Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

A Publisher Correction to this article was published on 11 May 2018

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Basal synaptic and AMPAR properties in GluA1C2KI and GluA2C1KI mice.
Fig. 2: Absence of NMDAR-LTP, but intact NMDAR-LTD, in GluA1C2KI mice.
Fig. 3: Absence of NMDAR-LTD, but intact mGluR-LTD, in GluA2C1KI mice.
Fig. 4: Restoration of both LTP and LTD in GluA1C2KI;GluA2C1KI double CTD replacement mice.
Fig. 5: Impaired glycine- and NMDA-induced AMPAR surface delivery and removal in GluA1C2KI and GluA2C1KI cultured neurons, respectively.
Fig. 6: Impaired glycine- and NMDA-induced AMPAR surface delivery and removal in GluA1C2KI and GluA2C1KI hippocampal slices, respectively.
Fig. 7: An increase in single channel conductance of AMPARs in WT and GluA2C1KI following LTP induction.
Fig. 8: Spatial and contextual fear memory deficits in GluA1C2KI and GluA2C1KI mice.

Similar content being viewed by others

Change history

  • 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.

References

  1. Bliss, T. V. P. & Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993).

    Article  PubMed  CAS  Google Scholar 

  2. Malenka, R. C. & Bear, M. F. LTP and LTD: an embarrassment of riches. Neuron 44, 5–21 (2004).

    Article  PubMed  CAS  Google Scholar 

  3. Kandel, E. R., Dudai, Y. & Mayford, M. R. The molecular and systems biology of memory. Cell 157, 163–186 (2014).

    Article  PubMed  CAS  Google Scholar 

  4. Henley, J. M. & Wilkinson, K. A. Synaptic AMPA receptor composition in development, plasticity and disease. Nat. Rev. Neurosci. 17, 337–350 (2016).

    Article  PubMed  CAS  Google Scholar 

  5. Malinow, R. & Malenka, R. C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126 (2002).

    Article  PubMed  CAS  Google Scholar 

  6. Collingridge, G. L., Isaac, J. T. & Wang, Y. T. Receptor trafficking and synaptic plasticity. Nat. Rev. Neurosci. 5, 952–962 (2004).

    Article  PubMed  CAS  Google Scholar 

  7. Kessels, H. W. & Malinow, R. Synaptic AMPA receptor plasticity and behavior. Neuron 61, 340–350 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Huganir, R. L. & Nicoll, R. A. AMPARs and synaptic plasticity: the last 25 years. Neuron 80, 704–717 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Lu, W. et al. Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron 62, 254–268 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Granger, A. J., Shi, Y., Lu, W., Cerpas, M. & Nicoll, R. A. LTP requires a reserve pool of glutamate receptors independent of subunit type. Nature 493, 495–500 (2013).

    Article  PubMed  CAS  Google Scholar 

  11. Granger, A. J. & Nicoll, R. A. LTD expression is independent of glutamate receptor subtype. Front. Synaptic Neurosci. 6, 15 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Hayashi, Y. et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287, 2262–2267 (2000).

    Article  PubMed  CAS  Google Scholar 

  13. Shi, S., Hayashi, Y., Esteban, J. A. & Malinow, R. Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105, 331–343 (2001).

    Article  PubMed  CAS  Google Scholar 

  14. Nishimune, A. et al. NSF binding to GluR2 regulates synaptic transmission. Neuron 21, 87–97 (1998).

    Article  PubMed  CAS  Google Scholar 

  15. Lüthi, A. et al. Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction. Neuron 24, 389–399 (1999).

    Article  PubMed  Google Scholar 

  16. Lüscher, C. et al. Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron 24, 649–658 (1999).

    Article  PubMed  Google Scholar 

  17. Lee, S. H., Liu, L., Wang, Y. T. & Sheng, M. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD. Neuron 36, 661–674 (2002).

    Article  PubMed  CAS  Google Scholar 

  18. Ahmadian, G. et al. Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD. EMBO J. 23, 1040–1050 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Jia, Z. et al. Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron 17, 945–956 (1996).

    Article  PubMed  CAS  Google Scholar 

  20. Zamanillo, D. et al. Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning. Science 284, 1805–1811 (1999).

    Article  PubMed  CAS  Google Scholar 

  21. Meng, Y., Zhang, Y. & Jia, Z. Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3. Neuron 39, 163–176 (2003).

    Article  PubMed  CAS  Google Scholar 

  22. Greger, I. H., Ziff, E. B. & Penn, A. C. Molecular determinants of AMPA receptor subunit assembly. Trends Neurosci. 30, 407–416 (2007).

    Article  PubMed  CAS  Google Scholar 

  23. Andrásfalvy, B. K., Smith, M. A., Borchardt, T., Sprengel, R. & Magee, J. C. Impaired regulation of synaptic strength in hippocampal neurons from GluR1-deficient mice. J. Physiol. (Lond.) 552, 35–45 (2003).

    Article  CAS  Google Scholar 

  24. Sans, N. et al. Aberrant formation of glutamate receptor complexes in hippocampal neurons of mice lacking the GluR2 AMPA receptor subunit. J. Neurosci. 23, 9367–9373 (2003).

    Article  PubMed  CAS  Google Scholar 

  25. Plant, K. et al. Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat. Neurosci. 9, 602–604 (2006).

    Article  PubMed  CAS  Google Scholar 

  26. Liu, S. J. & Zukin, R. S. Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death. Trends Neurosci. 30, 126–134 (2007).

    Article  PubMed  CAS  Google Scholar 

  27. Lee, H. K. et al. Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell 112, 631–643 (2003).

    Article  PubMed  CAS  Google Scholar 

  28. Kim, C. H. et al. Persistent hippocampal CA1 LTP in mice lacking the C-terminal PDZ ligand of GluR1. Nat. Neurosci. 8, 985–987 (2005).

