Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Antibodies used in the present study

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Recombinant, Immunohistochemistry-IF, Transduction

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The interaction of BRAG2a with endophilin 3 and PSD-95 is important for the mGluR-dependent decrease in AMPAR surface level. A, B, The effect of BRAG2 knockdown on basal surface GluA1 level. A, Representative immunofluorescence images of dendritic shafts and spines of cultured hippocampal neurons transfected with shBRAG2 (BRAG2-KD) or shControl (control) vectors at DIV14 and subjected to surface AMPAR labeling with anti-GluA1 (extracellular) antibody at DIV16. B, Quantification of surface GluA1 levels. Immunofluorescence intensity of surface GluA1 in BRAG2-KD-transfected neurons was normalized to that in the control-transfected neurons. Note that knockdown of BRAG2 did not affect the basal surface GluA1 levels. C, D, The effect of BRAG2 knockdown on surface GluA1 levels following DHPG treatment. D, Quantification of surface GluA1 levels. Note that BRAG2 knockdown blocked the decrease in surface GluA1 levels following 50 μm DHPG treatment (DHPG 50). Asterisks indicate statistical significance (one-way ANOVA; F(2,61) = 4.507, p = 0.0149; followed by Tukey–Kramer post hoc test, *p < 0.05). E, F, The effect of BRAG2 knockdown on surface GluN2B levels following DHPG treatment. E, Representative immunofluorescence images of dendritic shafts and spines of BRAG2-KD or control hippocampal neurons treated with 50 μm DHPG and subjected to surface NMDAR labeling with anti-GluN2B (extracellular) antibody. F, Quantification of surface GluN2B levels; the immunofluorescence intensities of surface GluN2B in BRAG2-KD and control neurons treated with DHPG were normalized to that in control and DHPG-untreated (DHPG 0) neurons. Note that DHPG treatment did not change surface GluN2B levels in BRAG2-KD or control neurons (one-way ANOVA: F(2,59) = 0.964, p = 0.387). Data for each surface GluN2B were obtained from 20–22 transfected neurons from three plates, and these results were confirmed by three independent experiments. G, H, The effect of coexpression of shBRAG2 and BRAG2a or its mutants on the DHPG-induced decrease in surface GluA1 levels. G, Representative immunofluorescence images of dendritic shafts and spines of neurons transfected with BRAG2-KD alone or with BRAG2-KD and sh-res BRAG2a-WT, BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. H, Quantification of surface GluA1 levels. Note that the BRAG2-knock-down phenotype on DHPG-induced surface GluA1 levels could be rescued by coexpression of shRNA-resistant BRAG2a-WT, but not BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. Asterisks indicate statistical significance (one-way ANOVA: F(5,138) = 12.822, p < 0.0001; followed by Tukey–Kramer post hoc test, *p < 0.05). Each surface GluA1 data were obtained from three independent experiments, and 20–28 transfected neurons from three plates were analyzed in each experiment. Scale bars, 2 μm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Immunofluorescence, Cell Culture, Transfection, Labeling, shRNA

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Lateral distribution of BRAG2a, PSD-95, GluA1/2, Arf6, endophilin 3, AP-2, and clathrin along the postsynaptic membrane. A, B, D, E, G, H, J, K, M, N, P, Q, S, and T, Representative postembedding immunoelectron microscopic images of the distribution of BRAG2a (A, B), PSD-95 (D, E), GluA1/2 (G, H), Arf6 (J, K), endophilin 3 (M, N), AP-2/α-adaptin (P, Q), and clathrin (S, T) in excitatory asymmetric synapses of the mouse hippocampal CA1 region. C, F, I, L, O, R, and U, Histograms showing lateral distribution of immunogold particles for BRAG2a (C), PSD-95 (F), GluA1/2 (I), Arf6 (L), endophilin 3 (O), AP-2/α-adaptin (R), and clathrin (U) along the postsynaptic membrane. The edge of the postsynaptic density (arrows) is defined as 0. The bin size of the histogram is 25 nm, and the synaptic and extrasynaptic sites are shown in the left and right sides, respectively. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques:

