Journal: IBRO Neuroscience Reports

Article Title: IL-33 relieves nerve injury by mediating microglial polarization in neuromyelitis optica spectrum disorders via the IL-33/ST2 pathway

doi: 10.1016/j.ibneur.2024.07.008

Figure Lengend Snippet: Microglial cells were activated and transformed to the M2 phenotype after treatment with IL-33. (a) Western blot images and the quantification of CD206 and CD40 levels after treatment with IL-33 or NMOD serum. (b) BV2 cells were labeled with Iba-1 and CD68 for immunofluorescence. (c) Expression levels of PSD95 were observed by immunofluorescence after treatment with IL-33 or NMOSD serum. One-way analysis of variance was used for statistical analyses. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: For protein immunoreactivity in cells, the primary cortical neurons or BV2 cells were fixed with 4 % paraformaldehyde for 30 min at RT and treated with 0.5 % Triton for 10 min. After being washed with PBS, the cells were blocked with 5 % bovine serum albumin (BSA) for 1 h and then incubated with a rabbit anti-PSD95 antibody (1:100 dilution, PA2295, Boster), anti-CD68 antibody (1:200 dilution, 28058–1-AP, Proteintech), anti-IBA1 antibody (1:100 dilution, CY7217, Abways), or anti-ST2 antibody (1:200 dilution, PRS3363, Sigma) at 4 °C overnight.

Techniques: Transformation Assay, Western Blot, Labeling, Immunofluorescence, Expressing

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP- (Green) and Q655-Syt2 (Red)-labeled vesicles; corresponding volume rendering of GFP terminal and SV detection (see ). ( B ) The number of labeled SVs detected in whole presynaptic terminal. ( C ) Live confocal imaging and SV tracking with the autoregressive motion (Red) or Brownian motion (Blue) algorithm (see ). ( D ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces, color-coded as in ( C ). ( E ) Comparison of SV displacements and diffusion coefficients for Q655-Syt2- (n = 12 terminals), Q585-Syt2- (n = 12) or C5E-Syt2- (n = 12) labeled vesicles, Q655-Syt2-labeled vesicles after chemical fixation (n = 12) and 40 nm beads (n = 12 ROI). Two-tailed unpaired t-test (*p<0.05; ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.003 10.7554/eLife.24845.004 Figure 1—source data 1. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.004 10.7554/eLife.24845.005 Figure 1—source data 2. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.005 10.7554/eLife.24845.006 Figure 1—source data 3. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.006 10.7554/eLife.24845.007 Figure 1—source data 4. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.007

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Volume rendering of GFP terminal and SV detection in calyceal terminals loaded with Q655 only or Q655-Syt2 after 16 hr. ( B ) Comparison of the number of SVs detected in calyceal terminals loaded with Q655 alone (Black, n = 5 terminals), Q655-Syt2 (Red, n = 5) or C5E-Syt2 (Magenta, n = 5) after overnight incubation. Two-tailed unpaired t-test (*p<0.05). ( C ) Fluorescence intensity distribution in 150 nm confocal spots for 40 nm FITC-beads (Green), SVs loaded with Q655-Syt2 (Red) or with C5E-Syt2 (Magenta). DOI: http://dx.doi.org/10.7554/eLife.24845.008

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Incubation, Two Tailed Test, Fluorescence

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP (Green) and C5E-Syt2 (Red)-labeled vesicles. ( B ) Visualization and quantification of exocytosis induced by bath application of 65 mM KCl. Upper left panel: confocal image before KCl application, lower left panel: confocal image after KCl application. Right panel: Measurement of C5E fluorescence intensity from ROI (white box on left panels) of five different terminals. Black trace: average of 5 traces (color coded), Red trace: Boltzman fitting. ( C ) Tracking of SVs in interconnected swellings before (upper panels) and after application of 65 mM KCl (lower panels). ( D ) Fluorescence recovery after photobleaching in swellings (upper panels) or finger-like structures (lower panels). ( E ) FRAP analysis showing fluorescence intensity recovery profile (ROI ~1.5 µm, white boxes) and estimation of the mobile and immobile fraction of SVs in swellings (Green) and finger-like structures (Red). DOI: http://dx.doi.org/10.7554/eLife.24845.009

