Non-image Forming Light Detection by Melanopsin, Rhodopsin, and Long-Middlewave (L/W) Cone Opsin in the Subterranean Blind Mole Rat, Spalax Ehrenbergi: Immunohistochemical Characterization, Distribution, and Connectivity

Abstract

The blind mole rat, Spalax ehrenbergi, can, despite severely degenerated eyes covered by fur, entrain to the daily light/dark cycle and adapt to seasonal changes due to an intact circadian timing system. The present study demonstrates that the Spalax retina contains a photoreceptor layer, an outer nuclear layer (ONL), an outer plexiform layer (OPL), an inner nuclear layer (INL), an inner plexiform layer (IPL), and a ganglion cell layer (GCL). By immunohistochemistry, the number of melanopsin (mRGCs) and non-melanopsin bearing retinal ganglion cells was analyzed in detail. Using the ganglion cell marker RNA-binding protein with multiple splicing (RBPMS) it was shown that the Spalax eye contains 890 ± 62 RGCs. Of these, 87% (752 ± 40) contain melanopsin (cell density 788 melanopsin RGCs/mm(2)). The remaining RGCs were shown to co-store Brn3a and calretinin. The melanopsin cells were located mainly in the GCL with projections forming two dendritic plexuses located in the inner part of the IPL and in the OPL. Few melanopsin dendrites were also found in the ONL. The Spalax retina is rich in rhodopsin and long/middle wave (L/M) cone opsin bearing photoreceptor cells. By using Ctbp2 as a marker for ribbon synapses, both rods and L/M cone ribbons containing pedicles in the OPL were found in close apposition with melanopsin dendrites in the outer plexus suggesting direct synaptic contact. A subset of cone bipolar cells and all photoreceptor cells contain recoverin while a subset of bipolar and amacrine cells contain calretinin. The calretinin expressing amacrine cells seemed to form synaptic contacts with rhodopsin containing photoreceptor cells in the OPL and contacts with melanopsin cell bodies and dendrites in the IPL. The study demonstrates the complex retinal circuitry used by the Spalax to detect light, and provides evidence for both melanopsin and non-melanopsin projecting pathways to the brain.

Keywords: Brn3a; Ctbp2; L/M cone opsins; RBPMS; amacrine cells; bipolar cells; calretinin; rhodopsin.

Figure 1

Confocal photomicrographs of melanopsin (green) RGC (mRGCs) and DAPI nuclear counterstaining (red) in the Spalax retina (A–C). Melanopsin RGCs were located in the ganglion cell layer (GCL) and few displaced RGCs were found in the inner nuclear cell layer (INL) (indicated by arrowhead). mRGCs projected mainly into the IPL, but also to the outer plexiform layer (OPL) (exemplified by open arrowhead) where they formed an outer plexus (indicated by single arrows in A–C). Few processes were also seen in the outer nuclear layer (ONL) (indicated by double arrows in A,B). (D–I) Ganglion cell marker RBPMS (red) in combination with melanopsin (green) and DAPI nuclear counterstaining (blue) in sagittal sections (D–F) and horizontal sections through the GCL (G–I). Scale bars: (A); 40 μm, (B,C); 15 μm, (D–F); 100 μm, (G–I); 50 μm.

Figure 2

Dendritic arborization of mRGCs in Spalax retina. (A) Representative image from a region of Spalax whole-mount retina was reconstructed in 3D after labeling with anti-melanopsin antibody. (B) Representative drawings of the dendritic field and soma of mRGCs from the 3D reconstruction picture in (A). (C) Minimal polygons fitted around the dendritic profile of each reconstructed mRGC in (B). Note the complexity of the mRGCs dendritic plexus. Scale bar: 100 μm.

Figure 3

Melanopsin (green) RGCs (mRGCs) and DAPI nuclear counterstaining (red) in flatmount Spalax retina. XYZ images of the ganglion cell layer (GCL) (A), inner plexiform/inner nuclear cell layer (IPL/INL) (B), and outer plexiform layer (OPL) (C) showing melanopsin immunoreactivity in a minor rest of the GCL (arrows in A) and a displaced mRGC in the INL (arrow in B) and the melanopsin plexus in the OPL in (C) (indicated by arrows). Note the projections X-Z and Y-Z showing the different sublayers. Scale bars: (A–C); 30 μm.

Figure 4

Representative retinal section of PACAP (red) and melanopsin (green) co-expressed in mRGCs (A–C). Note that PACAP and melanopsin were found in RGCs (indicated by thin arrows in B,C) and in distal dendrites in the inner nuclear layer (INL) and outer plexiform later (OPL) (indicated by double arrowheads in A–C). Calretinin (red) was found in the subpopulation of non-melanopsin RGCs and in amacrine cells in the INL. Brn3a (blue) was found in all non-melanopsin RGCs co-storing calretinin (D–F) (nuclear counterstaining with DAPI in gray in D). Panel (E) shows the frame indicated in (D) of the optic nerve containing melanopsin and calretinin positive axons. Panel (F) Shows Brn3a in non-mRGCs in high magnification. Scale bars: (A–C); 30 μm, (D); 50 μm, (E,F); 15 μm. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear layer.

