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GFP (enhanced GFP; Clontech) was inserted between the third and fourth amino acids after the predicted signal peptide cleavage site of rat GluR1-flop cDNA with standard molecular biology techniques. HEK cells were transfected with plasmid-based mammalian expression vector with lipofectin (Gibco-BRL Life Technologies). GluR1-GFP and GluR2 were cotransfected in 1:4 ratio. Protein immunoblotting was carried out with antibodies to GluR1 COOH-terminal (0.1 μg/ml; Chemicon International). Whole-cell recordings were obtained 2 to 5 days after transfection in Hepes (10 mM)-buffered Hanks' solution in the presence of 100 μM cyclothiazide. Kainate (1 mM) was applied through a puffer pipette positioned close to recorded cell Five to ten records were obtained at holding potentials of -80 to 60 mV (20-mV steps).
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unpublished results
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Whole-cell recordings from neurons infected with Sindbis virus for 1 to 4 days show normal passive membrane properties (for example, input resistance: uninfected, 276 ± 64, N = 13; GluR1-GFP infected, 302 ± 51, N = 8; P = 0.78) (S. Shi, Y. Hayashi, R. Malinow, unpublished results).
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Shi, S.1
Hayashi, Y.2
Malinow, R.3
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0344688657
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Dissociated cultured neurons were prepared as previously described (33).
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22
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0345550785
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note
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To estimate the level of recombinant GluR1-GFP expression, relative to endogenous GluR1 expression, we performed immunohistochemistry on fixed (14) dissociated hippocampal neurons (33) with a GluR1 COOH-terminal antibody (1 μg/ml; Chemicon International) that recognizes both proteins (Fig. 1B) as primary and Texas Red coupled as secondary (which does not overlap in fluorescence with GFP). In a field of infected and noninfected cells, dendrites (80 μm from cell body) of infected cells showed 2.7 ± 0.2 fold (mean ± SEM, N = 8) immunolabel compared with similar regions of uninfected cells.
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23
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0345119458
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note
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Tissue was fixed with freshly made 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) (dissociated neurons: 4 °C for 30 min; organotypic slices (76): 4 °C for 1 hour). With such fixation, immunohistochemistry (see below) detected only surface epitopes (dissociated cells: Fig. 2C; organotypic slices: Fig. 3G). To detect intracellular epitopes, tissue was further treated with 0.3% Triton X-100 in PBS (dissociated cells: 4 °C for 10 min; organotypic slices: 4 °C for 30 min). Immunohistochemistry: Cells were blocked in blocking solution (10% horse serum in PBS; 60 min) and then incubated with primary antibody (4 °C, overnight) in blocking solution. All primary rabbit polyclonal antibodies were visualized with biotin-conjugated antibody to rabbit immunoglobulin G (IgG) and Texas Red-avidin system. Mouse monoclonal antibodies were visualized with antibodies to mouse IgG labeled with Cy5 (Jackson ImmuoResearch). Images of dissociated neurons were collected with a cooled charge-coupled device (Photometrics) and analyzed with PMIS or V for Windows (Photometries); organotypic slices were imaged with TPLSM (21). Fraction of GluR1-GFP molecules on surface for a given region can be quantified by computing [(antibody to GFP signal)/(GFP signal)] in nonpermeabilized conditions for that region and normalizing by [(antibody to GFP signal)/(GFP signal)] obtained from similar tissue in permeabilized conditions. Signals in above computation indicate background subtracted values.
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24
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0345119457
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note
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2 at 28 °C. Responses to glutamate were evoked by adding (in μM) 40 L-glutamic acid γ-(α-carboxyl-2-nitrobenzyl)ester (Molecular Probes), 1 tetrodotoxin, 100 picrotoxin, 100 APV, and 1 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid. Ten to 25 ms of flash (100 W of Hg; quartz optics, Zeiss Axiovert 135) was delivered to a spot (∼50-μm diameter) positioned over dendrites.
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Organotypic slice culture of hippocampus was prepared as described [L. Stoppini et al., J. Neurosci. Methods 37, 173 (1991)]. Hippocampal slices (400 μm) were cut from postnatal 6-to 8-day-old rats with tissue chopper and cultured in medium as described [W. Musleh et al., Proc. Natl. Acad. Sci. U.S.A. 94, 9451 (1997)]. Slices were placed in a recording chamber and perfused with ACSF. Synaptic transmission was evoked (0.1 Hz, 100 μs, ∼10 V) with a glass stimulation electrode (1 to 2 megaohms, filled with 3 M NaCl), positioned in CA1 stratum radiatum. Tetanic stimuli consisted of two 100-Hz trains of 1-s duration separated by 20 s.
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note
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The distribution of GluR1 and GluR1-GFP was measured quantitatively with immuno-gold labeling (18). Sections from dendritic regions ∼100 μm from the cell bodies were examined. To identify a cell infected with GluR1-GFP virus, we identified a dendritic segment (∼1 μm in diameter) with immunolabeling. Comparable regions (with dendritic segments ∼1 μm in diameter) were chosen in tissue from uninfected P10 animals for analysis of endogenous GluR1. For each section, the label was identified as being in one of the following compartments: A, dendritic cytosol; B, dendritic surface; C, spine, non-PSD; and D, PSD. The total number of grains counted was 515 for endogenous GluR1 and 785 for GluR1-GFP. For background subtraction, 50 presynaptic terminals were randomly chosen, label was counted, and density was calculated (0.17 gold per square micrometer). This background density constituted 10 to 20% of the signal found in dendritic cytosol (0.92 gold per square micrometer) and was subtracted to reach dendritic cytosol value (A, above); this region is the only compartment with sufficient area to be affected by background labeling.
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2+ imaging studies with the same stimulus electrodes (tip resistance, 1 to 2 megaohms) and parameters indicate that synaptic excitation in this preparation is sparse. Thus, as in previous studies (21, 37), the tip of the stimulating electrode must be placed around 5 to 15 μm from imaged postsynaptic regions.
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Within 3 SD of the noise measured in background regions.
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50%) as an index of signal homogeneity.
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0345119456
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This decrease is 3.6 SD beyond those changes seen in the absence of stimulation, indicating a small likelihood that these changes occur from chance alone.
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We thank S. Schlesinger for Sindbis virus expression system; P. De Camilli for antibody against synapsin I; C. S. Zucker for antibody to GFP; J. Boulter, S. F. Heinemann, and P. H. Seeburg for cDNA clones; N. Dawkins-Pisani, B. Burbach, and P. O'Brian for technical assistance; and Y.-X. Wang for assistance in the immunogold studies. Y.H. is a recipient of research fellowships from Japan Society for the Promotion of Science and Uehara Memorial Foundation. S.H.Z. was a recipient of Wellcome Trust Fellowship. This study was supported by the NIH (R.M. and K.S.), the Mathers Foundation (R.M.), and the Human Frontier Science Program, Pew, and Whitaker Foundations (K.S).
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