pcl sep glua2  (Addgene inc)

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His and neurexin1-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-His or neurexin1-His. ( C and E) Violin, and cumulative probability plots of gold particle distribution at the normalized AZ. ( D and G) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances (dashed line). Insert depicts the residual percentage of the distance of gold particles to the next docked SV compared to their randomized distribution. ( E and H) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) to docked SVs. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for violin plots in C, D, E, F, G, and H was assessed by the Mann-Whitney test and for the cumulative distribution plots C, D, E, F, G, and H by Kolmogorov-Smirnov test. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His, neurexin-His, and neuroligin-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-HIS, neurexin1-His, or neuroligin1-His. ( C) Summary graph of docked SV number per AZ. ( D) Summary graph of the PSD length. ( E) Cumulative probability of docked SV distribution at the AZ. ( F) Summary graph of gold particles per AZ. ( G) Cumulative probability plot of gold particles per AZ. h) Relative frequency of gold particles in the synaptic cleft (0 = active zone membrane; 1 = post-synaptic membrane). ( I) Violin, and cumulative probability plots of gold the particle distribution at the normalized AZ for neuroligin-His expressing neurons compared to randomized data. ( J) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances. ( K) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) of gold particles to docked SVs. Data for bar graphs are individual values and means ± SEM. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for C, D, and F was assessed by Kruskal-Wallis test, for I, J, and K by Mann-Whitney test, and for the cumulative distribution plots in I, J, and K by the Kolmogorov-Smirnov test. *p < 0.05.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A-H) , Experiments were conducted in RIM/RBP deficient synapses (qKO) and their respective control (Δcre). ( A and C) Representative electron micrographs of cryo-fixed control and qKO primary hippocampal neurons expressing either GluA2-His or α2δ1-His. Scale bar, 200 nm. ( B and D) Number of gold particles per AZ. (E) Synaptic cleft width. ( F) Representative dSTORM images of GluA1 and GluA2 (green) and Homer (red) expressed in control and qKO neurons. Scale bars 1 µm. ( G) Number of GluA1 clusters per synapse area. ( H) Number of GluA2 clusters per synapse area. ( I) Sample traces of mEPSC events in control (grey) and qKO (red) derived from autaptic neurons. ( J) Miniature excitatory postsynaptic current (mEPSC) amplitude. ( K) mEPSC rise time. Data are individual values and means ± SEM. Statistical significance for B, D, E, J, K, G, and H was assessed by unpaired t-test and cumulative distribution plots in B and D by Kolmogorov-Smirnov test. *p < 0.05, ***p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Model of transsynaptic alignment of α2δ1-AP and GluA2-mSA by BirA co-expression in qKO synapses. ( B) Example traces of excitatory postsynaptic currents (EPSC). ( C) Excitatory postsynaptic current (EPSC) amplitudes. ( D) Example traces of synaptic responses to a 5 s application of hypertonic sucrose. ( E) Readily releasable pool (RRP) charge. ( F) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice. ( G) Absolute number of docked SV and docked SV per 100 nm AZ. ( H) Example images of SynGCaMP6f fluorescence in qKO neurons, obtained during trains of AP stimulation. Scale bars 5 µm ( I) Fluorescence changes (ΔF/F) upon single AP and APs trains. Data are individual values and means ± SEM. Statistical significance for C, E, and G was assessed by Kruskal-Wallis test and I by Two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice expressing α2δ1-AP and GluA2-mSA or α2δ1-AP, GluA2-mSA, and BirA. Scale bars 100 nm ( B) Cumulative probability plot and bar graph of the synaptic cleft width. ( C) Cumulative probability plot and bar graph of the PSD length. ( D) Example traces of miniature postsynaptic currents (mEPSCs).( E) Cumulative probability plot, and bar graph of mEPSC frequency. ( F) Cumulative probability plot, and bar graph of mEPSC amplitude. ( G) Cumulative probability plot, and bar graph of mEPSC rise time. ( H) Example images of Munc13-1 and synaptophysin immunostainings in neuronal cultures obtained from qKO mice. Scale bars 5 µm. ( I) Munc13-1 immunofluorescence intensities normalized to synaptophysin intensities. Data are means ± SEM. Statistical significance for B, C, E, F, G, and I was assessed by Kruskal-Wallis test. *p < 0.05, **p < 0.01.

Article Snippet: For the postsynaptic AMPA receptor GluA2, SEP-GluA2 (Addgene #24001) was used by exchanging the pHluorin cDNA with His and Gibson assembling into f(syn)NLS-GFP-P2A (BL-984).

