All the expression constructs were made by PCR. Internal deletion and point mutant were made either using QuikChange Site-Directed Mutagenesis Kit (Stratagene) or by megaprimer method (Barik, T. 2002.PCR Cloning Protocols, Vol 192, 2nd edn, Humana Press). The sequence of the primers used to generate each mutant will be supplied upon request. PCR products were cloned into expression vectorpRK5 (Genentech). All constructs were verified by sequencing.
All antibodies were previously described or were acquired commercially: Bassoon (Stressgen), GluR1-N (pAb, JH1816(Rumbaugh et al., 2003
)), GluR1-C (JH1710;(Ye et al., 2000
)), GluR2-C (pAb, JH1707 (Blackstone et al., 1992
)) pAb, Arc (pAb (Lyford et al., 1995
)), Arc (mAb, Santa Cruz),β-actin (mAb, Sigma),α-CaMKII (mAb, Boehringer Mannheim), PSD-95 (mAb, Affinity Bioreagents).
Recombinant Sindbis virus and infection
Arc ORF was first subcloned into pIRES2-EGFP and transferred into pSinRep5 (Invitrogen). At 14-21 days in vitro (DIV), cultured neurons were infected with virus. Experiments were usually performed 12-16 hrs after infection.
Preparation of crude synaptosomal fraction from Brain
The subcellular fractionation procedure was performed according to the technique of Huttner et al.(Huttner et al., 1983
). All procedures were performed at 4°C. In brief, rat brains were homogenized in 10 volumes of buffered sucrose (0.32 M sucrose, 4 mM HEPES/NaOH, pH 7.4, 1mM EDTA, 1mM EGTA and protease inhibitors cocktail) with a glass-Teflon homogenizer. The homogenate was centrifuged at 800Xg for 15 min and the supernatant was collected. The supernatant was again centrifuged at 9000Xg for 15 min and pellet was collected as crude synaptosomal fraction, P2.
Coimmunoprecipitation and Immunobloting
Six to eight weeks old Arc Wt and KO mice were sacrificed by decapitation and forebrain regions dissected. Protein concentration of an aliquot of total homogenate was measured. Rest of the homogenate used for P2 fractionation as described above and protein concentration of an aliquot of P2 measured. Equal amount of total protein from wt and ko was loaded. For coimmunoprecipitation, crude synaptosomal fraction (P2) was sonicated in PBS with 1% Triton X-100, 1mM EDTA, 1mM EGTA and protease inhibitors cocktail (Roche). The homogenate was centrifuged at 100,000Xg for 20 min at 4°C and supernatants with equal amount of protein were incubated with 2 ug of Rabbit polyclonal antibodies for GluR1 and GluR2. After 1.5 hr of mixing at 4°C Protein A agarose slurry was added and incubated for another hour. The beads were washed with PBS+1%Triton X-100 three times and eluted with SDS loading buffer. The samples were than analyzed by SDS-PAGE and western blotting.
For surface biotinylation, infected or drug-treated cortical neurons were cooled on ice, washed twice with ice-cold PBS containing 1mM CaCl2 and 0.5 mM MgCl2, and then incubated with PBS containing 1mM CaCl2, 0.5 mM MgCl2, and 1 mg/ml Sulfo-NHS-SS-Biotin (Pierce) for 30 min at 4°C. Unreacted biotin was quenched by washing cells three times with ice-cold 100mM Glycine (pH7.4). Cultures were harvested in RIPA buffer. Homogenates were centrifuged at 132,000 rpm for 20 min at 4°C. The resulting supernatant volume was measured and 15% of it separated as the total protein. The remaining 85% of the homogenate was rotated overnight at 4°C with Streptavidin beads (Pierce). Precipitates were washed with RIPA buffer and analyzed by immunoblotting with each antibody.
Cell Culture and Neuronal Transfection.
Low-density hippocampal neurons were prepared as described previously (Banker and Cowan, 1977
). High-density cortical cultures from embryonic day 18 (E18) rat pups were prepared as reported previously (ADD Refernce). Mouse cultures were prepared in a similar manner from E16.5-E17.5 mouse pups. Neuronal transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in DIV 12-14 neurons and were analyzed 16-24 hr after initial incubation.
Immunocytochemistry, Microscopy, and Data Analysis
Cells were fixed in 4% paraformaldehyde, 4% sucrose containing PBS solution for 20 min at 4°C and were subsequently permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were then blocked for 1 hr in 10% normal donkey/goat serum (NGS). Primary antibodies were diluted in 10% NGS and incubated with neurons for 1 hr at room temperature or overnight at 4°C. Alexa488, Alexa555, or Alexa647-conjugated secondary antibodies (1:500; Molecular Probes, Eugene, OR) to the appropriate species were diluted in 10% NDS and incubated at room temperature for 1 hr. Coverslips were mounted on precleaned slides with PermaFluor and DABCO.
