
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

39190352
202405138
10.1073/pnas.2405138121
datasetDatasetresearch-articleResearch ArticleneuroNeuroscience424
Biological Sciences
Neuroscience
A circuit motif for color in the human foveal retina
Kim Yeon Jin a https://orcid.org/0000-0002-6722-4063

Packer Orin a https://orcid.org/0000-0002-0885-0689

Dacey Dennis M. dmd@uw.edu
a b 1 https://orcid.org/0000-0001-8476-5585

aDepartment of Biological Structure, University of Washington, Seattle, WA 98195
bWashington National Primate Research Center, University of Washington, Seattle, WA 98195
1To whom correspondence may be addressed. Email: dmd@uw.edu.
Edited by Edward Callaway, University of California San Diego, La Jolla, CA; received March 12, 2024; accepted June 25, 2024

27 8 2024
3 9 2024
27 8 2024
121 36 e240513812112 3 2024
25 6 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

The human visual process begins in the fovea where diverse circuitries for color, form, and motion perception arise in an intricate synaptic network, whose details remain largely unknown. Here, we apply connectomic reconstruction of color-coding pathways that originate from the foveal blue cone bipolar cell interneuron. We find that circuits classically identified as red-green and blue-yellow are combined in an excitatory circuit motif that could play a singular role in human trichromatic color vision. Moreover, our findings suggest complete wiring diagrams of the human fovea are within reach and essential to identifying the neural circuits that start vision.

The neural pathways that start human color vision begin in the complex synaptic network of the foveal retina where signals originating in long (L), middle (M), and short (S) wavelength-sensitive cone photoreceptor types are compared through antagonistic interactions, referred to as opponency. In nonhuman primates, two cone opponent pathways are well established: an L vs. M cone circuit linked to the midget ganglion cell type, often called the red-green pathway, and an S vs. L + M cone circuit linked to the small bistratified ganglion cell type, often called the blue-yellow pathway. These pathways have been taken to correspond in human vision to cardinal directions in a trichromatic color space, providing the parallel inputs to higher-level color processing. Yet linking cone opponency in the nonhuman primate retina to color mechanisms in human vision has proven particularly difficult. Here, we apply connectomic reconstruction to the human foveal retina to trace parallel excitatory synaptic outputs from the S-ON (or “blue-cone”) bipolar cell to the small bistratified cell and two additional ganglion cell types: a large bistratified ganglion cell and a subpopulation of ON-midget ganglion cells, whose synaptic connections suggest a significant and unique role in color vision. These two ganglion cell types are postsynaptic to both S-ON and L vs. M opponent midget bipolar cells and thus define excitatory pathways in the foveal retina that merge the cardinal red-green and blue-yellow circuits, with the potential for trichromatic cone opponency at the first stage of human vision.

neural circuitry
human vision
color vision
connectomics
HHS | NIH | National Eye Institute (NEI) 100000053 EY-028282 Dennis Dacey
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pmcCirca 1980, the prevailing view for parallel visual pathway origins seemed driven toward a simple, unifying scheme whereby retinal ganglion cell properties were collapsed into three classes called X, Y, and W (1), but see also ref. 2. By contrast, the current view celebrates the complexities of cell type diversity, with nearly 40 visual pathways now described in the mouse retina (3, 4). Yet, this more advanced picture also remains a puzzle to interpret functionally, and visual neuroscience may have come full circle, returning to an early, and seminal, hypothesis (5, 6) that the multiplicity of postreceptoral visual pathways serve as complex feature detectors (7).

Human vision has a clear origin in the cells and circuits of the retina, yet the number and nature of the parallel pathways that talk to the brain remains difficult to access (8–10). Unlike in other mammals, in most human and nonhuman primates, color vision is trichromatic, originating in three cone photoreceptor types maximally sensitive at long (L), middle (M), and short (S) wavelengths (11–13). Chromatic processing is initiated in the retinal circuitry when L, M, and S cone signals are compared through antagonistic interactions, referred to as cone opponency (14). In nonhuman primates, two cone opponent pathways are well established: an L vs. M cone circuit, often referred to as the red-green pathway, linked to the midget ganglion cell type (15–17, e.g., ref. 18) and an S vs. L+M cone circuit often referred to as the blue-yellow pathway, linked to the small bistratified ganglion cell type (19–24). Together these cone opponent pathways have been taken to delineate cardinal directions in a trichromatic color space. Problems arise from this classical view, specifically with regard to retinal mechanisms and visual pathway origins. The first is that the midget ganglion cells must play a key role in both achromatic spatial vision and red-green color vision. By virtue of their private-line connectivity to single L or M cones, the foveal midget circuit sets the limit on the highest achromatic spatial resolution afforded by the photoreceptor mosaic (25) and also transmits a red-green chromatic signal (15, 17, 26–28). However, cortical mechanisms for extracting spatial and chromatic information from the midget pathway remain obscure, and a primary role for the midget circuit in chromatic processing has been questioned (29–31).

There is growing evidence for additional subcortical chromatic pathways that potentially complicates any simple link between retinal circuitry and color vision. In the macaque monkey retina, these additional chromatic pathways originate at the S cone. S cones comprise a minority 5 to 10% of the total cone population. This low sampling density is matched to the optical blurring of short wavelengths under normal viewing conditions, setting up the S cone’s limited use for achromatic spatial resolution and primary role in color vision (32). A foveal circuit linking S cones to OFF-midget bipolar cells and in turn to OFF-midget ganglion cells has been characterized by electron microscopic reconstruction from serial sections (33, 34). This midget circuit is clearly not an anatomical OFF counterpart of the small bistratified, S-ON pathway, and its physiological properties and are not well understood (34, 35). Recently, we used connectomic reconstruction to identify an S-OFF midget circuit in the human foveal retina (36). However, in the human retina, S cone circuit connectivity was distinctive, implying the possible merging of L vs. M opponency with S vs. LM opponency (see also refs. 18 and 34).