Techniques: Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His and neurexin1-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-His or neurexin1-His. ( C and E) Violin, and cumulative probability plots of gold particle distribution at the normalized AZ. ( D and G) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances (dashed line). Insert depicts the residual percentage of the distance of gold particles to the next docked SV compared to their randomized distribution. ( E and H) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) to docked SVs. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for violin plots in C, D, E, F, G, and H was assessed by the Mann-Whitney test and for the cumulative distribution plots C, D, E, F, G, and H by Kolmogorov-Smirnov test. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: As counterpart, the sequence of monomeric streptavidin (mSA) was amplified from Addgene #39860, fused after to the GluA2 sequence (Addgene clone #24001, GluA2-pH) by replacing the pHluorin sequence.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Graphic for the ultrastructural parameters analyzed in electron micrographs for GluA2-His, neurexin-His, and neuroligin-His expressing neurons. ( B) Representative electron micrographs of cryofixed primary hippocampal neurons expressing GluA2-HIS, neurexin1-His, or neuroligin1-His. ( C) Summary graph of docked SV number per AZ. ( D) Summary graph of the PSD length. ( E) Cumulative probability of docked SV distribution at the AZ. ( F) Summary graph of gold particles per AZ. ( G) Cumulative probability plot of gold particles per AZ. h) Relative frequency of gold particles in the synaptic cleft (0 = active zone membrane; 1 = post-synaptic membrane). ( I) Violin, and cumulative probability plots of gold the particle distribution at the normalized AZ for neuroligin-His expressing neurons compared to randomized data. ( J) Violin, and cumulative probability plots of the nearest distance between gold participles and the next docked SV compared to randomized distances. ( K) Violin, and cumulative probability plots of the measured data and null model data of mean nearest neighbor distance (NND) of gold particles to docked SVs. Data for bar graphs are individual values and means ± SEM. Data for violin plots are medians (solid lines) and quartiles (dashed lines). Statistical significance for C, D, and F was assessed by Kruskal-Wallis test, for I, J, and K by Mann-Whitney test, and for the cumulative distribution plots in I, J, and K by the Kolmogorov-Smirnov test. *p < 0.05.

Article Snippet: As counterpart, the sequence of monomeric streptavidin (mSA) was amplified from Addgene #39860, fused after to the GluA2 sequence (Addgene clone #24001, GluA2-pH) by replacing the pHluorin sequence.

Techniques: Expressing, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A-H) , Experiments were conducted in RIM/RBP deficient synapses (qKO) and their respective control (Δcre). ( A and C) Representative electron micrographs of cryo-fixed control and qKO primary hippocampal neurons expressing either GluA2-His or α2δ1-His. Scale bar, 200 nm. ( B and D) Number of gold particles per AZ. (E) Synaptic cleft width. ( F) Representative dSTORM images of GluA1 and GluA2 (green) and Homer (red) expressed in control and qKO neurons. Scale bars 1 µm. ( G) Number of GluA1 clusters per synapse area. ( H) Number of GluA2 clusters per synapse area. ( I) Sample traces of mEPSC events in control (grey) and qKO (red) derived from autaptic neurons. ( J) Miniature excitatory postsynaptic current (mEPSC) amplitude. ( K) mEPSC rise time. Data are individual values and means ± SEM. Statistical significance for B, D, E, J, K, G, and H was assessed by unpaired t-test and cumulative distribution plots in B and D by Kolmogorov-Smirnov test. *p < 0.05, ***p < 0.001.

Article Snippet: As counterpart, the sequence of monomeric streptavidin (mSA) was amplified from Addgene #39860, fused after to the GluA2 sequence (Addgene clone #24001, GluA2-pH) by replacing the pHluorin sequence.

Techniques: Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Model of transsynaptic alignment of α2δ1-AP and GluA2-mSA by BirA co-expression in qKO synapses. ( B) Example traces of excitatory postsynaptic currents (EPSC). ( C) Excitatory postsynaptic current (EPSC) amplitudes. ( D) Example traces of synaptic responses to a 5 s application of hypertonic sucrose. ( E) Readily releasable pool (RRP) charge. ( F) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice. ( G) Absolute number of docked SV and docked SV per 100 nm AZ. ( H) Example images of SynGCaMP6f fluorescence in qKO neurons, obtained during trains of AP stimulation. Scale bars 5 µm ( I) Fluorescence changes (ΔF/F) upon single AP and APs trains. Data are individual values and means ± SEM. Statistical significance for C, E, and G was assessed by Kruskal-Wallis test and I by Two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: As counterpart, the sequence of monomeric streptavidin (mSA) was amplified from Addgene #39860, fused after to the GluA2 sequence (Addgene clone #24001, GluA2-pH) by replacing the pHluorin sequence.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Functional architecture of the synaptic transducers at a central glutamatergic synapse

doi: 10.1101/2020.12.25.424391

Figure Lengend Snippet: (A) Representative electron micrographs of cryofixed primary hippocampal neurons from qKO mice expressing α2δ1-AP and GluA2-mSA or α2δ1-AP, GluA2-mSA, and BirA. Scale bars 100 nm ( B) Cumulative probability plot and bar graph of the synaptic cleft width. ( C) Cumulative probability plot and bar graph of the PSD length. ( D) Example traces of miniature postsynaptic currents (mEPSCs).( E) Cumulative probability plot, and bar graph of mEPSC frequency. ( F) Cumulative probability plot, and bar graph of mEPSC amplitude. ( G) Cumulative probability plot, and bar graph of mEPSC rise time. ( H) Example images of Munc13-1 and synaptophysin immunostainings in neuronal cultures obtained from qKO mice. Scale bars 5 µm. ( I) Munc13-1 immunofluorescence intensities normalized to synaptophysin intensities. Data are means ± SEM. Statistical significance for B, C, E, F, G, and I was assessed by Kruskal-Wallis test. *p < 0.05, **p < 0.01.

Article Snippet: As counterpart, the sequence of monomeric streptavidin (mSA) was amplified from Addgene #39860, fused after to the GluA2 sequence (Addgene clone #24001, GluA2-pH) by replacing the pHluorin sequence.

Techniques: Expressing, Immunofluorescence