Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP

Abstract

Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca2+ influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca2+ induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca2+-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca2+-dependent presynaptic vesicle exocytosis, also blocked Ca2+-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca2+-sensors for Ca2+-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.

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Figure 1: Inhibiting postsynaptic expression of Syt1 and Syt7 blocks LTP by a cell-autonomous mechanism.
Figure 2: Inhibiting postsynaptic Syt1 and Syt7 expression by various molecular manipulations blocks LTP.
Figure 3: Postsynaptic Syt1–Syt7 deficiency does not decrease AMPAR recruitment during homeostatic plasticity or surface AMPAR levels.
Figure 4: Syt1–Syt7 deficiency blocks AMPAR exocytosis during ‘chemical LTP’ in cultured hippocampal neurons.
Figure 5: Dominant-negative mutant Syt1 impairs presynaptic Ca2+-induced vesicle exocytosis and postsynaptic LTP-induced AMPAR exocytosis.

References

  1. 1

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    CAS  PubMed  Google Scholar 

  3. 3

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Morris, R. G. NMDA receptors and memory encoding. Neuropharmacology 74, 32–40 (2013)

    CAS  PubMed  Google Scholar 

  5. 5

    Opazo, P. & Choquet, D. A three-step model for the synaptic recruitment of AMPA receptors. Mol. Cell. Neurosci. 46, 1–8 (2011)

    CAS  PubMed  Google Scholar 

  6. 6

    Makino, H. & Malinow, R. AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron 64, 381–390 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    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)

    ADS  CAS  PubMed  Google Scholar 

  8. 8

    Lledo, P. M., Zhang, X., Südhof, T. C., Malenka, R. C. & Nicoll, R. A. Postsynaptic membrane fusion and long-term potentiation. Science 279, 399–403 (1998)

    ADS  CAS  PubMed  Google Scholar 

  9. 9

    Ahmad, M. et al. Postsynaptic complexin controls AMPA receptor exocytosis during LTP. Neuron 73, 260–267 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Jurado, S. et al. LTP requires a unique postsynaptic SNARE fusion machinery. Neuron 77, 542–558 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Südhof, T. C. Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80, 675–690 (2013)

    PubMed  Google Scholar 

  12. 12

    Reim, K. et al. Complexins regulate the Ca2+ sensitivity of the synaptic neurotransmitter release machinery. Cell 104, 71–81 (2001)

    CAS  PubMed  Google Scholar 

  13. 13

    Maximov, A., Tang, J., Yang, X., Pang, Z. P. & Südhof, T. C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994)

    CAS  PubMed  Google Scholar 

  15. 15

    Wen, H. et al. Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction. Proc. Natl Acad. Sci. USA 107, 13906–13911 (2010)

    ADS  CAS  PubMed  Google Scholar 

  16. 16

    Bacaj, T. et al. Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release. Neuron 80, 947–959 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Bacaj, T. et al. Synaptotagmin-1 and -7 are redundantly essential for maintaining the capacity of the readily-releasable pool of synaptic vesicles. PLoS Biol. 13, e1002267 (2015)

    PubMed  PubMed Central  Google Scholar 

  18. 18

    Schonn, J. S., Maximov, A., Lao, Y., Südhof, T. C. & Sørensen, J. B. Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells. Proc. Natl Acad. Sci. USA 105, 3998–4003 (2008)

    ADS  CAS  PubMed  Google Scholar 

  19. 19

    Xu, W. et al. Distinct neuronal coding schemes in memory revealed by selective erasure of fast synchronous synaptic transmission. Neuron 73, 990–1001 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Zhou, Q. et al. Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis. Nature 525, 62–67 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Wyllie, D. J., Manabe, T. & Nicoll, R. A. A rise in postsynaptic Ca2+ potentiates miniature excitatory postsynaptic currents and AMPA responses in hippocampal neurons. Neuron 12, 127–138 (1994)

    CAS  PubMed  Google Scholar 

  22. 22

    Kato, H. K., Watabe, A. M. & Manabe, T. Non-Hebbian synaptic plasticity induced by repetitive postsynaptic action potentials. J. Neurosci. 29, 11153–11160 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Kang, H. & Schuman, E. M. Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science 267, 1658–1662 (1995)