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The effect of DHPG treatment on BRAG2a, PSD-95, and GluA1/2 levels in the PSD and extrasynaptic region using acute hippocampal slice cultures. A, B, The effect of the DHPG treatment on the expression of GluA1/2 and Arc/Arg3.1, and the phosphorylation of p38 MAPK in acute hippocampal slice cultures. A, Representative immunoblots of acute hippocampal slices treated with or without DHPG with anti-GluA1/2, anti-Arc/Arg3.1, anti-phospho-p38 MAPK, anti-p38 MAPK, and anti-α-tubulin antibodies. B, Quantification of immunoreactive intensities of GluA1/2, Arc/Arg3.1, and phospho/total p38 MAPKs. Each immunoreactive intensity was normalized by the respective intensity for α-tubulin and expressed as the ratio with the control value. Note that the DHPG treatment induced the significant upregulation of Arc/Arg3.1 and the phosphorylation of p38 MAPK in hippocampal slices. Data for each group were obtained from three culture plates (n = 3). These results were confirmed by three independent experiments. C–T, Representative postembedding immunoelectron microscopic images (C–F, I–L, O–R) and histograms (G, H, M, N, S, T) of the lateral distribution of immunogold particles for BRAG2a (C–H), PSD-95 (I–N), and GluA1/2 (O–T) in axospinous asymmetric synapses in the CA1 stratum radiatum of acute mouse hippocampal slices following 50 μm DHPG treatment. The edge of the postsynaptic density (arrowheads) is defined as 0. The bin size of the histogram is 25 nm. U, V, Quantification of immunogold particles for BRAG2a, PSD-95, and GluA1/2 in the PSD (U) and extrasynaptic (V) region along the postsynaptic membrane. Values in U and V are expressed as the average immunogold particle numbers in the PSD of axospinous synapses and the percentage of immunogold particles in the extrasynaptic region in total immunoreactive particles along the postsynaptic membrane, respectively. Note that the DHPG treatment decreased the immunogold particles for GluA1/2 but not BRAG2a or PSD-95 without any changes in the proportion of BRAG2a, PSD-95, or GluA1/2 in the extrasynaptic region. *p < 0.05 (t test). Data for each group were obtained from three slices (n = 3). These results were confirmed by three independent experiments from different mice. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Expressing, Western Blot

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 1. Expression of 5-HT1A receptor and AMPA receptor subunit GluR1 in cortical neurons cultured for 3 days (A and B) and 14 days (C and D). (A) Neurons at 3 DIV were stained

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing, Cell Culture, Staining

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 2. Effects of 5-HT1A receptor agonist 8-OH-DPAT on the mRNA expression of BDNF and AMPA receptor subunits, GluR1 and GluR2, in cortical neurons in vitro. Neurons were

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing, In Vitro

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 3. Effects of 5-HT1A receptor agonist 8-OH-DPAT on the mRNA expression of BDNF and AMPA receptor subunits, GluR1 and GluR2, in the frontal cortex (A), and Tph2 and 5-HTT

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A, B ) Hippocampal neurons were subjected to ( A ) chemical LTP (cLTP) or ( B ) chemical LTD (cLTD). DKK3 levels were analyzed in the supernatant (extracell) and cell lysates by western blot. Phospho-GluA1 Ser845 was used as a readout of LTP and LTD induction, and tubulin as a loading control for the cell lysate. Graphs show the levels of DKK3 relative to control and the ratio of extracellular/lysate DKK3 levels (Mann-Whitney Test, n=3 independent cultures for cLTP and Student’s T-test, n=6 independent cultures for cLTD). ( C ) Representative immunoblots show DKK3 levels in the cell lysate and extracellular (extracell) fraction of acute WT hippocampal slices treated with vehicle (Ctrl) or NMDA (cLTD) and/or Brefeldin A (BFA) for 60 min. Actin was used as a loading control in homogenates. Graphs show densiometric quantifications of DKK3 relative to the control condition and the ratio of extracellular/lysate DKK3 levels (Kruskal-Wallis followed by Dunn’s multiple comparisons; n=5 animals). ( D ) Immunoblot showing DKK3 is less abundant in the extracellular fraction when compared to the cell lysate fraction. Representatives immunoblot of J20 brain slices treated with control or APV for 3 hr. Time exposure for obtaining DKK3 chemiluminescent images is indicated. Figure 2—figure supplement 3—source data 1. Uncropped western blot gels. Raw and annotated WB images. The representative western blot images for are indicated within a blue square.