A second unanticipated chromatic pathway arising from the S cones was first revealed by targeting ganglion cells retrogradely labeled from tracer injections placed in the LGN of macaque (37). These cells were bistratified at the same depths in the IPL as the small bistratified cells but showed much larger dendritic field sizes (Fig. 1 A and C) and they appeared similar in morphology to a ganglion cell type previously observed in the human retina (38, 39; Fig. 1D). Like the small bistratified cell, these large bistratified cells also responded to selective modulation of the S cone with an ON-type response (Fig. 1B). The response to L+M cone modulation was originally interpreted as an OFF-dominated response (37) but more recent, albeit limited recordings from the macaque large bistratified cells show a consistent ON–OFF type response (Fig. 1B and SI Appendix, Fig. S4) implying that L and/or M cone input occurs both in phase (synergistic) and out of phase (antagonistic) with the S-ON component.

Fig. 1. Dendritic morphology and light response of the large vs. small bistratified cell types in macaque and human retina. (A) Large and small bistratified cells both stratify at the same depths in the inner plexiform layer (Inset, IPL; GCL, ganglion cell layer border, INL, inner nuclear layer border). However, the dendritic tree of the large bistratified cell is larger, more sparsely branching, and mostly lacks the lobulated appendages that adorn the small bistratified dendrites. For both examples, the outer-stratifying dendritic branches are shown in red, inner branches in black. Both cells are from the retinal periphery in the macaque monkey. (B) The small bistratified cell (Upper traces) shows a sustained ON-response to selective S cone modulation (Left) and a sustained OFF-response to selective L+M cone modulation (Right). The large bistratified cell (Lower) also shows an S-ON light response, but the response to L+M cone modulation displays both ON and OFF response components. (C) Dendritic field size change with distance from the fovea (eccentricity) for the large (red symbols) and small bistratified cells (gold symbols) in macaque retina. (D) Similar data plotted for the human retina, which suggested that in humans, the large bistratified cell type is larger than its macaque counterpart. Data (A and C) from ref. 40, which is licensed under CC BY 4.0.; data in the Upper panel in (B) replotted from ref. 41; data in (D) replotted from ref. 38.

These data lead to a proposed circuitry for the large bistratified ganglion cell. First, dendritic costratification with the small bistratified cell, together with the S-ON light response suggests that the large bistratified cell would also receive a direct excitatory synaptic input from S-ON or “blue cone” bipolar (BB) cells. Second, the differences in the light response to L+M cone stimuli for the large vs. small bistratified cells predict that the wiring for this part of the circuit will be dissimilar. In the present study, we tested these hypotheses in three separate volumes of the human foveal retina by reconstructions of the BB cell output to ganglion cell types. We found, in addition to the small bistratified ganglion cell, two additional ganglion cell types postsynaptic to the BB cell, these are the large bistratified cell and a subpopulation of ON-midget ganglion cells. Reconstructions further revealed that both the large bistratified and ON-midget ganglion cells (but not the small bistratified cells) also received a major input from midget bipolar cells. Since the midget bipolar cells are expected to show L vs. M cone or red-green opponency both the large bistratified cell and some ON-midget ganglion cells would combine an S-ON input with L vs. M cone opponency revealing an unexpected circuit motif for color processing in the human foveal retina.

Results

Identification of the Primary S Cone Circuit and Small Bistratified Ganglion Cell Mosaic in the Human Foveal Retina.

In the macaque monkey retina, virtually all synapses made by BB cells on ganglion cell dendrites are directed at the small bistratified ganglion cell type (21). However, the S-ON light response for the large bistratified cell, together with its dendritic costratification with the small bistratified cell (Fig. 1) (37), suggests a direct input from BB cells. To determine whether large bistratified ganglion cells received input from BB cells, we reconstructed ganglion cells postsynaptic to BB cells in the human foveal retina. To do this, we first had to unequivocally identify the cell types of the primary S-ON pathway: the S cones, the BB cells, and the small bistratified ganglion cells.

We identified S cone circuitry in three retinal volumes from different locations within the central retina of a single donor (samples included both left and right eye foveas) (Materials and Methods and SI Appendix, Figs. S1–S3). One of these volumes (~600 µm retinal eccentricity) was used previously to compare aspects of S cone circuitry in marmoset, macaque, and human retinas to identify the human S-OFF midget pathway (36). At this retinal location S cone pedicles were relatively abundant, comprising ~9% of the total. In the present results, the two additional volumes were taken from more central locations on the foveal slope located at about 250 to 400 µm eccentricity, where cone pedicles first appear, displaced laterally from the foveal center (SI Appendix, Figs. S1–S3). At these locations, S cones were lower in density (15 of 316 total cone pedicles; 4.7% in one foveal volume) and irregularly distributed (see also ref. 42). Human S cones were more difficult to identify than their counterparts in either macaque or marmoset where the S cone pedicle is morphologically distinctive (34, 43). Thus, to reliably identify the S cone circuitry, we targeted the unique synaptic relationship of both S cones and small bistratified ganglion cells to the BB cells (Fig. 2 and SI Appendix, Fig. S5).