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Fluorescence

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Confocal images showing the co-localization of SVs labeled overnight with Q655-Syt2 (Red) and for 1 hr or 3 hr with Q585-Syt2 (Green), co-localization (white). ( B ) Comparison of the Pearson co-localization coefficient of Q655- and Q585-labeled vesicles after 1, 2, or 3 hr post-endocytosis. ( C ) Tracking of Q655- (Red) and Q585- (Green) labeled vesicles after 1 or 3 hr from terminals shown in ( A ). ( D ) Diffusion coefficient of Q655- and Q585-labeled vesicles after 1 (n = 3 terminals), 2 (n = 3), or 3 hr (n = 3) post-endocytosis. Two-tailed unpaired t-test (*p<0.05; ns, not significant). ( E ) Dynamic properties and displacement analysis of Q655- (Red) and Q585-labeled vesicles after 1 hr (Blue) or 3 hr (Green) post-endocytosis. DOI: http://dx.doi.org/10.7554/eLife.24845.010

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Labeling, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Live confocal imaging of a giant calyceal terminal expressing cytosolic GFP- and Q655-Syt2-labeled vesicles, with SV tracking, color-coded over time, or sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm and Red >4 µm, see ). ( B ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces (see ), color-coded as in ( A ). ( C ) Classification and quantification of SV movements in three groups based on their maximum speed and trajectory length (n = 6175 trajectories). ( D ) Displacement curves and displacement modalities (Red: diffusive motion, Blue: active motion) of identified traces (n = 6175 trajectories). ( E ) Diffusion coefficient of SVs at 37 o C (n = 12 terminals) or 25°C (n = 4); or in the presence of 2.5 µM OA at 37°C (n = 9). Two-tailed unpaired t-test (*p<0.05). DOI: http://dx.doi.org/10.7554/eLife.24845.013 10.7554/eLife.24845.014 Figure 2—source data 1. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.014 10.7554/eLife.24845.015 Figure 2—source data 2. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.015 10.7554/eLife.24845.016 Figure 2—source data 3. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.016

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles, and individual SV tracking sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm and Red >4 µm). ( B ) Speed variation profiles during short (Blue), medium (Green) or long (Red) trajectories. ( C ) Displacement modalities. Representative displacement curves showing different modes of movements. Each representative curve for diffusive motion and active motion (facilitated and impeded) was calculated and plotted from an average of 12 different curves extracted from displacement plots similar to . ( D ) Confocal imaging of a giant calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles. White circles and black lines represent swelling and finger areas, respectively. ( E ) Comparison of the diffusion coefficient of SVs in swellings or fingers in various conditions (Control, 30 µM nocodazole, 2.5 µM OA, 65 mM KCl, 500 mM sucrose or 1 Hz electrical stimulation, n = 6 terminals for each condition). ( F ) 3D tracking of SVs labeled with Q655-Syt2 in an individual swelling. Left panel: SV trajectories (time color-coded), right panel: Displacement vectors of SV trajectories. ( G ) Schematic diagram showing the proportion of SVs with displacement vectors going toward the synaptic cleft (Green), away from the synaptic cleft (Red), or moving laterally (Blue) in an individual calyceal swelling. ( H ) Comparison of SV mobilities between 2D and 3D tracking. Trajectory length analysis (Left panel) and diffusion coefficient (Right panel) in single 2D confocal section (Blue, n = 3) and 3D confocal z-stack (Red, n = 3). Two-tailed unpaired t-test (*p<0.05; ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.018

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Syt2-C5E-loaded SV tracking sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm and Red >4 µm) at three different image acquisition speed (0.5 s, 1 s and 2 s per image). Trajectory length analysis in single 2D confocal section 0.5 s per image (Blue, n = 3), 1 s per image (Black, n = 3) and 2 s per image (Red, n = 3). Two-tailed unpaired t-test for comparison between two groups and two-way ANOVA for multi groups comparison (ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.019

