Morphology and immunoreactivity of retrogradely double-labeled ganglion cells in the mouse retina

Abstract

Purpose: To examine the specificity and reliability of a retrograde double-labeling technique that was recently established for identification of retinal ganglion cells (GCs) and to characterize the morphology of displaced (d)GCs (dGs).

Methods: A mixture of the gap-junction-impermeable dye Lucifer yellow (LY) and the permeable dye neurobiotin (NB) was applied to the optic nerve stump for retrograde labeling of GCs and the cells coupled with them. A confocal microscope was adopted for morphologic observation.

Results: GCs were identified by LY labeling, and they were all clearly labeled by NB. Cells coupled to GCs contained a weak NB signal but no LY. LY and NB revealed axon bundles, somas and dendrites of GCs. The retrogradely identified GCs numbered approximately 50,000 per retina, and they constituted 44% of the total neurons in the ganglion cell layer (GCL). Somas of retrogradely identified dGs were usually negative for glycine, ChAT (choline acetyltransferase), bNOS (brain-type nitric oxidase), GAD (glutamate decarboxylase), and glial markers, and occasionally, they were weakly GABA-positive. dGs averaged 760 per retina and composed 1.7% of total GCs. Sixteen morphologic subtypes of dGs were encountered, three of which were distinct from known GCs. dGs sent dendrites to either sublaminas of the IPL, mostly sublamina a.

Conclusions: The retrograde labeling is reliable for identification of GCs. dGs participate in ON and OFF light pathways but favor the OFF pathway. ChAT, bNOS, glycine, and GAD remain reliable AC markers in the GCL. GCs may couple to GABAergic ACs, and the gap junctions likely pass NB and GABA.

Figure 1.

Retrograde labeling of the mouse retina. (a, b) Confocal micrographs of vertical retinal sections. (a) Low-power micrograph double-labeled by NB (red) and Goα (green). Goα antibody labels the IPL and OPL. NB is largely restricted in GCs. A brightly labeled optic nerve stump is outstanding. (b) Triple-labeling by LY (blue), NB (red), and the nuclear dye TO-PRO-3 (green). In the GCL, GC (purple) and AC (green) somas were usually arranged in a single layer and they were clearly distinguishable because of the labeling. Scale bar: (a) 100 μm; (b) 20 μm.

Figure 2.

Glial cells and GCs in the mouse retina. (a–c) Labeling with NB (red), LY (blue), and glial cell markers (green). (d₁, d₂) Tripled labeling with NB (red), TO-PRO-3 (blue, or white in the insets), and MHC II (green). (a₁) Confocal micrograph from a flat-mounted mouse retina, showing the morphology of astrocytes (GFAP-IR). In the same area, astrocyte somas (double arrows in a₁) are nearly invisible in the focal plane of GC somas (a₂). (b) Stacked confocal micrograph from a vertical retinal slice. Astrocytes were mostly located in a layer separated from the GCL (a, b), except those processes of astrocytes wrapping the blood vessels (). (c) A confocal micrograph from a vertical retinal slice shows that Müller cell somas (GS-IR) are located in the INL and are not to be confused with somas in the GCL. (d₁, d₂) Micrographs of a flat-mounted retina focused on the GCL (d₁) and IPL (d₂). TO-PRO-3 labeled nuclei (d₂, inset; d₁, arrow) of microglia cells (MHC II-IR) are more intense than that in the GCs (d₁, open arrow). NB and LY do not clearly reveal astrocytes, microglia, and Müller cells. GFAP-, GS- and MHC II-IR were not found in retrogradely labeled GCs. IPL, the inner plexiform layer; INL, the inner nuclear layer. Scale bar, 20 μm.

Figure 3.

Observation of retrogradely identified GCs and total neurons in the GCL. Stacked confocal micrographs were taken from the same area over the GCL, which had been stained with TO-PRO-3 (a, black: c, green) and retrograde dye NB (b, black; c red). GC somas were nearly absent in a blood vessel (b, ), where quite a few nuclei (12.5%, 88/705 nuclei) were revealed by TO-PRO-3 (a, c). These nuclei were either spindle shaped, as is typical of endothelial nuclei, or oval. They were not counted as neurons. Scale bar, 20 μm.

Figure 4.

Soma size of GCs in the GCL. (a) Low-power confocal micrograph of a flat-mounted retina retrogradely labeled with LY and NB (black), focusing on the GCL. (a) A progressive decrease of GC density toward the peripheral retina is obvious. The average GC soma size (major axis) in the central retina was significantly smaller than that in the peripheral retina (b). The soma size is well fit by a normal Gaussian function (line).

