Retinal ganglion cells with distinct directional preferences differ in molecular identity, structure, and central projections

Abstract

The retina contains ganglion cells (RGCs) that respond selectively to objects moving in particular directions. Individual members of a group of ON-OFF direction-selective RGCs (ooDSGCs) detect stimuli moving in one of four directions: ventral, dorsal, nasal, or temporal. Despite this physiological diversity, little is known about subtype-specific differences in structure, molecular identity, and projections. To seek such differences, we characterized mouse transgenic lines that selectively mark ooDSGCs preferring ventral or nasal motion as well as a line that marks both ventral- and dorsal-preferring subsets. We then used the lines to identify cell surface molecules, including Cadherin 6, CollagenXXVα1, and Matrix metalloprotease 17, that are selectively expressed by distinct subsets of ooDSGCs. We also identify a neuropeptide, CART (cocaine- and amphetamine-regulated transcript), that distinguishes all ooDSGCs from other RGCs. Together, this panel of endogenous and transgenic markers distinguishes the four ooDSGC subsets. Patterns of molecular diversification occur before eye opening and are therefore experience independent. They may help to explain how the four subsets obtain distinct inputs. We also demonstrate differences among subsets in their dendritic patterns within the retina and their axonal projections to the brain. Differences in projections indicate that information about motion in different directions is sent to different destinations.

Figure 1.

Transgenic markers of ooDSGCs that prefer different directions. A, Responses of a BD-RGC to a white rectangle moving across the receptive field center in eight different directions at 575 μm/s. Average responses are displayed in a polar plot and surrounding traces show raster plots for seven repeats. Note ON and OFF phases of spiking in rasters. V, Ventral; D, dorsal; N, nasal; T, temporal. B, Preferred directions of BD-RGCs recorded from <60% eccentricity. Arrow length indicates the extent of direction selectivity, calculated as in Kim et al. (2010). C, Preferred directions of W9-RGCs (black arrows) and DRD4-RGCs (gray arrows). D, E, Retina whole mount showing BD-RGCs (red) and DRD4-RGCs (green), in triple transgenic mouse (DRD4-GFP × FSTL4-CreER × ROSA-CAGS-STOP-tdTomato). Blue box in D shows region enlarged in E. RGCs express either green or red fluorescent proteins, but not both, demonstrating that DRD4- and BD-RGCs are distinct populations.

Figure 2.

Relationship between structure and function of ooDSGCs. A, B, Morphology of W9 RGCs does not correlate with their preferred direction. Confocal stack z-projection showing two W9 cells that were injected with Lucifer yellow following recording. Scale bar, 100 μm. C, DRD-4-RGC filled with Lucifer yellow following recording. Arrow indicates preferred direction, which is distinct from the orientation of its dendritic asymmetry. D, Sketch of part of a whole mounted retina (see inset at bottom left) showing dendritic asymmetry of the BD-RGCs. Arrows originate from the somas and point in the direction of dendritic asymmetry. Length of arrow is proportional to degree of dendritic asymmetry. Dot, Optic disc. E, Micrograph of two BD-RGCs from the retina. Scale bar: (in E) C, E, 100 μm. F, Polar plot summarizing the dendritic asymmetry of BD-RGCs from a retina similar to that shown in D and E. G, Relationship between dendritic asymmetry and direction selectivity of 22 BD-RGCs. The preferred direction is plotted relative to the direction of the dendritic arbor (dot). Black lines indicate dorsal-preferring cells from the dorsal margin of the retina.

Figure 3.

Molecular markers for subsets of ooDSGCs. A, B, In situ hybridization for Cdh6 RNA (red) combined with anti-GFP antibody staining (green) reveals expression of Cdh6 in BD- but not DRD4-RGCs. Yellow arrows in A–H indicate double-labeled cells. Red arrows in A–H indicate marker-positive cells that do not express GFP. C, D, In situ hybridization for Col25a1 RNA (red) shows expression in GFP-labeled (green) BD-RGCs but not DRD4-RGCs. E, F, In situ hybridization for Mmp17 RNA (red) shows expression in GFP-labeled (green) DRD4- but not BD-RGCs. G, H, CART is a marker of both BD- and DRD4-RGCs as shown by immunostaining with anti-CART (red) and anti-GFP (green). Laminae marked in H (INL, inner nuclear layer; IPL, inner plexiform layer) apply to A–H, and K. Scale bars: 10 μm. I, J, Quantification of the fraction of RGCs expressing Cdh6, Col25a1, or Mmp17 at P14 (I) and at P7 (J) (n ≥ 44 for BD-RGCs, ≥104 for DRD4-RGCs, ≥1198 for all RGCs). K, MMP17 immunoreactivity in section from P14 retina. Antibody labels the putative starburst layers of the IPL, suggesting immunoreactivity in the dendrites of starburst amacrines and/or ooDSGCs. A subset of RGC cell bodies (arrows) as well as putative starburst amacrines (arrowheads) are labeled in the GCL and INL. Scale bar, 25 μm.

Figure 4.

