Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 2. Two-color DyMIN STED reveals the colocalization pattern of diverse transporters on the same vesicle (A) Bar graph quantifying the proportion of SVs expressing a specific VT. Values are normalized to Syp, which acted as the reference for total SVs. The inset illustrates double immunolabeling of SVs against Syp and a VT. Error bars represent SEM (n = 3 experiments for each pair; >10,000 total SVs for each dual labeling). (B) Illustration depicting dual immunolabeling of SVs against two different VTs (VT1 and VT2). (C) Bar graph showing the degree of vesicular colocalization of VGLUT1 against the specific VTs on the x axis. Error bars represent SEM (n > 3 experiments for each pair; >10,000 total SVs for each dual labeling). (D) Representative images (inverse color map) of VGLUT1 and ZnT3 channels from the two-color DyMIN STED experiment shown in (C). Circles portray the SV area derived by a 2D Gaussian fit on VGLUT1 (green) and ZnT3 (magenta) puncta. The overlay shows merged SV circle profiles of the two channels (n > 3 experiments; >20,000 total SVs for both VGLUT1 and ZnT3). Scale bar, 200 nm. (E–J) Bar graphs showing the degree of vesicular colocalization of a specific VT indicated on top of each graph against the VTs on the x axis (n = 3 experiments for each pair; >10,000 total SVs for each dual labeling). No data are shown for the VMAT2 and VAChT combination. All values are means ± SEM. See also Figures S3–S6.

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Expressing, Immunolabeling, Labeling, Derivative Assay

Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 3. SVs are highly diverse, based on the phenotype of expressed vesicular transporters (A) Bar graph showing the relative abundance of different vesicle classes containing either one (below dashed red line) or two (above the red dashed line) distinct VTs, obtained from the colocalization degree of each VT pair and the respective proportion of each VT in the total SV pool as shown in Figure 2. The numbers next to each bar denotes the actual value. Inset: representative electron immunogold microscopy (IEM) image validating colocalization of VGLUT1 and ZnT3 by displaying gold nano-particles specific for VGLUT1 (5 nm) and ZnT3 (10 nm) (n = 2 IEM experiments, 4.14% of all SVs carried gold particles specific for both VGLUT1 and ZnT3; 35.6% and 6.7% of vesicles were positive for VGLUT1 and ZnT3 alone; negative controls that lacked primary antibodies exhibited negligible signals). Bar graph values are means ± SEM. Scale bar in the inset, 50 nm. (B) Pie diagram showing the broad composition of total SVs containing either one or two distinct VTs. (C) Left, pie diagram showing the composition of glutamatergic vesicles expressing either one of the three vesicular glutamatergic transporters (VGLUT1-3) or in association with another VT. Right, the small components are visualized by an exploding pie chart recalibrated to 100% for greater visibility. (D) Similar pie chart, as shown in (C), for GABAergic vesicles expressing VGAT. (E) Similar pie chart, as shown in (C), for SVs expressing one of the three VGLUTs and VGAT. All statistics above were obtained from two-color DyMIN STED images. See also Figure S1.

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Microscopy, Expressing

Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 4. ZnT3 is a highly abundant SV protein, with 4 copies per vesicle (A) An example quantitative immunoblot for ZnT3 in SVs and purified recombinant ZnT3 protein (rZnT3, standard) at specified concentrations. (B) Scatter plot showing the quantification of ZnT3 abundance in SVs using rZnT3 as standard (n = 2 biological samples). (C) SV proteins ranked by their abundance, as estimated by quantitative mass spectrometry (MS). Selected SV proteins are color-coded for visibility; purple for specified SV markers (Syb2, Syp, and Syt1) and red for the specified VTs. Inset: bar graphs showing the relative abundance (left) and copy number (right) of VGLUT1 and ZnT3 estimated from MS analysis. Error bars represent SEM (n = 2 biological samples). (D) Illustration of the labeling of purified SVs to determine the copy number of VGLUT1 and ZnT3 via single-vesicle imaging. The stoichiometric ratio between VGLUT1 (middle) or ZnT3 (right) molecules and the number of fluorophores was preserved using monoclonal primary antibody and secondary nanobody conjugated to a fixed number of fluorophores (Ab-Nb). (E) Representative DyMIN STED images (fire) of free Ab-Nb (left) and SVs labeled against VGLUT1 (middle) and ZnT3 (right), as described above. Scale bar, 100 nm. (F) Boxplot quantifying the copy numbers of VGLUT1 and ZnT3 in individual SVs. Box range represents 25th–75th percentile, and the whiskers represent outliers (n = 3, p < 0.001, unpaired t test).

