Fig. 4. Glial network integration of ES cell-derived astrocytes. (A) Epifluorescence microscopy during a 30-minute iontophoretic injection of LY into a GFP-labeled donor cell, located at the border of EC and subiculum (arrow, 25 days after transplantation; both LY and GFP signals are recorded in the FITC channel). Note the extensive dye coupling to adjacent host cells. (B) Confocoal microscopy and 3D reconstruction of (A), reveals a cluster of 50 coupled cells with total volume of 5.6x10^–3 mm³. The picture is taken following fixation and depicts the 3D nature of the endogenous glial network. (C-D) Subsequent serial sectioning and double labeling with an antibody to the mouse-specific antigen M2 (red) confirms the donor origin of the injected astrocyte depicted in A. Note the extensive arborization (arrowheads) and the perforation of the cell body (arrow). (E) Overlay of C and D (boxed area delineated in C), triple labeled with an antibody to connexin43 (blue). Patches of connexin43 immunoreactivity (arrowheads) are detected at the contact zones between the LY/M2+ donor cell and two adjacent LY-filled host-cell processes. (F) Prominent connexin43 expression (blue) is also observed on LY-filled processes connecting individual host cells of the same cluster (confocal 3D reconstruction). Note that figures A-F are from the same donor-host cell cluster. Scale bars: in A, 200 µm; in B, 50 µm; in C-F, 25 µm.