Article

The C-terminal tails of endogenous GluA1 and GluA2 differentially contribute to hippocampal synaptic plasticity and learning

  • Nature Neurosciencevolume 21pages5062 (2018)
  • doi:10.1038/s41593-017-0030-z
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Abstract

Long-term potentiation (LTP) and depression (LTD) at glutamatergic synapses are intensively investigated processes for understanding the synaptic basis for learning and memory, but the underlying molecular mechanisms remain poorly understood. We have made three mouse lines where the C-terminal domains (CTDs) of endogenous AMPA receptors (AMPARs), the principal mediators of fast excitatory synaptic transmission, are specifically exchanged. These mice display profound deficits in synaptic plasticity without any effects on basal synaptic transmission. Our study reveals that the CTDs of GluA1 and GluA2, the key subunits of AMPARs, are necessary and sufficient to drive NMDA receptor–dependent LTP and LTD, respectively. In addition, these domains exert differential effects on spatial and contextual learning and memory. These results establish dominant roles of AMPARs in governing bidirectional synaptic and behavioral plasticity in the CNS.

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Change history

  • Correction 11 May 2018

    In the version of this article initially published, the wrong version of Supplementary Fig. 10 was posted and the city for affiliation 4, the Co-innovation Center of Neuroregeneration, Nantong University, was given as Nanjing instead of Nantong. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Y.-T. Wang for GluA2-CTD antibodies, W. Lu for the use of the fear conditioning chambers, and L. Han, R. Mao and other members of Jia laboratory for technical assistance and comments on the manuscript. This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MOP119421, Z.J.; FDN154276, G.L.C.), Canadian Natural Science and Engineering Research Council (NSERC, RGPIN341498, Z.J.), Natural Science Foundation of China (NSFC 31571040, Z.Z.), NSFC and CIHR Joint Health Research Initiative Program (81161120543, W.X. and CCI117959, Z.J.), Brain Canada (Z.J. and G.L.C.) and the Hospital for Sick Children Foundation (Z.J.). S.X. was supported by the Scientific Research Foundation of Graduate School of Southeast University, China.

Author information

Author notes

  1. Zikai Zhou, An Liu, Shuting Xia and Celeste Leung contributed equally to this work.

Affiliations

  1. Neurosciences & Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada

    • Zikai Zhou
    • , Shuting Xia
    • , Celeste Leung
    • , Yanghong Meng
    •  & Zhengping Jia
  2. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

    • Zikai Zhou
    • , Shuting Xia
    • , Celeste Leung
    • , Yanghong Meng
    • , Pojeong Park
    • , Graham L. Collingridge
    •  & Zhengping Jia
  3. The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, China

    • Zikai Zhou
    • , An Liu
    • , Shuting Xia
    • , Junxia Qi
    •  & Wei Xie
  4. Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China

    • Zikai Zhou
    •  & Wei Xie
  5. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    • Pojeong Park
    •  & Graham L. Collingridge
  6. Centre for Synaptic Plasticity, Department of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, UK

    • Pojeong Park
    •  & Graham L. Collingridge

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Contributions

Z.J. conceived and supervised the study. A.L., Z.Z., G.L.C. and Z.J. designed the experiments. Z.Z., A.L., S.X., C.L., J.Q. and Y.M. performed experiments. Z.Z., A.L., S.X., C.L., P.P. and W.X. analyzed data. Z.J., G.L.C. and A.L. wrote the paper. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Graham L. Collingridge or Zhengping Jia.