Fig. 2. Identification of a large field bistratified ganglion cell type postsynaptic to S-ON bipolar (BB) cells. (A) A single BB cell (light blue) identified by its distinctive dendritic morphology and targeted connection with a sparsely distributed population of cone synaptic pedicles (violet; S cone 6) together with its axonal stratification deep in the IPL near the border with the GCL where it synapsed profusely with the small bistratified ganglion cell (SBGC, gold). This unique conjunction of morphological features established the identity of the three main cell types of the S-ON pathway: S cones, S-ON, or “blue-cone” bipolar (BB) cells, and small bistratified ganglion cells (SI Appendix, Fig. S5). To the right and overlapping this small bistratified cell is another bistratified ganglion cell (red) that received input from this BB cell; we identified this as a large bistratified cell based on its larger dendritic field and sparse input from 4 BB cells (SI Appendix, Table S3). (B and C) Reconstructions of the ganglion cell dendrites postsynaptic to the BB cell synaptic ribbons (small red filled arrowheads) revealed many dyad synapses occupied by two ganglion cell dendrites; most of these dendrites belonged to small bistratified ganglion cells (SBGC, white open arrowheads) but a small percentage, mostly of smaller caliber, arose from the large bistratified ganglion cell (LBGC, red open arrowheads) whose dendrites tended to be finer and more sparsely branching.

In the macaque monkey retina, BB cell dendrites extend long branches that seek out and densely contact the widely spaced S cone pedicles, forming all invaginating, central elements, at the S cone synaptic triad (44). A comparable pattern was present for the human BB cell type (Fig. 2A and SI Appendix, Fig. S5B). This connection, unlike the midget circuit linked to each L and M cone of the central retina (45–47) is not a private line connection; the sparsely distributed S cone pedicles can be presynaptic to up to four BB cells (48). In the macaque retina, the axonal arbor of the BB cells is likewise distinctive, stratifying near the inner border of the inner plexiform layer (IPL) where the small bistratified ganglion cell receives convergent excitatory input from multiple BB cells (21, 34, 35). This connectivity is also a distinctive feature of the BB cell in the human foveal retina (Fig. 2A and SI Appendix, Fig. S5 C and D).

The relative sparseness of S cones in the human foveal retina gives rise to an unusual spatial arrangement of the postreceptoral circuitry. The small bistratified ganglion cell dendritic trees postsynaptic to BB cells are relatively large and variable in shape, forming a highly distorted mosaic (SI Appendix, Fig. S6A). The explanation for this appears to lie in their selective connectivity to the reduced number of BB cell axon terminals, which do not tile the retina evenly and uniformly but are widely spaced, forming a patchy distribution (SI Appendix, Fig. S6B). In effect, some dendrites of neighboring small bistratified ganglion cells extend for long distances to receive selective input from the widely spaced axon terminals of the BB cells (dotted blue lines, SI Appendix, Fig. S6C). This connectivity creates a mosaic of ganglion cell dendritic trees that are very irregular in size and shape, with some cells extending dendrites long distances to connect densely to a given BB cell axon terminal. The result is not only an irregular mosaic of unusually large dendritic trees, but a distinctive clustering of ganglion cell terminal dendrites around single BB cell axon terminals (e.g., red arrowheads in SI Appendix, Fig. S6 A–C).

A characteristic feature of the BB cell output is a high density of dyad synapses in which two small bistratified dendrites (often arising from neighboring small bistratified cells) are postsynaptic at the ribbon; nearly 90% of the synaptic output of the BB cell was to the small bistratified cell (mean ± SD = 88.7 ± 11.3%, n = 16; Table 1 and Fig. 2C). Contacts at dyad synapses with amacrine cells were also identified but were not quantified in the current study. To determine the remaining output of BB cells to other ganglion cell types we reconstructed the postsynaptic dendrites that did not originate from small bistratified ganglion cells. We found that these synapses are made with two additional ganglion cell types, identified as the large bistratified and ON-midget ganglion cell types.

Table 1. Relative density of synaptic output from S-ON bipolar cells (blue cone bipolars, BBs) to identified ganglion cell dendrites (SBGC, small bistratified ganglion cells; IMGC, inner-ON midget ganglion cells; LBGC, large bistratified ganglion cells)

		SBGCs (%)	ON MGs (%)	LBGCs (%)	
1	BB 3	0.707	0.276	0.017	
2	BB 5	0.611	0.333	0.056	
3	BB 7	0.830	0.151	0.019	
4	BB 9	0.765	0.235	0.000	
5	BB 12	0.870	0.130	0.000	
6	BB 15	0.940	0.060	0.000	
7	BB 16	0.848	0.152	0.000	
8	BB 17	0.891	0.087	0.022	
9	BB 4	0.975	0.000	0.025	
10	BB 6	1.000	0.000	0.000	
11	BB 8	0.984	0.000	0.016	
12	BB 13	0.929	0.000	0.071	
13	BB 14	0.891	0.000	0.109	
14	BB 19	1.000	0.000	0.000	
15	BB 24	0.947	0.000	0.053	
16	BB 20	1.000	0.000	0.000	
AVERAGE		0.887	0.089 (0.178)	0.024 (0.043)	
STD		0.113	0.112 (0.095)	0.032 (0.032)	
Data are from the volume at ~300 µm from the foveal center (volume 2 to 300; see also SI Appendix, Fig. S2). The mean and SD for only those ON-midget ganglion cells (n = 8) and large bistratified ganglion (n = 9) cells that received input from S-ON bipolar cells is given are parentheses at the bottom.

S-ON Bipolar Input to the Large Bistratified Ganglion Cell Type.