To label surface GluR1-containing AMPA receptors, 2.5 μg of GluR1-N JH1816 pAb was added to neuronal growth media and incubated at 10°C for 20 min. The unbound excess antibody was quickly washed with fresh warmed growth medium and then fixed and mounted according to the methods described above.
Glycine stimulation was carried out as described before (Watt et al., 2004
). Neurons were incubated at 37 °C and 5% CO2
for 15 min in ACSF containing the following: NaCl, 126 mM; KCl, 5.5 mM; MgSO4
, 0.4 mM; NaH2
, 1 mM; NaHCO3
, 25 mM; CaCl2
, 2 mM; dextrose, 14 mM; glycine, 0.2 mM; bicuculline, 0.01 mM. Then, we replaced the medium in the culture dishes and returned them to the tissue culture incubator until immunocytochemistry was performed. Immunofluorescence was viewed and captured using a Zeiss LSM 510 confocal laser scanning microscope. Quantification of surface GluR1 puncta were carried out essentially as described (Rumbaugh et al., 2003
), using Metamorph imaging software (Universal Imaging, Downingtown, PA). Images were acquired and saved as multi-channel TIFF files with a dynamic range of 65536 gray levels (16-bit binary; MultiTrack acquisition for confocal). To measure punctate structures, neurons were thresholded by gray value at a level close to 50% of the dynamic range. Background noise from these images was negligible. After a dendrite segment was selected, all puncta were treated as individual objects and the characteristics of each, such as pixel area, average fluorescence intensity, and total fluorescence intensity, were logged to a spreadsheet. In addition, each dendrite length was logged in order to calculate puncta density and total intensity per dendritic length (all values shown are per 10 μm of dendrite). untransfected cells in individual coverslips. The average single pixel intensity from each region was calculated and averages from all regions were derived. Significance was determined by a paired Student’s T test.
Electrophysiology and mEPSC analysis
Whole-cell patch-clamp recordings were performed from forebrain cultures at the day in vitro (DIV) indicated. To isolate AMPAR-mediated mEPSCs, neurons were continuously perfused with artificial cerebral-spinal fluid (aCSF) at a flow rate of 1 ml/min. The composition of aCSF was as follows (in mM): 150 NaCl, 3.1 KCl, 2 CaCl2,1 MgCl2, 10 HEPES, 0.1 DL-APV, 0.005 strychnine, 0.1 picrotoxin, and 0.001 tetrodotoxin. The osmolarity of aCSF was adjusted to 305-310 and pH was 7.3-7.4. Intracellular saline consisted of (in mM): 135 Cs-MeSO4, 10 CsCl, 10 HEPES, 5 EGTA, 2 MgCl2, 4 Na-ATP, and 0.1 Na-GTP. This saline was adjusted to 290-295 mOsm, and pH was 7.2.
Transfected neurons were selected based on fluorescent (eGFP) signal. Once the whole-cell recording configuration was achieved, neurons were voltage-clamped and passive properties were monitored throughout. In the event of a change in series resistance (Rs) or input resistance (Ri) >15% during the course of a recording, the data were excluded from the set. mEPSCs were acquired through a MultiClamp 700A amplifier (Axon Instruments, Union City, CA), filtered at 2 kHz, and digitized at 5 kHz. Sweeps of 20 seconds with zero latency (essentially “gap free”) were acquired until a sufficient number of events were recorded (a minimum of 5 and no longer than 30 minutes). Data were recorded continuously only after a period of two minutes, during which the cell was allowed to stabilize. mEPSCs were detected manually with MiniAnalysis software (Synaptosoft Inc, Decatur, GA) by setting the amplitude threshold to √ RMS x 3 (usually 4 pA). Once a minimum of 100 events had been collected from a neuron, the amplitude, frequency, rise time (time to peak), decay time (10-90%), and passive properties were measured. In all electrophysiological experiments, a similar amount of data was acquired from both transfected and untransfected neurons on the same day. We have found that recording transfected neurons followed by recording an untransfected neuron in the immediate vicinity yields remarkably consistent results. This is likely a result of reducing errors that arise from slight changes in neuronal density between preps or changes in the density of neurons from different areas of a coverslip. These parameters were crucial for obtaining reliable, low variability data between experimental populations. Data from each group were then averaged, and statistical significance was determined by Student’s T test (unless noted otherwise). All electrophysiological experiments were performed from at least two individual platings of neurons from three different transfections.