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Western Blot, Recombinant, Mass Spectrometry, Labeling, Imaging

Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 5. Heterogeneous pools of SVs containing VGLUT1, ZnT3, or VGLUT1-ZnT3 reside at individual synapses (A) Illustration showing DNA PAINT labeling of VGLUT1 and ZnT3 with Piccolo as the AZ marker in hippocampal culture neurons. (B) A representative confocal image of a selected synapse region showing the Piccolo distribution. Scale bar, 400 nm. (C) An image of the zoomed-in region from (B) showing 2D MINSTED localization position estimates of VGLUT1 with Piccolo in green background. The purple circles represent the two-sigma range around the VGLUT1 cluster centers. Scale bar, 400 nm. (D) Histogram showing the size distribution of VGLUT1 clusters. (E) Lateral distance distribution of VGLUT1 clusters to their nearest neighbor. (F) Lateral distance distribution of VGLUT1 clusters to their nearest AZ.

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Labeling, Marker

Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 6. Zn2+ facilitates vesicular glutamate content by a mecha- nism mediated by ZnT3 (A) Bar graph showing enhanced basal vesicular glutamate uptake in SVs (LP2 fraction) isolated from ZnT3 WT mice (WT) in comparison to the KO (*p < 0.05, n = 6 experiments, unpaired t test). (B) Bar graph showing enhancement of glutamate uptake in increasing con- centrations of Zn2+ in the WT, which is absent in SVs prepared from ZnT3 KO mice. Error bars represent SEM (***p < 0.001, n = 6 experiments, two- way ANOVA). (C) Scatter plot showing dose-dependent facilitation of glutamate uptake by Zn2+. The data points were fitted with a sigmoidal function (Logistic5) to calculate the EC50 value. (D) Illustration of enhanced vesicular glutamate uptake by Zn2+.

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Isolation, Comparison

Journal: Neuron

Article Title: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

doi: 10.1016/j.neuron.2022.02.008

Figure Lengend Snippet: Figure 7. Ambient Zn2+ increases synaptic quantal size via ZnT3 (A) Scheme of whole-cell recordings performed on hippocampal CA3 neurons. (B) Representative traces of mEPSC activity during the baseline recording and after Zn2+ application in ZnT3 WT and KO mice. (C) Average traces showing the time course of mEPSCs during the baseline and after Zn2+ application in the WT (left, decay: 6.6 ± 0.4 ms during baseline versus 7.48 ± 0.7 ms after Zn2+ application, p > 0.2, unpaired t test; rise time: 1.0 ± 0.16 ms during baseline versus 1.1 ± 01 ms after Zn2+ application, p > 0.5, unpaired t test) and the KO (right, decay: 7.8 ± 1.2 ms during baseline versus 6.8 ± 0.7 ms after Zn2+ application, p > 0.20, unpaired t test; rise time: 1.15 ± 0.14 ms during baseline versus 1.1 ± 0.1 ms after Zn2+ application, p > 0.5, unpaired t test). (D) Violin plots comparing the peak amplitude (left) and frequency (right) of mEPSCs between baseline recordings and after Zn2+ application in ZnT3 WT and KO mice. Left: the baseline peak amplitude of mEPSCs was decreased in the KO compared with WT mice (24.9 ± 1.5 pA in WT, n = 10; 20.73 ± 0.7 pA in KO, n = 7; *p < 0.05, t test). Following Zn2+ application, the peak amplitude significantly increased in the WT (24.9 ± 1.5 pA during baseline; 31.2 ± 1.9 pA after Zn2+ application, n = 10, ***p < 0.002, unpaired t test) and not in the KO mice (20.7 ± 0.7 pA during baseline; 20.4 ± 1.2 pA after Zn2+ application, n = 7, p > 0.5, unpaired t test). Right: the baseline frequency was slightly reduced in the KO when compared with the WT (WT, 0.42 ± 0.16 Hz, n = 10; KO, 0.16 ± 0.06 Hz, n = 7, p < 0.05, unpaired t test). Following Zn2+ application, the frequency did not change in both the WT (0.42 ± 0.1 Hz during baseline; 0.28 ± 0.1 Hz after Zn2+ application, p > 0.1, unpaired t test, n = 10) and the KO (0.16 ± 0.06 Hz during baseline; 0.17 ± 0.03 Hz after Zn2+ application, n = 7, p > 0.5, unpaired t test). (E) Illustration showing the role of ZnT3 in the regulation of glutamate quantal size. Top left: colocalization of VGLUT1 and ZnT3 leads to co-uptake and corelease of glutamate and Zn2+ in resting conditions. Bottom left: In the presence of ambient Zn2+, more glutamate is taken up into SVs carrying both VGLUT1 and ZnT3,

