Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p < 0.001 versus the control peptide group (n = 5 samples per group, Mann-Whitney U test). Experiments were repeated 3 times (F), 4 times (A, D, and E), or 5 times (B and C).

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Control, Plasmid Preparation, Ligation, Construct, MANN-WHITNEY

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p < 0.001 versus the control vector group. (B) Original blots and quantification show that α2δ-1 reduces cell membrane expression of heteromeric GluA1/GluA2 receptors in HEK293 cells. Surface membrane proteins were isolated by biotinylation and were precipitated with an anti-GFP antibody (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the P3 group. (C) Original blots and quantification show that nerve injury increases α2δ-1 but reduces GluA2 in spinal cord synaptosomes (n = 5 rats per group). *p < 0.05, ***p < 0.001 versus the sham group. (D and E) Original blots and quantification show that nerve injury reduces heteromeric GluA1/GluA2 receptors in spinal cord synaptosomes (n = 5 rats per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group. Mann-Whitney U test was conducted in (A)–(E).

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Control, Plasmid Preparation, Membrane, Expressing, Isolation, MANN-WHITNEY

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p < 0.05, **p < 0.01 versus the control vector group. (C) Original blots and quantification show that nerve injury increases GluA2 retention in the ER of the spinal cord. ER-enriched fractions were isolated from dorsal spinal cords of sham control and SNL rats 3 weeks after surgery. Blotting was conducted using antibodies against GluA1, GluA2, and calreticulin (an ER protein marker). n = 5 rats per group. *p < 0.05 versus the sham group. (D and E) Original blots and quantification show that nerve injury diminishes heteromeric GluA1/GluA2 receptors in the ER extracts of spinal cords. n = 5 rats per group. **p < 0.01, ***p < 0.001 versus the sham group. (F) Original images and quantification show the cellular distribution of heteromeric GluA1-GluA2 PLA signals (red) in HEK293 cells cotransfected with GluA1/GluA2 and α2δ-1-IRES-GFP or GFP (green). Scale bar, 10 μm. n = 4 per group. ***p < 0.001 versus the GFP group. (G) Original images and quantification show the distribution of GluA1-GluA2 PLA signals (green) in the superficial dorsal horn of rats subjected to SNL or sham surgery. Thick white lines outline the lamina II region. Scale bar, 50 μm (left) and 10 μm (right). n = 4 per group. ***p < 0.001 versus the sham group. Mann-Whitney U test was conducted in (B)–(F).

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Control, Plasmid Preparation, Isolation, Marker, MANN-WHITNEY

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p < 0.001 versus GluA1/GluA2 only (n = 56 cells). One-way ANOVA followed by Tukey test. (D) Original PLA images and quantification show the effect of 100 μM gabapentin, 1 μM α2δ-1CT-Tat peptide, or 1 μM control peptide on heteromeric GluA1/GluA2 protein complexes (red) in HEK293 cells cotransfected with GluA1/GluA2 and α2δ-1-IRES-GFP (green). Scale bar, 10 μm. ***p < 0.001 versus control peptide (n = 4 per group). Kruskal-Wallis test. (E) Original images and quantification show the lack of an effect of gabapentin on heteromeric GluA1-GluA2 PLA signals diminished by α2δ-1R217A coexpression in HEK293 cells (n = 4 per group). Scale bar, 10 μm. (F and G) I-V plots of glutamate-elicited currents (F) and quantification of the rectification index (G) show the effect of gabapentin (GBP), α2δ-1CT-Tat peptide, or control peptide in HEK293 cells cotransfected with GluA1/GluA2(R) and α2δ-1 or α2δ-1R217A (n = 11 cells in GluA1/A2(R)+α2δ-1, n = 12 cells in α2δ-1+GBP, n = 11 cells in α2δ-1R217A+GBP, n = 12 cells in α2δ-1CT-Tat peptide, n = 12 cells in control peptide). *p < 0.05 versus the untreated group (GluA1/A2(R)+α2δ-1). One-way ANOVA followed by Dunnett test.