Integrated Supplementary Information

  1. Supplementary Figure 1 Creation of GluA1C2KI, GluA2C1KI and GluA1C2KI;GluA2C1KI mice

    (a) Schematic graph of the WT GluA1 gene locus, CTD replacement targeting vector and targeted GluA1 locus in GluA1C2KI mice. (b) Southern blot analysis of ES DNA using 5’ and 3’ probes showing the successful replacement of the DNA region containing the CTD of the GluA1 with that of the GluA2 in GluA1C2KI +/- clones. This experiment was repeated at least two times on independent clones with identical results. (c) PCR analysis of tail DNA of the offspring littermates from the heterozygous breeders showing wild type (+/+), heterozygous (+/-) and homozygous (-/-) for the CTD replacement. All the mice used in this study were homozygous (-/- = GluA1C2KI) and their wild type littermates (+/+ = WT). This experiment was repeated for all the mice used for the experiments. (d) PCR analysis of tail DNA of the offspring littermates from the heterozygous breeders using additional pairs of oligo-primers showing wild type (+/+), heterozygous (+/-) and homozygous (-/-) for the CTD replacement. This experiment was repeated for every mouse used for the present study. (e) Schematic graph of the WT GluA2 gene locus, CTD replacement targeting vector and targeted GluA2 locus in GluA2C1KI mice. (f) Southern blot analysis of ES DNA showing the successful replacement of the DNA region containing the CTD of the GluA2 with that of the GluA1 in GluA2C1KI mice, using the 5’ and 3’ probes. This experiment was repeated at least two times for each of the independent ES clones with identical results. (g) PCR analysis of tail DNA of the offspring littermates from the heterozygous breeders showing wild type (+/+), heterozygous (+/-) and homozygous (-/-) for the CTD replacement. All the mice used in this study were homozygous (-/- = GluA2CIKI) and their wild type littermates (+/+ = WT). This experiments was repeated for every mouse used for the present study. (h) PCR analysis of tail DNA of the offspring littermates from the heterozygous breeders for both CTD replacements showing various genotypes, including wild type (+/+ = WT) and double homozygous (-/-, -/-, indicated by purple box) for the CTD replacements (GluA1C2KI;GluA2C1KI, i.e. CTD swapped mice). See Supplementary Fig. S9 for full length blot/DNA gel scans.

  2. Supplementary Figure 2 Characterization of GluA1C2KI and GluA2C1KI mice

    (a) Western blot analysis of total brain protein lysate from GluA1C2KI and WT control littermates showing the successful replacement of the CTD of GluA1 with that of GluA2. Note in GluA1C2KI mice, no GluA1 band was detected with a GluA1-CTD antibody, but a normal GluA1 band was seen with a GluA1-NTD antibody. (b) Western blot analysis of total brain protein lysate from GluA2C1KI and WT control mice showing the successful replacement of the CTD of GluA2 with that GluA1. Note in GluA2C1KI mice, no GluA2 band was detected with a GluA2-CTD antibody, but normal GluA2 level with a GluA2-NTD antibody. (c) Quantifications of various proteins tested in (a,b) showing that phosphorylated GluA1 (Ser831 and Ser 845) was absent in GluA1C2KI, but increased in GluA2C1KI compared to WT mice, and that phosphorylated GluA2 (Tyr869/873/876) was absent in GluA2C1KI, but increased in GluA1C2KI compared to WT mice. Other proteins were not altered except that there was a small increase in GluA1NTD in GluA2C1KI mice (A1-NTD: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 0.86 ± 0.07, n = 4 mice, p = 0.085; GluA2C1KI = 1.20 ± 0.05, n = 4 mice, *p = 0.023; F(2, 9) =10.993, p = 0.004. A2-NTD: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 0.88 ± 0.04, n =4 mice; GluA2C1KI = 1.16 ± 0.18, n = 4 mice; F(2, 9) =1.743, p = 0.229. p-GluA1-831: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 0.08 ± 0.02, n = 3 mice, ***p = 0.00000002; GluA2C1KI = 4.00 ± 0.89, n = 4 mice, *p = 0.015; F(2, 8) =12.786, p = 0.003. p-GluA1-845: WT = 1.00 ± 0.00, n = 5 mice; A1C2KI = 0.10 ± 0.02, n = 4 mice, ***p = 0.0000006; GluA2C1KI = 4.84 ± 0.92, n = 5 mice, **p = 0.003; F(2, 11) =19.077, p = 0.000265. p-GluA2-Tyr: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 1.51 ± 0.18, n = 4 mice, **p = 0.007; GluA2C1KI = 0.15 ± 0.03, n = 4 mice, ***p = 0.000268; F(2, 9) = 43.860, p = 0.000022. GluA3: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 1.08 ± 0.26, n = 4 mice; GluA2C1KI = 1.09 ± 0.08, n = 3 mice; F(2, 8) = 0.085, p = 0.919. GluA4: WT = 1.00 ± 0.00, n = 5 mice; A1C2KI = 0.93 ± 0.11, n = 5 mice; GluA2C1KI = 1.00 ± 0.10, n = 5 mice; F(2, 12) = 0.621, p = 0.555. NR1: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 1.34 ± 0.09, n = 4 mice; GluA2C1KI = 0.85 ± 0.19, n = 4 mice; F(2, 9) = 4.180, p = 0.052. Syn1: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 1.04 ± 0.14, n = 4 mice; GluA2C1KI = 1.10 ± 0.19, n = 4 mice; F(2, 9) = 0.145, p = 0.867. PSD95: WT = 1.00 ± 0.00, n = 4 mice; A1C2KI = 0.69 ± 0.06, n = 4 mice; GluA2C1KI = 1.14 ± 0.19, n = 4 mice; F(2, 9) = 3.945, p = 0.059). (d) Whole brain protein lysates immunoprecipitated with an anti-GluA1NTD antibody and probed with anti-GluA1NTD and anti-SAP97 antibodies showing reduced or absence of SAP97 in the immunocomplex in GluA1C2KI mice. (e) Whole brain protein lysates immunoprecipitated with an anti-GluA2NTD antibody and probed with anti-GluA2NTD and anti-NSF antibodies showing reduced or absence of NSF in the immunocomplex in GluA2C1KI mice. (f,g) Fixed brain sections immunostained with anti-synapsin 1 (green) and the nuclear marker DAPI (blue) showing normal hippocampal anatomy and synapse distribution in GluA1C2KI (f) and GluA2C1KI (g) mice compared to WT littermates. (h,i) Fixed brain sections immunostained with anti-GluA2-NTD (green) and the nuclear marker DAPI (blue) showing normal distribution of AMPARs in the hippocampus of GluA1C2KI (h) and GluA2C1KI (i) mice compared to WT littermates. Scale bar: 200 μm. Experiments were repeated at least 3 times for a,b,d and i. One-way ANOVA test was used for Fig. S2c followed by post-hoc Fisher’s LSD multiple comparison test for A1-NTD and p-GluA2-Tyr analysis, and by two tailed t-test at 95% confidence interval for p-GluA1-831 and p-GluA1-845 analysis. Data were presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001. See Supplementary Fig. S10 for full length Western blot scans.