ISH for Drd4 RNA in P14 retina of wild-type or DRD4-GFP transgenic mice. A, Wild-type mice show Drd4 expression in photoreceptors in the outer nuclear layer (ONL) and in a subset of inner nuclear layer (INL) cells. Very low levels of transcript are detected in the GCL. B, DRD4-GFP transgenic mice show the same pattern of transcripts in photoreceptors and INL as in A but also exhibit Drd4 signals in DRD4-GFP-positive RGCs (arrowheads). The difference in labeling between wild-type and transgenic mice suggests that Drd4 transcripts in DRD4-RGCs of DRD4-GFP mice arise from the transgene, not the endogenous Drd4 gene. Scale bar, 25 μm.

Figure 5.

CART antibody labels ooDSGCs. A, CART and anti-GFP immunostaining identify double-positive RGCs in a retinal whole mount from line YFP-H. GFP channel shows the morphology of a YFP⁺ CART immunoreactive RGC (arrow; z-projection of confocal stack). CART channel (A′) shows CART⁺ RGCs in a single confocal plane through the GCL. Arrow indicates the soma of the YFP⁺CART⁺ double-positive cell. B, Morphological analysis of RGCs in line YFP-H that were CART⁺ and CART⁻ (n = 140). All CART-immunoreactive RGCs (n = 22) have the bistratified morphology of ooDSGCs. By contrast, none of the CART⁻ cells showed this morphology. C, D, Single confocal planes through the cell shown in A reveal morphological features that identify it as an ooDSGC. The cell dendrites (green) are bistratified, with both the ON (C) and OFF arbors (D) cofasciculating with the choline acetyltransferase (ChAT)-positive processes of starburst amacrines (red). E, Rotation of a 3-D reconstruction of the dendrites of this cell show that it has bistratified projections to the OFF and ON starburst IPL sublaminae (arowheads). GFP (green) and ChAT (red) channels are shown. Scale bars: 20 μm.

Figure 6.

Cadherin 6-positive ooDSGCs prefer vertical motion. A, Cdh6-RGCs and starburst amacrine cells labeled with YFP (green) in retina sections from a cdh6 knock-in heterozygote (Cdh6-CreER × Thy1-stop-YFP). Choline acetyltransferase (ChAT) (red) labels starburst amacrine cell somas and dendrites. Cdh6-RGC dendrites project to starburst IPL layers. Blue, Fluorescent Nissl stain. B, Retina whole mount from a cdh6 knock-in heterozygote. C, Responses of Cdh6-RGCs, showing that the vast majority prefer dorsal or ventral motion (n = 23 cells from 7 retinas, 7 mice). D, Molecular markers expressed by Cdh6-RGCs. Scale bars: A, 50 μm; B, 200 μm.

Figure 7.

Lamina-specific projections of different ooDSGC functional subtypes. A, Cholera toxin subunit B (CTB)-labeled axons in the dLGN arising from the contralateral (red) and ipsilateral (blue) eyes. A′, CART⁺ axons (green) overlap with CTB-labeled axons from the contralateral eye (red). B, After monocular enucleation, the CART⁺ axon terminals (green) disappear from the dLGN contralateral to the eye removed, indicating that these terminals originate from RGCs. C, Projections of CART⁺ RGCs to superior colliculus (SC). After monocular enucleation CART⁺ axons (green) are present only in the SC contralateral to the remaining eye. CTB-labeled axons in the contralateral (left) SC (red) overlap with CART⁺ axon terminals. No CART⁺ RGC terminals are seen in the CTB-labeled ipsilateral projection (right). D, BD- (red) and DRD4-RGC (green) axons in the dLGN. Overlay in D″ shows distinct laminar targeting of the two RGC subsets. E, BD- (red) and DRD4-RGC (green) axons in the SC. Scale bars: A–D, 200 μm; E, 400 μm.

Figure 8.

ooDSGCs project to the vLGN and the pretectal system. A, BD-RGC axons (green) arborize in the vLGN. A′, Merge of CTB (red) and BD-RGC (green) axons. B, DRD4-RGC axons (green) arborize in the vLGN. B′, Merge of CTB (red) and DRD4-RGC (green) axons. C, BD-RGC axons (green) project to the MTN. Blue, fluorescent Nissl stain. D, DRD4-RGC axons (green) are not present in the MTN. D′, Merge of DRD4-RGC (green) and CTB (red) axons in the MTN. Blue, Fluorescent Nissl stain. E, BD-RGC axons (green) arborize in the nucleus of the optic tract (NOT; arrowhead). CTB (red) labels the optic tract and the nucleus. F, DRD4 axons (green) are absent from the NOT (arrowhead), labeled with CTB (red). Scale bars: A–E, 200 μm.

Figure 9.

Markers that identify the four ooDSGC subsets and distinguish ooDSGCs from other RGCs. The schematic includes genes identified in the microarray analysis (CART, Col25a1, Cdh6, and Mmp17) as well as the transgenes, which do not correspond to endogenous genes(BD, DRD4, and W9). *Some of the Cdh6-RGCs ooDSGCs that prefer temporal motion may be Mmp17 positive.