Figure 5.

Patch-clamp analysis of cells in intact organoids at different developmental stages show functional neurons

(A) Picture showing a glass pipette during a patch-clamp recording; note the edge position of the cell under recording. In the inset, a scheme shows the glass pipette used for patch-clamp recording of a whole mount organoid placed on a glass coverslip.

(B–D) Passive membrane properties: resting membrane potential (RMP) (B), membrane capacitance (Cm) (C), and input resistance (Rin) (D) of cells in intermediate (11–13d), early mature (19–26d) and mature (33–42d) organoids. In all graphs, the median is represented by the longest horizontal red lines, whereas the 25–75 confidence interval is marked by the vertical red bars.

(E) Representative traces of K⁺ and Na⁺ currents elicited by depolarizing voltage steps in cells recorded from the whole mount organoid. At intermediate (11d, left panel) and early (19d, middle panel) developmental stages, cells show outward currents but do not exhibit inward Na⁺ current, consistent with the absence of action potentials. At more mature developmental stages (34d, right panel), cell exhibits both inward Na⁺ and outward K⁺ currents. In the inset (left panel), the stimulus protocol applied is shown. • indicates the point where delayed rectifier K⁺ current (I_DR) amplitude was measured; ○ indicates point where A-type K⁺ current (I_A) amplitude was measured; ○ indicates point where Na⁺ current (I_Na) amplitude was measured.

(F) Graph showing the percentage of cells exhibiting I_DR, I_DR + I_A, and I_DR + I_A + I_Na in intermediate (11–13d), early mature (19–26d) and mature (33–42d) organoids.

(G) Averaged current-voltage (I-V) relationship for I_A(left) and I_DR(right). Black line refers to intermediate organoid cells (11–13d); red line refers to early mature organoid cells (19–26d); light blue line refers to mature organoid cells (33–42d). Data are presented as mean of current density (pA/pF) ± SEM. ∗p<0.05, §<0.01.

(H) Averaged I-V relationship for I_Na. Light blue line refers to mature organoid cells (33-42d), while I_Na was not observed in intermediate (11–13d) and early mature (19–26d) organoids. Data are presented as mean of current density (pA/pF) ± SEM.

(I) Representative current-clamp traces evoked in a mature organoid cell (34d) by 500 ms current pulses; depolarization induces action potentials that are blocked by TTX (1 μM). In the inset (right panel), the stimulus protocol applied is shown.