1. Volume 23, Issue 10, Pages 2955-2966 (June 2018)
Crucial Role of Postsynaptic Syntaxin 4 in Mediating Basal Neurotransmission and Synaptic Plasticity in Hippocampal CA1 Neurons 
Na-Ryum Bin, Ke Ma, Hidekiyo Harada, Chi-Wei Tien, Fiona Bergin, Kyoko Sugita, Thomas T. Luyben, Masahiro Narimatsu, Zhengping Jia, Jeffrey L. Wrana, Philippe P. Monnier, Liang Zhang, Kenichi Okamoto, Shuzo Sugita 
Cell Reports 
Volume 23, Issue 10, Pages (June 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 2955-2966DOI: (10.1016/j.celrep.2018.05.026)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Generation of Tissue-Specific Syntaxin 4 cKO Mice
(A) Genotyping of syntaxin 4 wild-type or flox and Cre recombinase DNA. The wild-type band is 366 bp, while the flox band is 472 bp. The Cre band is 319 bp.
(B) PCR to confirm the specificity of the CaMK2a-Cre-mediated deletion of syntaxin 4 in different hippocampal regions. The wild-type band is 945 bp, while the flox band is 1,013 bp and the KO band is 295 bp. See also Figure S1A for locations of PCR primers.
(C) CA1-specific deletion of syntaxin 4 does not lead changes in gross morphology of hippocampus. Coronal sections (30 μm) of hippocampus from control and syntaxin 4 cKO mice were prepared, and gross morphology was visualized by Nissl staining. Scale bars, 500 μm.
(D) Quantification of CA1 cell body area and density. Using ImageJ (NIH, Bethesda, MD), the morphological CA1 cell body layer was manually selected, and area and intensity were measured. Two parameters were then normalized to the control (n = 30 for both groups). ∗p < 0.05, two-sample t test.
(E) Schematic diagram illustrating two-photon fluorescence imaging. ChR2-YFP-positive neurons are sparsely present throughout hippocampus and the primary apical dendrite regions of CA1 pyramidal neurons were observed for dendritic spine density and size assays.
(F) Representative images of dendritic spines of CA1 neurons in hippocampal slices from both control and syntaxin 4 cKO mice. Scale bar, 0.5 μm.
(G) Cumulative frequency plots of dendritic spine volumes from control and syntaxin 4 cKO mice.
(H) Average volume of dendritic spines and number of spines from the imaging area.
Error bars indicate SEM (dendritic fluorescence images: n = 87 for control, 118 for syntaxin 4 cKO; slices: n = 18 for control, 23 for syntaxin 4 cKO; n = 4 animals for both groups). ∗p < 0.05; two-sample t test.
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4. Figure 2
Drastic Decrease in fEPSPs in Tissue-Specific Syntaxin 4 cKO Mice
Schaffer collateral axonal fibers were given two successive stimulations from 10 to 150 μA, and the resulting local fEPSPs from apical dendrites of CA1 pyramidal neurons were recorded.
(A and B) Averaged traces of dendritic fEPSPs from syntaxin 4 flox/flox (control) (A) and syntaxin 4 flox/flox; CaMK2a-Cre (cKO) mice (B).
(C and D) fEPSP amplitudes (C) or slopes (D) were plotted against presynaptic fiber volley amplitudes.
(E and F) Paired-pulse ratios of amplitudes (E) or slopes (F) were plotted against presynaptic fiber volley amplitudes.
Error bars indicate SEM (slices: n = 98 for control, 94 for syntaxin 4 cKO; n = 10 animals for both groups). See also Figure S2 for recordings from 8-week-old animals.
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5. Figure 3
Tissue-Specific Syntaxin 4 Deletion Causes a Decrease in Both AMPAR- and NMDAR-Mediated fEPSPs
(A) Dendritic fEPSP recordings from CA1 neurons of control and syntaxin 4 cKO mice in various indicated bath solutions. APV, (2R)-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; PTX, picrotoxin.
(B) Normalized percent AMPAR- and NMDAR-mediated charge transfer in control and syntaxin 4 cKO mice. The recordings obtained using 100 μM PTX + Mg2+-free ACSF were analyzed for this purpose. The area under the curve during this AMPAR activity was calculated as the AMPAR-mediated charge transfer, while the remaining response was calculated as the NMDAR-mediated charge transfer. The charge transfer of both AMPARs and NMDARs was then normalized to the control group.
(C) The charge transfer of both AMPARs and NMDARs was normalized to respective fiber volley amplitudes.
(D) Ratio of percent charge transfer of AMPARs and NMDARs in control and syntaxin 4 cKO mice. Contribution of AMPARs and NMDARs to the total charge transfer was calculated and expressed.
