glun2a n terminus  (Alomone Labs)

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Schematic diagram of the mechanism underlying detection of exocytotic pH-sensitive SEP-GluN2 receptors at the neuronal surface. (B) Acidification test of SEP-GluN2A subunits expressed in DIV21 hippocampal neurons. pH values of the imaging solution are ∼7.4 and ∼5.5, respectively. Fluorescence intensity is color coded, bar=10 μm. (C) Schematic diagram showing bleaching assay in TIRF mode to detect SEP-GluN2 exocytosis. Individual exocytotic events are indicated with red arrow. (D) Representative time-lapse images of hippocampal neurons transfected with Homer1c-DsRed and SEP-GluN2A are shown to illustrate the actual detection steps of the bleaching assay for SEP-GluN2 exocytosis (step 1 and 2). Boxed regions are amplified in the upper right corner. The neurons after bleaching are outlined in yellow (step 3). An exocytosis event is annotated with red arrow in step 4. Bar=5 μm. (E) Representative maximum projection of the time-lapse image stacks showing exocytic events at 5 min. Bracketed area is amplified in upper right box. Bar=5 μm. (F) y-t projection of the region bracketed in (E) , with time-lapse frames of an exocytotic event shown in bottom panels. Scale bar as indicated. (G) Quantification of average duration of GluN2A exocytic fusion events in neurons before (control) and after 5 to 10 min of cLTP treatment (cLTP). Results are shown in mean±SEM, n.s. no significant difference, n as indicated. Neurons were from three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Imaging, Fluorescence, Bleaching Assay, Transfection, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Schematic diagram of the cLTP model used in this study. Mature hippocampal neurons (DIV>21) were pre-incubated in Buffer A for 60 min and then flushed with 200 nM of Glycine (Buffer B) for 10 min to induce glycine-dependent cLTP. They were then returned to resting condition in Buffer A for 10 to 30 min. 50 μM APV was added to the bathing medium as indicated. Surface levels of GluN2 receptors were detected at indicated time points. (B) Calcium fluctuations of dendritic spine regions visualized in GCaMP6-expressing hippocampal neurons, which were treated with the above cLTP protocol. Fluorescence intensity of bracketed area is amplified in lower left boxes. Bar=20 μm. Kymograph (C) and intensity profile (D) of the Ca 2+ intensity in individual dendritic spines before (control) and during the 10 min of cLTP treatment (+cLTP). (E) Quantification of Ca 2+ fluctuations induced by the cLTP protocol. ⊗F/F of the GCaMP6-expressing neurons was used to show the Ca 2+ intensity change before (control), and during the cLTP treatment without (-) or with (+APV) the existence of 50 μM APV. Results are shown in scatter plot with mean±SEM, * p <0.05. n=6 (control), 7 (cLTP), 6 (+APV) cells from two independent cultures, two tailed student’s t -test. (F) Representative western blots showing the levels of biotin labeled surface GluN2A receptors in cultured rat cortical neurons treated as indicated. (G) Quantification of results in (F) . Results are shown in scatter plot with mean±SEM, * p <0.05. n=9 (ctrl), 7 (cLTP) repeats from at least seven independent preparations, two tailed student’s t -test. (H) Confocal images showing surface immunostaining for GluN2A receptors in cultured rat hippocampal neurons treated as indicated. Bar=20 μm. (I-J) Quantification of immunostaining of surface GluN2A receptor levels (I) , total GluN2A receptor levels, which is the GluN2A immunostaining signal after cell membrane permeabilization. (J) and the ratio of surface to total GluN2A receptor levels (K) before or after cLTP treatment for indicated time. Results are shown in mean±SEM, * p <0.05, ** p <0.01, *** p <0.001. Results were from n=39 (ctrl), 51 (cLTP), 42 (+APV) cells of three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Incubation, Expressing, Fluorescence, Amplification, Two Tailed Test, Western Blot, Labeling, Cell Culture, Immunostaining

