Extended Data Fig. 6: Optogenetic stimulation of GABAergic interneurons expressing DLX-ChRmine.
From: GABAergic neuron-to-glioma synapses in diffuse midline gliomas
a. Immunostaining and representative confocal micrographs of DLX-ChRmine (red) expression colocalized with GAD65 (green) and/or GAD67 (white). Arrows indicate co-labelled interneurons. Scale bar, 100 µm. b. Inward current and corresponding depolarization of GABAergic interneurons expressing DLX-ChRmine in response to optogenetic stimulation were recorded using patch clamp electrophysiology. c. Unbiased stereological quantification of GAD67+ interneurons in tumor-affected frontal cortex and contralateral control frontal cortex of mice bearing patient-derived DMG xenografts, SU-DIPGVI (n = 4 mice, p = 0.1147) and SU-DIPGXIII-FL (n = 3 mice, p = 0.829). Paired two-tailed student’s t-test. Right, representative confocal images of GAD67+ interneurons (white, arrows) near H3K37M+ DMG cells (red). Scale bar, 100 µm. d. Optogenetic stimulation of interneurons expressing DLX-ChRmine (red) induces neuronal activity, indicated by cfos expression (green). Arrows indicate co-labeled interneurons. Scale bar, 100 µm. Above, experimental timeline. Right, quantification of cfos expression in interneurons 90 min after optogenetic stimulation in the stimulated hemisphere (stim) indicates greater numbers of cfos-labeled interneurons than in the unstimulated hemisphere (unstim); n = 5 mice per group; p = 0.0078. Paired two-tailed student’s t-test. e. Optogenetic stimulation of interneurons expressing DLX-ChRmine (red) in the microenvironment of DMG xenografts increases cfos expression (white) in GFP+/HNA+ glioma cells (green). Arrows indicate co-labeled DMG cells. Scale bar, 100 µm. Above, experimental timeline. Right, quantification of cfos expression in glioma 24 h after optogenetic stimulation of interneurons in the stimulated group (stim) indicates greater numbers of cfos-labeled DMG cells than in the mock stimulated group (mock stim); n = 3 mice per group; p = 0.0264. Two-tailed student’s t-test. All data are mean ± s.e.m. *P < 0.05, **P < 0.01. The diagrams in panels b,d,e were created using BioRender (https://biorender.com).
