Fig. 1.

Grafted mNSC and host cell interactions in fluctuating networks are blocked by inhibiting gap junction function. (A) Communication between grafted mNSCs (GFP⁺, green arrowhead) and endogenous OC cells tracked by ratiometric [Ca²⁺]_i imaging. (B) Typical [Ca²⁺]_i response, after 14 DIV, of mNSC (no. 1) and endogenous OC cells (nos. 2–6) in A. Note that no spontaneous [Ca²⁺]_i events are detected during control conditions (aCSF) or during ATP application. Glutamate causes an instantaneous rise in [Ca²⁺]_i and induces temporary coupled oscillations in both mNSCs and OC cells. [Ca²⁺]_i traces (gray box in B) are enlarged in C. The mNSC trace is green; overlapping events are marked with dotted lines I–VII. (D) Activity plot showing even variations in Ca²⁺ transients in all parts of the field (Movie S3). (E and F) mNSC (green cells in E and green arrowhead in F) and OC cells (white arrowheads in F) characterized by time lapse [Ca²⁺]_i imaging (F). Curves in G depict the [Ca²⁺]_i response of cells 1–3 in F. Overlapping events are marked with dotted lines I–V. Glutamate causes an instantaneous rise in [Ca²⁺]_i and induced temporary coupled oscillations in both mNSCs and OC cells. Addition of gap junction inhibitor CBX disrupts the synchronous communication and uncouples the successive low frequency fluctuations. (H and I) Intercellular gap-junctional contacts (here Cx26) between GFP+ (green) mNSCs themselves (red punctate immunopositivity in H); between mNSCs and OC cells; and between host OC cells themselves (white punctate immunopositivity in I) (Movie S4). White arrows in I mark gap-junctional plaques between Tuj1+ mNSC-derived cells and OC cells. (Bars: 20 μm, A, E, F, and I; 5 μm, H).