    ADS  CAS  PubMed  Google Scholar 

  24. 24

    Harward, S. C. et al. Autocrine BDNF-TrkB signalling within a single dendritic spine. Nature 538, 99–103 (2016)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Arendt, K. L. et al. Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis. Proc. Natl Acad. Sci. USA 112, E5744–E5752 (2015)

    CAS  PubMed  Google Scholar 

  26. 26

    Dudek, S. M. & Bear, M. F. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-d-aspartate receptor blockade. Proc. Natl Acad. Sci. USA 89, 4363–4367 (1992)

    ADS  CAS  PubMed  Google Scholar 

  27. 27

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

    CAS  PubMed  Google Scholar 

  28. 28

    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)

    CAS  PubMed  Google Scholar 

  29. 29

    Passafaro, M., Piëch, V. & Sheng, M. Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat. Neurosci. 4, 917–926 (2001)

    CAS  PubMed  Google Scholar 

  30. 30

    Lin, D. T. & Huganir, R. L. PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization. J. Neurosci. 27, 13903–13908 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Kwon, O. B., Longart, M., Vullhorst, D., Hoffman, D. A. & Buonanno, A. Neuregulin-1 reverses long-term potentiation at CA1 hippocampal synapses. J. Neurosci. 25, 9378–9383 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Lee, J., Guan, Z., Akbergenova, Y. & Littleton, J. T. Genetic analysis of synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release. J. Neurosci. 33, 187–200 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996)

    CAS  PubMed  Google Scholar 

  34. 34

    Malinow, R., Schulman, H. & Tsien, R. W. Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP. Science 245, 862–866 (1989)

    ADS  CAS  PubMed  Google Scholar 

  35. 35

    Malenka, R. C. et al. An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. Nature 340, 554–557 (1989)

    ADS  CAS  Google Scholar 

  36. 36

    Silva, A. J., Stevens, C. F., Tonegawa, S. & Wang, Y. Deficient hippocampal long-term potentiation in α-calcium-calmodulin kinase II mutant mice. Science 257, 201–206 (1992)

    ADS  CAS  PubMed  Google Scholar 

  37. 37

    Thiagarajan, T. C., Piedras-Renteria, E. S. & Tsien, R. W. α- and βCaMKII. Inverse regulation by neuronal activity and opposing effects on synaptic strength. Neuron 36, 1103–1114 (2002)

    CAS  PubMed  Google Scholar 

  38. 38

    Pang, Z. P., Cao, P., Xu, W. & Südhof, T. C. Calmodulin controls synaptic strength via presynaptic activation of calmodulin kinase II. J. Neurosci. 30, 4132–4142 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Chetkovich, D. M., Chen, L., Stocker, T. J., Nicoll, R. A. & Bredt, D. S. Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors. J. Neurosci. 22, 5791–5796 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Opazo, P. et al. CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin. Neuron 67, 239–252 (2010)

    CAS  PubMed  Google Scholar 

  41. 41

    Maximov, A. et al. Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis. Proc. Natl Acad. Sci. USA 105, 3986–3991 (2008)

    ADS  CAS  PubMed  Google Scholar 

  42. 42

    Xu, J., Pang, Z. P., Shin, O. H. & Südhof, T. C. Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nat. Neurosci. 12, 759–766 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Tang, J. et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006)

    CAS  Google Scholar 

  44. 44

    Maximov, A., Pang, Z. P., Tervo, D. G. & Südhof, T. C. Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation. J. Neurosci. Methods 161, 75–87 (2007)

    PubMed  Google Scholar 

  45. 45

    Gähwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A. & Thompson, S. M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997)

    PubMed  Google Scholar 

  46. 46

    Aoto, J., Martinelli, D. C., Malenka, R. C., Tabuchi, K. & Südhof, T. C. Presynaptic neurexin-3 alternative splicing trans-synaptically controls postsynaptic AMPA receptor trafficking. Cell 154, 75–88 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from the NIH (P50 MH086403 to R.C.M., T.C.S. and L.C., and F32 MH100752 and K99 MH107618 to D.W.).

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D.W., T.B., W.M., D.G. and K.L.A. performed the experiments; all authors planned the experiments, participated in data analyses, and wrote the paper.

