quantum dot-based single-gaba a r tracking  (Quantum Dot Inc)

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: GABAergic neurotransmission affected by gephyrin mutant ( A ) Example traces from L2/3 pyramidal cells expressing gephyrin variants or GFP (control). Colored traces: averaged currents from one neuron. Grey traces: all events measured from one neuron. ( B ) Miniature inhibitory postsynaptic currents (mIPSCs) amplitude. ( C ) inter-event interval (IEI) in L2/3 pyramidal neurons expressing individual gephyrin variants. Data are reported as mean ± SEM. Statistics: eGFP, n = 23; K148R, n = 13; gephyrin-DN, n = 13; gephyrin-SSA, n = 10 neurons, from 4–5 mice in each group. One-way ANOVA with Tukey post-hoc test. Bars: mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Mutagenesis, Expressing, Control

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: Co-expression of RCaMP and eGFP-gephyrin variants. ( A ) An illustration of different gephyrin mutations used in the study, along with the labels of signaling pathways that are affected by the mutated residue. The gephyrin-K148R (SUMO1 conjugation site mutant) facilitates scaffolding. The gephyrin-S303A and S305A (PKA and CaMKIIα phospho-null mutant) hamper NMDA receptor activity-induced scaling at GABAergic postsynaptic sites. The gephyrin-DN (lacks part of E domain) disrupts endogenous gephyrin scaffolds in neurons. ( B ) Representative images of neuron co-expressing RCaMP and eGFP-gephyrin variants after injection of AAVs in L2/3 barrel cortex in vivo. The following combination of viruses were injected into the barrel cortex (same combination used in 2P Ca 2+ imaging): AAV6-hSyn1-flex-gephyrin variants, AAV6-CaMKIIα-CreER T2 , AAV6-CaMKIIα-RCaMP1.07. All brain sections were stained for CaMKIIα and eGFP. Scale bar: 20 µm. ( C ) The averaged percentages of L2/3 pyramidal neurons from the field of views that were RCaMP-positive and RCaMP-positive + gephyrin variant-positive. Quantification of average expression of RCaMP-expressing neurons, gephyrin variant-expressing neurons, and neurons co-expressing RCaMP and eGFP-gephyrin mutants after normalization to total CaMKIIα-positive neurons.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Expressing, Protein-Protein interactions, Residue, Conjugation Assay, Mutagenesis, Scaffolding, Activity Assay, Injection, In Vivo, Imaging, Staining, Variant Assay

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: The expression of gephyrin mutants differentially modulates L2/3 pyramidal neuron spontaneous activity. ( A ) An overview of the viral infection in vivo. The control site received only AAV6-CaMKIIα-RCaMP1.07, while the experimental site received a combination of RCaMP/CaMKIIα- Cre/gephyrin flex viruses. ( B ) Absolute values for calcium event amplitude, duration and number of events over 40 trials (response probability) comparing values before and after tamoxifen (overexpression) for control (grey), gephyrin WT (dark blue), K148R (green), SSA (turquoise) and DN (pink) mutants. ( C ) Average percentage changes during spontaneous activity for calcium event amplitude, duration, and number of events over 40 trials (response probability) after gephyrin-mutant expression for individual neurons. The relative changes in Ca 2+ transient amplitude, duration, and number of events of individual neurons by normalizing these parameters after gephyrin mutant expression to the activity before tamoxifen (TAM) injection [(After-Before)/Before]. Control, n = 491 neurons; gephyrin WT, n = 643 neurons; gephyrin-K148R, n = 308 neurons; gephyrin-SSA, n = 249 neurons; gephyrin-DN, n = 204 neurons; 3–4 mice per group. Statistics: linear mixed-effects models and Tukey post hoc tests. All bar graphs are represented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Expressing, Activity Assay, Infection, In Vivo, Control, Over Expression, Mutagenesis, Injection

