Specific roles of AMPA receptor subunit GluR1 (GluA1) phosphorylation sites in regulating synaptic plasticity in the CA1 region of hippocampus

Abstract

Activity-dependent changes in excitatory synaptic transmission in the CNS have been shown to depend on the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). In particular, several lines of evidence suggest that reversible phosphorylation of AMPAR subunit glutamate receptor 1 (GluR1, also referred to as GluA1 or GluR-A) plays a role in long-term potentiation (LTP) and long-term depression (LTD). We previously reported that regulation of serines (S) 831 and 845 on the GluR1 subunit may play a critical role in bidirectional synaptic plasticity in the Schaffer collateral inputs to CA1. Specifically, gene knockin mice lacking both S831 and S845 phosphorylation sites ("double phosphomutants"), where both serine residues were replaced by alanines (A), showed a faster decaying LTP and a deficit in LTD. To determine which of the two phosphorylation sites was responsible for the phenotype, we have now generated two lines of gene knockin mice: one that specifically lacks S831 (S831A mutants) and another that lacks only S845 (S845A mutants). We found that S831A mutants display normal LTP and LTD, whereas S845A mutants show a specific deficit in LTD. Taken together with our previous results from the "double phosphomutants," our data suggest that either S831 or S845 alone may support LTP, whereas the S845 site is critical for LTD expression.

FIG. 1.

Generation of GluR1–S831A and GluR1–S845A mutant mice. A: schematics showing the specific mutations made to generate the GluR1–S831A targeting construct and the GluR1–S845A targeting constructs. Sal I or Sac II was included to each construct as depicted. B: normal gross cytoarchitecture of the hippocampus as revealed by Nissl staining (left column) and normal distribution of glutamate receptor 1 (GluR1, middle column) and GluR2/3 (right column) as shown using immunohistochemical labeling. Top rows: wildtype (WT) hippocampal sections. Middle rows: S831A homozygous hippocampal sections. Bottom rows: S845A homozygous hippocampal sections.

FIG. 2.

Quantitative analysis of phosphorylation of GluR1 on S831 and S845 in young and adult S831A and S845A mutants. A: no significant changes in the relative S845 phosphorylation or the total GluR1 levels in hippocampal samples obtained from young (3-wk-old) S831A mutants. Representative immunoblots are shown on the top panels. S831A mutant samples specifically lack signal when probed with S831 phosphospecific antibody (S831-p Ab). The relative S845 phosphorylation level (bottom left: S845 signal normalized to total GluR1 levels) and the total GluR1 level (bottom right) did not differ between WT and S831A. B: a significant reduction in the relative S845 phosphorylation (bottom left) and a significant increase in the total GluR1 level (bottom right) in hippocampal samples from adult (≥3-mo-old) S831A mutants. The observed decrease in the relative S845 phosphorylation level was largely due to the increase in the total GluR1 levels. *P < 0.001, t-test). Representative immunoblot images are shown in the top panel. S831A mutant samples specifically lack signal when probed with S831 phosphospecific antibody (S831-p Ab). C: no changes in the relative S831 phosphorylation (bottom left: S831 signal normalized to total GluR1 levels) and the total GluR1 levels (bottom right) in hippocampal samples of young (3-wk-old) S845A mutants. Representative immunoblot images are shown in the top panel. S845A mutant samples specifically lack signal when probed with S845 phosphospecific antibody (S845-p Ab). D: no significant changes in the relative S831 phosphorylation (bottom left) and GluR1 levels (bottom right) in hippocampal samples of adult (≥3-mo-old) S845A mutants. Representative immunoblot images are shown in the top panel. S845A mutant samples specifically lack signal when probed with S845 phosphospecific antibody (S845-p Ab).

FIG. 3.

Characterization of basal synaptic transmission and plasticity in the Schaffer collateral synapses onto CA1 in the young (3-wk-old) S831A mutants. A: normal basal synaptic transmission in the young S831A mutants. Input–output (I-O) curves were generated by increasing stimulation intensities. Measured field potential (FP) slope was plotted against the amplitude of presynaptic fiber volley. There was no difference in the I-O curve of S831A mutants (closed symbols) and that of WT littermates (open symbols). B: normal paired-pulse facilitation (PPF) in the young S831A mutants. PPF ratios measured at different interstimulus intervals (ISIs) did not differ between S831A mutants (closed symbols) and WT littermates (open symbols). C: GluR1–S831A mutants exhibit normal long-term potentiation (LTP) induced by delivering 4 trains of theta burst stimulation (TBS). WTs: open symbols; S831A mutants: closed symbols. Representative traces taken right before (thin line) and 2 h after LTP induction (thick line) are shown to the right. D: the average magnitude of long-term depression (LTD) induced by a 15-min train of 1 Hz was slightly larger in the S831A mutants (closed symbols), but was not statistically significant from that of WT littermates (open symbols) at 1 h. S831A mutants displayed a significantly larger initial depression (average of the first 10 min post-1 Hz). Representative traces taken right before (thin line) and 1 h after the onset of 1-Hz stimulation (thick line) are shown to the right.

