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. 2019 Jan 2;39(1):78-95.
doi: 10.1523/JNEUROSCI.1784-18.2018. Epub 2018 Oct 30.

Molecular Fingerprinting of On-Off Direction-Selective Retinal Ganglion Cells Across Species and Relevance to Primate Visual Circuits

Onkar S Dhande  1 , Benjamin K Stafford  2 , Katrin Franke  3   4 , Rana El-Danaf  2 , Kumiko A Percival  5 , Ann H Phan  2 , Peichao Li  6 , Bryan J Hansen  6 , Phong L Nguyen  2 , Philipp Berens  3   4 , W Rowland Taylor  5 , Edward Callaway  6 , Thomas Euler  7   8 , Andrew D Huberman  1   7   8   9
Affiliations

Molecular Fingerprinting of On-Off Direction-Selective Retinal Ganglion Cells Across Species and Relevance to Primate Visual Circuits

Onkar S Dhande et al. J Neurosci. .

Abstract

The ability to detect moving objects is an ethologically salient function. Direction-selective neurons have been identified in the retina, thalamus, and cortex of many species, but their homology has remained unclear. For instance, it is unknown whether direction-selective retinal ganglion cells (DSGCs) exist in primates and, if so, whether they are the equivalent to mouse and rabbit DSGCs. Here, we used a molecular/circuit approach in both sexes to address these issues. In mice, we identify the transcription factor Satb2 (special AT-rich sequence-binding protein 2) as a selective marker for three RGC types: On-Off DSGCs encoding motion in either the anterior or posterior direction, a newly identified type of Off-DSGC, and an Off-sustained RGC type. In rabbits, we find that expression of Satb2 is conserved in On-Off DSGCs; however, it has evolved to include On-Off DSGCs encoding upward and downward motion in addition to anterior and posterior motion. Next, we show that macaque RGCs express Satb2 most likely in a single type. We used rabies virus-based circuit-mapping tools to reveal the identity of macaque Satb2-RGCs and discovered that their dendritic arbors are relatively large and monostratified. Together, these data indicate Satb2-expressing On-Off DSGCs are likely not present in the primate retina. Moreover, if DSGCs are present in the primate retina, it is unlikely that they express Satb2.SIGNIFICANCE STATEMENT The ability to detect object motion is a fundamental feature of almost all visual systems. Here, we identify a novel marker for retinal ganglion cells encoding directional motion that is evolutionarily conserved in mice and rabbits, but not in primates. We show in macaque monkeys that retinal ganglion cells (RGCs) that express this marker comprise a single type and are morphologically distinct from mouse and rabbit direction-selective RGCs. Our findings indicate that On-Off direction-selective retinal neurons may have evolutionarily diverged in primates and more generally provide novel insight into the identity and organization of primate parallel visual pathways.

Keywords: direction selectivity; mouse vision; primate; retina; retinal ganglion cells; visual circuits.

