
==== 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

39231211
202413104
10.1073/pnas.2413104121
research-articleResearch ArticleneuroNeuroscience424
Biological Sciences
Neuroscience
Cellular and circuit remodeling of the primate foveal midget pathway after acute photoreceptor loss
Akiba Ryutaro a b c
Lind Boniec Shane a
Knecht Sharm a
Uyama Hirofumi b
Tu Hung-Ya b d
Baba Takayuki c
Takahashi Masayo b e
Mandai Michiko b e
Wong Rachel O. wongr2@uw.edu
a 1 https://orcid.org/0000-0001-5089-6296

aDepartment of Biological Structure, University of Washington, Seattle, WA 98195
bLaboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan
cDepartment of Ophthalmology and Visual Sciences, Chiba University Graduate School of Medicine, Chiba 260-8677, Japan
dLaboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan
eResearch Center, Kobe City Eye Hospital Research Center, Kobe, Hyogo 650-0047, Japan
1To whom correspondence may be addressed. Email: wongr2@uw.edu.
Contributed by Rachel O. Wong; received June 30, 2024; accepted August 1, 2024; reviewed by Daniel Kerschensteiner and Wei Li

4 9 2024
10 9 2024
4 9 2024
121 37 e241310412130 6 2024
01 8 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

Restoring high-acuity vision upon photoreceptor loss requires understanding the ensuing cellular and neural circuit remodeling in the damaged primate fovea. Using serial electron microscopy, we show that foveal midget circuits, which are responsible for high-acuity vision, remodel after acute cone photoreceptor loss in a nonhuman primate model. Unlike some species, however, foveal midget bipolar cells (MBCs) disconnected from photoreceptors are largely unable to extend dendrites far enough to rewire with surviving cones. Disconnected OFF MBCs lose output connections before ON MBCs. These primary visual pathways that arise within the retinal fovea thus show differential responses to injury at their output synaptic layer. Our findings have implications for designing photoreceptor replacement therapy to restore foveal vision.

The retinal fovea in human and nonhuman primates is essential for high acuity and color vision. Within the fovea lies specialized circuitry in which signals from a single cone photoreceptor are largely conveyed to one ON and one OFF type midget bipolar cell (MBC), which in turn connect to a single ON or OFF midget ganglion cell (MGC), respectively. Restoring foveal vision requires not only photoreceptor replacement but also appropriate reconnection with surviving ON and OFF MBCs and MGCs. However, our current understanding of the effects of cone loss on the remaining foveal midget pathway is limited. We thus used serial block-face electron microscopy to determine the degree of plasticity and potential remodeling of this pathway in adult Macaca fascicularis several months after acute photoreceptor loss upon photocoagulation. We reconstructed MBC structure and connectivity within and adjacent to the region of cone loss. We found that MBC dendrites within the scotoma retracted and failed to reach surviving cones to form new connections. However, both surviving cones and ON and OFF MBC dendrites at the scotoma border exhibited remodeling, suggesting that these neurons can demonstrate plasticity and rewiring at maturity. At six months postlesion, disconnected OFF MBCs clearly lost output ribbon synapses with their postsynaptic partners, whereas the majority of ON MBCs maintained their axonal ribbon numbers, suggesting differential timing or extent in ON and OFF midget circuit remodeling after cone loss. Our findings raise rewiring considerations for cell replacement approaches in the restoration of foveal vision.

