Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of hippocampal slices following electrophysiology stained for NeuN (grey, AF-647), GluA1 (blue, AF-488), GluA2 (red, AF-594), and DAPI (green) and corresponding merged images for naïve, cLTP, sLTP, cDEP, and sDEP slices (maximum intensity projection). The dotted white line shown in the merged image represents the region of interest (stratum radiatum, SR) for each slice. Scalebar = 200 µm. ( B ) After normalization to naïve slices, the GluA1/GluA2 ratio in the SR was higher following cLTP compared to sLTP (unpaired Student’s t test, mean difference = 0.277 ± 0.084 times the naïve GluA1/GluA2 ratio, p = 0.022). Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. ( C ) No differences in the GluA1/GluA2 ratio were observed following cDEP compared to sDEP (unpaired Student’s t test, mean difference = −0.002 ± 0.186 times the naïve GluA1/GluA2 ratio, p = 0.993). Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. ( D ) Fluorescence intensity profiles normalized to the maximum fluorescence intensity as a function of distance along the SR (normalized to 1) for GluA1 (left) and GluA2 (right) in naïve slices. Data are means ± SEM from 10 biological replicates. ( E ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cLTP or sLTP. Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. Differences in GluA1 fluorescence intensity along the SR relative to the maxima were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.11, p = 0.239; main effect of LTP type F(1,5) = 9.43, p = 0.028). No statistically significant differences in GluA2 distribution were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.23, p = 0.081, LTP type F(1, 5) = 3.11, p = 0.138). ( F ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cDEP or sDEP. Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. No statistically significant differences were observed between cDEP and sDEP for GluA1 (distance x DEP F(99, 591) = 0.459, p > 0.999, DEP type F(1, 6) = 2.07, p = 0.200) or GluA2 (distance x DEP F(99, 591) = 0.680, p = 0.991, DEP type F(1, 6) = 1.69, p = 0.242) using mixed effects analysis. Data from male (closed circles) and female (open circles) mice combined, 1-2 technical replicates per biological replicate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Confocal Microscopy, Staining, Fluorescence

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) GluA1 normalized to naïve hippocampal slices following cDEP and sDEP in control conditions (black), in the presence of 100 µM 7-CK (purple) and in the presence of 50 µM APV (blue). DEP x treatment F(2, 15) = 0.240, p = 0.790. ( B ) GluA2 normalized to naïve slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.220, p = 0.805. ( C ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.583, p = 0.571, main effect of DEP type F(1, 15) = 12.27, p = 0.003. Post-hoc cDEP+7-CK vs. sDEP+7-CK least squares (LS) mean difference = 1.3 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.034; cDEP vs. sDEP LS mean difference = 1.4 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.021. ( D ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 1.62, p = 0.231, main effect of DEP type (F(1, 15) = 5.88, p = 0.028) and drug treatment (F(2, 15) = 6.57, p = 0.0089). Post-hoc cDEP+7CK vs. cDEP+APV LS mean difference = 3.7 ± 1.4 times naïve pGluA1 S845/GluA1 expression, p = 0.054; sDEP vs. sDEP+7-CK LS mean difference = −4.4 ± 1.6 times naïve pGluA1 S845/GluA1 expression, p = 0.043; cDEP vs. sDEP LS mean difference = 4.3 ± 1.5 times naïve pGluA1 S845/GluA1 expression, p = 0.012. Data in (A-D) are means ± SEM from 4 (cDEP) and 3 (sDEP) biological replicates. ( E-H ) AMPAR expression and phosphorylation states are unaltered following cDEP in the presence of C1.1 compared to C1.1Scr. ( E ) GluA1 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr (black) versus C1.1 (grey) (p = 0.555). ( F ) GluA2 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr versus C1.1 (p = 0.876). ( G ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.238) ( H ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.067). Data in (E-G) are means ± SEM from 4 biological replicates per group. 1-2 technical replicates per biological replicate. Blots were cut to probe for each protein (see Fig. S5A-C). Each drug treatment condition was normalized to a naïve slice run in the same blot. Statistical comparisons were made using ordinary two-way ANOVA followed by Holm-Šídák post-hoc comparisons within and across drug treatment conditions (A-D) or unpaired Student’s t test (E-H) as appropriate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Full blots stained for GluA1 (top left-hand side of blots), pGluA1 S831 (top right-hand side of blots) and GAPDH (bottom section of blots) after membranes were cut as indicated along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown. Samples were treated during electrophysiology experiments with 7-CK or APV as indicated. ( B ) Full blots stained for GluA2 (top left-hand side of blots), pGluA1 S845 (top right-hand side of blots) and GAPDH (bottom of blots) after blots were cut along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown with corresponding treatments indicated. ( C ) Full blots stained for GluA1 (top left-hand side of left blot), pGluA1 S831 (top right-hand side of left blot), GluA2 (top left-hand side of right blot) and pGluA1 S845 (top right-hand side of right blot). Cropped blots from main are indicated with the corresponding colour-coded boxes. Images of blots stained for pGluA1 S831 and S845 were mirrored in for clarity.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Staining

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: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

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: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

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: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

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