Visualization of Subunit-Specific Delivery of Glutamate Receptors to Postsynaptic Membrane during Hippocampal Long-Term Potentiation  Hiromitsu Tanaka, Tomoo Hirano  Cell Reports  Volume 1, Issue 4, Pages 291-298 (April 2012) DOI: 10.1016/j.celrep.2012.02.004 Copyright © 2012 The Authors Terms and Conditions

Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure 1 NRX-Coated Glass-Induced PSLM Formation (A) Schematic drawing of NRX-coated glass and PSLM. NRX is immobilized on the glass surface through biotin-streptavidin and antigen-antibody linkers (beige zone). NRX binds to NL expressed on the neuronal membrane. Postsynaptic scaffold proteins (magenta) and receptors (AMPAR) accumulate near NL in the TIRFM visualization zone (yellow). Yellow arrows indicate excitation laser beam. See also Figure S1. (B) Representative images of HA-tagged NL-expressing hippocampal neurons cultured on the NRX-coated glass (left) or on the mutated NRX-coated glass (right). (C) PSD95-EGFP (arrows) was clearly observed with TIRFM on the NRX-coated glass, but not on the mutated NRX-coated glass. (D) Endogenously expressed Homer (magenta) was colocalized (arrows) with PSD95-EGFP (green) in the TIRFM visualization zone. (E) The localization of immunostained PSD95-EGFP (green) and Vglut1 (magenta) in the same field was observed with TIRFM (left) or with epi-fluorescence (right). Only PSD95-EGFP (green) was observed (arrows) with TIRFM, whereas other PSD95-EGFP signals were colocalized with Vglut1 (arrowheads) when observed with epi-fluorescence. Scale bars, 20 μm (B) and 5 μm (C–E). Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure 2 Changes of AMPAR Subunit Number by LTP-Inducing Stimulation (A–C) Averaged time courses of GluA1–3 fluorescence intensity in PSLM (red) and in non-PSLM (black) measured every 10 min before and after the field stimulation (arrows). Data in the presence of APV (+APV) are also shown (dotted lines). See also Figures S2, S3A, and S3B. (D–F) Averaged time courses of GluA1–3 fluorescence intensity measured every 4 min. Error bars indicate SEM. Significant differences between −APV and +APV (Dunnett's test), or before and after the stimulation (Steel's test) are marked (n = 8–13 cells; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). (G–I) GluA-SEP signals (green) and PSD95-RFP signal (magenta) are shown. PSD95-RFP was recorded before the stimulation, and images of detection of the two signals were overlaid. GluA-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. Scale bar, 2 μm. Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure 3 Exocytosis and Lateral Movement of GluA1 and GluA2 during LTP (A) Two examples of GluA1-SEP (green) exocytosis (arrows) shown together with PSD95-RFP (magenta). The numbers indicate time (seconds) after the field stimulation. The dotted rectangle is enlarged in (C). See also Figures S4A–S4D. (B) Representative time courses of GluA1-SEP signal intensity before and after the exocytosis. (C) The left image shows GluA1-SEP appearance in the periphery of PSD95-RFP-positive area (dotted area). (D) The number of GluA1 exocytosis events within 1 min after the stimulation. (E) An example of GluA2-SEP (green) exocytosis (arrow) in non-PSLM shown together with PSD95-RFP signal (magenta). See also Figures S4E and 4G. (F) An example of lateral movement (arrows) of a cluster of GluA2-SEP to PSLM. See also Figures S4F and S4H. Scale bars, 500 nm (A, E, and F) and 200 nm (C). Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure 4 Frequency of GluA1–3 Exocytosis during LTP (A–F) The time courses of exocytosis frequency of GluA1-SEP (A and D), GluA2-SEP (B and E), and GluA3-SEP (C and F) in PSLM (red) and in non-PSLM (black) are presented (n = 8–13 cells; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, compared to no stimulation [No stim.], Dunnett's test). In (A)–(C), GluA1-, GluA2-, or GluA3-SEP was expressed alone. In (D), GluA1-SEP was coexpressed with GluA2 labeled with myc tag (GluA2-myc), in (E), GluA2-SEP was coexpressed with GluA1-myc, and in (F), GluA3-SEP was coexpressed with GluA2-myc. See also Figure S3C. (G) A hypothetical scheme of postsynaptic delivery of AMPARs during LTP. Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure S1 NRX Coating of Glass, Related to Figure 1 (A) Schematic drawing of NRX coating. Fc-tagged NRX was immobilized on the glass through biotin-streptavidin linkers and Fc antigen-antibody reaction. (B) Observation of the coated NRX signal (green) by immunostaining (left). NRX-Fc was purified from HEK cells expressing NRX-Fc. No NRX signal was detected when non-transfected HEK cells were used (right). (C–E) HEK cells expressing NL were cultured on the glass coated with NRX or the mutated NRX, and NRX (green) and NL (magenta) were immunostained after membrane permeabilization. The number of HEK cells on the mutated NRX-coated glass was much smaller than that on the NRX-coated glass (C), which was quantified in (D) (n = 12 for each, ∗∗∗p < 0.001, two-tailed Student's t test). Higher magnification views (E) show fan-shaped swellings of HEK cells (arrows), which was characteristic for NL-expressing cells on the NRX-coated glass. (F) Staining of HEK cells without membrane permeabilization. NRX was not stained under a HEK cell on the NRX-coated glass, but stained on the mutated NRX-coated glass. These data suggest that the coated NRX interacted with NL expressed in a HEK cell so tightly that anti-Fc antibody could not reach the NRX under the cell. Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure S2 Effects of Electrical Field Stimulation and Stabilization of PSLM, Related to Figure 2 (A) Representative fluorescence ratio images of Fura-2 (F340/F380) 2 s before, during, and 10 s after the field stimulation in pseudo color. (B) An example of the time course of the Fura-2 fluorescence ratio in a primary dendrite (arrow in A). The stimulation (Stim.) started at 0 s. (C) Representative fluorescence images of GCaMP3 (green) and PSD95-RFP (magenta) in dendrites before and after the field stimulation recorded with TIRFM. The elevation of intracellular Ca2+ concentration increases the signal intensity of GCaMP3. Yellow circles indicate application of the field stimulation. Images recorded every 1 s are shown. (D) An example of the time course of GCaMP3 fluorescence intensity in PSLM (arrow in C). The stimulation (Stim.) started at 0 s. (E) Representative miniature excitatory postsynaptic current (mEPSC) traces without (- Stim.) and 10-20 min after (+ Stim.) the field stimulation. (F) Examples of mEPSC amplitude distribution without and after the stimulation. Right-shift of the amplitude distribution was observed after the stimulation. (G) The time courses of changes of amplitude and frequency of mEPSCs after the stimulation (n = 12-14 cells). The amplitude, but not the frequency, was significantly larger after the stimulation. (∗p < 0.05, compared to No stim., Dunnett's test). (H) Averaged time courses of GluA1 fluorescence intensity in PSLM (red) and in non-PSLM (black) recorded every 10 min without the field stimulation (n = 10 cells). Presumably, photobleaching contributed to the signal decay. (I) Representative images without the field stimulation. GluA1-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. (J) PSD95-RFP clusters recorded 10 min before (green) and 30 min after (magenta) the field stimulation. 91 ± 6.1% of PSD95 clusters (n = 6 cells; 248 clusters) were colocalized, as indicated by arrows. Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure S3 Chemical Induction of LTP, Related to Figures 2 and 4 (A) Averaged time courses of GluA1-SEP fluorescence intensity in PSLM (red) and in non-PSLM (black) measured every 10 min before, during and after the 10 min application of the glycine containing external solution without Mg2+ (n = 7 cells, ∗p < 0.05 and ∗∗p < 0.01, compared to No stim, Steel's test). (B) Representative images before and after the chemical stimulation. GluA1-SEP signals in PSLM and non-PSLM are indicated by arrows and an arrowhead, respectively. (C) The time courses of exocytosis frequency of GluA1-SEP in PSLM (red) and in non-PSLM (black) before, during and after the 10 min application of the solution. (∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, compared to No stim, Dunnett's test). Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions

Figure S4 Estimation of the Number of Exocytosed GluA1-SEP Subunits, and Exocytosis and Lateral Movement of GluA1 and GluA3 in Non-PSLM, Related to Figure 3 (A) Sequential images of SEP signal recorded every 100 ms with TIRFM. (B) The time course of SEP fluorescence intensity presented in (A), showing single-step photobleaching characteristic of a single molecule. (C) Distribution of presumptive single SEP fluorescence intensities. Mean ± SEM was 5.5 ± 0.1 (n = 256, arrowhead). Background signal was subtracted. (D) The distribution of exocytosed GluA1-SEP fluorescence intensities (76 ± 11, n = 34, arrowhead). (E and G) Representative images GluA1-SEP (E) and GluA3-SEP (G) (green) exocytosis (arrows) in non-PSLM shown with PSD95-RFP signal (magenta). The numbers indicate time (sec) after the field stimulation. (F and H) Lateral movement (arrows) of a cluster of GluA1-SEP (F) and GluA3-SEP (H) to PSLM. Scale bar, 500 nm (E–H). Cell Reports 2012 1, 291-298DOI: (10.1016/j.celrep.2012.02.004) Copyright © 2012 The Authors Terms and Conditions