retinal remodeling
foveal circuit plasticity
injury and neural circuit remodeling
retinal degeneration
HHS | NIH | National Eye Institute (NEI) 100000053 EY033447 Rachel O. Wong HHS | NIH | National Eye Institute (NEI) 100000053 EY01730 Rachel O. Wong MEXT | Japan Society for the Promotion of Science (JSPS) 501100001691 Overseas Fellowship Ryutaro Akiba Japan Agency for Medical Research and Development (AMED) 100009619 JP20bm0204002 Michiko Mandai
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pmcIn primates, including humans, high-acuity vision relies on a specialized circuit arrangement localized to a small region within the retina called the “fovea.” At the fovea, cone photoreceptors are densely packed, reducing the sampling aperture of the cones and facilitating high spatial resolution. Across much of the primate retina, individual cone photoreceptors diverge their signals to several bipolar interneurons of the same type, which in turn can contact many ganglion cells and ganglion cell types. But, within the primate fovea, a single cone generally connects to a pair of depolarizing (ON) and hyperpolarizing (OFF) midget bipolar cells (MBC), which in turn wire with an ON and an OFF midget ganglion cell (MGC), respectively. This so-called midget “private-line” connectivity preserves the spatial resolution of the cone photoreceptor mosaic and is the first step in a pathway for high-acuity vision.

Degeneration of cone photoreceptors, particularly in the fovea, such as that which occurs in disease, results in visual acuity loss and severely impairs the quality of life. Cell replacement therapy is one strategy to restore visual function (1–3). Embryonic stem (ES) cells (4–6) or induced pluripotent stem cells (7–12) are ideal sources of new photoreceptors because these approaches can introduce large numbers of rod and/or cone photoreceptors in a single procedure. Transplantation of photoreceptor (precursor) cells has been shown to recover visual function in congenital stationary night blindness (13) and animal models of retinal degeneration (1, 2, 14). Stem cell–derived retinal sheets introduced into mouse models of retinal degeneration have been shown to integrate into the host retina (7, 8), form synapses (15), and can exhibit improvement of light responsiveness in rodents (16) and primates (17). Recent studies also demonstrated that compared to conventional organoid transplantation, genetic modification of retinal organoids results in improved host-graft synaptogenesis and functional recovery (18, 19). Transplantation of purified cone photoreceptors has also been observed to restore some visual function (20, 21). However, restoration of vision with significantly high visual acuity will require not only successful photoreceptor replacement but also that new photoreceptors connect with the surviving neurons in a manner that restores the midget private line in the fovea.

To this end, we sought to better understand the connectivity of the surviving MBCs in response to acute photoreceptor loss. Previous studies suggest that retinal neurons can remodel their structure and connectivity after becoming disconnected with photoreceptors, thus potentially posing significant challenges to the restoration of the original circuit arrangements (22–24). However, to date, it is not known whether the adult primate foveal midget pathway exhibits structural and circuit changes when retinal neurons no longer receive input from the photoreceptors. Past studies in nonprimates have demonstrated changes in adult connectivity of surviving photoreceptors and their postsynaptic bipolar cells after acute photoreceptor loss, induced either by laser photocoagulation (25) or by transgenic expression of the diphtheria toxin receptor (26, 27). Photocoagulation of photoreceptors in the rabbit retina resulted in rod but not cone bipolar cells extending dendrites to form synapses with surviving photoreceptors in their vicinity (28). Partial loss of cones in the mature mouse retina also triggered dendritic changes and formation of connections with new cones but in only one of four types of cone bipolar cell types studied (27). Type 6 mouse bipolar cells with small dendritic arbors extended dendritic processes longer than normal after losing connections with some cones, but these processes failed to capture new partners within the survival period (26, 27). When cone bipolar cells are able to extend dendrites to connect synaptically with novel partners, such as the S-cone bipolar cells in the cone-dominant squirrel retina, these bipolar cells target specifically their preferred cone type (29). Collectively, these studies suggest that upon acute photoreceptor loss, adult mammalian cone bipolar cells can demonstrate plasticity and rewire, sometimes specific in their partner targeting. However, the degree and nature of rewiring appear to vary across bipolar cell types and across species. Thus, we used a photocoagulation approach to cause cell death of patches of cone photoreceptors in vivo within the Macaca fascicularis fovea followed by serial block-face scanning electron microscopy (SBFSEM) to directly uncover the circuit arrangements of the foveal midget pathway after acute cone loss.