Error bars indicate SEM (n = 4 slices for both groups; n = 2 animals for both groups).
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6. Figure 4
Amplitude, but Not Frequency, of Spontaneous EPSCs of CA1 Pyramidal Neurons Is Reduced upon Syntaxin 4 cKO
(A) Examples of current traces collected from whole-cell voltage patch clamp on CA1 pyramidal neurons of control and syntaxin 4 cKO brain slices at −70 mV holding voltage.
(B) Cumulative frequency plot of spontaneous EPSC amplitude distribution from control and syntaxin 4 cKO mice.
(C and D) Average amplitude (C) and frequency (D) of spontaneous EPSCs from control and syntaxin 4 cKO mice.
(E) Kinetic properties of spontaneous EPSCs from control and syntaxin 4 cKO mice.
Error bars indicate SEM (n = 28 cells for control, n = 31 cells for syntaxin 4 cKO; n = 5 animals for both groups). ∗p < 0.05; two-sample t test. See also Figure S3 for intrinsic properties of CA1 neurons and Figure S4 for cultured hippocampal neuron recordings.
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7. Figure 5
Loss of Syntaxin 4 Leads to Reduced Surface Expression of Glutamate Receptors in Cultured Hippocampal Neurons
(A and B) Syntaxin 4 flox/flox hippocampal neurons expressing EmGFP alone (control) or EmGFP with Cre recombinase (syntaxin 4 KO) were fixed on DIV13 and stained with GluA2 and synaptophysin antibodies (A) or GluN1 antibody (B) in non-permeabilized (surface) or permeabilized (total) conditions. Scale bars, 10 μm.
(C–E) Quantification of synaptophysin puncta (C), surface GluA2 puncta (D), and synaptic GluA2 puncta (based on co-localization with synaptophysin puncta) (E) were analyzed from a 50 μm length of neuropils and normalized to control.
(F) Expression of total GluA2 was analyzed and normalized to control.
(G and H) Quantification of surface GluN1 puncta (G) and total GluN1 (H) analyzed and normalized to control.
Error bars indicate SEM (for surface GluA2, n = 49 cells; for control, n = 47 cells for syntaxin 4 KO; for total GluA2, n = 24 cells for control, n = 23 cells for syntaxin 4 KO; for surface GluN1, n = 44 cells for control and n = 49 cells for syntaxin 4 KO; for total GluN1, n = 25 cells for control, n = 21 cells for syntaxin 4 KO; n = 3 animals for both groups). ∗p < 0.05; two-sample t test. See also Figure S4 for whole-cell patch-clamp recordings of cultured hippocampal neurons.
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8. Figure 6
Syntaxin 4-Mediated Trafficking of Glutamate Receptors Is Essential for Long-Term Potentiation
(A and B) Average fEPSP slope (A) and normalized (against baseline) fEPSP slope (B) in control and syntaxin 4 cKO slices 30 min before theta-burst stimulation (15 bursts of 4 pulses at 100 Hz with interburst internal of 200 ms) and 60 min after. Throughout recording, stimulation intensity was set to obtain a baseline fEPSP slope 30% of maximum evoked slopes.
(C) Representative recordings of fEPSP of control and syntaxin 4 cKO at baseline (1), immediately after LTP induction (2), and 50 min after LTP induction (3), as illustrated in (B).
(D) Normalized fEPSP slope control and syntaxin 4 cKO at times 2 and 3.
Error bars indicate SEM (slices: n = 13 for control, n = 20 for syntaxin 4 cKO; n = 3 animals for both groups). ∗p < 0.05; two-sample t test.
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9. Figure 7
Tissue-Specific Syntaxin 4 KO Mice Exhibit Impaired Spatial Memory
(A) Protocol used for Morris water maze test. Each animal underwent 4 trials per day, with randomized entry points to the pool. Both distance and latency to find platform was measured with a visible platform (days 1–3) and a hidden platform (days 4–15). The location hidden platform was change from the visible platform. The location of hidden platform was changed again during reversal training (days 12-15). During the probe test, the platform was removed from the pool, and the percentage of time spent in the quadrant where the platform was previously located was calculated from the total recording time of 60 s.
(B and C) The learning curves for training to find the visible (days 1–3) or hidden (days 4–15) platform in the Morris water maze. The distance (B) and latency (C) taken to find the platform in 4 daily trials were averaged.
(D) Time spent in the correct quadrant was measured at days 6, 9, 12, and 15. Error bars indicate SEM (n = 6 animals for both groups). ∗p < 0.05; two-sample t test.
Cell Reports  , DOI: ( /j.celrep )
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