Synaptic output from BB cells to non-small bistratified ganglion cells comprised ~11.3% of the total synapses averaged across the sample of 16 cells (3 BB cells synapsed exclusively with small bistratified cells; Table 1, volume at ~300 µm from the foveal center). One of the additional ganglion cell types postsynaptic to BB cells is also bistratified at the same IPL depths as the small bistratified ganglion cell, but shows a larger, more sparsely branching dendritic tree (Figs. 2A and 3 A and B). We identified this cell as the large bistratified type (38). This identification follows from the current classification of ganglion cell types in the primate retina (40, 49) where, aside from the small and large bistratified types, the only other bistratified ganglion cell recognized is the recursive bistratified cell, which stratifies narrowly near the center of the IPL, shows a distinctive morphology, and corresponds to the ON–OFF direction selective type (40). The BB input to these large bistratified cells was sparsely distributed (Fig. 3). Of the 16 bipolar cells sampled, 7 did not contact a large bistratified cell, and of those that did, only 4.3% of the total synaptic output was directed at this ganglion cell type (mean ± SD = 4.3 ± 3.2%, n = 9; Table 1).

Fig. 3. ON and OFF midget bipolar inputs sum with S-ON bipolar inputs to the large bistratified ganglion cell dendritic tree. (A) The dendritic tree of a small bistratified cell (SBGC, gold) and a part of the dendritic tree of a large bistratified cell (LBGC, red) are shown in horizontal view with the locations of synaptic inputs (50 total) from 12 BB cells to the LBGC indicated by the small blue balls (SI Appendix, Table S5). (B) The ganglion cell dendritic trees shown in A are rotated 90 deg to provide a vertical view of the bistratification in the IPL. The distribution of synaptic inputs from invaginating-ON midget bipolar cells (IMB; small green balls; 43 synapses from 31 IMB cells) and flat-OFF midget bipolar cells (FMB; small yellow balls; 33 synapses from 16 FMB cells) to the LBGC are shown; IMB synapses are present deep in the IPL overlapping with synapses from BB cells; FMB synapses appear on the outer dendrites in the outer third of the IPL. An additional 60 synapses from a number of distinct ON and OFF “diffuse” bipolar types were also annotated (Dataset S1). (C) Ultrastructure of IMB synaptic ribbon input (small red filled arrowheads) to the large bistratified cell. IMB terminals (green) are large and globular and invested with the large dendritic terminals of an inner-ON midget ganglion cell (IMGC). (D and E) Finer processes of the large bistratified cell (LBGC, white open arrowheads) are often postsynaptic at dyad synapses made by midget bipolar with midget ganglion cells (zoomed views of red boxes in C). (F and G) Ultrastructure of FMB synaptic ribbon input. Like IMB, FMB terminals are also large and globular and dominated by synapses with an outer-OFF midget ganglion cell (OMGC); and show a similar motif at dyad synapses with the smaller large bistratified cell dendrites (LBGC, white open arrowheads). (H and I) Zoomed view of red boxes shown in (F and G).

Although a synapse from any single BB cell to a large bistratified cell is infrequent, their large dendritic trees collected convergent input from several BB cells (SI Appendix, Tables S3–S5). This connectivity is evident from reconstructions of the total BB cell input to three large bistratified cells, each from different retinal volumes; two of these volumes (SI Appendix, Figs. S1 and S2) were 200 to 300 µm retinal eccentricity where the laterally displaced cone pedicles arise from cones at the foveal center (50–52). At this central location, S cones and BB cells are very sparsely distributed (SI Appendix, Fig. S6C) and each of two large bistratified cells collected input from 4 BB cells (SI Appendix, Tables S3 and S4), comprising 7.1 and 13.0% of the total bipolar input to these two cells (SI Appendix, Table S1). By contrast, a partial dendritic tree of a third large bistratified cell, located at ~600 µm (SI Appendix, Fig. S3) from the foveal center receives 50 synaptic inputs from 12 different BB cells (SI Appendix, Table S5 and Fig. 3A), making up 26.9% of the total bipolar input to this cell (SI Appendix, Table S1). Thus, the large bistratified cell lacked the selective and targeted connection with the BB cell that is hallmark of the BB-small bistratified pathway, but instead drew a sparse and convergent input over a much larger dendritic tree (Fig. 3 A and B).

The Large Bistratified Cell Is Postsynaptic to ON and OFF Midget Bipolar Cells.

The large bistratified ganglion cell is also distinct from the small bistratified ganglion cell in its connectivity to other bipolar cell types. For the small bistratified ganglion cells most of the excitatory synaptic input to the inner stratifying dendrites originates in the BB cell (mean ± SD = 84.4 ± 15.9% of total, n = 12, SI Appendix, Table S6), with a minor input from ON diffuse bipolar cells (mean ± SD = 11.1 ± 16.2% of total input, n = 12). In addition, we found that the outer dendrites are sparse and input from OFF diffuse bipolar types is minor (mean ± SD = 0.8 ± 2.9% of total input, n = 12). Input from either ON-invaginating (IMB) or OFF-flat (FMB) midget bipolar cells is also rare [mean ± SD = 3.2 ± 5.3% and 0.5 ± 1.8% respectively, n = 12; (see also refs. 21, 23, and 53)]. By contrast, all three large bistratified cells in our sample receive abundant, convergent input from previously identified IMB and FMB cells, comparable in density to the BB cell synapses. IMB cells were presynaptic to inner stratified dendrites comprising ~ 30% of the total input (mean ± SD = 30.3 ± 10.2%, n = 3; Fig. 3 C–E and SI Appendix, Table S1). Similarly, FMB cells provide input to the outer dendrites (mean ± SD = 11.0 ± 8.2%, n = 3; Fig. 3 F–H and SI Appendix, Table S1). In common with the synapses from BB cells, synapses from IMB and FMB cells are sparsely distributed and derive from many bipolar cells (21 and 23 from the two foveal large bistratified cells and 47 from the larger cell shown in Fig. 3 A and B and SI Appendix, Tables S3–S5). We can infer that due to their high density, many midget bipolar cells would not be positioned to contact and synapse with the sparsely branching dendrites of the large bistratified ganglion cells. However, more data are needed to determine the proportion of midget bipolar cells that provide input to the large bistratified cell. Finally, the large bistratified cells are also postsynaptic to other non-midget or “diffuse” bipolar types, both inner and outer stratifying, which, taken together, also formed a major input to these cells (mean ± SD = 42.9 ± 19%, n = 3, SI Appendix, Table S1). We did not attempt to identify the number of distinct bipolar cell types in this group at this time or analyze them further.