Figure 5.

Immunocytology of dGCs. Stacked confocal micrographs of flat-mounted retinas were triple-labeled with retrograde dyes and specific antibodies, focusing on the INL. dGCs, LY (yellow) and NB (blue) double-labeled (a–c, green) or brightly NB labeled (d–f, blue), are often negative for GABA (a, pink, e, yellow), glycine (d, yellow), ChAT (f, yellow), and bNOS (b, d, e, pink), but they are frequently found to accumulate 5-HT (c, pink, arrows and the inset showing tripled-labeled GCs in black and green). (a, arrow and inset) a GC strongly LY- and NB-positive but weakly GABA-IR; (a–c, open arrow) tracer-coupled ACs (blue); (e, arrows, inset) ACs double-labeled for bNOS and GABA (red). Scale bar: 20 μm.

Figure 6.

Expression of GAD in the mouse retina. A confocal micrograph from a vertical retinal section labeled by NB (blue), LY (yellow), and GAD (pink) shows that retrogradely labeled GCs (green) were not colocalized with GAD-IR. GAD primarily labeled IPL, OPL, and somas in the proximal INL. Scale bar, 20 μm.

Figure 7.

Topographic distribution of dGCs in the INL. A flat-mounted whole retina labeled by LY (black). Stacked confocal micrographs were taken from the INL and recomposed. dGCs were counted, and their soma locations were depicted with dots. dGCs were preferentially distributed in the temporal and peripheral retina.

Figure 8.

Comparison of retrogradely labeled dGCs in whole-mounted retinas and retinal slices. (a) Image scanning with a confocal microscope. A set of consecutive optical sections in x–y plane (ai and aii) is obtained first and is reconstructed into a single three-dimensional image (aiii). (b, c, e) Retinas are double labeled by LY and NB (white). (b) An x–y view of a stacked image of a dGC from a whole-mounted retina; an x–z view (x:y:z = 1:1:2) of the cell is depicted in (c). (e) A stacked image of a dGC in a retinal slice, whose soma size, ramification level of dendrites, and dendritic field are similar to those of the cell in (c). The morphology of the cell in (b) and the cell in (e) are manually traced and presented in (d) and (f), respectively; but they are scaled down to fit the space. (c, e, g, arrow) axon. Scale bar, 20 μm.

Figure 9.

Morphology of dGCs in vertical sections. Stacked confocal micrographs of the 16 subtypes of dGC in retinal slices double-labeled by LY and NB (white). The images optimize the visibility of the GC dendrites, thus axon bundles and some GC somas, especially in thicker stacks of images, are saturated. Dendritic fields of some cells are not fully visible because of image trimming. dGCs have somas in the IPL (7) and INL. Their axons are visible (arrow). (5, ) A diffuse dGC. Scale bar, 20 μm.

Figure 10.

Morphology of dGCs in flat-mounted retinas. The 16 subtypes of dGCs retrogradely labeled by LY and NB are included: dG1 to -16. The morphology of the cells was manually traced from stacked confocal micrographs. Their axons are clear in x–z views of the stacked images (Fig. 8) but not clear in x–y views. Scale bar, 20 μm.

Figure 11.

Morphology of bistratified (dG13) and tristratified (dG14) dGCs in flat-mounted retinas. The cells were manually traced from x–y views of stacked confocal micrographs labeled by LY and NB. For dG13 (a), the first level of dendrites is near 25% of the IPL depth (red) and the second (blue) is near 70% of the IPL depth. For dG14 (b), the first (red), second (blue), and third (green) levels of dendrites are near 10%, 40%, and 80% of the IPL depth, respectively. Scale bar, 20 μm.

Figure 12.

Summary of 16 morphologic types of dGCs in the mouse retina. dGCs are named dG1 to -16. Their dendritic arbors are arranged in an artificial 10-layer IPL. Dendritic arbors in IPL are presented in strata with 100% as the proximal margin (nearest the GCL) of the IPL. Most GC dendrites are stratified in the IPL, in three levels. Monostratified cells have only one level (the first level) of dendrites. The soma size illustrated in the artificial IPL is relevant to the original size, but the dendritic fields are not in some cells. Soma size: large (L), >17.5 μm; medium (M), 13 to 17.5 μm; and small (S), <13 μm. Dendritic field size: large (L), >400 μm; medium-large (M_L), ≤400 μm and > 250 μm; medium-small (M_S), 100 to 250 μm; and small (S), <100 μm; −, not applicable; Y, observed; N, rarely found; arrows axons.