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Live confocal imaging of a giant immature terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles, with SV tracking color-coded over time, or sorted according to trajectory length (Blue <2 µm, Green 2–4 µm and red >4 µm). ( B ) Confocal imaging of a giant mature terminal as described in ( A ). ( C ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces from immature (left panel) or mature (right panel) calyceal terminals, color-coded as in ( A ). ( D ) Classification and quantification of SV movements in three groups based on their maximum speeds and trajectory lengths in immature (Red, n = 9 terminals) and mature (Blue, n = 9) terminals. ( E ) Displacement modalities and diffusion coefficients of SVs in immature (Red, n = 9) and mature (Blue, n = 9) terminals. Two-tailed unpaired t-test (*p<0.05). DOI: http://dx.doi.org/10.7554/eLife.24845.024 10.7554/eLife.24845.025 Figure 4—source data 1. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.025 10.7554/eLife.24845.026 Figure 4—source data 2. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.026

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Confocal z-stack imaging of a calyceal terminal labelled with antibodies against de-tyrosinated α-tubulin (Red), VGLUT1 (Green) and DAPI (Blue). ( B ) Live confocal imaging of a calyceal terminal over-expressing GFP and labeled with SiR-Tubulin before and after treatment with 30 µM Nocodazole. ( C ) Quantification of GFP- and SiR-Tubulin fluorescence intensity during nocodazole treatment. ( D ) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2 labeled vesicles, and SV tracking (long tracks displayed only). ( E ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control (left panel) or nocodazole-treated terminals (right panel), color-coded (Blue <2 µm, Green 2–4 µm and red >4 µm). ( F ) Classification and quantification of SV movements in three groups based on their maximum speeds and trajectory lengths in control (Red, n = 8 terminals) and nocodazole-treated (Blue, n = 8) terminals. ( G ) Diffusion coefficient of SVs in control (Red, n = 8) and nocodazole-treated (Blue, n = 8) terminals. Two-tailed unpaired t-test (*p<0.05; ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.031 10.7554/eLife.24845.032 Figure 6—source data 1. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.032

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Imaging, Expressing, Labeling, Fluorescence, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: Analysis of C5E-Syt2-labeled SVs in giant calyceal terminals. ( A ) KCl stimulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals (Red) or terminals incubated with 65 mM KCl (Blue). ( B ) Sucrose stimulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals (Red) or terminals incubated with 500 mM sucrose (Blue). ( C ) Electrical simulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals (Red) or terminals during 1 Hz electrical field stimulation for 30 s (Blue). ( D ) Trajectory length analysis in control (Red) and KCl-treated terminals, sucrose-treated terminals, or 1 Hz-stimulated (Blue) terminals. ( E ) Displacement modality analysis in control (Red) and KCl-treated terminals, sucrose-treated terminals, or 1 Hz-stimulated (Blue) terminals. ( F ) Diffusion coefficient analysis in control (Red) and KCl-treated terminals, sucrose-treated terminals, or 1 Hz-stimulated (Blue) terminals. (KCl treatment: n = 6; sucrose treatment: n = 6; 1 Hz stimulation: n = 6 in D, ( E and F ). Two-tailed unpaired t-test (*p<0.05; ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.035 10.7554/eLife.24845.036 Figure 7—source data 1. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.036 10.7554/eLife.24845.037 Figure 7—source data 2. Data and statistics for . DOI: http://dx.doi.org/10.7554/eLife.24845.037

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Labeling, Incubation, Diffusion-based Assay, Two Tailed Test

Journal: eLife

Article Title: Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses

doi: 10.7554/eLife.24845

Figure Lengend Snippet: ( A ) Upper panels: confocal images of C5E-Syt2 labeled vesicles loaded during spontaneous activity for 1 hr or during train of 1 Hz electrical stimulation. Lower panel: Number (N) and fluorescence intensity (FI) of C5E-Syt2-labeled vesicles loaded spontaneously or during 1 Hz stimulation (Blue bar). ( B ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces during spontaneous (Red) or stimulated activity (Blue). ( C ) Diffusion coefficients of C5E-Syt2-labeled vesicles loaded during spontaneous (Red, n = 3 terminals) or stimulated (Blue, n = 3) activity. Two-tailed unpaired t-test (ns, not significant). DOI: http://dx.doi.org/10.7554/eLife.24845.038

Article Snippet: Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).

Techniques: Labeling, Activity Assay, Fluorescence, Diffusion-based Assay, Two Tailed Test