Figure 4.

AAV2/1-syn1-GCaMP3 labels a subset of neurons in the ganglion cell layer and permits imaging with tolerably low laser power. A–D, Two-photon fluorescence images of neuronal somas in the ganglion cell layer (GCL) (A, B) and their dendrites in the IPL (C, D). A, B, Image at the level of the GCL showing somas of GCaMP3-expressing ganglion cells (A) and somas of ganglion cells 60 min after bulk loading with OGB-1-AM (B). C, D, Inner plexiform layer of a retina with GCaMP3-expressing ganglion cells (C) and a retina 60 min after bulk loading with OGB-1-AM (D). E, Distribution of soma sizes of neurons in the ganglion cell layer labeled with GCaMP3 (green) and OGB (black). Soma size is expressed as the equivalent soma diameter (i.e., the diameter of a disc with surface area equal to that of the soma in the fluorescence image). F, Distribution of baseline (nonstimulated) fluorescence intensity for each soma, at typical laser power (∼25 mW after objective). G, Extracellularly recorded spike responses (peristimulus time histogram) of an α-type (brisk transient) ganglion cell expressing GCaMP3 (top), one bulk loaded with OGB-1-AM (middle), and a control cell (nontransfected retina; bottom), stimulated with a brief light flash (458 nm, 125 ms duration, 100 μW/cm²; indicated in blue). For the GCaMP3- and OGB-labeled cells, graphs include the simultaneously recorded fluorescence response (7 Hz frame rate; green circles and black curve). H, Spatiotemporal filters calculated from OFF-type (top) and ON-type ganglion cells (bottom) stimulated with a binary white noise stimulus. Curves show spike temporal response characteristic of the receptive field center; X–Y plots represent the spatial weighting function measured at the temporal response peak of each cell. Scale bar, 200 μm. I, Filter time-to-peak across the recorded ganglion cell population (all brisk transient-type; each group n = 6). n.s., Not significant (t test, p = 0.34). *Significant (t test, p = 0.002). Error bars indicate SEM.