To quantify the relative abundance of osteopontin-expressing M1 and M3 ipRGCs, we performed retinal flatmount staining, colabeling with melanopsin and osteopontin antibodies, and acquired confocal volume scans. On average, an area of 0.22 ± 0.04 mm
2 per retina (
n = 5; 1.1 mm
2 in total) was analyzed. Through this double-labeling, we identified altogether 147 melanopsin-positive (M1–M3) ipRGCs and 143 osteopontin-positive cells. Seventy-eight cells colabeled for melanopsin and osteopontin, thus representing 53 % of all M1 to M3 ipRGCs. Performing confocal volume scans on these retinal flatmounts moreover allowed us to trace the dendritic arbor of individual cells, enabling us to determine dendritic stratification, which characteristically distinguishes between M1, M2, and M3 subtypes. Per flatmount stained, we traced the dendritic tree of one osteopontin
+/melanopsin
+ cell (“index”) and one neighboring osteopontin
−/melanopsin
+ cell (“control”;
n = 6 each). All index cells were monostratifying in the ON sublamina of the inner plexiform layer exclusively, suggesting they were M2 ipRGCs. By contrast, control cells were either monostratifying in the OFF layer or bistratifying in ON and OFF layers (
Figs. 4,
5A), therefore most likely representing the ipRGC subtypes M1 (OFF) and M3 (ON–OFF). In agreement with these findings, we observed that the index cells had a large dendritic field diameter of 253.91 ± 20.3 µm and many (20 ± 3) dendritic branching points, while the control cells had a smaller dendritic field diameter of 243.47 ± 44.6 µm and fewer (12 ± 3) branching points (
Figs. 5B,
5C). Also, in accordance with these findings, the mean intensity of the melanopsin immunofluorescence measured over the somata of index cells was significantly lower compared to that of control cells. As for the osteopontin intensity, there was moderately but significantly weaker signal in the osteopontin
+/melanopsin
+ cells compared to cells that only expressed osteopontin but no melanopsin (i.e., mostly αRGC; relative fluorescence intensity in osteopontin
+/melanopsin
+ was 73% [inter quartile range, 65%–79%] of that observed in osteopontin
+/melanopsin
− cells;
P = 0.032).