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Figures

Figure 1.
Figure 1.
The vast majority of posterior-tuned On–Off DSGCs express Satb2. A, Schematic of logic for molecularly identifying On–Off DSGCs in the mice, rabbits, and primates. B–C″, Virtually all Trhr-RGCs (B–B″, green) and Drd4-RGCs (C–C″, green) express Satb2 (magenta). White arrowheads indicate colocalization of GFP and Satb2 and dashed circles indicate lack thereof. D–E″, DSGCs tuned for upward motion (Hb9-RGCs, D–D″, green) and those DSGCs that form the accessory optic system (Hoxd10-RGCs, E–E″, green) do not express Satb2 (magenta). F, Quantification of expression of Satb2 by different types of DSGCs. A, Anterior; P, posterior; U, upward; D, downward; PR, photoreceptors; BC, bipolar cell; AC, amacrine cell; IPL, inner plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer. Scale bars, 10 μm in B″, C″, D″, and E″. Figure 1-1 demonstrates the molecular analysis of Satb2-RGCs.
Figure 2.
Figure 2.
Satb2 is preferentially enriched in On–Off DSGCs encoding motion along the anterior–posterior axis. A–A″, En face view of a dye-filled posterior-tuned On–Off DSGC (A). The cell was identified physiologically based on directional tuning to drifting gratings (A′, top) and based on On–Off light responses (A′, bottom). The dendrites of the filled cell (red) co-stratify with starburst amacrine cell dendrites (ChAT, A″ left, green), a hallmark of DSGCs. The recorded DSGC expressed Satb2 (A″, right). B–D″, Same as in A for an upward-tuned On–Off DSGC that did not express Satb2 (B), an anterior-tuned On–Off DSGC that expressed Satb2 (C), and a downward-tuned On–Off DSGC that did not express Satb2 (D). A, Anterior; P, posterior; U, upward; D, downward; ChAT, choline acetyltransferase; PR, photoreceptor; BC, bipolar cell; AC, amacrine cell; IPL, inner plexiform layer; RGC, retinal ganglion cell. Scale bars, 50 μm (A–D); 25 μm (A″, B″, C″, D″).
Figure 3.
Figure 3.
Satb2 expression is conserved in rabbit On–Off DSGCs. A–A″, Colocalization of Satb2 (magenta, A) with RBPMS marker (green, A′) in the rabbit retina. B, En face view of a dye-filled posterior-tuned On–Off DSGC. C, D, Cell in B was identified physiologically based on directional tuning to drifting gratings (C) and based on On–Off light responses (D). E, Dendrites of the filled cell (red) co-stratify with starburst amacrine cell dendrites (ChAT, green). F, Recorded posterior-tuned rabbit DSGC (red) expressed Satb2 (cyan). G–I, Example of a recorded, dye-filled and Satb2 stained Off-sustained RGC. This non-DSGC does not express Satb2 (I). A, Anterior; P, posterior; U, upward; D, downward; ChAT, choline acetyltransferase. Scale bars, 25 μm (A″), 50 μm (B, G).
Figure 4.
Figure 4.
Satb2 is expressed by a restricted subset of macaque RGCs. A, Photomicrograph of an example macaque retina (left) compared with a mouse retina (right). B–B″, Satb2 (red) expression completely overlaps with the expression of RBPMS, a RGC-specific marker (cyan), in the macaque retina. White arrowheads indicate colocalization of Satb2 and RBPMS. C–C″, Example density plot of Satb2-RGCs. Schematic (C) shows area of Satb2-immunostained-retina imaged (blue) relative to major retinal landmarks [fovea: black circle; optic nerve head (ONH): pink circle]. Satb2-RGC (blue dots) locations plotted within a 1 mm wide strip along the temporal-nasal axis (C′). Insets show Satb2-RGCs (blue dots) within the peripheral (∼8 mm) to central (∼4 mm) retina (C″). D, Quantification of Satb2-RGC density as a function of retinal eccentricity. E, Distribution of the distances between nearest-neighbor Satb2-RGCs. F, Regularity index of Satb2-RGC (from this study) compared with parasol RGCs and melanopsin RGCs from Liao et al. (2016). Scale bars, 2 mm (A); 10 μm (B″).
Figure 5.
Figure 5.
Macaque Satb2-RGCs are synaptically connected to geniculate neurons. A, Schematic demonstrating injection of G-deleted rabies virus (Rb-ΔG) encoding fluorescent proteins in the dLGN. A′, A″, Local infection/spread of Rb-ΔG-GFP (green) within the dLGN (A″). dLGN layers visualized by VGlut2 staining (A′, white). B–B″, Injection of Rb-ΔG-GFP in dLGN results in infection of RGCs (B) that form synapses with geniculate neurons. Satb2 (magenta, B′) is expressed by some dLGN projecting RGCs (green, B). White arrowheads indicate colocalization of GFP and Satb2 and dashed circles indicate lack thereof. C, Example en face view of Satb2-RGC morphology recovered from rabies GFP infection. D–D″, Higher magnification of Satb2-RGC soma (D, green) expressing Satb2 (D′, magenta). E, 3D reconstruction of Satb2-RGC dendritic morphology (black) shown in C. Soma and axon shown in red. XFP: GFP or mCherry fluorescent protein; M, magnocellular layer; P, parvocellular layer. Scale bars, 1 mm (A″), 25 μm (B″), 100 μm (C, E).
