Fig. 9: Functional neurons and neural networks in 5-month-old SNR-derived organoids. | Nature Communications

Fig. 9: Functional neurons and neural networks in 5-month-old SNR-derived organoids.

From: Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes

Fig. 9

a Low- (top) and high- (bottom) resolution images of a 5-month-old organoid slice used for electrophysiology. b Image of a biocytin-filled control neuron with dendritic spines in organoid slice after electrophysiology. c Traces of membrane potentials recorded from cells (marked with asterisk in d) in different clusters in response to Δ5 pA somatic current injections. d Heatmap of unsupervised hierarchical clustering of recorded neurons based on non-redundant electrophysiological characteristics (n = 103 cells from 4 2242-5, 3 EP2-15, 11 H9, and 2 EYQ organoids). e, f Organoid section immunostained for presynaptic marker BASSOON. e BASSOON and SHANK3, SHANK2, SHANK1, VGLUT1, or GEPHYRIN (f). g Quantification of excitatory and inhibitory synaptic puncta (n = 8 [Bassoon:VGluT1], 28 [Bassoon:SHANK3], 20 [VGluT1:PSD-95], 20 [Bassoon:Homer], 8 [Bassoon:SHANK2], 24 [Bassoon:Gephyrin], and 8 [Bassoon:SHANK1] sections from 1–3 organoids produced in one batch from 2242-5 line). h, i Traces of spontaneous excitatory postsynaptic currents (EPSCs) (h) and inhibitory postsynaptic currents (IPSCs) (i) recorded at different holding membrane potentials without inhibitors and with 3 mM kynurenic acid (glutamate receptor antagonist) or 100 μM picrotoxin (GABA receptor antagonist). j Image of a 5-month-old SNR-derived organoid implanted with a 16-channel multi-electrode array. k–n Spontaneous electrical activity recorded from different electrodes (E1–16) before (k) and after tetrodotoxin (TTX, 1 µM) application (n). Waveforms extracted after filtering of low-frequency components with a 300-Hz high-pass filter (l). Extracellular potentials recorded from different electrodes after delivery of stimuli of varying intensity at E7 (m). o, p Spectral analysis of recorded signals (o) and average rate of high-frequency events (>300 Hz) before (n = 80 fields obtained from five organoids [16 fields per organoid] produced in one differentiation batch from H9 line) and after TTX application (n = 16 fields obtained from 1 H9 organoid) (p). Data are mean ± s.e.m.; *P = 0.047, unpaired two-sided Kolmogorov-Smirnov test. Scale bars = 50 and 10 (zoom in) (b), 200 and 20 (zoom in) (e), and 20 and 10 (zoom in) (f) μm. Source data are provided as a Source Data file.

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