Results

Targeted Photocoagulation Results in Local Photoreceptor Loss and Disconnection in the Fovea.

Like other primates, the M. fascicularis fovea is characterized by a local indentation or pit in the temporal retina when visualized in vivo by optical coherence tomography (OCT) (Fig. 1A). At the fovea, generally, an individual cone photoreceptor contacts an ON and an OFF MBC, which in turn connects with an ON or OFF MGC respectively, forming the “private line” (Fig. 1B). We ablated photoreceptors in vivo at sites within the fovea (Fig. 1C) by laser photocoagulation. OCT images of the photocoagulated area two weeks later revealed that the outer nuclear layer (ONL) was ablated (Fig. 1D). To examine the morphology of the photoreceptors, we obtained images of the ultrastructure of the photocoagulation site 6 mo postablation. Transmission electron microscopy (TEM) of the photocoagulated area confirmed that photoreceptors were selectively ablated, whereas the inner retina appeared morphologically intact (SI Appendix, Fig. S1A). Photoreceptors adjacent to the ablation site retained both inner and outer segments (SI Appendix, Fig. S1B). SBFSEM reconstructions of individual photoreceptors demonstrated that compared to the control (nonlasered retina of a different animal; SI Appendix, Fig. S1C), photoreceptors adjacent to the photocoagulation area had, not surprisingly, skewed morphology (SI Appendix, Fig. S1E), presumably due to the absence of mechanical support from neighboring photoreceptors and tissue distortion due to the ablation (SI Appendix, Fig. S1A). The observation that photoreceptors two to three cell bodies away from the photocoagulation boundary retained straight inner segments further suggests the lack of mechanical support might contribute to changes to the morphology of photoreceptors at the ablation boundary. Notably, the photoreceptors adjacent to the photocoagulation area still retained inner and outer segments, suggesting that these cells likely remained light-sensitive (SI Appendix, Fig. S1 D–E).

Fig. 1. Local photocoagulation of the fovea and midget circuit reconstructions. (A) (Upper panel) Fundus image of an adult Macaca fascicularis (Upper panel) eye. The asterisk marks the location of the fovea. ONH, optic nerve head. (Lower panel) Optical coherence tomography (OCT) image of the fovea in A. (B) Schematic of the foveal midget pathway showing a cone synapsing with an ON and an OFF midget bipolar cell (MBC) in the outer plexiform layer (OPL). Each ON and OFF MBC makes synapses with an ON or an OFF midget ganglion cell (MGC) respectively, in the inner plexiform layer (IPL). The midget ON and OFF pathways generally consist of connections from 1 cone to 1 MBC to 1 MGC. (C) Fundus image of photocoagulated regions at the fovea, rotated 90° clockwise from (A). The yellow pentagon outlines the region processed for electron microscopy in the current study. The purple dashed line indicates the orientation of the OCT image in (D). The red dashed line corresponds to a location where cone cell bodies are ablated, schematized in (E). The blue dashed line corresponds to a region where the axon terminals of the ablated cones are missing (see E). (D) OCT image of the photocoagulated area (yellow dashed line) near the foveal pit (asterisk). (ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (E) Schematic illustrating photoreceptor cell loss in the retinal fovea. The Henle fibers of the photoreceptors extend away from the foveal centralis to synapse with distal bipolar cells. Axon pedicle-depleted zone (blue dashed line) of ablated cone cell bodies (red dashed line). (F) Transmission electron microscopy image of a pedicle-depleted zone (PDZ) next to a pedicle full zone (PFZ, arrowhead) in the outer plexiform layer (OPL). INL, inner nuclear layer. (G) Examples of photoreceptor synapses imaged by the serial block-face scanning electron microscopy (SBFSEM) in the nonlasered retina, the PFZ/PDZ border, and the PDZ of a photocoagulated retina 6 mo postablation. Photoreceptor pedicles (p), horizontal cells (h), ON bipolar cells (b), and OFF bipolar cells (o) are colored yellow, blue, purple, and green, respectively. (H)SBFSEM reconstructions of the ON and OFF midget circuits in the PFZ, PDZ, and the PFZ/PDZ border region. Yellow: cone pedicle, purple: ON MBC, green: OFF MBC, orange and red: OFF MGCs, cyan and light yellow: ON MGCs.

