Fig 1.
Transverse sections of the Monodelphis domestica retina.
(A) The electron micrograph of an ultrathin transverse section shows a typical retinal layering as seen in nocturnal placental mammals. The thickest layer is the outer nuclear layer (ONL), containing the photoreceptor somata. (B) Immunolabeling of a transverse cryostat section for rod opsin (yellow) shows the densely packed rod outer segments; counterstaining with DAPI (blue) shows the retinal layers. (C) Double-immunolabeling of a transverse cryostat section for shortwave-sensitive SWS1 (green) and middle-to-longwave-sensitive LWS cone opsin (red) shows the opsin-containing cone outer segments of the sparse cone populations; counterstaining with DAPI (blue). Images in (B) and (C) are maximum intensity projections of confocal image stacks. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer, GCL, ganglion cell layer. Scale bar in (B) applies to (B, C).
Fig 2.
PKCα-ir bipolar cells in M. domestica.
(A-C) Double-immunolabeling for PKCα and ChAT. (A) PKCα immunostaining labels bipolar cells with dendrites in the OPL, somata in the INL and thick axon terminations in the IPL. (B) ChAT immunostaining labels the two populations of cholinergic amacrine cells, one with somata in the INL and dendrites stratifying at the boundary between strata S1 and S2 of the IPL, and one with somata in the GCL and dendrites stratifying in S4. (C) The merged image of the two labels localizes the majority of PKCα-ir axon terminals to stratum S5, but there also are some side-branches and en passant varicosities above the inner ChAT band (two indicated by arrowheads). (D-F) Double-immunolabeling for PKCα and the general ON bipolar cell marker Gγ13. (D) Same bipolar cell morphologies as in (A). (E) The Gγ13 label shows that presumed ON bipolar cell axon terminals occupy strata S3-S5 of the IPL. (F) The merged image shows that the axon terminals and varicosities of the PKCα-ir presumed RBCs occupy S3-S5, characterizing them as presumed ON cells. Counterstaining with DAPI (blue) localizes the PKCα-ir somata to the outer part of the INL. The white IPL tick marks in (C, F) divide the IPL into five strata S1 (top) to S5 (bottom) of equal width, the white tick marks in (E) divide the IPL into the presumed functional OFF (S1-S2) and ON (S3-S5) sublayers. Abbreviations as in Fig 1. Images are maximum intensity projections of confocal image stacks. The inner ChAT band in (B, C) appears broader than the outer one, and there are apparent ChAT-ir structures that also are PKCα-ir. Some of this may be a projection artifact, because single-labeled structures that are located behind each other in the z-axis appear to be double-labeled in the projection plane. Some of the apparent double-label is bleedthrough between the fluorescence filters, because it is strongest in the most intensely PKCα-ir elements. Scale bar applies to (A-F).
Fig 3.
PKCα-ir bipolar cells in Sminthopsis crassicaudata.
(A-C) Double-immunolabeling for PKCα and ChAT. (A) PKCα immunostaining labels bipolar cells with dendrites in the OPL, somata in the INL and globular axon terminations in the IPL. The axon terminals and varicosities form at least two bands in the IPL. (B) ChAT labeling of the two cholinergic amacrine cell populations, one with somata in the INL and dendrites stratifying at the S1/S2 boundary of the IPL, and one with somata in the GCL and dendrites stratifying in S4. Counterstaining with DAPI (blue) shows the retinal layers and localizes the PKCα-ir somata to the outer part of the INL. (C) The merged image of the two labels localizes the PKCα-ir axon terminals and varicosities below and above the inner ChAT band. (D-F) Immunolabeling for PKCα and the general ON bipolar cell marker Gγ13. The antisera used here were both from rabbit, hence double-immunolabeling was not feasible and single-labeling was performed on equivalent sections. (D) Same bipolar cell morphologies as in (A). (E) Gγ13 label shows that presumed ON bipolar cell axon terminals occupy strata S3-S5 of the IPL. (F) Overlay (montage) of (D, E) shows that the axon terminals and varicosities of the PKCα-ir presumed RBCs also occupy S3-S5, characterizing them as presumed ON cells. White IPL tick marks as in Fig 2, abbreviations as in Fig 1. Images are maximum intensity projections of confocal image stacks. Scale bar applies to (A-F).
Fig 4.
