Fig. 4: Midbrain-Striatum assembloids develop electrical connectivity and directionality.

a Bright field image of MO and StrO cultured onto an 8 × 8 electrode MEA chip. Individual electrode location is identified by line and column numbered between 1 and 8. The microfluidic system laying on top of the MEA is schematized in the lateral inset. b Raster plot of individual spike events detected during a representative 3 min recording for each active electrode. c Given two electrodes (e1 and e2), the Spike Time Tiling Coefficient (STTC) is calculated as the probability to detect at least one spike (red coloured) in e2 within a temporal window (Δt) centered around a given spike in e1. d Cross-correlograms of three pairs of electrodes (A-B, C-D, E-F) of a selected assembloid. When significant, the cross-correlogram maximum can be interpreted as the (most probable) time delay t required by the signal to propagate between the two electrodes. The signal speed V can be estimated as the ratio of the distance r between the two electrodes and the time delay t. e The mean signal directionality for a given electrode can be expressed as a vector. In this representative experiment, only electrode vectors (black arrows) having STCC higher than 0.8 are shown, together with the mean firing rate (in Hz) for the whole MEA. f Eleven assembloids (92%) were found to form a stable connection between MO and StrO within 40 days in vitro (D40). Four assembloids (33%) formed connections already by D4. g The mean electrical signal directionality of the assembloid can be computed as a vector with vertical (y) and lateral (x) components. In the 12 assembloids analyzed at different maturation stages, the positive sign of the mean vertical component (± standard deviation, STD) indicates that the electrical signal travels more from the MO to the StrO than the opposite direction. Statistical analysis was performed by Kruskal–Wallis post hoc analysis between all groups ** p < 0.01. h The mean lateral directionality (calculated as the absolute value of x ± STD) yielded a significant increase during the assembloid maturation (*** p < 0.001).