Using SBFSEM, we reconstructed the midget circuitry in regions where the axon pedicles of the photoablated cone photoreceptors had degenerated 2 or 6 mo after the injury. These EM volumes were located distal to the photocoagulation site because foveal cone photoreceptors extend several hundred micrometer long axons, called Henle fibers, toward the peripheral retina to connect with the MBCs (Fig. 1E). The pedicle-depleted zone (PDZ) was readily apparent by a lack of axon pedicles adjacent to the pedicle full zone (PFZ)(Fig. 1F). For the lasered retina, cells in the PFZ that were within 30 µm from the PDZ were categorized as “Border” cells.

Cone synapses, with the classical “triad” formation of control (nonlasered) retinas, were apparent at the PFZ and at the PDZ border (Fig. 1G). These synaptic triads comprised a synaptic ribbon, two invaginating horizontal cell processes, and one invaginating ON MBC process (Fig. 1G). Within the SBFSEM volume of the region shown in Fig. 1F, we identified ON and OFF MBCs, and ON and OFF MGCs based on their compact morphology and axonal (MBC) or dendritic (MGC) arborization and stratification within the inner or outer halves of the inner plexiform layer, respectively (30, 31). ON and OFF MBC axon terminals and MGC dendrites in the PDZ remained stratified as in control. This preserved feature enabled us to identify and reconstruct the ON and OFF midget pathways within the PFZ, at the PDZ border, and within the PDZ (Fig. 1H).

Outer Retinal Morphology Alters After Acute Photoreceptor Loss.

We compared the morphology of the cone pedicles, their synapses, and the dendrites of the MBCs of the nonlasered control retina (approximately 650 µm away from the fovea centralis) with cells in the photocoagulated retina. Foveal cone pedicles in the control had numerous ribbons, localized at presynaptic sites apposed to dendrites of an ON and an OFF MBC (Fig. 2A, control). In the lasered retina, the axon pedicle morphology of the surviving cones at the PFZ and the PDZ border were similar across these regions (Fig. 2A). However, some of the pedicles at the PDZ border showed morphological alterations such as extension of an appendage-like process toward the PDZ (SI Appendix, Fig. S2 A and B).

Fig. 2. Outer retinal reorganization near and within cone pedicle-depleted zone. (A) Three-dimensional SBFSEM reconstructions of cone pedicles and their synaptic ribbons, and the dendrites of ON or OFF MBCs in the control (nonlasered) retina, at the pedicle full zone (PFZ), border of the pedicle-depleted zone (PDZ), and the PDZ 6 mo postablation. MBC dendrites within the PDZ are also shown. (B) Total dendritic length of ON and OFF MBCs in nonlasered control (Ctrl), the PFZ, the PDZ border, and within the PDZ two (2 M) and six (6 M) months after laser damage. Black asterisks indicate statistically significant differences compared to the control (P < 0.05). Red asterisks indicate significant difference between the two groups (Mann–Whitney U test P < 0.05). (C) The total number of ribbons within a pedicle and the number of ribbons opposite ON MBC dendritic invaginations within each cone pedicle at two and six months postablation. (D) Examples of synaptic motifs of cone pedicles in lasered retina. Synaptic ribbons are indicated by white arrowheads, ON MBC processes are colored purple, and horizontal cell processes are colored blue. NI, noninvaginating. (E) Proportions of synapses showing different motifs observed in cone pedicles in the PFZ and the PDZ border 6 mo postablation. NI, noninvaginating. n = number of pedicles reconstructed.