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative confocal images of surface GluN2A immunostaining in cultured rat hippocampal neurons co-transfected with SEP-GluN2A and Homer1c-DsRed, which were treated as indicated. Boxed regions were amplified in the bottom rows. Extrasynaptic NMDARs were indicated with arrowheads. Bar=1 μm. (B) Quantification of immunostaining for surface GluN2A receptor levels. Data represent mean±SEM, n=17 (0’), 19 (-) and 22 (+APV) neurons. * p <0.05, ** p <0.01, two-tailed student’s t -test, data were derived from three independent cultures. (C) Representative 3D-SIM images of endogenous surface GluN2A and Homer immunostaining in cultured rat hippocampal neurons treated as indicated. Wide-field images are shown in the upper left insets. Boxed regions of interest (ROIs) are shown in lower panels. Extrasynaptic NMDARs were indicated with arrowheads. Bar=1 μm. (D) Cluster size of Surface GluN2A receptor were determined using the ‘surface’ function of Imaris. The frequency distribution of cluster size were shown in control, cLTP 10 min and cLTP 40 min neurons. Clusters with a diameter of 0.01 to 0.1 μm 2 were characterized as ‘small’ clusters, whereas all others were characterized as ‘big’ clusters. Data represent mean±SEM, n=6 (control), 5 (10 min) and 7 (40 min) cells were analyzed. * p <0.05, ** p <0.01, two-tailed student’s t -test, data were derived from three independent cultures. (E) Quantification of the average density of small GluN2A clusters. Data represent mean±SEM, n=12 (0’), 10 (cLTP 10’) and 14 (cLTP 40’) neurons. * p <0.05, two-tailed student’s t -test, data were derived from three independent cultures. (F) The dual-colour SIM images were analysed using Imaris software, and the representative Imaris-rendered surfaces were shown. GluN2A and Homer were extracted as separate surfaces to detect the overlap between surface of GluN2A receptors and that of the postsynaptic marker Homer. Boxed regions are amplified in lower panels. Bar=1μm. (G) Representative spatial separation efficiency of the Imaris surface method in extracting the synaptic and extrasynaptic GluN2A receptors. Non-Homer1c overlapping (Homer-) GluN2A receptors were characterized as extrasynaptic, and Homer1c overlapping (Homer+) GluN2A receptors were characterized as synaptic. Boxed regions are amplified in the bottom panels with extrasynaptic or synaptic GluN2A receptors marked using red and green surfaces, respectively. (H-I) Cumulative frequency distribution of the size of extrasynaptic (H) and synaptic (I) GluN2A receptors surfaces in cultured hippocampal neurons before (control) or after 10 min of cLTP treatment (cLTP). (J) Average cluster sizes of the synaptic and extrasynaptic surface GluN2A receptors, in cultured hippocampal neurons before (control) or after 10 min of cLTP treatment (cLTP). Data represent mean±SEM, n=606, 800, 981 and 1323 clusters from at least five neurons were compared. ** p <0.01, *** p <0.001, two-tailed student’s t -test, data were derived from three independent cultures.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Immunostaining, Cell Culture, Transfection, Amplification, Two Tailed Test, Derivative Assay, Software, Marker

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) In DIV21 rat hippocampal neurons, surface GluN2A NMDA receptors were stained with specific antibodies against their extracellular domain before fixation, followed by immunostaining using Homer-1c antibody. Confocal microscopy was used to visualize the distribution of fluorescent signals at dendritic spines. Scale bar= 5 μm. (B) The ratio of GluN2A receptors localized in the extrasynaptic region was defined as the R extra , which was calculated from the ratio of GluN2A not overlapping with Homer-1c (See Methods). Data represent mean±SEM, * p <0.05, ** p <0.01. N represents the number of cells analyzed and are shown on the bar. Data were measured from two independent cultures, two-tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Staining, Immunostaining, Confocal Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) DIV21 rat hippocampal neurons were fixed and stained with specific antibodies against endogenous GluN2A, SNAP-23 and SNAP-25. Confocal microscopy was used to detect the distribution of signals from three different fluorescent channels. To the right boxed region is amplified with channels separated. Bracketed regions are further amplified. Spine annotated by arrows were positive for GluN2A, both SNAP-23 and SNAP-25, whereas spines annotated by arrowheads shows the overlapping between GluN2A and only SNAP-23. Scale bar as indicated on the panels. (B) DIV21 rat hippocampal neurons were live-stained for surface GluN2A expression, followed by fixation and staining with specific antibodies against endogenous SNAP-23 and SNAP-25. 3D-SIM was used to visualize the distribution of fluorescent staining signals at dendritic spines. ROIs were amplified in top and right panels, with 3-color line-profile performed across the single synapse shown in right panel. Scale bar as indicated. (C) Line profiles showing the colocalization of GluN2A (green), SNAP-23 (red) and SNAP-25 (magenta). (D) Colocalization level between GluN2A and SNAP25 or SNAP23 were quantified with Pearson’s coefficient. Data represent mean ± SEM, n=43 ROIs with SNAP-25 and SNAP-23, respectively. *** p <0.001, two-tailed student’s t -test, data were measured from two independent cultures.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Staining, Confocal Microscopy, Amplification, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) PC12 cells were transfected with plasmids expressing the catalytic light-chains (Lc) of BoNT/A or BoNT/E, respectively. Cleavage of SNAP-23 and SNAP-25 was detected using their specific antibodies, full-length of SNAP-25 and SNAP-23 are indicated with arrows. (B) Representative images showing the surface level of endogenous GluN2A following 10 min cLTP stimulation in DIV21-28 rat hippocampal neurons, which were co-transfected with Homer-DsRed and BoNT/A-Lc. Boxed regions showing the level of surface GluN2A subunits were amplified in the bottom panels. Scale bar=20 μm. (C) Quantification of the surface level of endogenous GluN2A in BoNT/A-Lc transfected groups. (D) PC12 cells were transfected with shSNAP-23 or shSNAP-25 plasmids to verify knock down efficiencies. Endogenous SNAP-23 and SNAP-25 was detected using their specific antibodies. (E) Representative images showing the surface level of endogenous GluN2A following 10 min cLTP stimulation in DIV21-28 rat hippocampal neurons co-transfected with Homer-DsRed and shSNAP-23, shSNAP-25 plasmids. Boxed regions showing the level of surface GluN2A subunits were amplified in the bottom panels. Scale bar=20 μm. (F) Quantification of the surface level of endogenous GluN2A in shSNAP-23, shSNAP-25 transfected groups. Results are shown in mean±SEM, *** p <0.001, n.s. no significant difference. For E, n=47, 41, 26 and 21 cells from three independent preparations, two tailed student’s t -test. For F, n=60, 82, 25, 28, 32 and 39 cells from three independent preparations, two tailed student’s t -test.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Transfection, Expressing, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: Mature rat hippocampal neurons were transfected with BoNT/E-Lc-GFP on DIV14-17, treated with cLTP stimulation on DIV21, followed by surface GluN2A immunostaining before fixation. Representative confocal images show the typical neurodegeneration phenotype of beading axons and round cell bodies in all BoNT/E-Lc-GFP expressing neurons treated as indicated. Bar = 20 μm.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Transfection, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative images showing each step of the bleaching assay for the automatic analysis of exocytosis. Boxed regions are amplified in the lower left corner. Bar= 10 μm. Inset Bar = 1 μm. (B) The last step (Step 4) of the image preparation of the boxed region in (A) shows effects of standard deviation filter (see Methods). For comparison, time-lapse images of unfiltered (top panels) and SD-filtered (bottom panels) are shown. (C) Comparison of improvement in signal to noise ratio averaging of image sequence (red) or when SD-filter was applied (black). (D) The trajectories detected using TrackMate plugin of Image J. One exocytosis event is marked out with red arrow. Bar as indicated on the figures. (E) Histogram of the average duration of SEP-GluN2A exocytotic trajectories detected by automatic method. The average duration of control condition is shown as mean ± SEM, from n=50 neurons of three different preparations. R 2 =0.983 with the least squares Gaussian curve fitting method.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Bleaching Assay, Amplification, Standard Deviation, Sequencing