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Transfection, Control

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p < 0.01 versus the sham group. Mann-Whitney U test. (C) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of heteromeric GluA1/GluA2 protein levels in spinal cord synaptosomes of sham and SNL rats (n = 6 rats per group). **p < 0.01 versus the sham group. Kruskal-Wallis test. (D and E) Original traces (D), I-V plots, and mean rectification index (E) of AMPAR-EPSCs of lamina II neurons in rats 3 weeks after SNL or sham surgery (n = 12 neurons). Spinal cord slices of SNL rats were treated with 100 μM gabapentin (n = 12 neurons), vehicle (n = 10 neurons), 1 μM α2δ-1CT-Tat peptide (n = 13 neurons), or 1 μM control peptide (n = 15 neurons). *p < 0.05 versus the sham group. One-way ANOVA followed by Tukey test. (F and G) Original traces (F), I-V plots, and mean rectification index (G) of AMPAR-EPSCs of lamina II neurons in naive rats (n = 12 neurons) and diabetic rats 4 weeks after diabetic induction. Spinal cord slices were treated with 100 μM gabapentin (n = 17 neurons), vehicle (n = 14 neurons), or 1 μM α2δ-1CT-Tat peptide (n = 16 neurons). *p < 0.05 versus naive control. One-way ANOVA followed by Tukey test.

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Control, MANN-WHITNEY

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The GFP-tagged GluA1 (C terminus), GFP-tagged GluA2 (C terminus), α2δ-2 (encoded by Cacna2d2 ), α2δ-3 (encoded by Cacna2d3 ), and HA-tagged α2δ-1, HA-tagged α2δ-2, and HA-tagged α2δ-3 constructs were obtained from Addgene (Watertown, MA).

Techniques: Recombinant, Modification, Control, DC Protein Assay, Western Blot, Lysis, Isolation, Plasmid Preparation, In Situ, Mutagenesis, Cloning, Software

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B, representative IgpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C, effects of anti-ClC-3 Ab on IgpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D, mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Fluorescence, Transfection, Transferring

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, time course of VSOAC currents from two PASMCs dialysed with either anti-ClC-3 Ab (5 μg ml−1) preabsorbed with antigen (50 μg ml−1; ○), or anti-ClC-3 Ab alone (5 μg ml−1; •). Inset: Western blot analysis of native ClC-3 expression in isolated canine PASMCs. B, mean current densities in cells dialysed with either standard intracellular solutions (n = 11), preabsorbed anti-ClC-3 Ab (n = 4) or anti-ClC-3 Ab alone (n = 5).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Western Blot, Expressing, Isolation

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions. Aa, pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). Ab, mean current densities for preabsorbed anti-ClC-3 Ab (□, n = 3) or anti-ClC-3 Ab alone (▪, n = 5) at times indicated. B, same as A, except cells were preswelled by pre-exposure to hypotonic bath solutions prior to membrane rupture and dialysis; preabsorbed anti-ClC-3 Ab (□, n = 4), and anti-ClC-3 Ab alone (▪, n = 5).

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Transferring

Journal:

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: A, Western blot analysis of native ClC-3 expression in oocytes. B, 4-phorbol 12,13-dibutyrate (PDBu) inhibition of native VSOAC currents in oocytes (-100 to +120 mV). C, time course of VSOAC activation in response to hypotonic bath solutions from a non-injected control oocyte (○) and an oocyte injected with anti-ClC-3 Ab (15 μg ml-1; •) at time shown. Cells were held at -30 mV for 30 ms, hyperpolarized to -100 mV for 210 ms and then depolarized to +100 mV for 210 ms, repetitively at 2 Hz. D, mean current densities from control oocytes (n = 10), oocytes injected with anti-ClC-3 Ab alone (15 μg ml-1) (n = 4) or injected with preabsorbed anti-ClC-3 Ab (150 μg ml-1) (n = 4). Peak current densities were measured after 5 min exposure to isotonic solutions, and after 85 min exposure to hypotonic solutions.

Article Snippet: Furthermore, in cardiac tissues endogenous ClC-3 protein expression has been confirmed using the same anti-ClC-3 Ab (Alomone Labs) in immunoblot and immunofluorescence experiments ( Britton et al. 2000 ). and illustrate the effects of intracellular dialysis of anti-ClC-3 Ab alone or antigen-preabsorbed anti-ClC-3 Ab on native VSOACs in guinea-pig cardiac and canine pulmonary arterial smooth muscle myocytes, respectively. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions.

Techniques: Western Blot, Expressing, Inhibition, Activation Assay, Injection

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