FIG. 4.

Basal synaptic transmission and plasticity in the Schaffer collateral synapses onto CA1 in the adult (≥3-mo-old) S831A mutants. A: normal basal synaptic transmission in the adult S831A mutants. I-O curves were generated by plotting the FP slope against the amplitude of presynaptic fiber volley. WTs: open symbols; S831A mutants: closed symbols. B: PPF ratios measured at different ISIs did not differ between S831A mutants (closed symbols) and WT littermates (open symbols). C: adult GluR1–S831A mutants exhibited normal LTP induced by delivering 4 trains of TBS. WTs: open symbols; S831A mutants: closed symbols. Representative traces taken right before (thin line) and 2 h after LTP induction (thick line) are shown to the right. D: adult GluR1–S831A mutants display normal LTP induced by a one-train TBS (1×TBS) protocol. WTs: open symbols; S831A mutants: closed symbols. Superimposed representative traces taken right before (thin line) and 2 h after LTP (thick line) are shown to the right. E: the magnitude of LTD induced by a 15-min train of paired-pulse 1 Hz (PP-1-Hz, ISI = 50 ms) was not significantly different at 1 h between adult S831A mutants (closed symbols) and WT littermates (open symbols). S831A mutants displayed a significantly larger initial depression (average of the first 10 min post PP-1-Hz). Representative traces taken right before (thin line) and 1 h after the onset of PP-1-Hz stimulation (thick line) are shown to the right.

FIG. 5.

Normal depotentiation and dedepression in the adult S831A mutants. A: depotentiation was induced by delivering a train of 1 Hz (15 min) at 30 min following LTP induction by 4 trains of TBS. Left: the entire experiment normalized to the pre-LTP baseline. Middle: superimposed representative traces taken right before LTP induction (thin line), 30 min after LTP (thick solid line), and 1 h after the onset of 1-Hz stimulation (thick dashed line). Right: the magnitude of depotentiation when renormalized to the pre-1-Hz baseline. B: dedepression was induced 1 h following the onset of PP-1-Hz stimulation by delivering 4 trains of TBS. Left: the entire experiment normalized to the pre-LTD baseline. Middle: superimposed representative traces taken right before LTD induction (thin line), 1 h after LTD (thick solid line), and 1 h after TBS (thick dashed line). Right: the magnitude of depotentiation when renormalized to the pre-TBS baseline.

FIG. 6.

Young (3-wk-old) S845A mutants display a specific deficit in LTD. A: I-O curves generated by plotting FP slope against the amplitude of presynaptic fiber volley were not different between S845A mutants (closed symbols) and WT littermates (open symbols). B: PPF ratios measured at various ISIs did not differ between S845A mutants (closed symbols) and WT littermates (open symbols). C: LTP induced by delivering 4 trains of TBS was normal in the young S845A mutants. WTs: open symbols; S845A mutants: closed symbols. Representative traces taken right before (thin line) and 2 h after LTP induction (thick line) are shown to the right. D: LTD induced by a 15-min train of 1 Hz was virtually abolished in the young S845A mutants (closed symbols), but was normal in the WT littermates (open symbols). Representative traces taken right before (thin line) and 1 h after the onset of 1-Hz stimulation (thick line) are shown to the right.

FIG. 7.

Adult (≥3-mo-old) S845A mutants also display a specific deficit in LTD. A: I-O curves generated by plotting FP slope against the amplitude of presynaptic fiber volley did not differ between S845A mutants (closed symbols) and WT littermates (open symbols). B: PPF ratios measured at various ISIs were not different between S845A mutants (closed symbols) and WT littermates (open symbols). C: LTP induced by delivering 4 trains of TBS was normal in the adult S845A mutants. WTs: open symbols; S845A mutants: closed symbols. Representative traces taken right before (thin line) and 2 h after LTP induction (thick line) are shown to the right. D: one train TBS (1×TBS) induced LTP was also normal in the adult S845A mutants. WTs: open symbols; S845A mutants: closed symbols. Superimposed representative traces taken before (thin line) and 2 h after LTD induction (thick line) are shown to the right. E: a 15-min train of PP-1 Hz (ISI = 50 ms) failed to induce LTD in hippocampal slices from the adult S845A mutants (closed symbols), while producing normal LTD in the WT littermates (open symbols). Representative traces taken right before (thin line) and 1 h after the onset of PP-1-Hz stimulation (thick line) are shown to the right.

FIG. 8.

Normal depotentiation in the adult S845A mutants. Depotentiation induced by delivering a train of 1 Hz (15 min) at 30 min following LTP induction was normal in the S845A mutants (closed symbols) compared with WT littermates (open symbols). Left: the entire experiment normalized to the pre-LTP baseline. Middle: superimposed representative traces taken right before LTP induction (thin line), 30 min after LTP (thick solid line), and 1 h after the onset of 1-Hz stimulation (thick dashed line). Right: the magnitude of depotentiation when renormalized to the pre-1-Hz baseline.