    Article  PubMed  CAS  Google Scholar 

  29. Gerlai, R., Henderson, J. T., Roder, J. C. & Jia, Z. Multiple behavioral anomalies in GluR2 mutant mice exhibiting enhanced LTP. Behav. Brain Res. 95, 37–45 (1998).

    Article  PubMed  CAS  Google Scholar 

  30. Collingridge, G. L., Peineau, S., Howland, J. G. & Wang, Y. T. Long-term depression in the CNS. Nat. Rev. Neurosci. 11, 459–473 (2010).

    Article  PubMed  CAS  Google Scholar 

  31. Lüscher, C. & Huber, K. M. Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease. Neuron 65, 445–459 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Kemp, N., McQueen, J., Faulkes, S. & Bashir, Z. I. Different forms of LTD in the CA1 region of the hippocampus: role of age and stimulus protocol. Eur. J. Neurosci. 12, 360–366 (2000).

    Article  PubMed  CAS  Google Scholar 

  33. Lu, W. et al. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29, 243–254 (2001).

    Article  PubMed  CAS  Google Scholar 

  34. Benke, T. A., Lüthi, A., Isaac, J. T. & Collingridge, G. L. Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 393, 793–797 (1998).

    Article  PubMed  CAS  Google Scholar 

  35. Clem, R. L. & Huganir, R. L. Calcium-permeable AMPA receptor dynamics mediate fear memory erasure. Science 330, 1108–1112 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Humeau, Y. et al. A pathway-specific function for different AMPA receptor subunits in amygdala long-term potentiation and fear conditioning. J. Neurosci. 27, 10947–10956 (2007).

    Article  PubMed  CAS  Google Scholar 

  37. Mitsushima, D., Ishihara, K., Sano, A., Kessels, H. W. & Takahashi, T. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus. Proc. Natl. Acad. Sci. USA 108, 12503–12508 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Rudy, J. W., Barrientos, R. M. & O’Reilly, R. C. Hippocampal formation supports conditioning to memory of a context. Behav. Neurosci. 116, 530–538 (2002).

    Article  PubMed  Google Scholar 

  39. Lu, W. & Roche, K. W. Posttranslational regulation of AMPA receptor trafficking and function. Curr. Opin. Neurobiol. 22, 470–479 (2012).

    Article  PubMed  CAS  Google Scholar 

  40. Kim, C. H., Chung, H. J., Lee, H. K. & Huganir, R. L. Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression. Proc. Natl. Acad. Sci. USA 98, 11725–11730 (2001).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Reisel, D. et al. Spatial memory dissociations in mice lacking GluR1. Nat. Neurosci. 5, 868–873 (2002).

    Article  PubMed  CAS  Google Scholar 

  42. Schmitt, W. B. et al. Spatial reference memory in GluR-A-deficient mice using a novel hippocampal-dependent paddling pool escape task. Hippocampus 14, 216–223 (2004).

    Article  PubMed  CAS  Google Scholar 

  43. Bannerman, D. M. et al. Hippocampal synaptic plasticity, spatial memory and anxiety. Nat. Rev. Neurosci. 15, 181–192 (2014).

    Article  PubMed  CAS  Google Scholar 

  44. Moser, E., Moser, M. B. & Andersen, P. Spatial learning impairment parallels the magnitude of dorsal hippocampal lesions, but is hardly present following ventral lesions. J. Neurosci. 13, 3916–3925 (1993).

    Article  PubMed  CAS  Google Scholar 

  45. Frankland, P. W., Cestari, V., Filipkowski, R. K., McDonald, R. J. & Silva, A. J. The dorsal hippocampus is essential for context discrimination but not for contextual conditioning. Behav. Neurosci. 112, 863–874 (1998).

    Article  PubMed  CAS  Google Scholar 

  46. Wiltgen, B. J., Sanders, M. J., Anagnostaras, S. G., Sage, J. R. & Fanselow, M. S. Context fear learning in the absence of the hippocampus. J. Neurosci. 26, 5484–5491 (2006).

    Article  PubMed  CAS  Google Scholar 

  47. Burwell, R. D., Saddoris, M. P., Bucci, D. J. & Wiig, K. A. Corticohippocampal contributions to spatial and contextual learning. J. Neurosci. 24, 3826–3836 (2004).

    Article  PubMed  CAS  Google Scholar 

  48. Liu, X., Gu, Q. H., Duan, K. & Li, Z. NMDA receptor-dependent LTD is required for consolidation but not acquisition of fear memory. J. Neurosci. 34, 8741–8748 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Meng, Y. et al. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 35, 121–133 (2002).

    Article  PubMed  CAS  Google Scholar 

  50. Zhou, Z., Hu, J., Passafaro, M., Xie, W. & Jia, Z. GluA2 (GluR2) regulates metabotropic glutamate receptor-dependent long-term depression through N-cadherin-dependent and cofilin-mediated actin reorganization. J. Neurosci. 31, 819–833 (2011).

    Article  PubMed  CAS  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Graham L. Collingridge or Zhengping Jia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated Supplementary Information

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Supplementary text and Figures

Supplementary figures 1–10.

Life Sciences Reporting Summary

Life sciences reporting summary.

Statistics data reporting by figure

Statistics data reporting by figure.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, Z., Liu, A., Xia, S. et al. The C-terminal tails of endogenous GluA1 and GluA2 differentially contribute to hippocampal synaptic plasticity and learning. Nat Neurosci 21, 50–62 (2018). https://doi.org/10.1038/s41593-017-0030-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-017-0030-z

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing