Fig. 3: Depolarizing GABAergic neuron-to-glioma synapses in DMG.
From: GABAergic neuron-to-glioma synapses in diffuse midline gliomas
a, Schematic depicting the electrophysiological recording of DMG cells xenografted into hippocampal CA1 in response to local stimulation. The schematic was created using BioRender (https://biorender.com). b, Representative traces (grey) of currents elicited by electrical stimulation in the presence of NBQX in two patient-derived DMG xenografts (SU-DIPG-VI and SU-DIPG-XIII-FL). c, Xenografted DMG (SU-DIPG-VI) cell dye filled (Alexa 568; red) during recording and co-labelled with GFP (green) and human nuclear antigen (HNA; white) post-recording. Scale bar, 10 µm. n = 5 biological replicates. d, Representative voltage-clamp trace of a tetrodotoxin (TTX; red)-sensitive DMG (SU-DIPG-VI) cell current in the presence of NBQX (grey). e, Representative trace of stimulation-evoked voltage change in a DMG (SU-DIPG-VI) cell in the presence of NBQX (grey) using current clamp at −70 mV. f, Representative voltage-clamp trace of the picrotoxin (PTX; red)-sensitive GABAergic postsynaptic current (PSC) in a DMG (SU-DIPG-VI) cell in the presence of NBQX (grey; left), and quantification of the current amplitude (right; n = 7 cells from 5 mice; P = 0.0341). Paired two-tailed Student’s t-test. g, Representative voltage-clamp trace of GABAergic PSC in a DMG (SU-DIPG-VI) cell with NBQX + D-AP5 (grey) and bicuculline (Bic; red; left), or no inhibitors (black trace), and quantification of the current amplitude (right; n = 5 cells from 3 mice; P = 0.1787 (control versus D-AP5), P = 0.1093 (control versus NBQX) and P = 0.0287 (control versus Bic)). Repeated measures one-way ANOVA with Dunnett’s post-hoc test. h, Representative voltage-clamp trace of a DMG (SU-DIPG-VI) cell response to stimulation demonstrating Bic-sensitive GABAergic PSC (left) and NBQX-sensitive glutamatergic PSC in the same DMG cell (right). Red trace, in the presence of Bic; grey trace, in the presence of NBQX; black trace, no inhibitors. i,j, Representative traces of perforated patch recordings from xenografted patient-derived DMG (SU-DIPG-VI) and hemispheric high-grade glioma (SU-pcGBM-2) cells in voltage clamp (i) and current clamp (j) in response to GABA. Black traces, H3K27M+ DMG with no inhibitors; red traces, H3K27M+ DMG in the presence of PTX; blue traces, hemispheric high-grade glioma. k, GABA current–voltage relationship of perforated patch recordings in DMG cells (SU-DIPG-VI; black; n = 6 cells from 5 mice) and H3/IDH WT hemispheric high-grade glioma cells (SU-pcGBM-2; blue; n = 6 cells from 5 mice), and whole-cell patch with high internal Cl− concentration in DMG cells (SU-DIPG-VI; grey; n = 4 cells from 4 mice). Representative traces of a DMG cell response to GABA at membrane potentials from −70 mV to +30 mV are also shown (inset). l, GABA current–voltage relationship of perforated patch recordings in DMG (SU-DIPG-VI) cells with no inhibitor (black) or in the presence of 100 μM bumetanide (red; n = 5 cells from 3 mice). Representative traces of membrane potentials from −70 mV to +10 mV with bumetanide are also shown (inset). m, GABA current–voltage relationship in DMG (SU-DIPG-VI) cells in whole-cell patch clamp with low internal Cl− concentration (negative Cl− load) in the presence (red) or absence (black) of 10 µM bumetanide (n = 5 cells from 3 mice). n, Representative voltage-clamp trace of the GABA current in DMG (SU-DIPG-VI) cells at membrane potentials from −70 mV to +10 mV under negative Cl− load conditions. o, Representative time course of bumetanide (10 µM) effect on GABA current in DMG (SU-DIPG-VI) cells at −70 mV under negative Cl− load conditions. Dashed line indicates baseline amplitude before addition of bumetanide. All data are mean ± s.e.m. NS, not significant, *P < 0.05.