The convergent input from many midget bipolar cells to large bistratified ganglion cells is unexpected since previous reconstructions from the macaque retina show that synaptic output from midget bipolar cells to ganglion cell types other than midget ganglion cells is either absent or negligible (34, 45, 54, 55). Previous reconstructions of human parafoveal midget circuits (56) agree with this conclusion. Again, since these sparse connections are not the dominant synaptic output of midget bipolars, it is uncertain whether they were overlooked previously in the macaque retina.

ON-Midget Ganglion Cells Receive S-ON Bipolar Input.

A third ganglion cell type postsynaptic to BB cells was identified as the inner stratifying ON midget ganglion cell (IMGC) by their uniquely small dendritic tree sizes and dominant input from a single midget bipolar cell postsynaptic to an L or M cone (Fig. 4A and SI Appendix, Tables S2 and S7). Only ~9% of the BB cells in our sample (mean ± SD = 8.9 ± 11.2%; n = 16; Table 1) synapsed with an inner-ON midget ganglion cell, but when these contacts occur, they comprise 17.8% of the output of that BB cell (mean ± SD = 17.8 ± 9.5%; n = 8; Table 1). To judge the functional significance of this connection, we asked what the relative weight is of this synaptic contact for the individual inner-ON midget ganglion cells that receive it, and for the midget ganglion cells as a population. We first determined the relative input strength (number of ribbon synaptic inputs expressed as a % of total ribbon synaptic input) of IMB and BB cells to inner-ON midget ganglion cells that received BB input in our sample (11 of 25 cells). We found that the BB cells provided ~14% (mean ± SD = 13.7 ± 9.2%, n = 11; SI Appendix, Table S2) of the total input to these midget ganglion cells (IMB synapses: mean ± SD = 84.7 ± 11.5%, n = 11; diffuse bipolar synapses: 1.6 ± 3.2%, n = 11). In addition, the BB input was spatially restricted with each midget ganglion cell postsynaptic to a single BB cell (SI Appendix, Table S7; see also Fig. 4). This connectivity contrasts with that for the large bistratified cell where input from many BB cells converge across the large dendritic tree (SI Appendix, Tables S3–S5). Because of the sparse distribution of BB axon terminals in the foveal retina, it appeared that only a minority of midget ganglion cells receives this input from BB cells. At one retinal location, we sampled all the inner-ON midget ganglion cells in a small patch and found that 2 of 16 cells receive a BB cell input. Such a direct, albeit infrequent, input from BB cells to inner-ON midget ganglion cells could account for results from a much larger sample taken from multielectrode array recordings in the macaque monkey retinal periphery that showed a small but measurable input from S cones for about 15% of the ON-midget ganglion cells (18). However, to understand whether this reflects a consistent connection for a distinctive minority population of ON-midget ganglion cells in the human fovea requires a complete connectome of all S cone bipolars and midget ganglion cells across a larger area of the retina and awaits further study.

Fig. 4. S-ON bipolar cells are also presynaptic to a sparse population of ON-midget ganglion cells. Many inner-ON midget ganglion cells (IMGCs) do not receive an input from the BB cell, but when this synapse occurs it provides a substantial fraction of the input to a given ON-midget ganglion cell (SI Appendix, Tables S1 and S2). (A) In this example, a single BB cell (light blue; partial transparency) is presynaptic to two neighboring inner-ON midget ganglion cells (IMGC 1, violet, and IMGC 2, gold). The ganglion cells receive 15.9% and 19.1% of their total bipolar input from this BB cell (ribbon synapses indicated by the small red balls) respectively (the remaining 84.1 and 80.9% arise from L or M cone connecting midget bipolar cells) (SI Appendix, Table S2). (B–D) These three panels show examples of BB cell (light blue) synaptic inputs (small red filled arrowheads) to IMGC 1 and IMGC 2 (white open arrowheads) in addition to inputs to small bistratified ganglion cell dendrites (SBGCs, black open arrowheads).

Discussion

The bipolar cell connectivity of the large bistratified ganglion cell type shown here is consistent with what is currently known about this cell type’s light-evoked response to cone type-selective stimuli in the macaque retina (Fig. 1B). In accord with an ON depolarization to selective S cone modulation this ganglion cell type is postsynaptic to S-ON bipolar (BB) cells. The reconstructions in the human retina reveal that while a synapse from a given BB cell to a large bistratified cell dendrite rarely occurs, and is thus difficult to detect, the total input from all the BB cells that overlap the large dendritic tree comprises a significant fraction of the total excitatory input (SI Appendix, Tables S1 and S3–S5).