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Correspondence to Robert C. Malenka or Thomas C. Südhof.

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Reviewer Information Nature thanks T. Blanpied, D. Choquet and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Overview of experimental questions, approaches, results and controls.

The diagram organizes the goals of the present study into six logically connected questions labelled A–F, and provides an overview of the experimental approaches, results and controls.

Extended Data Figure 2 Schematic overview of all viral vectors used in the current study.

a, Lentiviral vectors used. Vector names are listed on the left. Backbones are indicated with various insert elements in coloured boxes; promoters and encoded sequences (shRNAs or cDNAs) are colour-coded. Key restriction enzyme sites are shown for mapping and cloning purposes. Figures describing the experiments in which these vectors are used are listed on the right. Drawings are not to scale. Most vectors were described in refs 16, 17 and 19. b, Same as a, but for AAV vectors. c, Legend of vector components and restriction sites found in a and b.

Extended Data Figure 3 Control experiments for LTP measurements and characterization of a new Syt7 mutant mouse.

a, Cumulative frequency plots of LTP magnitude for acute slices from AAV-infected mice expressing control (black), Syt1 knockdown (green), Syt7 knockdown (blue) or Syt1–Syt7 DKD virus (red). Data are from Fig. 1b. b, AP5 (50 μM) in the extracellular solution or AIP (20 μM) in the pipette solution impair LTP. Left, representative traces and LTP time course; centre and right, cumulative frequency plots and summary graphs of LTP magnitude. c, Cumulative frequency plots of LTP magnitude in acute slices recorded from control (black) and lentivirally infected neurons (red) from mice stereotactically injected with lentiviruses expressing the Syt1–Syt7 DKD. Data are from Fig. 1c. d, Lentiviral in vivo Syt1–Syt7 DKD with a second, independent shRNA against Syt7 impairs LTP. e, Cumulative frequency plots of LTP magnitude for the experiments shown in Fig. 2a–d. In all experiments, black denotes control conditions; in the first and last panels, red denotes Syt1–Syt7 DKD; in the first panel, blue signifies rescue with wild-type Syt7; in the second panel, green and red indicate rescue with the Syt7 C2A-domain mutant with and without Syt1–Syt7 DKD, respectively; in the third panel, red denotes Syt1–Syt7 DKO. f, LTP is blocked by postsynaptic knockdown of Syt1 in constitutive Syt7 KO mice, but rescued by wild-type shRNA-resistant Syt7. g, Schematic showing the design of the new Syt7 mutant alleles. Using homologous recombination, exon 2 of the mouse Syt7 gene that encodes the transmembrane region was modified to introduce a haemagglutinin (HA) tag between the Syt7 protein N-terminal and the transmembrane region; in addition, a loxP site was introduced into the 5′ intron and a neomycin resistance cassette (NEO) that was flanked by frt1 sites and was followed by a second loxP site introduced in the 3′ intron. The initial mouse mutant was named 7SN; FLP recombination removed the NEO cassette to produce strain 7SF that should have expressed HA-tagged Syt7 per design but failed to do so (see i). 7SF was also designed to serve as a conditional knockout (cKO) in which Cre recombination deletes exon 2 to produce mouse strain 7SC, which represents a true constitutive Syt7 knockout because exon 2 is out-of-frame and encodes the vital transmembrane region. h, Lentiviral Cre expression in cultured hippocampal 7SF neurons reduced Syt7 mRNA levels by about 90%, demonstrating that the 7SF neurons express Syt7 mRNA and that the 7SF locus is a conditional knockout. i, Immunoblotting of brain homogenates from adult mice with the indicated genotypes using antibodies specific to the HA epitope, Syt7, VCP or actin (the latter two as loading controls) shows that 7SF does not express Syt7 protein. Neither HA antibodies nor Syt7 antibodies detected Syt7 protein in 7SF mice designed to express HA-tagged but otherwise normal Syt7 (see g). Immunoblots of proteins from wild-type CD1 mice and from another strain of Syt7 knockout mice were included as positive and negative controls for Syt7, respectively; immunoblots of Nrxn1–HA knockin mice (unpublished) were used as a positive control for the HA epitope immunoblot. Molecular weight markers are indicated on the right. For gel source data, see Supplementary Fig. 1. Data are mean ± s.e.m. (numbers in bars show number of neurons and mice analysed). Statistical significance was assessed in b and f with the Kruskal–Wallis test followed by pairwise comparisons with the Mann–Whitney U test, and in d by the Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001). Calibration bars: 50 pA, 50 ms for b, d and f. Source data