Article Snippet: Cultured hippocampal neurons, fixed (4%PFA for 10min), quenched (100mMNH4Cl2 for 10min), permeabilized (5%TritonX-100 for 5min) and blocked (5%NGS for 1 h), was indirectly immunolabeled against VGLUT1, ZnT3 andPiccolo (135308, 197011, SySy, 1:200 dilution) and Piccolo (142104, SySy, 1:500 dilution).

Techniques: Activity Assay

Journal: Neuropsychopharmacology Reports

Article Title: Preliminary analysis of hippocampus synaptic apoptosis and microglial phagocytosis induced by severe restraint stress

doi: 10.1002/npr2.12298

Figure Lengend Snippet: We acquired images from CA3 stratum lucidum region in control and stressed male C‐57 BL6 mice using confocal laser scanning microscopy, subsequently for analysis converted and binarize them using image converter/analyzer. Scale bar, 5 μm. (A) representative converted (left side) and binarized (right side) images in control and stressed mice. (B) ratios of the area where VGLUT1 co‐localizes with caspase 3 to the VGLUT1+ area.10‐d water immersion restraint stress significantly increase the value (control: n = 4 animals, stress: n = 3 animals, t5 = 3.0265, P = .0292). Error bars, SD

Article Snippet: For evaluating local synaptic apoptosis, sections were double stained with rabbit anti‐cleaved caspse‐3 (1:300; Cell Signaling, #9664) and guinea pig anti‐ vesicular glutamate transporter1 (VGLUT1) antibody (1:2000; synaptic systems, #135304) for 6 days at 4°C.

Techniques: Control, Confocal Laser Scanning Microscopy

Journal: Neuropsychopharmacology Reports

Article Title: Preliminary analysis of hippocampus synaptic apoptosis and microglial phagocytosis induced by severe restraint stress

doi: 10.1002/npr2.12298

Figure Lengend Snippet: (A) Representative 3D reconstruction images obtained from CA3 stratum lucidum region in control and stressed male C‐57 BL6 mice. Iba1+ microglia and LAMP1+ structures are visualized at left side. Iba1+ microglia, LAMP1+ structures and VGLUT1+ structures are visualized at right side. Scale bar, 5 μm. (B) Ratios of the volume of the LAMP1+ structures contained in the Iba1+ microglia to the volume of the Iba1+ microglia. There was no significant difference between control and stressed groups in the ratio (control: n = 4 animals, stress: n = 3 animals, t5 = 0.8946, P = .412). Error bars, SD . (C) Ratios of the volume of engulfed VGLUT1 within LAMP1+ structures contained in the Iba1+ microglia to the volume of the Iba1+ microglia. There was no significant difference between control and stressed groups in the ratio (control: n = 4 animals, stress: n = 3 animals, t5 = 0.4414, P = .6773). Error bars, SD

Article Snippet: For evaluating local synaptic apoptosis, sections were double stained with rabbit anti‐cleaved caspse‐3 (1:300; Cell Signaling, #9664) and guinea pig anti‐ vesicular glutamate transporter1 (VGLUT1) antibody (1:2000; synaptic systems, #135304) for 6 days at 4°C.

