Figure 2.

Photosensitivity of melanopsin-panned RGCs. A, Differential interference contrast image of two cells from a melanopsin-panned culture and pseudocolored images of fura-2 fluorescence ratios for these cells before and after light stimulation. B, The full optical recording for these cells showing that the larger melanopsin-panned RGC (cell 1) exhibited an increased fura-2 ratio (indicative of increased [Ca²⁺]_i) after each of two 20 s light pulses. The [Ca²⁺]_i of the smaller “non-RGC-like” cell (cell 2) was unaffected by light. C, Multiple light responses could be elicited from the cultured cells (left trace), although some cells (6 of 18 cells) did not respond if the second stimulus was given 5 min after its preceding pulse (right trace; compare responses in two traces denoted by arrows). D, Data summary shows effect (mean Δfura-2 ratio ± SEM; n = 18 RGCs) of the interstimulus interval (5, 10, or 20 min) on the Ca²⁺ signals evoked by 30 s light pulses (normalized to each ipRGC's initial light response; dashed line). *p < 0.05, one-way repeated-measures ANOVA, Tukey's test, compared with responses elicited after 20 min intervals. E, Melanopsin-panned RGCs responded to both light and glutamate (30 s application) over the first few days in culture (recording on left from ipRGC on DIV 2), but their photosensitivity diminished with longer culture periods (recording on right from melanopsin-panned RGC on DIV 4). F, Representative melanopsin-panned RGCs on DIV 7 that were immunoreactive for Thy1 (green; bottom row) but not melanopsin (red; top row). These cells were from the same culture as the DIV 1 melanopsin-panned RGCs depicted in Figure 1A (top row; note that Thy1-positive cells also melanopsin-positive), indicating that melanopsin protein expression had decreased.