Article Figures & Data
Figures
- Figure 1.
Generation and analysis of a GluR2 knock-out mouse. A, I, Allele generated by homologous recombination of the GluR2-targeting construct, showing the loxP sites that surround exon 11 and Neo inserted in the intron between exons 11 and 12. Exons 10, 11, and 12 are also shown. II, LoxP sites are indicated by open arrows; these contain BamHI restriction sites in the loxP cassette. 5′ and 3′ probes used for Southern blotting are shown as gray shaded boxes. B, BamHI; S, Spel. B, Southern blots demonstrating successful insertion of the construct at the targeted site. A 12.5 Kb BamHI fragment and 5.5 Kb SpeI fragment are seen in the wild-type mouse with the 5′ probe (lanes 1, 2). Excision of the floxed fragment in the knock-out (-) allele moves the SpeI site closer to the 5′ probe, resulting in a 3.5 Kb fragment (lane 4). Insertion of the loxP cassette BamHI restriction site shortens the BamHI fragment seen by the 3′ probe to 6 Kb in the knock-out allele (lane 6). C, RNase protection assay for exon 11 of the GluR2 allele, demonstrating loss of expression of the GluR2-deleted region in the global knock-out animal (-/-) and normal expression in wild-type (+/+) animals. Sense probe control is included (sense). RNA extracted from whole brains is shown. D, Hippocampus proteins from GluR2+/+, GluR2+/-, and GluR2-/- mice (10 μg per lane) were analyzed by SDS-PAGE and immunoblotted with the C terminus polyclonal antibody indicated. Samples analyzed with different antibodies were obtained from the same preparation of the hippocampus. Histograms show the relative average amount of protein (percentage of GluR2+/+) of three sets of experiments. Levels were measured by densitometric scanning of Western blots.
- Figure 2.
Immunoprecipitation of AMPAR subunits (bound fraction). Ten microliters of bound immunoprecipitate fractions was separated by SDS-PAGE, immunoblotted, and incubated with the indicated C terminus polyclonal antibodies.
- Figure 3.
AMPAR subunits remaining (unbound fraction) after immunoprecipitation of detergent-solubilized hippocampal neurons from GluR2+/+, GluR2+/-, and GluR2-/- mice. The two left lanes of each panel show the nonimmunoprecipitated Triton-solubilized fraction (Tx sol). The 100% lane represents 10 μl of sample applied, whereas the 5% lane represents 0.5 μl of sample applied (after 1:10 dilution with sample buffer). These lanes represent the range of labeling for quantification of immunoreactivity in the depleted fractions. For each gel, standards of 75, 50, 25, and 10% of the solubilized fraction were also analyzed (data not shown). To determine the amount of immunoprecipitated proteins, after GluR1, GluR2-R3, GluR4, GluR1 plus GluR2-R3, GluR1 plus GluR4, and GluR2-R3 plus GluR4 immunoprecipitation (as indicated on top), 10 μl of the depleted fraction was analyzed, equivalent to an equal volume of the solubilized fraction (100%). The percentage of immunostaining remaining in the depleted fractions is shown below each band (mean ± SEM of three separate experiments).
- Figure 4.
A, B, C, Immunogold localization of AMPARs in the CA1 stratum radiatum of GluR2+/+ (WT) and GluR2-/- (KO) mice, using GluR2-R3 (A), GluR1 (B), and GluR3 (C) antibodies. Note that in these micrographs, there is less gold labeling in GluR2-/- than in GluR2+/+ mice. D, Double labeling for GluR1 (10 nm gold particles) and GluR3 (5 nm gold particles). Arrowheads indicate 5 nm of gold in the postsynaptic process. Arrows in A--D indicate postsynaptic membranes or densities. Scale bars: A-C, 200 nm; D, 100 nm. E, Quantification of gold labeling for gold per synapse using GluR2/3, GluR2, GluR3, and GluR1 antibodies expressed as the percentage decrease in GluR2-/- mice, relative to GluR2+/+ mice.
- Figure 5.
Immunogold localization with GluR2/3 antibody on interneuron synapses [i.e., found on a dendrite shaft (den), compared with spine synapses, in two GluR2-/- animals]. Arrowheads indicate postsynaptic labeling. Note that the labeling is prevalent in the dendrite shaft synapses but not in the spine synapses. Scale bar, 200 nm.
- Figure 6.
Intracellular receptors in GluR2+/+ and GluR2-/- mice. A, Glycosylation state of AMPARs in the GluR2+/+ and GluR2-/- mice. Membrane homogenates from the hippocampus of GluR2+/+ and GluR2-/- mice were solubilized with 0.5% SDS in the presence of 1% β-mercaptoethanol. After dilution with 1% NP-40, soluble extracts were incubated in the absence of enzyme (control samples) or in the presence of Endo-H or PNGaseF. GluR1 and GluR3 have a small but distinct population that is Endo-H sensitive in the GluR2+/+ and GluR2-/- mice. B, Quantification of gold labeling per square micrometer using GluR1 antibody in the cell body (light gray; n = 38 for GluR2+/+; n = 35 for GluR2-/-; p = 0.96) and dendrites (dark gray; n = 21 for GluR2+/+; n = 17 for GluR2-/-; p = 0.50) of GluR2+/+ and GluR2-/- mice. C-J, Representative micrographs of GluR1 labeling in the cell body of GluR2+/+ (C,D) and GluR2-/- (G,H) mice and in dendrites of GluR2+/+ (E,F) and GluR2-/- (I,J) mice. Arrowheads indicate labeling associated with ER or reticular-like structures. Scale bar, 200 nm.
Tables
- Table 1.
Quantification of immunogold localization of AMPA receptors in the CA1 stratum radiatum
Antibody Animal CA1-stratum radiatum gold per synapse Number of synapses GluR2 gold per synapse WT (2 animals; 2 experiments using 1 antibody) 0.13 480 KO (2 animals; 2 experiments using 1 antibody) 0.005 372 GluR2/3 gold per synapse WT (2 animals; 4 experiments using 1 antibody) 1.23 860 KO (2 animals; 4 experiments using 1 antibody) 0.18 856 GluR3 1/100 gold per synapse WT 2 animals 0.25 460 KO 2 animals 0.18 385 GluR3 1/25 gold per synapse WT 2 animals 0.55 379 KO 2 animals 0.41 339 GluR1 gold per synapse WT (3 animals; 7 experiments using 2 antibodies) 0.55 1346 KO (3 animals; 7 experiments using 2 antibodies) 0.31 1324 GluR1 gold per micrometer WT (3 animals; 3 experiments using 2 antibodies) 3.5 641 KO (3 animals; 3 experiments using 2 antibodies) 2.2 664 Control WT (1 animal; 1 experiment using 1 antibody) 0 117 KO (1 animal; 1 experiment using 1 antibody) 0 109 All differences between WT (GluR2+/+) and KO (GluR2−/−) are highly significant, except for GluR3, for which the decreases in KOs are slightly significant (p<0.05 for 1/100; p<0.02 for 1/25). Average synapse length for GluR1-labeled sections is 0.20 μm (n=641) and 0.19 μm (n=664) for WT and KO, respectively. This 5% decrease in length is significant. For the latter study, the same area of neuropil was examined (>2000 μm2) for WT and KO, although the area contributed by large structures, especially dendrites, was not subtracted. This indicates that there are, at most, only small changes in synapse number and length.
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