The additional input to large bistratified cells from both ON and OFF midget bipolar cells is unexpected. In the macaque monkey retina, midget bipolar cells make virtually all synaptic output to midget ganglion cells (34, 45) and the very large dendritic trees of this ganglion cell type would seem an unlikely target for the high resolution, private-line midget pathway. Though this synapse is a relatively minor occurrence, like the connection to BB cells the total input is significant, with midget bipolars comprising nearly half the input to the large bistratified ganglion cell (41.3%; see SI Appendix, Table S1).

The connection from a BB cell to a given ON-midget ganglion cell is also sparse, perhaps occurring in 10 to 15% of the ON-midget ganglion cells, but when the connection occurs, multiple BB ribbon synapses, always arising from a single BB cell, provide as great as 36.7% of the total synaptic input to a single ON-midget ganglion cell (mean ± SD = 13.7 ± 9.2%, n = 11, Fig. 4 and SI Appendix, Tables S2 and S7). Thus, from the connectomics alone, it is possible to conclude that for both the large bistratified ganglion cell and the ON-midget ganglion cell, rare, apparently nonselective synapses (by contrast to more selective and powerful synaptic connections) can sum to create functionally distinctive circuitries only revealed by detailed, synapse-by-synapse analysis (Fig. 5A) and that for many ganglion cell types no single bipolar pathway dominates: Rather, the spiking output is shaped by integration of multiple diverse synaptic inputs.

Fig. 5. Origin of parallel chromatic pathways from the S-ON bipolar (BB) cell synaptic output. (A) Reconstruction of the synaptic output of a single BB cell (ribbon synapse locations indicated by small balls within the axon terminal), exemplifies the main findings of this study. Most synapses are presynaptic to the small bistratified cell (gold balls; 69 synapses; 78.4% of total). A smaller number of synapses distribute to either the large bistratified ganglion cell (red balls; 12 synapses; 13.6% of total) or the inner-ON midget ganglion cell (green balls, 7 synapses; 8% of total). Note that not every BB cell contacted all three ganglion cell types (Table 1) and that the majority of inner-ON midget ganglion cells do not receive BB cell input, but when this synapse occurs it provides a significant fraction of the total input to a given midget ganglion cell (SI Appendix, Tables S2 and S7). (B–D) Hypothesized receptive field spatiochromatic 2D gaussian profiles for the two novel chromatic pathways, compared to that for the small bistratified cell. (B) Small bistratified cell shows S-ON vs. L+M-OFF cone opponency (Fig. 1B). (C) large bistratified ganglion cell, receptive field center will combine moderate S-ON input with L vs. M opponency derived from midget bipolar input. L vs. M opponency assumes surround cancellation of the weaker cone input to the receptive field surround (for details of mechanism see refs. 15 and 16). (D) ON-midget ganglion cell, S+L vs. M or S+M vs. L opponency, assumes a small S-ON input combined with L vs. M cone opponency (15).

One question this study raises is the degree to which bipolar ribbon synapses that make divergent output to multiple ganglion cell types could be considered equivalent. The general assumption is that the compact bipolar cell axonal arbor would be isopotential and transmitter release in response to a voltage change would be similar at all synapses. However bipolar axon terminals show a complex lobulated morphology and BB bipolars contain ~50 synaptic ribbons. Modulation of the electrotonic properties of axon terminal branches to vary the synaptic output may be possible when the branches are thin (0.3 µm), and the synapses are >10 µm apart. Several lines of recent evidence suggest subcellular functional divergence within a bipolar axon terminal which may be attributed to the presence of voltage-gated channels and/or interaction with specific and localized inhibitory inputs (57–60). Further, glutamate release from bipolar cell axon terminal branches that contain calcium-permeable postsynaptic receptors can be locally modulated by amacrine cells due to the limited diffusion of calcium (58, 61). Thus, the location of BB cell synaptic outputs from different segments of their axonal branches to ON-midget and small and large-bistratified ganglion cells shown in Figs. 2 and 4 could represent as yet unexplored subcellular specializations that might locally alter synaptic output. Finally, given divergent bipolar output to multiple ganglion cells it will be important to recognize the potential impact of variation in the glutamate receptors present at these functionally distinct synaptic loci and the possibility that postsynaptic conductances can be modulated by activity-dependent trafficking of the channels (62).

It is surprising that previous studies of the underlying circuitry for L vs. M opponency in the midget and S vs. L+M opponency in small bistratified circuits showed that antagonistic interactions were fundamentally transmitted by the excitatory bipolar synapses. However, there is a precedent for an amacrine cell to play a critical role in opponent S cone circuitry in the ground squirrel retina (63, 64) and we have not yet explored how amacrine cells might contribute to cone opponency in large bistratified cells. Hypotheses can nevertheless be made about how the excitatory input from midget bipolar cells contributes to the light response of the large bistratified ganglion cell type. Small bistratified cells show a sustained L+M cone-driven OFF response opposing a similarly sustained S-ON light response (Fig. 1B) (22) and this OFF response derives from OFF diffuse bipolar types that contact L and M cones nonselectively (19, 21, 22) (Fig. 5B). However, large bistratified cells in macaque show an ON–OFF type response to L+M cone modulation (Fig. 1B and SI Appendix, Fig. S4). The simplest hypothesis is that this largely reflects the input from ON and OFF midget bipolar cells (as well as diverse ON and OFF diffuse bipolar types). Midget bipolar cells should show strong L vs. M cone opponency, and center-surround receptive field organization (65). The midget bipolar and midget ganglion cell receptive field center are driven primarily by either an L or M cone and a surround derived from some combination of L and M cones. L vs. M cone opponency arises from a probabilistic imbalance of L and M cone input to a small center vs. large surround (15, 16, 27). The hypothesis, which remains to be demonstrated directly, would be some form of L vs. M cone opponency in the large bistratified cell combined with an S-ON input (Fig. 5C). Alternatively, the summation of midget bipolars with L and M cone centers may instead degrade the opponent signal. However, midget ganglion cells in the retinal periphery, like the large bistratified cells in the fovea, collect input from many midget bipolar cells and can retain strong L vs. M opponency (15, 18, 66). It remains unclear however what impact the convergence of both ON and OFF midget bipolar input would have on the L and M cone signal; for example, it could create a form of double opponency with combined L-ON-M-OFF and L-OFF-M-ON response components. In addition, the input from sparsely distributed ON and OFF midget circuits raises the question of whether there is any L or M cone selectivity in this synaptic connection.

