Fig 1.
Negative and preabsorption tests in the R. ferrumequinum retina.
(A, B) The negative test. (C, D) The preabsorption test. These tests were performed to assess the specificity of the rabbit polyclonal melanopsin antibody in the R. ferrumequinum retina. Melanopsin-IR cells were not detected in the R. ferrumequinum retina. IR, immunoreactive; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar = 50 μm.
Fig 2.
Melanopsin-IR cells in 50 μm vertical sections of R. ferrumequinum retina.
Melanopsin-IR cell types were revealed through stacked confocal images and drawings. (A-F) The M1c type showed monostratified dendritic arborization at the uppermost OFF sublayer of the IPL. M1c cell bodies lay in the GCL. (G-H) M1 displaced RGCs. The M1d type also displayed monostratified dendritic arborization at the uppermost OFF sublayer of the IPL and cell bodies lay in the INL. (I-J) The M2 type showed monostratified dendritic arborization at the bottommost ON sublayer of the IPL, M2 cell bodies lay in the GCL. (K-N) The M3c type displayed dendritic arborization at both the ON and OFF sublayers of the IPL. The M3c cell bodies lay in the GCL. (O-P) The M3d type had bistratified dendritic arborization and cell bodies in the INL. (Q-T) The M3c-crv type had a curved dendrite and cell bodies in the GCL. (A, C, E, G, I, K, M, O, Q, S) Fluorescence photomicrographs. (B, D, F, H, J, L, N, P, R, T) Drawings of melanopsin-IR cells, distinguished by different colors; red (M1c), pink (M1d), brown (M2), yellow-green (M3c), green (M3d), blue (M3c-crv). IR, immunoreactive; RGC, retinal ganglion cell; IPL, inner plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar = 10 μm.
Fig 3.
Drawings of melanopsin-IR cells somato-dendritic profiles.
The colors used differently for each stratum is as follows: black (GCL), blue (ON sublayer of the IPL), red (OFF sublayer of the IPL), and green (INL). (A and B) Tracing drawings of the M1c type. (C) A tracing drawing of the M1d type. (D) A tracing drawing of the M2 type. (E and F) Tracing drawings of the M3c type. (G) A tracing drawing of the M3d type. (H) A tracing drawing of the M3c-crv type. IR, immunoreactive; RGC, retinal ganglion cell; IPL, inner plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer. Scale bar = 50 μm.
Fig 4.
Descriptions of M3c-crv type of ipRGCs on confocal images.
(A) Two white arrowheads indicate the dendrite located in the ON sublayer of the IPL. The cell body in the GCL is highlighted by the asterisk. (B) The white arrow shows the dendrite located in the OFF sublayer of the IPL. (C) The stacked confocal image. IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 20 μm.
Fig 5.
Histogram of the soma and dendritic field diameters of melanopsin-IR cells and differences in soma, dendritic field diameter and dendritic length of the M1, M2 and M3 types in the R. ferrumequinum retina.
The fields were positioned in the mid-peripheral retina and the best labeled neurons were analyzed. (A) A total of 180 neurons labeled with melanopsin were measured in the three retinas. The soma diameter ranged from 8.15 to 16.16 μm. (B) Soma diameters of the different cell types were measured in the three retinas. The different cell types were marked using different shapes as follows: a diamond-square for the M1 type, square for the M2 type, and triangle for the M3 type. The lines represent the mean soma diameter. (C) A total of 120 neurons labeled with melanopsin were measured for the three retinas. The dendritic field diameter ranged from 142.35 to 238.29 μm. (D) The dendritic field diameters and dendritic lengths of the different cell types were measured in the three retinas. The different cells types have been marked by the following shapes: the diamond-square for the M1 type, square for the M2 type, and triangle for the M3 type. The crosses with different colors represent the mean dendritic field diameters and dendritic lengths. IR, immunoreactive.
Fig 6.
Distribution patterns of melanopsin-IR cells in the R. ferrumequinum retina.
Confocal images were acquired as z-stacks from a mid-peripheral area in the R. ferrumequinum retina. (A-D) Each image was obtained at different magnifications. (A-C) Melanopsin-IR cells were not evenly distributed in the retina; complex dendrites were entangled with one another. Arrows show closely positioned cell bodies. (B) Medium magnification photomicrograph of the inset square of (A) clearly shows the positions of the cell bodies. (C) The varicosities along the sparsely branched dendrites of the whole-mount retina are indicated by arrow heads. (D) A dendritic complexity is shown in one location of the whole-mount retina. The curving and stretching processes and the multiple subdivided dendritic branches are displayed. IR, immunoreactive. (A and B) Scale bar = 50 μm, (C and D) Scale bar = 10 μm.
Table 1.
The density of melanopsin-IR RGCs in microbat, R. ferrumequinum.
Fig 7.
Distribution of each melanopsin-IR cell type on whole-mount retinas.
Cell types are represented by different colors and symbols as follows: pink circle (M1c); pink asterisk (M1d); yellow circle (M2); green circle (M3c); green asterisk (M3d); blue circle (M3c-crv); and a sky-blue X (unidentified cells). Two different retinas were used to show the distribution of cell types. (A, B) and (C, D) show the same areas in different retinas. (B, D) Display the whole-mount retina showing the different cell types. IR, immunoreactive. Scale bar = 50 μm.
Table 2.
Density of each melanopsin-IR cell type in the R. ferrumequinum retina.
Table 3.
The total number of neurons in the GCL and total optic nerve axons in the R. ferrumequinum.
Fig 8.
Electron micrograph from sections of R. ferrumequinum optic nerve.
Large and small myelinated axons are shown. The two images were observed at different magnifications: (A) 100× objective; (B) 700× direct mag, 6680× print mag. The whole optic nerve is shown. (A) Scale bar = 20 μm. (B) Scale bar = 2 μm.