  3. Supplementary Figure 3 Intact mGluR-LTD in GluA1C2KI and GluA2C1KI mice

    (a) Intact PP-LFS-induced LTD in GluA1C2KI mice (WT = 65.05 ± 9.27%, n = 5 slices from 5 mice; A1C2KI = 68.58 ± 8.64%, n = 6 slices from 6 mice, two tailed t-test at 95% confidence interval, t(9) = -0.278, p = 0.787). (b) NMDAR independence of PP-LFS-LTD (WT = 66.06 ± 7.09%, n = 7 slices from 7 mice; A1C2KI = 65.13 ± 9.00%, n = 5 slices from 5 mice; A2C1KI = 58.28 ± 4.81%, n = 5 slices from 5 mice, F(2, 14) = 0.326, p = 0.737). (c) Dependence of PP-LFS-LTD on mGluR (WT = 92.84 ± 3.65%, n = 6 slices from 6 mice; A1C2KI = 97.17 ± 13.89%, n = 4 slices from 4 mice; A2C1KI = 88.56 ± 15.20%, n = 4 slices from 4 mice, F(2, 11) = 0.148, p = 0.864). One-way ANOVA test was used for b, c. Scale bar: 50 pA/25 ms. Data were presented as mean ± s.e.m.

  4. Supplementary Figure 4 Intact DHPG-induced LTD in GluA2C1KI mice

    (a) Lack of effect of AP5 on DHPG-LTD (WT = 75.81 ± 5.33%, n = 4 slices from 4 mice; A2C1KI = 67.52 ± 4.75%, n = 4 slices from 4 mice, t(6) = 1.161, p = 0.290). (b) Blockade of DHPG-LTD by MPEP (WT = 111.37 ± 5.70%, n = 4 slices from 4 mice; A2C1KI = 118.55 ± 9.49%, n = 4 slices from 4 mice, t(6) = −0.648, p = 0.541). Two-tailed t-test at 95% confidence interval. Scale bar: 50 pA/25 ms. Data were presented as mean ± s.e.m.