We then compared the dendritic morphologies of the MBCs within the PFZ, at the PDZ border, and within the PDZ with that of the control. In all sampled regions of the lasered retina, including the PDZ, the MBC dendrites remained confined to the outer plexiform layer. However, the dendritic complexity of both ON and OFF MBCs decreased progressively toward the PDZ (Fig. 2A). We quantified the ON and OFF MBC’s total dendritic length starting from the first branching point of the dendrite to the dendritic tips (Fig. 2B). Compared to the nonlasered control retina, ON and OFF MBCs at the PDZ border had significantly shorter total dendritic lengths, retracting increasingly with their proximity to PDZ (Fig. 2B and SI Appendix, Fig. S2C) (SI Appendix, Table S1 for statistical analysis). These differences were already apparent at 2 wk and 2 mo postablation, although dendritic loss in ON MBCs within the PFZ continued in the ensuing months (Fig. 2B, ON PFZ and SI Appendix, Fig. S2D). However, ON MBCs in the PDZ appeared to increase their total dendritic length between 2 and 6 mo postablation (Fig. 2B, PDZ), implying that these neurons re-extended dendrites. Dendritic loss of MBCs is unlikely to be caused directly by laser damage because the photocoagulation area was outside the region of synaptic contact. Although it is expected that disconnected bipolar cells retract their dendrites (22), it was unusual to find that the MBCs that remained connected to cones surrounding the lesioned area also demonstrated progressive dendritic loss (see Discussion).

Outer Retinal Synapses and Connectivity Adjacent to the Lesion Remodel.

To determine whether there were synaptic changes in the surviving cones surrounding the lesion, we quantified the number of ribbons per cone pedicle and the number that were associated with invaginating ON MBC dendrites. We found that both the total number of ribbons within a pedicle and the number of ribbons per pedicle associated with ON MBC dendritic tips were both significantly decreased compared to the nonlasered control (Fig. 2C and SI Appendix, Table S2). Moreover, much of the loss in ribbons appeared to be those closely associated with ON MBC dendrites. Pedicles at the PDZ border had significantly fewer ribbons compared to those at the PFZ. The ribbons that were not opposed to ON MBC were still anchored at the active zone; no floating ribbons were observed within three pedicles we reconstructed fully (n = 43 ribbons). There were no significant differences in ribbon numbers between 2 and 6 mo after photocoagulation, suggesting that synaptic changes occurred within 2 mo. In fact, examining pedicles at the PDZ border 2 wk after the photocoagulation revealed that ribbons were already lost at this time, in parallel with dendritic loss at this stage (SI Appendix, Fig. S2E).

The architecture of the photoreceptor synapses adjacent to the lesion was also altered. In addition to the classical synaptic triad, we found unconventional motifs in the 6-mo postlasered retina, including dyads and monads involving invaginating ON MBC dendritic tips. Some synapses comprised noninvaginating ON MBC dendritic tips that also formed triads or dyads (Fig. 2D). We charted the motifs with respect to distance from the PDZ border and found that unconventional motifs became more common at the PDZ border (Fig. 2E). We also noticed that some pedicles near the PDZ border contained densely packed tumbleweed-like processes within the cone pedicle not observed in the other conditions (Fig. 2D, lower right panel). These structures most likely have originated from horizontal cells, because the processes were invaginating and possessed a morphology that was distinct from the dendrites of the ON bipolar cells, extending extremely long distances and could not be traced to MBCs. We created the 3D reconstruction of a horizontal cell from the nonlasered control (SI Appendix, Fig. S3 A–D), and also traced the processes of the presumed horizontal cell in the lasered retina (SI Appendix, Fig. S3 E-F). Although we could reconstruct the horizontal cell completely in the control, the processes of the presumed horizontal cell in the lasered retina extended beyond the volume and thus we could not identify its cell body. We also attempted to identify other horizontal cells within our imaged region of the lasered retina, but we could not find any cells resembling the horizontal cell observed in the control block. This finding is consistent with another study where photocoagulation resulted in loss of horizontal cells (32). We surmise that the presumed cell processes arise from cells far distal to the imaged region. If so, horizontal cell bodies may be absent within the PDZ.