If the synaptic connectivity of large bistratified cells found in the human retina is similar in the macaque retina, then recordings from these cells applying L and M cone isolating stimuli along with selective blockade of ON and OFF pathways could begin to address these questions. However, given the possibility of species differences at the circuit level (36), it may also be worthwhile to build realistic, biophysically based neural simulations that incorporate morphology and connectivity (67).

Finally, the same prediction holds for the subset of ON-midget ganglion cells that also receive a direct input from BB cells. These ganglion cells should sum an S-ON input with strong L vs. M cone opponency based on their dominant input from a single midget bipolar cell (Fig. 5D and SI Appendix, Table S7). Thus, for both the large bistratified and ON-midget ganglion cells, the result is a clearly noncardinal merging of an S-ON with L vs. M opponent circuitry. Another commonality is that both cell types should show poor spatial resolution, but for different reasons: the large bistratified cell due to its large dendritic field size and the S cone connected ON-midget cells due to their sparse sampling of the cone mosaic.

Do these results reflect departures from the expected S cone circuitry found in the macaque monkey retina? A previous study provided evidence in the macaque central retina for a large field ganglion cell that received abundant synaptic input from BB cells (68). These cells were described as monostratified and proposed equivalent to the inner Melanopsin-expressing ganglion cells (69, 70). However, previous physiological recordings from identified inner Melanopsin-expressing ganglion cells to selective, high contrast modulation of S-cone output did not reveal an S-ON response (69), whereas large bistratified cells show a consistent S-ON response (see Fig. 1; 37, 41).

These previous results, along with the present finding that the synaptic output from BB cells to a large field ganglion cell was to the S-ON, large bistratified type makes the postulated melanopsin cell observation (68) enigmatic. The Melanopsin and large bistratified cells both show large dendritic fields but otherwise differ substantially in morphology. The inner Melanopsin cell has a large cell body and a very sparsely branched dendritic tree which is narrowly stratified along the inner border of the IPL (70–72). Moreover, the inner melanopsin-expressing ganglion cells receive a major cone bipolar input from the DB6 bipolar type and immunohistochemical staining of synaptic puncta shows that DB6 cells likely provide the entire cone bipolar input to inner melanopsin cells (71). It thus appears highly unlikely that in either the macaque or marmoset retina, inner melanopsin ganglion cells receive significant input from BB cells. This observation would be consistent with the present finding in the human retina that beyond a major output to the small bistratified ganglion cell the remaining output of BB cells is to the large bistratified ganglion cell and to a small subpopulation of ON-midget ganglion cells.

How does the unexpected circuitry of the large bistratified cell and the sparse subpopulation of ON-midget ganglion cells with S cone input fit with current hypotheses about the origins of chromatic signals in the human visual system? Psychophysical studies generally conclude that multiple independent chromatic channels are present, though the number of pathways, their chromatic tuning, and their neural basis all remain uncertain (73–81). Nevertheless, one point of agreement is that at some point along the visual pathway S cone signals must be summed with an L vs. M opponent signal. Moreover, for the red-green channel the S cone contribution is relatively small, and for the blue-yellow channel, the S cone contribution is large (76, 82–84; for example, see figure 14 in refs. 85 and 86). This result mirrors, at least qualitatively, the pathways that emerge from the BB cell synapse. The S-ON small bistratified ganglion cell and the S-OFF foveal midget ganglion cell (34) receive a dominant and selective drive from S cones. By contrast the large bistratified and the ON-midget ganglion cells receive a relatively smaller input from BB cells (15.7 and 13.7% respectively) and a larger input from midget bipolar cells (SI Appendix, Tables S1 and S2), which by virtue of their private-line connectivity, show L vs. M opponency.

The convergence of the two major cone opponent signaling pathways, the BB cell and the midget bipolar, at the dendritic tree of the large bistratified ganglion cell provides a neural locus for a noncardinal chromatic signal in the retina, for an identified LGN projecting pathway (40). On the other hand, a role for this pathway in color processing remains unclear. For example, the large bistratified cell exists at a low density, accounting for an estimated 1.5% of the total ganglion cells in the retinal periphery (40). Thus, these cells would be unlikely to play a role in setting the resolution limit along any chosen dimension of human color vision (87, 88). Indeed, as opposed to the private-line, “double-duty” role posited for the midget pathway in chromatic and achromatic processing (15, 17, 27), the low density and large receptive field constructed from widely distributed S-ON and L vs. M circuitry points to a singular or pure role in color vision.

