ampar blocker  (Alomone Labs)

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity.

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: Fig. 3 | BDNF regulates trafficking of AMPAR to the glioma postsynaptic membrane. a, Schematic depicting AMPAR trafficking downstream of BDNF– TrkB–CAMKII signalling46. b, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c, Quantification of cell surface GluA4 in b (n = 3 independent biological replicates). d, Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e, Quantification of cell surface GluA3 in d (n = 3 independent biological replicates). f, Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g, Quantification of cell surface GluA4 data in f (n = 3 independent biological replicates). h, Schematic showing GluA2–SEP experiments. i,j, Validation of pHluorin approach. i, Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture

Article Snippet: For EdU proliferation assays, 70,000 wild-type or NTRK2-KO glioma cells were plated and incubated for 48 h, before treatment with EdU (10 μM) with or without the AMPAR blocker NBQX (10 μM, Tocris) and incubated for a further 24 h. Following incubation, the cultures were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and stained for immunofluorescence analysis.

Techniques: Membrane, Western Blot, Biomarker Discovery, Expressing, Co-Culture Assay

Journal: Neuron

Article Title: A Distance-Dependent Distribution of Presynaptic Boutons Tunes Frequency-Dependent Dendritic Integration

doi: 10.1016/j.neuron.2018.06.015

Figure Lengend Snippet: Increases in Short-Term Facilitation with Distance along a Dendrite Boost Distal Synaptic Integration (A) Whole-cell patch-clamp technique was used to record synaptic currents and fill CA1 pyramidal cells with a fluorescent dye to image its structure. Two stimulating pipettes were placed in the proximal (red) and distal (blue) extracellular domains of the basal dendritic tree of pyramidal cells to stimulate the local fibers. (B) Single cell example of average EPSC responses (average of 20 individual sweeps) to trains of 5 pulses at 20 Hz delivered to the proximal region (red trace) and the distal region (blue trace). The distal response shows greater facilitation compared to the proximal one. (C) Normalized average peak EPSC amplitudes for distal and proximal responses show greater sustained facilitation during a 5 pulse train (20 Hz) for distal synapses, n = 35 cells, multiple t tests with p values adjusted with the Holm-Sidak method, p < 0.05. (D) Paired-pulse ratios (PPRs) for each individual cell recorded at distal and proximal synapses. The majority (29/35) of cells display greater facilitation in the distal domain, n = 35 cells, p = 0.0003 two-tailed paired t test. (E) Distal increase in PPR is not ascribable to postsynaptic AMPA receptor desensitization (prevented by CTZ application) or to AMPA receptor saturation (avoided with γDGG application). Distal PPR is greater than proximal PPR with CTZ (n = 9 cells, p = 0.01), and γDGG (n = 13 cells, p < 0.01), two-tailed paired t test. Two-way ANOVA to test PPRs in control (D), CTZ, and γDGG conditions together shows no significant interaction, p = 0.84, indicating that the drugs have no effect on STP properties. (F) Full Synaptotagmin7 KO eliminates facilitation and proximo-distal STP differences. Triangles in lighter colors are from Syt7KO mice, n = 9 cells, circles in darker colors are littermate wild-type mice, n = 12 cells. For WT mice, proximal facilitation is lower than distal, multiple t tests, p < 0.05. WT facilitation is greater than Syt7KO facilitation, p < 0.01 multiple t tests. (G–I) Distal synaptic EPSCs take longer to reach the soma. (G) Left panel: normalized trace for a proximal and distal EPSC response showing the delayed kinetics of the distal compared to the proximal synaptic current. Right panel: the rise time constant of the EPSCs was significantly higher in distally triggered events, n = 49 cells, p < 0.001 Wilcoxon signed rank test. (H) Longer rise times correlate with the amount of facilitation, Spearman’s correlation. (I) PPR is higher when the stimulation electrode is placed further away from the soma, measured as distance along the dendrite, Spearman’s correlation. (J) Proximal synapses display greater P r than distal synapses. After MK-801 bath application, the normalized amplitude of EPSCs from proximally stimulated synapses decay faster, following successive stimulations, than distal ones; n = 8 cells. Data points were fit with a double exponential function (filled lines). Insets are example traces of 7 successive NMDA mediated EPSCs. (K) Frequency tuning curve showing PPRs for all frequencies tested. Distal PPRs (second stimulus only) increase significantly in the 20 Hz (n = 35) same as (D), and 50 Hz range (n = 20), p < 0.05, multiple t tests with Holm-Sidak adjusted p values. 5 Hz (n = 13 cells), 10 Hz (n = 16), 80 Hz (n = 9). (L–M) Short-term facilitation contributes to dendritic non-linear events in distal domains. (L) Current-clamp example traces (red proximal, blue distal stimulation) in response to a paired pulse, of increasing stimulus intensity (lighter color shades represent lower intensity). (M) The proportion of supra-linear events (at least 2 mV above the expected response) for distal synapses is greatly increased for the second pulse (P2) following facilitation at 20 Hz (0 events in P1, 8 events in P2, n = 10), while supra-linear events were detected to the first pulse (P1) for proximal stimulations (n = 10, 4 in P1, 5 in P2). At 5 Hz, where STP is absent, distal synapses had fewer supra-linear events (n = 5, 0 in P1, 2 in P2). See also and . Data are represented as mean ± SEM.

Article Snippet: For the NMDAR depletion experiments the extracellular solution contained the AMPAR channel blocker NBQX 10 μM (Santa Cruz Biotechnology) instead of AP-5.

Techniques: Patch Clamp, Two Tailed Test, Control

Journal: Neuron

Article Title: A Distance-Dependent Distribution of Presynaptic Boutons Tunes Frequency-Dependent Dendritic Integration

doi: 10.1016/j.neuron.2018.06.015

Figure Lengend Snippet:

Article Snippet: For the NMDAR depletion experiments the extracellular solution contained the AMPAR channel blocker NBQX 10 μM (Santa Cruz Biotechnology) instead of AP-5.

Techniques: Recombinant, Software