Techniques: Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Layer-Specific Vesicular Glutamate Transporter 1 Immunofluorescence Levels Delineate All Layers of the Human Hippocampus Including the Stratum lucidum

doi: 10.3389/fncel.2021.789903

Figure Lengend Snippet: Layers of the human hippocampus determined by VGLUT1 immunofluorescence and Nissl staining. (A) Lipofuscin (pigment, purple) and Nissl substance (red) were stained with aldehyde fuchsine and Darrow red in a human hippocampal section, occipital part. The asterisk marks adjoining parts of the gyrus fasciolaris . Scale bar, 700 μm. (B) Zoom-in of panel (A) to display layer boundaries. Alv = alveus , SO = stratum oriens , SP = stratum pyramidale , SR = stratum radiatum , SLM = stratum lacunosum-moleculare , oml = outer molecular layer, iml = inner molecular layer, PML = polymorphic layer, GCL = granule cell layer, CA4 = cornu Ammonis region 4. Scale bar, 500 μm. (C) A subsequent section from the tissue block used in panel (A) was immunostained with VGLUT1 and acquired at a confocal microscope. LAS-X Navigator was used to create and stitch the tilescan. Dashed squares (white) are shown in panel (E) as high-resolution z-stacks. The asterisk marks adjoining parts of the gyrus fasciolaris . Scale bar, 700 μm. (D) Zoom-in of panel (C) to display layer boundaries. The yellow arrows mark the borders of the stratum lucidum (SL), i.e., CA3. Scale bar, 500 μm. (E) Maximum intensity projections (MIPs) of high-resolution z-stacks (stack size = 2.64 μm) acquired in the SP of CA3 (1), SL and SR of CA3 (2), dentate gyrus (3), and CA4 (4). Numbers refer to position 1–4 in the tilescan from panel (C) . Scale bar, 30 μm. Images in panels (A–E) are derived from case 1.

Article Snippet: The following primary antibodies were used: SPO-rabbit (1:200, Synaptic Systems, Cat. No. 102003, RRID:AB_2619748 ), VGLUT1-guinea pig (1:500, Synaptic Systems, Cat. No. 135304, RRID:AB_887878 ), VGLUT2-rabbit (1:200, Synaptic Systems, Cat. No. 135402, RRID:AB_2187539 ), and VGLUT2-mouse (1:100, Millipore, MAB5504, RRID:AB_2187552 ).

Techniques: Immunofluorescence, Staining, Blocking Assay, Microscopy, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Layer-Specific Vesicular Glutamate Transporter 1 Immunofluorescence Levels Delineate All Layers of the Human Hippocampus Including the Stratum lucidum

doi: 10.3389/fncel.2021.789903

Figure Lengend Snippet: Co-staining of VGLUT1 and the mossy fiber-enriched protein synaptoporin in the human hippocampus. (A) Synaptoporin (SPO, magenta) and VGLUT1 (green) co-staining in a hippocampus section, areas of overlap appear in white (left). Individual channels are shown in the middle (SPO) and right (VGLUT1) column. Dashed squares (white) are shown in higher magnification in panels (B–D) as images with dashed frames. The yellow arrows mark the borders of the stratum lucidum , i.e., CA3. Scale bar, 300 μm. Zoom-in images of stratum lucidum (B) , stratum radiatum (C) , and CA4 (D) are displayed in the same manner as outlined in panel (A) . Contrast adjustment was performed identically for panels (B–D) . Insets at the lower right of each image represent MIPs of high-resolution z-stacks (z = 1.98 μm) in the respective layer, imaging parameters and contrast adjustments were kept constant. (B) White arrowheads in the inset mark non-co-localizing SPO puncta. Scale bar overview, 20 μm; inset, 2 μm. (C) Scale bar overview, 10 μm; inset, 2 μm. (D) White arrowheads in the inset mark perisomatic SPO puncta of a mossy cell in CA4. Scale bar overview, 10 μm; inset, 3 μm. Images in panels (A–D) are derived from case 1.