Classic multistage models for human color vision make the assumption that parallel retinal output falls along the cardinal L vs. M and S cone axes and is combined at the cortical level (reviewed in refs. 85 and 89), possibly as early as primary visual cortex (90). The fundamental assumption of these approaches is a neural representation of color that falls into distinct color opponent categories: red vs. green, blue vs. yellow, linking color appearance to a relatively simple and fixed neural substrate. By contrast, a more recent model assumes that early cone opponency is more related to the constraints of extracting both spatial and chromatic information from the cone mosaic (91) than to fixed pathways for color appearance (92, 93), and unit recordings from extrastriate visual areas reveal cells narrowly tuned to many directions in color space (94, 95). Our connectomics results suggest that this diversity begins to emerge early in the visual pathway.

Returning to the origin of human visual pathways in the multiplicity of retinal ganglion cell types and their distinctive circuitry, a complete accounting of the number and nature of human visual pathways remains to be achieved, though a full connectome for the neural complexity of the human foveal retina may be in reach (96–99), and could reveal not only the total number of visual pathways but lead to discoveries about foveal circuit organization and the nature of individual variability in the human central nervous system (100–102). Regarding circuits that code for color, the division of S-ON signal output into diverse cone-opponent pathways with distinctive circuit motifs suggests that a nanoscale, synaptic level view will be essential for a full understanding of how color is ultimately represented in the human visual system (103, 104).

Materials and Methods

Human Tissue Preparation and Acquisition.

Human eyes (52-y-old, white male; brain-dead organ donor) were acquired at the time of death by surgical enucleation from the Medical University of Vienna, Vienna, Austria. The eyes were immersion fixed for electron microscopy in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH7.3-7.4 for ~1 h at room temperature. Medical history confirmed no abnormalities of the visual system recorded and no driving limitations; the ocular status was defined medically as unremarkable at the time of enucleation.

Serial Block-Face SEM Sample Preparation and Image Acquisition.

Three retinal pieces within the central retina from different locations (~200, ~300, and ~600 µm eccentricities; SI Appendix, Figs. S1–S3) were dissected and prepared for EM and ultrathin sectioning in the vertical plane as described previously (40). The embedded tissue blocks were mounted in a Volumescope SEM (ThermoFisher) for serial block-face scanning electron microscopy. Regions of interest (ROIs) from each block were defined that encompassed the full depth of the retinal circuitry (~200 × 200 µm ROI divided into 40 × 40 µm tiles) from cone pedicles to the ganglion cell layer. The blocks were sectioned at 50 nm (~200 µm: 2902 sections, ~300 µm: 3028 sections, ~600 µm: 1904 sections) and scanned at 5 nm x-y resolution after each section (voxel: 5 × 5 × 50 nm). The resulting set of TIFF images was filtered, contrast normalized, montaged, and aligned using procedures available with TrakEM2 (NIH Fiji) software.

Volume Reconstruction and Analysis.

All cell and circuit reconstructions were also performed using TrakEM2 to first create arbor skeletons; nodes within these skeletons were used to tag pre- and postsynaptic synaptic structures so that the locations and number of synapses for a given cell could be determined. In selected neurons, complete volume rendering was also performed with TrakEM2 tools and converted to 3D mesh objects that could be viewed or moved to other programs designed for display of 3D images (e.g., Dragonfly, Object Research Systems or Amira, ThermoFisher).

In this study, we needed to clearly identify ribbon synapses made by cone pedicles and bipolar cell axon terminals. Synaptic ribbons were easily discernable platelike structures, ~25 nm thick and ~250 nm wide and associated with a halo of synaptic vesicles; it was thus possible to identify them unequivocally in our volumes (e.g., Fig. 2 B and C). We used multiple annotators to confirm basic skeleton structure, total ribbon numbers, and synaptic connectivity during the process of skeletonization and circuit reconstruction.

In Vitro Physiological Recordings.

The extracellular loose patch recordings shown in Fig. 1 and SI Appendix, Fig. S4 were acquired from an in vitro preparation of the macaque monkey (macaque nemestrina) retina of either sex. Details of the standard macaque in vitro preparation, recording methods, and chromatic stimulus generation have been previously reported in detail (15, 22, 34). In brief, for the records shown here red, green, and blue primary lights (presented at high photopic light levels) were used to create both S-cone isolating and L+M cone isolating stimuli (max, 80% cone contrast) that were either square wave modulated in contrast at 2 Hz temporal frequency as a spot that covered the entire field (800 µm diam) or sinusoidally modulated in contrast grating, drifting at 2 Hz spatial frequency.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Dataset S05 (XLSX)

Dataset S06 (XLSX)

Dataset S07 (XLSX)

This work was supported by NIH grant EY-028282 to D.M.D. and by NIH Grant RR-00166 to the Tissue Distribution Program of the Washington National Primate Research Center (WaNPRC), grant P51 OD010425 from the NIH Office of Research Infrastructure Program to the WaNPRC, and EY01730 to the Vision Research Core at the University of Washington. We thank Dr. Andreas Pollreisz for human tissue acquisition and Sharm Knecht for tissue preparation for electron microscopy and managing connectomics data acquisition, and Paul Martin, Ulrike Grünert, Qasim Zaidi, and Rob Smith for comments and helpful discussions.

Author contributions

Y.J.K. and D.M.D. designed research; Y.J.K. and D.M.D. performed research; Y.J.K., O.P., and D.M.D. analyzed data; and Y.J.K. and D.M.D. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information.

Supporting Information

This article is a PNAS Direct Submission.
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