Extended Data Figure 4 Dendroaxonal localization of Syt1 and Syt7 in cultured hippocampal neurons.

a, Representative image of a wild-type cultured hippocampal neuron transfected with a vector co-expressing V5-tagged Syt1 and GFP. Neurons were stained for V5 (red), MAP2 (blue) and VGluT1 (magenta) with GFP in green; the image shows the merged staining for all four markers. b, Enlarged images of a segment of the dendrite marked by a yellow box in a, illustrating the distribution of individual markers. c, d, Same as a, b, but for V5-tagged Syt7. Note that overexpressed Syt1 and Syt7 enters the entire dendritic extensions of neurons as well as their axons.

Extended Data Figure 5 Double deletion of both Syt3 and Syt5 (Syt3–Syt5 DKO) does not alter LTP, and BDNF does not rescue the blocked LTP in Syt1–Syt7 double-deficient neurons.

a, Field EPSP (fEPSP) recordings from heterozygous (Syt3,5 Het) and homozygous constitutive Syt3 and Syt5 double knockout mice (Syt3,5 DKO). Left, LTP time course; centre and right, cumulative frequency plots and summary graphs of LTP magnitude. b, LTP recordings were performed in Syt1–Syt7 double knockdown cells from acute hippocampal slices as described for Fig. 1. BDNF (2 nM) was applied as indicated. Scale bars below sample EPSCs are 50 pA, 50 ms. All data are mean ± s.e.m.; numbers in bars indicate the number of neurons and mice analysed. Statistical significance in a was assessed by the Mann–Whitney U test. Source data

Extended Data Figure 6 Postsynaptic Syt1–Syt7 ablation does not impair basal synaptic transmission or alter short-term plasticity, and does not affect the amplitude or frequency of spontaneous mEPSCs in CA1-region pyramidal neurons.

a, b, Fluorescence image of a slice with patched adjacent non-fluorescent uninfected and fluorescent infected neurons (a), and schematic of simultaneous whole-cell recordings from uninfected and infected Syt1–Syt7 DKD pyramidal neurons (b). c, Syt1–Syt7 DKD does not decrease NMDAR- and AMPAR-mediated synaptic transmission (left, representative AMPAR- and NMDAR-EPSCs; right, scatter plots of dual recordings; red crosses show mean ± s.e.m.). d, Normal AMPAR/NMDAR ratios in Syt1–Syt7 DKD neurons (left, summary graphs of AMPAR- and NMDAR-mediated EPSCs; right, summary graphs of AMPAR/NMDAR ratios). e, Syt1–Syt7 DKD does not cause major changes in the paired-pulse ratio (left, representative traces; right, summary plot of the paired-pulse ratio versus inter-stimulus interval). fh, Lentiviral in vivo Syt1–Syt7 DKD has no significant effect on the frequency or amplitude of spontaneous mEPSCs. Representative mEPSC traces are shown in f; cumulative plots of the inter-event interval and summary graphs of the mEPSC frequency are displayed in g, and cumulative plots and summary graphs of the mEPSC amplitude in h. Data are mean ± s.e.m. (numbers in bars or graphs show number of neurons and mice analysed; numbers for c also apply to d). Statistical significance in d was assessed by Wilcoxon signed rank test for normalized amplitude and the Mann–Whitney U test for AMPAR/NMDAR ratios, and in e, g and h by the Mann–Whitney U test. Source data

Extended Data Figure 7 Retinoic acid-dependent homeostatic plasticity and LTD are normal in Syt1–Syt7 double-deficient neurons.