  5. Supplementary Figure 5 Dendritic spines and basal AMPARs in GluA1C2KI and GluA2C1KI mice

    (a) Sample dendritic segments of cultured hippocampal neurons stained for MAP2 and F-actin (phalloidin) showing dendritic spines. Scale bar: 2 μm. (b) Summary graph of spine length (μm) (WT = 1.05 ± 0.02, n = 32 neurons from 3 independent cultures; A1C2KI = 1.07 ± 0.04, n = 12 neurons from 3 independent cultures, p = 0.701 compared to WT; A2C1KI = 1.07 ± 0.02, n = 19 neurons from 3 independent cultures, p = 0.647 compared to WT; F(2, 60) = 0.138, p = 0.872). (c) Summary graph of spine density (spines/20 μm) (WT = 12.33 ± 0.41, n = 24 neurons from 3 independent cultures; A1C2KI = 12.92 ± 0.63, n = 12 neurons from 3 independent cultures, p = 0.418 compared to WT; A2C1KI = 13.11 ± 0.45, n = 19 neurons from 3 independent cultures, p = 0.219 compared to WT; F(2, 52) = 0.842, p = 0.436). (d) Cultured hippocampal neurons stained for basal surface GluA2 (GluA2-NTD) and synaptophysin. Scale bar: 20 μm. (e) Normalized basal surface (non-permeabilized) GluA2 fluorescence intensity (WT = 1.01 ± 0.03, n = 55 neurons from 3 independent cultures; A1C2KI = 1.01 ± 0.05, n =29 neurons from 3 independent cultures, p = 0.293 compared to WT; A2C1KI = 0.76 ± 0.04, n = 35 neurons from 3 independent cultures, ***p = 0.000003 compared to WT; F(2, 116) = 16.797, p = 0.0000004). (f) Normalized basal total (permeabilized) GluA2 fluorescence intensity (WT = 1.00 ± 0.05, n = 16 neurons from 3 independent cultures; A1C2KI = 0.91 ± 0.06, n = 12 neurons from 3 independent cultures, p = 0.319 compared to WT; A2C1KI = 0.92 ± 0.06, n = 16 neurons from 3 independent cultures, p = 0.051 compared to WT; F(2,41) = 0.718, p = 0.494). (g) Normalized basal total synaptophysin fluorescence intensity (WT = 1.00 ± 0.05, n = 42 neurons from 3 independent cultures; A1C2KI = 1.02 ± 0.05, n = 34 neurons from 3 independent cultures, p = 0.728 compared to WT; A2C1KI = 1.00 ± 0.05, n = 30 neurons from 3 independent cultures, p = 0.969 compared to WT; F(2,103) = 0.083, p = 0.921). One-way ANOVA followed by post-hoc Fisher’s LSD multiple comparison test was used for b, c,e-g. Data were presented as mean ± s.e.m. ***p < 0.001.

  6. Supplementary Figure 6 Locomotor activity and anxiety in GluA1C2KI and GluA2C1KI mice

    (a) Total travel distance (meters) in the open field test (WT = 14.66 ± 1.17, n = 12 mice; A1C2KI = 20.29 ± 1.65, n = 13 mice, **p = 0.003 compared to WT; A2C1KI = 14.21 ± 0.83, n = 16 mice, p = 0.873 compared to WT; F(2,38) = 7.508, p = 0.002). (b) Total time (seconds) spent in the center arena of the open field (WT = 17.17 ± 2.71, n = 12 mice; A1C2KI = 16.25 ± 2.41, n =13 mice; A2C1KI = 11.53 ± 3.18, n = 16 mice; F(2, 38) = 1.172, p = 0.321). (c) Ratios of open/closed arm entries in the elevated plus maze test (WT = 0.89 ± 0.11, n = 12 mice; A1C2KI = 1.19 ± 0.14, n = 13 mice; A2C1KI = 1.07 ± 0.15, n = 16 mice; F(2, 38) = 1.075, p = 0.352). (d) Ratios of time spent in open/closed arms (WT = 0.36 ± 0.06, n = 12 mice; A1C2KI = 0.65 ± 0.20, n =13 mice; A2C1KI = 0.53 ± 0.10, n = 16 mice; F(2, 38) = 1.079, p = 0.350). One-way ANOVA followed by post-hoc Fisher’s LSD multiple comparison test was used for a-d. Data were presented as mean ± s.e.m. **p<0.01.

