPS, pH 7.2, 25 mm
-glycerol phosphate, 5 mm
EGTA, 1 mm
sodium orthovanadate, 1 mm
DTT, and 2 μ
g of p70 S6 kinase (T412E). The reaction was initiated by the addition of ATP/MgCl2
[final concentrations: 100 μm
ATP, 10 μ
Ci of [γ
P]ATP (GE Healthcare, Piscataway, NJ), 15 mm
]. After a 40 min incubation at 32°C, the peptide microarrays were washed six times, alternating between 0.1 m
phosphoric acid and distilled water. γ
P incorporation in the immobilized peptide spots was detected on a Typhoon 8600 PhosphorImager (50 μ
m resolution; Amersham Biosciences). Image analysis and signal quantification were performed using Image-Quant TL (GE Healthcare), and positive signals were defined after background subtraction. The following recombinant kinases were used (all obtained from Upstate Biotechnology): protein kinase A (PKA), catalytic subunit, Akt1/(ΔPH, S473D), protein kinase Cα
, PKCζ, phosphoinositide-dependent kinase 1 (PDK-1), Cdk5, Raf-1, p38α
, mitogen-activated protein kinase kinase 1 (MEK1), extracellular signal-regulated kinase 1 (Erk1), Erk2, Rsk2, c-Jun N-terminal kinase 3 (JNK3), glycogen synthase kinase 3β
), CaMKII, casein kinase 1, casein kinase 2, p70 S6 kinase (T412E), Rho GTPase-Rho kinase II (ROCK-II), TANK binding kinase 1 (TBK1), Src, Fyn, Pyk2, and Fes.
Paired and unpaired t tests or one-way ANOVAs followed by Student–Newman–Keuls tests for multiple pairwise comparisons were used to assess statistical significance. Nonparametric versions of these tests (Mann–Whitney rank sum tests and Friedman repeated-measures ANOVAs on ranks) were used where appropriate. Statistical tests were performed using SigmaStat (Systat Software, Richmond, CA). All results are reported as mean ± SEM.