Figure 5. Electrophysiological spontaneous activity of control and FGF8-treated 3D organoid networks.

(A–C) Spontaneous activity maps showing mean spike amplitude in 4-month-old (day136; upper row) and 7-month-old (day215; lower row) control (WNTi; A) and treated organoids (WNTi + FGF8; B), 2–3 weeks post-plating on high-density MEA chips. Mean activity metrics from three independent batches are summarized in (C), showing a general reduction in spike amplitude, percentage of active electrodes, and firing rate in FGF8-treated organoids. White arrowheads in electrode images indicate organoids fully or partially adhered to the MEA recording surface; note that high activity levels are observed at the organoid edges, where axonal tracts extend. (D–F) Temporal raster plots displaying firing events recorded by the 1024 most active electrodes (upper graphs) and their synchronicity indicative of network activity (lower graphs) in 7-month-old controls (WNTi; D) and FGF8-treated organoids (WNTi + FGF8; E), 3 weeks post-plating. Note that the instrument automatically sets the detection threshold (black bar) at varying levels, based on the average baseline activity specific to each sample (see Materials and methods). Graphs in (F) show network metrics (Burst frequency; Spikes within bursts) and burst metrics (number of spike per burst, burst peak firing rate, burst duration, inter-spike interval within bursts). (G–I) Overview fields of axon tracts (upper row) and representative images of individual neuronal tracts (lower rows) extending from organoids on the MEA surface, as detected using the automatic axon tracking function in WNTi (G) and WNTi + FGF8 (H) samples at day215. Graphs in (I) present axon tracking metrics (network conduction velocity, total axon or branch lengths, longest signal latency, maximum distance from initiation site, and signal amplitude), showing reduced signal amplitude and spatial propagation in FGF8-treated organoids. Additional axon tract images at different stages of differentiation are provided in Figure 5—figure supplement 1. (J) Immunostaining for GAD1 (red) and SLC17A6 (green) in day 145 control (WNTi) and treated (WNTi + FGF8) organoids, following detachment from MEA chips post-recording. Percentages of GAD1- and SLC17A7-positive tissue in organoids are indicated. For all graphs: data represent n=3 distinct batches (each with 2–4 organoids on the MEA chip, see Materials and methods).

Figure 5—figure supplement 1. Representative spike traces and axonal maturation of WNTi and WNTi + FGF8 organoids on HD-MEA.

(A, B) Footprints of action potentials in control WNTi (A; left column) and treated WNTi + FGF8 (B; right column) organoids, as automatically detected by MaxOne software. Progressive higher magnifications of propagating spikes are displayed in red and blue boxes, while representative traces displaying the shape of action potentials are shown in the bottom row (green boxes). (C, D) Overview fields of axon tracts extending from organoids on MEA surface, as detected by the automatic axon tracking function, in WNTi (C) and WNTi + FGF8 (D) samples at day136. (E, F) Exemplificative images of single neuronal tracts extending from organoids on MEA surface, in WNTi (left) and WNTi + FGF8 (right) samples at day134 (E) and at day213 (F). Note the progressive increase in complexity and length of axons, suggesting maturation of the organoids, which is more limited after treatment with FGF8. (G) Graphs show axon tracking metrics (network conduction velocity, total axon or total branch lengths, longest signal latency, longest distance from and signal amplitude at the initiation site), in control (orange columns) and in FGF8-treated organoids (blue columns) at different developmental stages, as indicated. For graphs in G: data from n=2 distinct batches at two different differentiation times (each batch including 2–4 organoids on the MEA chip, see Materials and methods).

Figure 5—figure supplement 2. Bicuculline-mediated inhibition of GABA-A receptors and its effect on WNTi + FGF8 organoid spontaneous activity.

(A–C) Spontaneous activity maps showing the mean spike amplitude of 6-month-old WNTi + FGF8 organoids, before (left) or after (right) treatment with 10 µM Bicuculline, a chemical inhibitor of GABA-A receptor. Mean values of activity metrics are shown in C and highlight a slight but significant increase of spike amplitudes. White arrowheads in the electrode image on the top left point to organoids adhering totally or partially on the MEA recording surface. (D–F) Temporal raster plots showing firing events recorded by the 1024 most active electrodes (upper graphs) and their synchronicity suggestive of network activity (lower graphs), as detected from untreated (left) or Bicuculline-treated (right) WNTi + FGF8 organoids. Note that the background noise due to random activity falling outside of synchronous bursts (red dotted boxes) is decreased upon treatment with Bicuculline, as also visible in high magnification insets (* and **; before and after Bicuculline treatment, respectively). Graphs in F depict network metrics (Burst frequency and Spikes within bursts), highlighting a significant increase of percentage of spikes within synchronous network bursts. For all graphs: data from n=2 distinct batches (each including 2–4 organoids on the MEA chip, see Materials and methods).