Figure 5

Melanopsin (green), L/M cone opsin (red) and rhodopsin (blue) and DAPI counter staining (gray) in Spalax (A–E). Melanopsin was expressed in the RGCs located in the GCL and in the INL (B). L/M cone opsin was expressed in photoreceptor cells in the ONL, and displaced in the INL (A,C,E). Rhodopsin was expressed in photoreceptor cells located in the ONL and in displaced cells in the INL (A,D,E). The strongest immunostaining in both rods and cones was found in the degenerated outer segment (arrows in C–E). Scale bars: (A); 70 μm, (B–E); 35 μm. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear cell layer, OPL; outer plexiform layer, ONL; outer nuclear cell layer, OS; outer (photoreceptor) segment.

Figure 6

Melanopsin dendrites were found in close apposition with rods and cones indicating synaptic contact. Melanopsin (green), L/M cone opsin (red) and rhodopsin (blue/gray) stainings in Spalax (A). Rod and cone opsins were expressed in the cell membrane of the soma, in the inner segment and in the outer segments (B–D). In the OPL melanopsin dendritic processes are found in close apposition with rod pedicles (framed area in A is shown in high power in B) and co-localization indicating synaptic contacts (determined by IMARIS® colocalization module) as shown in purple. Ribbon containing synapses visualized by Ctbp2 (white in C,D can be found in L/M-cone pedicles of the OPL). Likely synaptic contact between L/M cone pedicles and melanopsin distal processes can be found in the OPL. The frame area in C shown L/M cone opsin photoreceptor cells costoring Ctbp2. By using IMARIS colocalization module overlap of L/M cone opsin, CtBp2, and melanopsin are indicated by arrows in (D) and in the insert in Panel (D) (yellow dots corresponding to the area in C indicated by ^*). Double arrows in (D) show the degenerated outer segment in two L/M-cone photoreceptor cells. The grid in Panel (B–D) indicates that the image was created in 3D. Scale bars: (A); 40 μm, (B); 5 μm, (C); 50 μm, (D); 10 μm and insert in (D); 4 μm. INL; inner nuclear cell layer, OPL; outer plexiform layer, ONL; outer nuclear cell layer.

Figure 7

Melanopsin (green) RGCs are innervated by recoverin (red) expressing bipolar cells in the IPL/GCL (yellow frame in A and in higher magnification in H,I) and have direct synaptic contact with recoverin containing photoreceptor cells in the OPL expressing Ctbp2, a marker for synaptic ribbons (white) (white frame in A and ultra-high magnification in C,G). Panels (A–D) and panels (H,I) are 3D reconstructions representing 65 sections separated by 0.2 μm. For better illustration, single digital section (E–G) representing x-y, x-z, and y-z views are shown of the distal melanopsin process (indicated by arrowhead in E,G) and contact with recoverin containg photoreceptor pedicles (indicated by double arrow heads) also containing the synaptic marker Ctbp2 (shown in white in F). The synaptic contacts in which melanopsin have direct overlap with the co-localization of recoverin/Ctbp2 are indicated by arrows in blue (C–G). Synaptic contact between melanopsin and recoverin containing bipolar terminals co-storing Ctbp2 are indicated by arrows in blue in I. Scale bars: (A); 10 μm, (B,C), and (H,I); 5 μm, (D), (E–G); 2 μm.

Figure 8

Melanopsin (green) RGCs are innervated by calretinin expressing amacrine cells (red). (A) Calretinin immunoreactivity in the Spalax was found in amacrine cells in the inner part of the INL and in cells of the outer part of the INL, which could be horizontal cells and in processes innervating the OPL in close contact with rod photoreceptors (blue). In the IPL/GCL calretinin processes have close contact to melanopsin processes. Using the IMARIS co-localization module on 73 digital sections separated by 0.2 μm used for 3D reconstruction co-localization indicating synaptic contact was found between melanopsin soma and proximal processes and calretinin axons. Panels (D,E) are 3D images and for better illustration, single digital section (F–I) representing x-y, x-z, and y-z views are shown of the melanopsin cell indicated by ^* as well as single channels. The calculated co-localization of melanopsin and calretinin in I indicated by arrows. Scale bars: (A); 50 μm, (B), and (D); 20 μm, (C); 15 μm, (E) and (G); 10 μm., (F–I); 5 μm. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear cell layer, OPL; outer plexiform layer, ONL; outer nuclear cell layer.