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: The expression of gephyrin mutants differentially modulates L2/3 pyramidal neuron excitability evoked by whisker stimulation. ( A ) Illustration of a trial with single whisker stimulation. ( B ) Average trace examples from imaging sessions for whisker stimulation trials (40 trials in one session) of both before and after gephyrin mutant expression. Grey bar: whisker stimulation. Lighter grey: standard deviation. ( C ) Calcium event amplitude, duration, and number of events (response probability) over 40 trials with whisker stimulation comparing values before and after tamoxifen for control (grey), gephyrin WT (dark blue), K148R (green), SSA (turquoise) and DN (pink) mutants. ( D ) Average percentage changes amplitude, duration and number of events after gephyrin-mutant expression for individual neurons during whisker stimulation. The relative changes in Ca 2+ transient amplitude, duration, and number of events of individual neurons by normalizing these parameters after gephyrin mutant expression to the activity before tamoxifen (TAM) injection [(After-Before)/Before]. ( E ) Onset and ( F ) decay time for before and after expression of the gephyrin mutants. Control, n = 491 neurons; gephyrin WT, n = 643 neurons; gephyrin-K148R, n = 308 neurons; gephyrin-SSA, n = 249 neurons; gephyrin-DN, n = 204 neurons; 3–4 mice per group. Statistics: linear mixed-effects models and Tukey post hoc tests. All bar graphs are represented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Expressing, Whisker Assay, Imaging, Mutagenesis, Standard Deviation, Control, Activity Assay, Injection

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: Spontaneous and whisker stimulation-induced activity changes in L2/3 pyramidal neurons expressing gephyrin variants. ( A ) Proportions of responding neurons before and after gephyrin-mutant expression in vivo are categorized based on Ca 2+ transient amplitude. ( B ) Proportions of responding neurons before and after gephyrin-mutant expression are categorized based on the number of events (response probability). Control, n = 491 neurons; gephyrin WT, n = 643 neurons; gephyrin-K148R, n = 308 neurons; gephyrin-SSA, n = 249 neurons; gephyrin-DN, n = 204 neurons. TAM: tamoxifen. The numbers on the pie charts are rounded to one decimal place.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Whisker Assay, Activity Assay, Expressing, Mutagenesis, In Vivo, Control

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: Interaction of gephyrin variants and synaptic α1- or α2-GABA A R subtypes. The primary cortical neurons were transfected with eGFP-gephyrin WT, eGFP-gephyrin DN, eGFP-Gephyrin K148R or eGFP-gephyrin SSA mutant at 13 days in vitro (13 DIV) and stained for α1 and α2 subunits of GABA A Rs at 20 DIV. ( A ) Example images of transfected neurons with gephyrin mutants. Lower panels: magnified images of selected dendrites (white box). Scale bar: 25 μm in full images, 10 μm in zoom-in images. White arrows: eGFP-gephyrin clusters without GABRA2 staining. ( B – E ) Cluster analysis was performed to show the size, density, and colocalization of eGFP-gephyrin variants with α1-GABA A Rs. ( F – I ) Cluster analysis was performed to show the size, density and colocalization of eGFP-gephyrin variants with α2-GABA A Rs. eGFP-gephyrin DN was excluded from cluster analysis as the GFP signal and the staining of α1-GABA A Rs and α2-GABA A Rs were diffused. ( J – K ) eGFP-gephyrin variant size and density. N = 20–22 neurons per group, 3 independent experiments. Statistics: One-way ANOVA, with Tukey post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Transfection, Mutagenesis, In Vitro, Staining, Variant Assay

Journal: Scientific Reports

Article Title: The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

doi: 10.1038/s41598-024-54720-7

Figure Lengend Snippet: Summary of the effects of different mutants used in this study.

Article Snippet: Quantum dot–based single-GABA A R tracking studies have shown that the gephyrin scaffold influences GABA A R surface mobility and synapse retention .

Techniques: Inhibition