Figure 4.

Functional graft-to-host synapses formed by C-Kit⁺ RPCs in RCS rats

(A–D) Integrated C-Kit⁺ RPCs formed graft-to-host synapses and expressed presynaptic marker synaptophysin (A), presynaptic marker CtBP2 (C), and postsynaptic marker PSD95 (B and D) (all red), counterstained with RGC marker Tuj1, bipolar marker PKCα, and photoreceptor marker Rhodopsin. White arrows mark outer segments (C).

(E and F) Immunofluorescence of the integrated cells showing the co-expression of GCaMP5 and synaptophysin, indicating the generation of synaptic connections. (F) Terminals responding to light (white arrows) confirm the activities of presynaptic calcium transient using two-photon live imaging.

(G) Fluorescence image of integrated cell expressing GCaMP5.

(H) Region of interest corresponding to single synaptic bouton in the confocal image.

(I and J) The GCaMP5 fluorescence responses (left panels, gray traces and their average in green trace) and the raster plots showing fluorescence responses (right panels) from 25 terminals of integrated cells (I) and control cells (J), respectively, elicited by a brief light flash shown as a blue bar. Imaging frequency, 1 Hz; 256 × 256 pixels.

(K) Filter time-to-peak across the recorded integrated cells (green bar) and control cells (black bar) (each group n = 25, t test, p = 0.34, not significant). Error bars indicate SEM.

(L) Cumulative frequency histograms of fluorescence response kinetics for GCaMP5-labeled integrated cells (green) and control cells (black). Left, rise kinetics; right, decay kinetics. Insets, fits for t_1/2 (rise) and t_1/2 (decay). Scale bars, 20 μm (A, B, and D) and 10 μm (C and F).