Cones and ON or OFF MBCs normally demonstrate a 1:1 connection in the nonlasered control (Fig. 3A, upper panel). However, 6 mo postablation, some cones near or at the PDZ showed increased convergence and divergence at their synapses (Fig. 3A, middle and lower panels) (SI Appendix, Tables S3 and S4). Cone convergence onto ON MBCs remained 1:1 at 2 and 6 mo postablation (Fig. 3B, upper panel), whereas individual cones contacted more than one OFF MBC, suggesting an increase in divergence, which was apparent by 2 mo (Fig. 3B, lower panel). Alteration of cone to MBCs divergence was more apparent than convergence for both the ON and OFF pathways, observed as early as 2 mo postablation (Fig. 3C). Thus, in addition to changes in their synaptic arrangement, cones adjacent to the region devoid of cones remodeled their connectivity with the MBCs.

Fig. 3. Divergence and convergence changes in the outer retina after acute cone loss. (A) Example 3D SBFSEM reconstruction of the 1:1 connection between a cone and an ON (purple) and an OFF (green) MBC in control retina, an example of two cones (orange/yellow) converging onto a single OFF MBC in the photocoagulated retina at the PDZ border after 6 mo postablation, and the example of one cone diverging onto 2 ON (purple/light purple) and 3 OFF (cyan, light green, green) MBCs. (B and C) Convergence and divergence of cones onto ON or OFF MBCs in control and at the PDZ border 2 and 6 mo after photocoagulation. Plotted are the percentages of MBCs contacted by 1, 2, or 3 cones (B) and percentages of cones sampled contacting none (0), 1, 2, or 3 MBCs (C).

Cone-Disconnected MBC Remodel Connectivity with MGCs.

Previous work suggested that loss of input to bipolar cells can lead to alterations in connectivity of the bipolar cells to retinal ganglion cells. For example, in Crx−/− mice with functionally and morphologically impaired photoreceptors, retinal ganglion cells are hyperactive and demonstrate increased synaptic contact with bipolar cells (33). Also, inner retinal circuits remodel in the late phase of retinitis pigmentosa in humans (34). Whether cone loss in the fovea triggers inner retinal remodeling in the mature midget pathway is unknown. We thus addressed this question next.

We traced the axon terminals of the ON and OFF MBCs in the lasered and nonlasered retina (examples provided in Fig. 4A and SI Appendix, Fig. S4A). Axonal stratification of ON and OFF MBCs was not altered in the lasered retina after 6 mo (see also Fig. 1H). We compared quantitatively ON and OFF MBC axonal morphologies and their synaptic ribbons between the nonlasered control, and the PFZ and PDZ of the 6 mo postlasered retina (Fig. 4B and SI Appendix, Table S5). The general axonal structure and the total axonal lengths were similar between control and the PDZ for OFF MBCs, but the ON MBCs had relatively shorter total axonal length in the PDZ (SI Appendix, Fig. S4B). Ribbon synapse numbers per axon were lower within the PDZ of the 2-mo and 6-mo lasered retina compared to the control (Fig. 4C and SI Appendix, Table S6). The decrease was observed in ON MBCs (2 mo: P = 0.018, 6 mo: P = 0.048), and was more prominent in the OFF MBCs (2 mo: P = 0.0044, 6 mo: p=0.00082). These results suggest that cone loss triggers elimination of MBC ribbons, particularly for the OFF MBCs. We found dyads, monads, and triads in synapses of MBCs that were disconnected from cones (Fig. 4D). Although the number of ribbons per MBC axon in the PDZ was relatively lower at 6 mo, the proportion of the various MBC synaptic motifs was largely unchanged, and dyad synapses remained the dominant motif (Fig. 4 D−E).

