Extended Data Fig. 6. Heterogeneity and specificity of glioma electrophysiological response to glutamate.

a, Representative image of Alexa 568 (red)- filled GFP+ glioma cell following whole-cell patch clamp recording. Co-labelled with GFP (green) and human nuclear antigen (HNA, grey). Scale bars = 10 µm. b, Representative voltage-clamp traces of whole cell patch-clamp electrophysiological recordings in glioma cells. Hippocampal slices were perfused with ACSF containing tetrodotoxin (TTX, 0.5 µM), and response to a local puff (250 msec) application of 1 mM glutamate (black square) was recorded from xenografted glioma cells with sequential application of NMDAR blocker (AP-5, 100 µM), TBOA (200 µM), AMPAR blocker (NBQX, 10 µM). c, Quantification of data in b (n = 7 glioma cells, 4 mice, P = 0.0165). d, Whole cell patch-clamp electrophysiological recording of glioma cell with ACSF puff, representative voltage clamp trace. e, Representative traces of glutamate-evoked inward currents (black square) in patient-derived glioma xenografted cells before (grey) and after 30-minute perfusion with NLGN3 recombinant protein (100 ng/ml) in ACSF (containing TTX, 0.5 µM) (purple). f, Quantification of data in e (n = 5 glioma cells, 3 mice). g, Model of calcium imaging of tdTomato nuclear tagged (red nuclei), GCaMP6s-expressing (green calcium transients) glioma cells xenografted into the mouse hippocampal region. h, Quantification of number of xenografted SU-DIPG-XIII-FL or SU-DIPG-VI cells glioma cells demonstrating a calcium transient (as depicted in Fig. 2i, j and Extended Data Fig. 6j) in response to a glutamate puff (responders, grey, non-responders, white). i, Baseline GCaMP6s intensity in SU-DIPG-VI glioma cells before and 30-min after BDNF exposure, in the absence of glutamate puff (n = 7 cells, 3 mice). j, GCaMP6s intensity trace of SU-DIPG-VI glioma cells response to glutamate puff before (3 cells, 3 mice: light grey, average: dark grey) and after BDNF perfusion (three cells: light blue, average intensity: dark blue). k, SU-DIPG-VI GCaMP6s cell response to glutamate puff at baseline and after BDNF perfusion (100 ng/ml, 30 min, n = 7 cells, 4 mice, P = 0.0174). l, Duration of calcium transient response to glutamate puff in SU-DIPG-VI hippocampal xenografted cells, before and after perfusion with BDNF (100 ng/ml, 30 min, n = 6 cells, 4 mice, P = 0.0302). m, Representative traces of SU-DIPG-XIII glioma GCaMP6s intensity in the presence of BDNF (100 ng/ml, 30 min). Response to glutamate application (black) recorded with BDNF perfusion (3 cells, 2 mice: light blue, average: dark blue) or with BDNF and NBQX (10 µM, 3 cells: light red, average: red). n, Response of GCaMP6s cells to glutamate puff with BDNF application, in the presence and absence of NBQX (n = 6 cells, 3 mice, P = 0.0002). Data are mean ± s.e.m., *P < 0.05, ***P < 0.001, ns = not significant, two-tailed paired Student’s t-test for k, l, n, and two-tailed Wilcoxon signed pairs matched rank test for d, f and i.