guinea pig anti-glua2  (Synaptic Systems)

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: ( A - C ) Surface biotinylation was used to isolate endogenous surface (s) GluA1 and sGluA2 in acute striatal slices derived from wildtype (WT) and Lrrk2 G2019S (GS) mice. A ) Representative Western blot images of sGluA1, sGluA2, and actin from dataset quantified in B and C . Scatterplot/bar graphs plot values normalized to WT ± SEM (n = 7 - 8 mice/genotype, 3 slices/mouse). Unpaired t test *p= 0.0492, compared to WT. D ) Confocal image of DARPP32-labeled (white) co-cultured SPN (left) and super-resolution, STED images (right) of dendritic processes. Punctate sGluA1 (magenta), sGluA2 (green) labeling (tagged prior to permeabilization) associates largely, but not completed with PSD95 labeling (blue). E and F ) Violin plots compare surface AMPAR area ( E ) and intensity ( F ) within masks defined by PSD95 labeling in SPNs. Unpaired t test, ****p<0.0001; ***p<0.0001; Mann Whitney test *p=0.04. n= 3 preps and 15 ROIs/genotype.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Derivative Assay, Western Blot, Labeling, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A ) Schematic outlines antibody feeding assay used to monitor GluA1 internalization in wildtype (WT) and Lrrk2 G2019S (GS) corticostriatal co-cultures (DIV16-18) and serves as a key for the colors used to show data. B and C ) Overlay images show labeled surface (s) GluA1 (green mask + red mask) and internalized (i) GluA1 (red mask only) signal contained within DARPP-32 labeled SPNs (shown at a reduced intensity to permit visualization of puncta. Masks were generated in Image J and magnification is shown in B. D - G ) Intensity distribution of green and red labeling along a 60 µm line scan. H ) Quantification of the internalization index of GluA1 receptors in WT and GS SPNs at 0 and 60 min (n = 12 - 16 cells, 3 preps/genotype). Two-way ANOVA (F ( , ) = 9.809, p = 0.0029), post hoc Šidák test **p = 0.0049.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Feeding Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A, C ) Bar graph/scatterplots comparing functional contribution of CP-AMPARs using ratios of EPSCs evoked in the presence/absence of NASPM in D 1 R ( A; Drd1tdTom+ ) and D 2 R ( C; Drd1tdTom- ) SPNs in WT and GS mice (P70 - P90) in acute slices through dorsal striatum. Bars are mean ± SEM (n = 11 cells, 5 - 6 mice/group, unpaired t test *p = 0.0122). Example traces ( B ) show AMPAR currents before (black) and after (lavender) bath application of NASPM (200µM, 10 min). D - G , Superresolution (tau-STED) images ( D, E ) and quantification ( F, G ) of synaptic and extrasynaptic receptors in 21 DIV WT and GS D 1 R SPNs expressing tdTomato (white, Drd1 Cre/+; Ai14 , in D, E, and used to segment D1R SPNs for F, G) and co-cultured with unlabeled cortical neurons of the same genotype. sGluA1 ( D , magenta, STED) and sGluA2 ( E , green, STED) puncta in relation to PSD95 labeled postsynaptic sites (blue, confocal). Circled zones are enlarged in the center panels. F, G ) Bar graph/scatterplots show synaptic ( F , within a mask defined by PSD95) and extrasynaptic ( G , outside a PSD95 mask) sGluA1 (lavender) and sGluA2 (green) nanoclusters in D 1 R SPNs. F : Unpaired t test **p = 0.0369; n = 16 ROIs/genotype). G : Unpaired t test ****p<0.0001; n = 16 ROIs/genotype. H - K ) Examples ( H ) and quantification ( I - K ) of FRAP experiments. (H) Time lapse confocal images pre- and postphotobleaching (dotted circles approximate ROIs) in WT and GS D 1 R SPNs labeled and co-cultured as above. Table ( I ) compares time constant, diffusion (D) and % mobile receptors. D was calculated using: D = 0.25 (r 2 /τ 1/2 ), where r refers to the bleach radius and τ 1/2 to the time constant 46 . Graph ( J ) plots normalized SEP-GluA1 fluorescence recov-ery in WT and GS neurons imaged every 2.5 s. Lighter shading is ± SEM. Two-way RM-ANOVA (F(89, 3115) = 17.79, **p < 0.001, n = 19-20 spines/genotype. Scatterplot ( K ) compares recovery of SEP-GluA1 intensity at T222.5 s time point relative to post-bleach in WT and GS D 1 R SPNs. One-way ANOVA (F(3, 70) = 24.15, p < 0.0001), post hoc Tukey’s multiple comparison test ****p<0.0001.

Article Snippet: After blocking, coverslips were incubated with primary antibodies targeting surface epitopes (GluA1;1:500, Alomone AGP-009, RRID:AB_2340961 or GluA2; 1:500, Alomone AGC-005-GP, RRID:AB_2756617) at 4°C overnight.

Techniques: Functional Assay, Expressing, Cell Culture, Labeling, Diffusion-based Assay, Fluorescence, Comparison

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Quantitative RT-PCR, Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Cell Culture, Immunolabeling, Labeling, Infection, shRNA

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Immunolabeling, Activity Assay, Labeling, Expressing, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Western Blot, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet:

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Infection, In Vivo

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Nucleic Acid Electrophoresis, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Immunolabeling, Microscopy, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling, Microscopy