Fig. 4. Synaptic changes in the inner retina after acute cone loss. (A) Three-dimensional SBFSEM reconstruction of ON and OFF MBC axon terminals and ribbons in the control (Ctrl) retina and in the pedicle-deprived (PDZ) zone of the lasered retina 6 mo postablation. (B) Total axonal terminal (arbor) length of MBCs in control, pedicle full zone (PFZ), and PDZ 6 mo (6 M) after the lesion. (C) Number of axonal ribbons of ON and OFF MBCs in control and the PDZ where ablation occurred 2 wk (2 W), 2 mo (2 M), or 6 M prior to eye fixation. Asterisks indicate statistically significant differences (Mann–Whitney U test) compared to control. (D) Example of MBC synaptic motifs in control and lasered retina. Bipolar cells (BC) are colored yellow, and postsynaptic partners (P1-3) are colored blue, red, or green. Synaptic ribbons are indicated by the white arrowheads. (E) Proportions of the various synaptic motifs of ON or OFF MBC synapses in the inner plexiform layer of control and the PDZ. n = 3 MBCs for each group.

We also examined whether the private line between MBCs and MGCs was preserved in all regions of the 6-mo lasered retina. This appeared to be so for many pairs of MBCs and MGCs although we did observe changes in convergence and divergence of some connections (SI Appendix, Fig. S4B). Some MGCs received input from more than one and up to three MBCs (Fig. 5A, PDZ), although even in the control retina, not all connections formed a private line (Fig. 5A, Control). Statistical comparisons across the reconstructed cells do not suggest a significant change in convergence or divergence between the MBCs and MGCs (SI Appendix, Tables S7 and S8). However, Fig. 5 B and C demonstrates clearly that there was a significant loss in the number of MBC-to-MGC OFF ribbon connections in the 6 mo PDZ border region and within the PDZ (see SI Appendix, Tables S7−S10). This result suggests that the overall decrease in ribbon number per axon of the OFF MBCs (shown in Fig. 4B) was largely attributed to synapse loss between OFF MBCs and OFF MGCs, although we have not quantified the synapse between OFF MBCs and amacrine cells and nonmidget ganglion cells. Synapse loss in ON MBCs onto MGCs was less apparent, although examples of MBCs with decreased connectivity with their primary MGC were observed (Fig. 5B and SI Appendix, Table S9).

Fig. 5. Wiring alterations of foveal midget bipolar with ganglion cells upon cone loss. (A) Examples of synaptic convergence and divergence of ON or OFF MBCs and MGCs in the inner plexiform layer of control fovea and the pedicle-deprived zone (PDZ) 6 mo postablation. The number of synapses formed between each bipolar cell and the target ganglion cell(s) is indicated by the number of matching colors. For example in control, ON convergence, the green-colored ON MBC makes 47 synapses, and the magenta MBC forms 2 synapses with the ganglion cell (gray). (B) Number of synapses formed between each MBC and a MGC. In control and lasered retina, some MGCs receive input from more than one bipolar cell, but often one MBC provides the dominant input (partner rank 1). Synapses from each of the presynaptic MBCs onto a single MGC are colored the same (linked by a line). Asterisks indicate a statistically significant differences (P < 0.05, Mann–Whitney U test) between the number of synapses made by the primary MBCs in control and MBCs at the PDZ border or PDZ of the lasered retina. (C) Number of synapses formed by a single MBC onto target MGCs. Synapses that diverge from the same MBC are colored similarly. Asterisks indicate statistically significant differences (P < 0.05, Mann–Whitney U test) between the dominant MGC synaptic partners (rank 1) in control and different locations of the lasered retina.

Discussion

Foveal MBC Dendrites Fail to Reconnect After Acute Cone Loss.

