Expression of transgenic proteins and inactivation of RhoA in RGCs. RGCs were transfected in vivo with AAV carrying a gene encoding C3 and GFP after an IRES. a, The C3 transcript is detected by RT-PCR in transfected retinas (right) but not in normal retinas. RT-PCR reactions include primers for GAPDH as a control (C3 band at 615 bp; GAPDH at 300 bp). b-d, Low-power photomicrographs of flat-mounted whole retina stained for βIII tubulin (b) and the GFP reporter (c). d, The merged image shows that most RGCs express the transgene. e, f, Higher-magnification of areas from c and d, respectively. Note intense staining in RGC somata and their axons, which coalesce to form the optic nerve. g-l, C3 transfection decreases RhoA activation. Sections through the retina were prepared 2 weeks after transfection with either AAV-GFP (g, i, k) or AAV-C3-IGFP (h, j, l) and were stained with TUJ1 to visualize all RGCs (g, h) and RBD-GST, which binds to active (GTP-bound) RhoA, followed by antibodies to detect GST (i, j). Note low levels of active RhoA in C3-transfected RGCs (j) compared with control RGCs (i). k, l, Merged images showing colocalization of RBD binding in TUJ1-positive RGCs. Scale bars: b-d, 50 μm; e, f, 25 μm.