Article Snippet: The following primary antibodies were used: SPO-rabbit (1:200, Synaptic Systems, Cat. No. 102003, RRID:AB_2619748 ), VGLUT1-guinea pig (1:500, Synaptic Systems, Cat. No. 135304, RRID:AB_887878 ), VGLUT2-rabbit (1:200, Synaptic Systems, Cat. No. 135402, RRID:AB_2187539 ), and VGLUT2-mouse (1:100, Millipore, MAB5504, RRID:AB_2187552 ).

Techniques: Staining, Imaging, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Layer-Specific Vesicular Glutamate Transporter 1 Immunofluorescence Levels Delineate All Layers of the Human Hippocampus Including the Stratum lucidum

doi: 10.3389/fncel.2021.789903

Figure Lengend Snippet: Distribution of SPO at VGLUT1-positive synapses in CA3 and DG layers of a human hippocampus. (A–E) High-resolution z-stacks (z = 1.98 μm) were acquired in a hippocampus section co-stained for SPO and VGLUT1. A tilescan of the VGLUT1 channel served as a map to identify the layers and define the z-stacks. Overlay of SPO (magenta), VGLUT1 (green), and DAPI (blue) is shown in the left, individual channels are shown in the middle (SPO) and right (VGLUT1) column. White squares are shown as higher magnified insets at the lower right of each image. Imaging parameters and contrast adjustments were kept constant for all images. Scale bars overviews, 30 μm; scale bars insets, 2 μm. Images in panels (A–E) were derived from case 1.

Article Snippet: The following primary antibodies were used: SPO-rabbit (1:200, Synaptic Systems, Cat. No. 102003, RRID:AB_2619748 ), VGLUT1-guinea pig (1:500, Synaptic Systems, Cat. No. 135304, RRID:AB_887878 ), VGLUT2-rabbit (1:200, Synaptic Systems, Cat. No. 135402, RRID:AB_2187539 ), and VGLUT2-mouse (1:100, Millipore, MAB5504, RRID:AB_2187552 ).

Techniques: Staining, Imaging, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Layer-Specific Vesicular Glutamate Transporter 1 Immunofluorescence Levels Delineate All Layers of the Human Hippocampus Including the Stratum lucidum

doi: 10.3389/fncel.2021.789903

Figure Lengend Snippet: Detectable VGLUT2 immunofluorescence in the human hippocampus. (A) Immunofluorescence staining with VGLUT2 (magenta) and VGLUT1 (green) in the SP of CA3 is represented as overlay (left) and separately for VGLUT2 (gray, right). Staining results obtained with two different VGLUT2 antibodies are opposed, one antibody was raised in rabbit (upper row), the second one was raised in mouse (lower row). Images represent MIPs of high-resolution z-stacks (z = 1.98 μm), imaging parameters and contrast adjustments were kept constant per antibody. (B) In the same manner as explained in panel (A) , immuno-reactivity for the GCL is displayed. Scale bars (A,B) , 10 μm. (C) Rabbit anti-VGLUT2 immunofluorescence in the SO, SR, oml, and iml. Acquired images are shown in a dual fashion; the upper left corner solely shows the VGLUT2 channel, for the lower right corner, the VGLUT1 channel was overlaid. Images represent MIPs of z-stacks (z = 1.98 μm) and imaging parameters as well as contrast adjustments are constant per antibody. Co-staining did not reveal co-localizing puncta, arguing for specific labeling of both proteins. Scale bar, 5 μm. Images in panels (A,B) are derived from case 1, (C) represents case 2.

Article Snippet: The following primary antibodies were used: SPO-rabbit (1:200, Synaptic Systems, Cat. No. 102003, RRID:AB_2619748 ), VGLUT1-guinea pig (1:500, Synaptic Systems, Cat. No. 135304, RRID:AB_887878 ), VGLUT2-rabbit (1:200, Synaptic Systems, Cat. No. 135402, RRID:AB_2187539 ), and VGLUT2-mouse (1:100, Millipore, MAB5504, RRID:AB_2187552 ).

Techniques: Immunofluorescence, Staining, Imaging, Labeling, Derivative Assay