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of SYNPLA targeting presynaptic myc-NRXN and. cLTP induces mobilization of GluA1 from an extrasynaptic pool and insertion into the post-synaptic density, decreasing the distance between the targeted proteins and permitting PLA. b ) Representative images of SYNPLA reactions performed on myc-NRXN expressing cultured rat hippocampal neurons at 14 days in vitro for the indicated conditions; PLA shown in gray. Scale bar: 10 μm. c ) Quantification of a single SYNPLA experiment (as in f ). The number of SYNPLA puncta detected in each field of view (round) and the average (square) ± SEM across all fields of view are shown; *** p<0.001, 1-way ANOVA followed by Tukey-Kramer post-hoc test. d ) Quantification of six independent experiments (indicated by different colors); the average number of SYNPLA puncta across 10-12 fields of view for each experiment (circles, normalized to CTRL) and the average PLA signal across experiments (square) ± SEM are shown; ** p<0.01, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) Diagram of SYNPLA in rat organotypic hippocampal slices. Sindbis virus expressing myc-NRXN is injected in the presynaptic CA3 region SYNPLA between myc-NRXN and endogenous GluA1 is measured in the postsynaptic CA1 region. f ) Representative images of SYNPLA under the indicated conditions in region CA1 of organotypic slice cultures. Scale bar: 10 μm. g ) Quantification of SYNPLA puncta in organotypic slices, from three independent experiments (indicated with different colors, 4 slices per condition (one circle per slice)); squares indicate average ± SEM across experiments; *** p<0.001, paired t-test.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Expressing, Cell Culture, In Vitro, Injection

Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of a general PLA reaction. Note that A and B must be sufficiently close to permit complementary binding and ligation of ABo1 and ABo2 and subsequent amplification to make a rolony. b ) Diagram of PLA targeting recombinant presynaptic myc-NRXN and postsynaptic HA-NLGN. c ) PLA rolonies (white dots, right) formed between postsynaptic cultured mouse hippocampal neuron expressing HA-NLGN + mCherry (red; middle) and co-cultured presynaptic neurons expressing myc-NRXN + GFP (green; left). Scale bar: 10 μm. d ) PLA puncta in each sample (circles) and average (squares) ± SEM for indicated expression and days in vitro (color indicates data acquired on the same day). *** p<0.001, 2-way ANOVA (see Methods); * p<0.05, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) PLA reaction between endogenous GluA1 and recombinant myc in cultured rat hippocampal neurons (exposed to cLTP, see ) expressing myc-NRXN and cytosolic fluorescent protein (green), subsequently immunostained for endogenous PSD-95 (red). Scale bars: 10 μm (left); 5 μm (right). f ) PLA puncta localize to synapses. In PSD-95 and myc-NRXN channels (from e , right), pixels were set to zero (masked) for values below a progressively increasing threshold (x axis). Puncta (PLA or randomly placed) display colocalization if non-zero pixels exist within 0.14 μm in both thresholded channels. For indicated threshold, ∼95% of PLA, while only ∼50% of randomly placed (mean ± SEM of 30 placements) puncta colocalized with pre- and post-synaptic markers. See Supplementary Fig. 3 and Methods for details.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Binding Assay, Ligation, Amplification, Recombinant, Cell Culture, Expressing, In Vitro

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