Journal: Neurobiology of disease

Article Title: Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and is associated with altered regional microglial morphology

doi: 10.1016/j.nbd.2025.107191

Figure Lengend Snippet: ( A ) Representative traces of AMPAR-mediated eEPSCs at −60 mV holding potential and NMDAR-mediated eEPSCs at +40 mV holding potential in WT and Shank3 KO mice. ( B ) Average peak amplitudes of AMPAR-mediated eEPSCs. ( C ) Average amplitudes of NMDAR-mediated eEPSCs at +40 mV, 15 ms after stimulation. ( D ) Ratio of average AMPAR to NMDAR amplitudes. ( E ) Representative traces of paired-pulse responses (inter-stimulus interval [ISI]: 20 ms) in Shank3 WT and KO mice. ( F ) Quantification of the paired-pulse ratio in both genotypes. ( G ) Representative traces of normalized AMPAR-mediated eEPSC illustrating decay kinetics in both genotypes. ( H ) Weighted decay tau values of AMPAR responses evoked by 100 μA stimulation in both genotypes. For panels B – D, F, and H , individual data points are shown as circles, and bars represent the mean ± SEM. Shank3 WT: n = 17–23 cells from N = 10–11 mice; Shank3 KO: n = 16–24 cells from N = 13–18 mice. Statistical significance was determined using an unpaired t-test for B, D, and F , and a Mann-Whitney test for C and H . * p < 0.05 indicates a significant difference between WT and KO; ns: not significant.

Article Snippet: In the same eEPSC experimental setup using CsCl-based internal solution, the AMPAR-mediated response was recorded at −60 mV after stimulating with single-pulse stimulation of 100 μA at a frequency of 0.2 Hz in the presence of gabazine (10 μM; Tocris Bioscience, catalog #1262) followed by the supplemental application of selective calcium-permeable AMPAR blocker IEM-1460 (100 μM; Tocris Bioscience, catalog #1636).

Techniques: MANN-WHITNEY

Journal: Neurobiology of disease

Article Title: Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and is associated with altered regional microglial morphology

doi: 10.1016/j.nbd.2025.107191

Figure Lengend Snippet: ( A ) Schematic diagram of glutamate uncaging experiment illustrating brief exposure of cerebellar slice to blue LED light for photolytic cleavage of Rubi-glutamate supplied in ACSF (0 mM Mg 2+ ) while recording from a CGC at −70 mV. ( B ) Representative current traces of combined AMPA + NMDA (red: Shank3 WT, light red: Shank3 KO) current recorded upon light exposure in the presence of Rubi-glutamate, gabazine, and TTX. Subsequent addition of NBQX isolated NMDA current (gray). AMPA current trace (black) was obtained by subtracting NMDA component from the composite AMPA + NMDA current. ( C ) Average peak amplitudes of the combined AMPA + NMDA response. ( D ) Average peak amplitudes of the AMPAR response. ( E ) Average peak amplitudes of the NMDA response. ( F ) Ratio of average AMPAR to NMDAR amplitudes. ( G ) Average current density of combined AMPA + NMDA response. ( H ) Average current density of the AMPA response. ( I ) Average current density of the NMDA response. For panels C – I , individual data points are shown as circles, and bars represent the mean ± SEM. Shank3 WT: n = 13–21 cells from N = 7–8 mice; Shank3 KO: n = 14–22 cells from N = 6–8 mice. Statistical significance was determined using an unpaired t-test for the data in panels C-E, H, I and a Mann-Whitney test for the data in panels F and G . * p < 0.05 indicates a significant difference between Shank3 WT and KO mice; ns: not significant.

Article Snippet: In the same eEPSC experimental setup using CsCl-based internal solution, the AMPAR-mediated response was recorded at −60 mV after stimulating with single-pulse stimulation of 100 μA at a frequency of 0.2 Hz in the presence of gabazine (10 μM; Tocris Bioscience, catalog #1262) followed by the supplemental application of selective calcium-permeable AMPAR blocker IEM-1460 (100 μM; Tocris Bioscience, catalog #1636).

Techniques: Isolation, MANN-WHITNEY

Journal: Neurobiology of disease

Article Title: Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and is associated with altered regional microglial morphology

doi: 10.1016/j.nbd.2025.107191

Figure Lengend Snippet: ( A ) Schematic diagram of the cerebellar circuit, illustrating the stimulation of MFs and recording a CGC in the presence of intracellular spermine. ( B ) Representative current traces of AMPAR-mediated response from CGCs of WT and Shank3 KO at −60 and + 60 mV. ( C ) Mean ± SEM of normalized (to response at −60 mV) peak eEPSC current and a range of holding potentials (I-V). ( D ) eEPSC rectification index values for CGC responses to MF stimulation in Shank3 WT and KO mice. ( E, F ) Example ( E ) and group average ( F ) AMPAR-mediated EPSC responses before (ACSF) and during IEM-1460 (100 μM) application, normalized to the average baseline response recorded over 5 min prior to drug application. ( G ) Percentage of the initial baseline eEPSC response amplitude calculated from the average of the last 3 min of recording in IEM-1460 from both genotypes. In panels D and G , individual data points are shown as circles, and bars represent the mean ± SEM. Shank3 WT: n = 10–13 cells from N = 6–7 mice; Shank3 KO: n = 11–13 cells from N = 3–7 mice. Statistical significance was determined using an unpaired t-test for the data in panels B, D, and F . * p < 0.05 indicates a significant difference between Shank3 WT and KO mice. Abbreviations: ML: Molecular layer, PCL: Purkinje cell layer, GCL: Granule cell layer.

Article Snippet: In the same eEPSC experimental setup using CsCl-based internal solution, the AMPAR-mediated response was recorded at −60 mV after stimulating with single-pulse stimulation of 100 μA at a frequency of 0.2 Hz in the presence of gabazine (10 μM; Tocris Bioscience, catalog #1262) followed by the supplemental application of selective calcium-permeable AMPAR blocker IEM-1460 (100 μM; Tocris Bioscience, catalog #1636).

Techniques:

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