  7. Supplementary Figure 7 Spatial and fear memory deficits in GluA1C2KI and GluA2C1KI mice

    (a) Visible platform water maze test showing all three genotypes (WT, n = 11 mice; GluA1C2KI, n = 9 mice; GluA2C1KI, n = 11 mice) reached the platform equally well (between genotypes: F(2, 28) = 0.981, p = 0.388; repeated two way ANOVA). (b) Probe test at 2 h after the last training session showing that both WT and GluA2C1K, but not GluA1C2KI mice, spent significantly more time swimming in the target quadrant (WT: target = 23.68 ± 2.52, other = 11.70 ± 0.94, n = 11 mice, t(10) = 3.537, **p = 0.005; A1C2KI: target = 15.28 ± 3.76, other = 14.91 ± 1.25, n = 9 mice, t(8) = 0.689, p = 0.510; A2C1KI: target = 22.44 ± 2.87, other = 12.52 ± 0.96, n = 11 mice, t(10) = 3.028, *p = 0.012) compared other quadrants. (c) Probe test at 24 h after the last training session showing that both WT and GluA2C1K, but not GluA1C2KI mice, spent significantly more time swimming in the target quadrant (WT: target = 24.34 ± 3.85, other = 11.89 ± 1.29, n = 11 mice, t(10) = 2.424, *p = 0.036; A1C2KI: target = 15.36 ± 2.77, other = 14.94 ± 0.941, n = 9 mice, t(8) = 0.113, p = 0.913; A2C1KI: target = 18.96 ± 1.49, other = 13.69 ± 0.50, n = 11 mice, t(10) = 2.654, *p = 0.024) compared other quadrants. (d) Probe test at 2 h after the enhanced training protocol showing that both WT and GluA2C1KI, but not GluA1C2KI mice, spent significantly more time swimming in the target quadrant (WT: target = 23.31 ± 1.46, other = 12.24 ± 0.49, n = 9 mice, t(8) = 6.78, ***p = 0.00002; A1C2KI: target = 15.23 ± 1.91, other = 14.93 ± 0.64, n = 8 mice, t(7) = 0.116, p = 0.911; A2C1KI: target = 30.60 ± 5.04, other = 9.81 ± 1.68, n = 8 mice, t(7) = 3.095, *p = 0.017). (e) Probe test 24 h after the enhanced training protocol showing that both WT and GluA2C1KI, but not GluA1C2KI mice, spent significantly more time swimming in the target quadrant (WT: target = 29.71 ± 2.02, other = 11.96 ± 0.63, n = 9 mice, t(8) = 7.08, ***p = 0.00005; A1C2KI: target = 20.70 ± 4.11, other = 13.32 ± 0.97, n = 8 mice, t(7) = 1.679, p = 0.137; A2C1KI: target = 30.66 ± 5.42, other = 13.10 ± 0.73, n = 8 mice, t(7) = 3.475, *p = 0.010). (f) Cued memory test 2 h after the three shock conditioning (WT: before = 21.56 ± 2.68%, after = 40.08 ± 3.52%, n = 12 mice, t(11) = −9.071, ***p = 0.000002; A1C2KI: before = 20.43 ± 3.36%, after = 33.67 ± 3.74%, n = 12 mice, t(11) = −2.995, **p = 0.012; A2C1KI: before = 23.79 ± 2.21%, after = 38.00 ± 3.11%, n = 12 mice, t(11) = −7.310, ***p = 0.00002). (g) Cued memory test 24 h after conditioning (WT: before = 32.91 ± 4.49%, after = 46.18 ± 4.55%, n = 12 mice, t(11) = −3.719, **p = 0.003; A1C2KI: before = 34.41 ± 4.94%, after = 50.83 ± 4.88%, n = 12 mice, t(11) = −1.600, p = 0.138; A2C1KI: before = 33.23 ± 3.49%, after = 50.07 ± 4.90%, n = 12 mice, t(11) = −4.043, **p = 0.002). (h) Cued memory test 2 h after the two shock conditioning (WT: before = 18.68 ± 2.99%, after = 39.87 ± 5.55%, n = 9 mice, t(8) = −5.432, ***p = 0.0006; A1C2KI: before = 18.22 ± 4.01%, after = 32.92 ± 9.12%, n = 8 mice,t(7) = −1.682, p = 0.137; A2C1KI: before = 20.89 ± 2.71%, after = 37.81 ± 6.65%, n = 9, mice,t(8) = −3.899, **p = 0.005). (i) Cued memory test 24 h after the two shock conditioning (WT: before = 21.71 ± 2.52%, after = 36.61 ± 6.40%, n = 9 mice, t(8) = −2.549, *p = 0.034; A1C2KI: before = 33.24 ± 7.57%, after = 41.87 ± 9.58%, n = 8 mice, t(7) = −0.688, p = 0.514; A2C1KI: before = 25.94 ± 2.26%, after = 41.11 ± 7.03%, n = 9 mice, t(8) = −2.594, *p = 0.032). Paired t-test at 95% confidence interval was used for b-i. Data were presented as mean ± s.e.m. *p<0.05, **p<0.01,***p<0.001.

  8. Supplementary Figure 8 Full-length western blot scans for the cropped images presented in Fig. 6

    Full length Western blot scans for the cropped images presented in Fig. 6g–m6.

  9. Supplementary Figure 9 Full length Southern blot and DNA gel scans for the cropped images presented in Supplementary Fig. S1.

    Full length Southern blot and DNA gel scans for the cropped images presented in Supplementary Fig. S1.

  10. Supplementary Figure 10 Full length western blot scans for the cropped images presented in Supplementary Fig. S2.

    Full length Western blot scans for the cropped images presented in Supplementary Fig. S2.

Supplementary information

  1. Supplementary text and Figures

    Supplementary figures 1–10.

  2. Life Sciences Reporting Summary

    Life sciences reporting summary.

  3. Statistics data reporting by figure

    Statistics data reporting by figure.