Figure 6.
Figure 6.
Parasol, midget, and melanopsin RGCs in the macaque retina do not express Satb2. A, B, Example en face view of On parasol RGC (A) and Off midget RGC (B) morphology recovered from rabies GFP infection. Insets show lack of expression of Satb2. C, Example en face view of melanopsin RGC morphology recovered from rabies GFP infection. Inset (left) shows lack of expression of Satb2. D–D″, RGCs expressing melanopsin photopigment (B, cyan) do not express Satb2 (B′, magenta). Scale bars, 100 μm (C); 50 μm (A); 10 μm (B, D″).
Figure 7.
Figure 7.
Macaque Satb2-RGCs are morphologically divergent from mouse On–Off DSGCs. A–D, Example en face reconstructions of Satb2-RGC dendrites recovered from rabies GFP infection from three different retinal eccentricities [(central (B), midperipheral (C), and peripheral (D)]. B–D, Location of Satb2-RGCs schematized in A. E, Example en face reconstruction of dendrites of parasol (left) and midget (right) RGCs. F, Quantification of the dendritic diameter of Satb2-RGCs. G, Quantification of dendritic complexity as measured by Sholl analysis and the average number of branch points (inset). H, Side view of dendrites of Satb2-RGC shown in D demonstrating that the dendrites of Satb2-RGCs stratify close to the inner nuclear layer. I, Examples of z-projection of Off parasol, Off midget, and Satb2-RGC dendrites recovered from rabies XFP infection (green) within the inner plexiform layer (IPL). The intensity profile (green line) of the dendrite (plotted to the left) throughout the IPL is shown. Nonspecific background fluorescent signal in the tissue is marked by a white arrows. J, Quantification of Satb2-RGC dendritic stratification depth within the inner plexiform layer. ONH, Optic nerve head; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm (D). *p < 0.05; **p < 0.01; ***p < 0.001 Student's t test. Figure 7-1 demonstrates exemplars of the maximum intensity projection and corresponding reconstruction of Satb2-RGCs.
Figure 8.
Figure 8.
Mouse Satb2-RGCs comprise three functionally distinct RGC types. A, Schematic of imaging setup for recording light-evoked calcium signals form RGCs. B, En face view of ganglion cell layer cells labeled with the synthetic calcium indicator Oregon Green BAPTA-1 (green) after bulk electroporation and blood vessels visualized with sulforhodamine-101 (red). White crosses indicate Satb2-expressing RGCs shown in C. Responses of cells highlighted with blue and magenta circles are shown in D1 and F1, respectively. C Experimental retinal tissue in B post hoc immunostained for RBPMS (green) and Satb2 (blue), with blood vessels in red. Dotted rectangles outline the two scan fields shown in B. D, Functional clustering of Satb2-RGCs. The majority of Satb2-positive cells were allocated to three functional RGC groups (Baden et al., 2016): On–Off DS (Group 12), Off DS (Group 2), and Off sustained (Group 7). E1, Calcium responses of Satb2-expressing On–Off DSGCs highlighted (blue circle) in B in response to three different light stimuli: full-field chirp, bright bars moving in eight directions (including traces sorted by motion direction and polar plot with vector sum in red), and binary noise for space-time kernels. Single trials are shown in gray, averages in black. E2, Average calcium responses of Satb2 expressing On–Off DSGCs, with SD shading in gray and group average from Baden et al. (2016) in red. The retinocentric polar plot shows the distribution of preferred motion directions of Satb2-RGCs (black) assigned to the On–Off DSGC group (Group 12). The preferred motion directions of cells that were not Satb2 expressing are shown in gray. F, G, Calcium responses of Satb2-expressing Off DSGCs (F1, F2) and Satb2-expressing Off-sustained RGCs (G1, G2). The preferred motion directions of cells that were not Satb2-expressing are shown in gray (F2). Scale bars, 20 μm (A, B).
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