Journal: bioRxiv

Article Title: Long-Term Potentiation Induces Extrasynaptic Exocytosis of Glun2A-containing NMDA Receptors that is Mainly Controlled by SNAP23

doi: 10.1101/746404

Figure Lengend Snippet: (A) Representative figures of cultured hippocampal neurons with indicated treatments showing sites of GluN2A exocytosis. Postsynaptic regions are labelled with Homer-DsRed. Boxed regions showing the exocytic events near the spine are amplified in the upper right corner. Bar=5 μm. (B) Quantification of the relative number and (C) extrasynaptic/synaptic ratio of SEP-GluN2A exocytosis in neurons before (control) and after 5 to 10 min of cLTP treatment (cLTP). (D) Quantification of exocytosis frequency in neurons before (control) and after 5-10 min of cLTP treatment (cLTP), which were compared to those of APV (50 μM)-treated or BoNT/A-Lc (A/Lc) transfected neurons. Results are shown in mean±SEM, ** p <0.01, *** p <0.001, n as indicated. Neurons were from at least three independent preparations, two tailed student’s t -test. (E) Fluorescence intensity recorded from each exocytic fusion event decayed in step-like manner. Each step corresponds to one inserted and bleached SEP-GluN2A subunit. Most intensity-time traces showed one bleaching step (left graph) and smaller number of traces had two bleaching steps (right graph). The steps are indicated with arrows. (F) A histogram showing fluorescence intensity distribution of fusion events before step bleaching has occurred. The two peaks are due to traces with one (light gray Gaussian fit) and two (dark gray Gaussian fit) bleaching steps. (G) A histogram showing the number of traces that had one, two or three bleaching steps in control (white) and cLTP treated neurons (black). Maximum number of steps observed was three. 163 (control) and 111 (cLTP) exocytic fusion events were analyzed.

Article Snippet: Rabbit anti-GluN2A N-terminus polyclonal antibody was obtained from Invitrogen (#480031) and mouse anti-GluN2B N-terminal monoclonal antibodies from NeuroMab (#75-101).

Techniques: Cell Culture, Amplification, Transfection, Two Tailed Test, Fluorescence

Journal: Cell Reports

Article Title: The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences

doi: 10.1016/j.celrep.2018.09.089

Figure Lengend Snippet:

Article Snippet: anti-GluN2A (N terminus) , Thermo Fisher Scientific , Cat# 480031; RRID: AB_2532251.

Techniques: Recombinant