a, Representative traces of evoked AMPAR- and NMDAR-mediated EPSCs in dual recordings of infected Cre and uninfected adjacent neurons in the same cultured hippocampal slice that had been incubated for 36 h in DMSO and CNQX. b, Summary graph of AMPAR/NMDAR ESPC ratios calculated from the EPSCs monitored in a. c, d, Scatter plots of individual dual recordings of AMPAR and NMDAR EPSCs in Syt1–Syt7 DKD and control neurons in slices that had been incubated in DMSO or CNQX. e, Syt1–Syt7 DKD does not alter NMDAR-dependent LTD (left, representative traces and time course of induced LTD; centre and right, cumulative frequency plots and summary graphs of the LTD magnitude). All data are mean ± s.e.m.; numbers in bars indicate the number of neurons and mice analysed. Statistical significance was assessed by the Mann–Whitney U test comparing the test conditions to the controls (*P < 0.05; **P < 0.01). Source data

Extended Data Figure 8 Measurements of GluA1 and GluA2 endocytosis in control and Syt1–Syt7 double-deficient cultured hippocampal neurons.

a, Representative traces showing that AMPA-puff-induced net currents in nucleated outside-out patch are blocked at a 0-mV holding potential. b, Cumulative frequency plot of the mean peak current amplitude induced by AMPA puffs in nucleated outside-out patches. Note that the only condition that decreases such currents in all independent experiments is the homozygous GluA1 KO (GluA1−/−). c, Experimental procedure flowchart for the endocytosis assay. See methods for detailed protocol. d, Representative images of GluA1 endocytosis in control and Syt1–Syt7 DKD neurons (left), and quantification of GluA1 endocytosis (right). Endocytosis was measured as the ratio of the internal GluA1 fraction (red) to the total GluA1 fraction (internal (red) and surface (green)), and was normalized to the wild type. e, Same as d, but for GluA2. All data are mean ± s.e.m.; numbers in bars indicate the number of neurons and mice analysed. Statistical significance was assessed by the Mann–Whitney U test comparing test conditions to the control. Source data

Extended Data Figure 9 Presynaptic and postsynaptic expression of dominant-negative Syt1-C2A*B*.

a, b, Action potential-evoked (a) and sucrose-induced IPSCs (b) from cultured hippocampal wild-type neurons infected with control lentiviruses or lentiviruses encoding the indicated Syt1 or Syt7 constructs. In culture, lentiviruses infect all neurons uniformly; thus, recordings reflect conditions in which lentiviruses had infected both pre- and postsynaptic cells. c, Evoked EPSCs recorded in cultured wild-type neurons that were infected either with a control lentivirus or with lentiviruses encoding the equivalent mutants of Syt1 (Syt1-C2A*B*) or Syt7 (Syt7-C2A*B*). Note that only the Syt1 but not the Syt7 mutant is dominant negative (left, representative traces; right, summary graph of the EPSC amplitude). d–h, In vivo expression of dominant-negative Syt1-C2A*B* in postsynaptic neurons alone does not affect basal transmission. Postsynaptic overexpression of dominant-negative mutant Syt1-C2A*B* in a subset of CA1-region pyramidal neurons by stereotactic injection of lentiviruses does not cause a major change in paired-pulse ratios of AMPAR EPSCs at different inter-stimulus intervals (d), AMPAR/NMDAR ratio (e), or frequency or amplitude of mEPSCs (f, sample traces; g, cumulative probability plot of the mEPSC inter-event interval with the summary graph of the mean frequency; h, cumulative probability plot of the mEPSC amplitude with the summary graph of the mean amplitude). All data are mean ± s.e.m.; numbers in bars indicate the number of neurons and mice analysed. Statistical significance was assessed in c by the Kruskal–Wallis test followed by pairwise comparisons by the Mann–Whitney U test (*P < 0.05; ***P < 0.001), and in dh by the Mann–Whitney U test only. Source data

Extended Data Figure 10 Summary graphs of the combined effects of all Syt1–Syt7 loss-of-function manipulations on postsynaptic LTP.

a, Cumulative data for all LTP and voltage-pulse LTP experiments. Statistical significance was assessed by the Mann–Whitney U test (***P < 0.001) comparing the test conditions to the control. The dotted line represents the 100% mark corresponding to a lack of change in the synaptic strength as a function of LTP induction. b, Normalized frequency of the data in a grouped in bins of 50%. Source data

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Wu, D., Bacaj, T., Morishita, W. et al. Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP. Nature 544, 316–321 (2017). https://doi.org/10.1038/nature21720

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