Synaptic connections of PKCα-ir bipolar cells with photoreceptors in M. domestica (A-E) and S. crassicaudata retinae (F). (A) Fluorescence double-labeling for PKCα (green) and the synaptic ribbon marker CtBP2 (red). In the OPL, PKCα-ir bipolar cell dendrites terminate in close proximity to the single-ribbon rod spherules (two marked by white arrows), but commonly not to the multi-ribbon cone pedicles (yellow arrow). (B) Electron micrograph of rod spherules (RS) in the OPL, where PKCα-ir dendrites (black staining) form invaginating contacts with the rod spherules (arrowheads). However, we did not observe any PKCα-ir dendrites in close proximity to the ribbon band of rod spherules (arrows). Nuc, rod nuclei in the ONL. (C-E) Double-labeling for PKCα (green) and CtBP2 (red) in flatmounted retina, where the focus is on the OPL. (C) The CtBP2 label shows the larger single ribbons of rod spherules and the somewhat smaller, clustered ribbons of two cone pedicles (positions marked in D). (D) Dense plexus of PKCα-ir dendrites, mostly sparing the position of the cone pedicles (circles). However, one dendrite ends at a cone pedicle (arrowhead). (E) The merge of (C, D) shows this dendritic tip in close proximity to cone ribbons, suggesting synaptic contact. Many of the other dendrites terminate close to rod ribbons. (F) Double-labeling for PKCα (green) and CtBP2 (red) in a S. crassicaudata retinal section, showing the PKCα-ir dendrites in close proximity to the single-ribbon rod spherules (three marked by white arrows), but not to the multi-ribbon cone pedicles (four marked by yellow arrows).
Fig 5.
Synaptic connections of PKCα-ir RBCs with calretinin-ir AII cells.
(A) Calretinin immunolabeling reveals amacrine cells with the typical bistratified AII morphology of processes in the outer IPL (arrow heads) and in the inner IPL (arrows). (B-D) Triple immuno-labeling for PKCα, calretinin and CtBP2. (B) Overview showing that RBC axon terminals and their en passant varicosities (green) are in close apposition to AII cell processes (red; CtBP2 label not displayed). (C, D) Magnified view of the two fields outlined in (B), showing all three labels. Labeled axon terminals have ribbon synapses, hence the CtBP2 label (white) at the contact points between RBCs and AII cells indicate that these are synaptic contacts (some marked by arrows). White CtBP2 puncta not associated with contact points between RBCs and AII cells are ribbon synapses of cone bipolar cells with ganglion cells or other amacrine cells. (B-D) is a single focus image from a confocal image stack. The IPL is divided into five equal strata S1 –S5. All scale bars are 10 μm.
Fig 6.
Flat view of the photoreceptor pattern in M. domestica retina.
Differential interference contrast image (DIC) focused on the photoreceptor inner and outer segments in a flatmounted retina. The cobblestone-like mosaic of smaller profiles are the rods, the sparse larger profiles are the cones (some indicated by arrowheads). The twin arrowheads point to a double cone. As the photoreceptor outer segments were slightly squashed by the coverslip, they are bent sideways in patches, blurring the image. Rod density could be determined in the small patches where the rods are seen face-on. The retina was somewhat undulated, hence the focal plane of the micrograph grazes different levels of the photoreceptors, showing larger cone cross-sections in the upper when compared to the lower part of the image.
Fig 7.
Flat view of RBC and AII cell populations in M. domestica.
The images show one field in a retina double-labeled for PKCα and calretinin. (A) Focus on the densely packed RBC somata in the outer part of the INL; (B) focus on the vertically running RBC axons in the inner part of the INL; (C) focus on the AII somata in the inner part of the INL. Images are maximum intensity projections of confocal image stacks. The scale bar applies to (A-C).
Fig 8.
Density distributions of RBCs and AII cells in M. domestica retinae.
For the two analyzed retinae md1 and md2, densities in the sampling fields were entered in photographs of the retinal flatmounts. Each number pair is positioned at the location of the sampling field, the top number is the AII cell density and the bottom number the RBC density in cells/mm². The broken red lines mark the temporal-nasal (T-N) and dorsal-ventral (D-V) transects along which the data of Fig 9 were sampled. The scale bar applies to both retinae.
Fig 9.
Gradients of RBC densities, AII densities and RBC/AII ratios across M. domestica retina.
For retinae md1 and md2, the graphs show the density and ratio changes in relation to distance from the optic nerve head along the temporal-nasal (T-N) and dorsal-ventral (D-V) transects marked in Fig 8.
Fig 10.
Comparison of mammalian RBC morphologies.
Drawings of RBCs of the gray short-tailed opossum Monodelphis domestica and the fat-tailed dunnart Sminthopsis crassicaudata (PKCα immunolabel, present study), of the house mouse Mus musculus (dye-injected cell, modified from Fig 8 in [45]), and of Seba's short-tailed bat Carollia perspicillata (dye-injected cell, modified from Fig 7 in [40]). The gray lines divide the IPL into five equal strata S1 –S5. In Monodelphis and mouse, the OFF (outer) sublayer of the IPL is made up of the top two strata S1 and S2 (gray shading), the ON (inner) sublayer is made up of strata S3–S5. In Sminthopsis, the OFF sublayer includes S1 and a large part of S2, and in Carollia, it includes S1 and only a small part of S2. The positions of the outer (OFF) and the inner (ON) band of cholinergic amacrine cell processes (ChAT bands) are indicated in red. Drawings are scaled to equal IPL thickness.