In species such as the rabbit, after local photoreceptor loss, deafferented rod bipolar cells extend their dendrites to form synapses with surrounding photoreceptors, but cone bipolar cells do not (28). In ground squirrel, cone ablation triggers S-cone bipolar cells to extend their dendrites and selectively make synapses with novel S-cone partners (29). Although we observed some plasticity in the mature Macaque foveal MBC connections with surviving cones near and at the scotoma boundary, MBCs well within the region of cone pedicle loss retracted their dendritic arbor, and remained disconnected up until 6 mo postablation. Mature foveal MBCs are thus, perhaps unsurprisingly, much less plastic than their counterparts in development (35). In this regard, the foveal MBCs resemble other mammalian bipolar cells, which also demonstrate a loss of plasticity with age (27).

Failure of MBCs to reconnect in the adult macaque fovea may be exacerbated by these bipolar cells being unable to extend long processes to reach the surviving cones. Additionally, there may be a loss of cellular and molecular processes that support dendrite extension or growth from MBCs with age. However, even during development, MBCs do not need to elaborate large dendritic arbors to contact and connect with their cone partners. Indeed, foveal MBCs establish synaptic connections with one or a few cones before the foveal pit emerges (36) and maintain connected as the cone axons extend to form the fibers of Henle with maturation. Therefore, MBCs may not possess the cellular and molecular mechanisms to drive extension of their dendrites beyond their typically small arborizations. Whether adult foveal MBCs elaborate dendrites more extensively after 6 mo of disconnection, however, remains to be determined. Photoreceptor precursor cells have been shown to elaborate neurites after transplantation (37), which might provide some hope that such photoreceptors can seek out MBCs to form new connections, if the MBCs are unable to extend their dendrites. Future analysis of diffuse bipolar cell types in the nonhuman primate fovea after photoablation will reveal whether or not failure of midget dendrites to extend after disconnection is limited to these bipolar cell types.

Surviving Midget Circuits at the Scotoma Boundary Exhibit Remodeling.

We observed that Macaque foveal cones and MBCs at the scotoma boundary can demonstrate changes in synaptic convergence or divergence. The degree of these changes differed somewhat between the ON and OFF pathways, with cone to OFF MBC convergence altering before changes are detected in the ON pathway. At the synaptic level, we found a consistent decline in ribbon numbers in cones, apparent by 2 wk postablation, at and near the PDZ. This ribbon loss was not due to disconnection with bipolar cell partners (divergence), as observed during development (35), but rather due to a loss of active zones at membrane appositions with bipolar cell dendrites. The reduction in presynaptic ribbons in some surviving cones largely parallels the decrease in their dendritic contact with individual MBCs (SI Appendix, Fig. S3G). Ribbon loss, however, occurred regardless of whether or not the cones increased in their divergence, contacting more than their usual number of MBC partners. Whether ribbons disassemble before or after dendrites shrink is not yet evident from our current observations.

These observations in the primate fovea differ from the homeostatic increase in invagination of the cone pedicles by the dendrites of some types of cone bipolar cells in the mouse retina after acute cone loss (27). However, like the mouse retina, Macaque bipolar cells in the fovea may also respond differentially upon acute loss of neighboring cones. This is because the majority of cone ribbon loss appears to be associated with synapses with MBCs, raising the possibility that foveal cone connectivity with non-MBCs may be relatively more stable. Although we have not investigated the non-MBCs in this study, future studies focusing on these bipolar cells and ganglion cells will reveal the remodeling of the nonmidget pathways after photoreceptor loss. Future comparisons with scotoma-induced remodeling of midget cell type orthologues in mice (38) would also be informative. We also found that a few cone pedicles at the scotoma edge extended a small appendage into the pedicle-depleted zone, and in one instance contacting an OFF MBC. Cone pedicles in the adult fovea may thus have the ability to be structurally plastic. However, as yet we do not know to what extent cone terminals can rearrange compared to human rod photoreceptors, which have been shown in retinitis pigmentosa, to extend their processes as far as into the inner plexiform layer (39). Future comparisons between cones and rods in the more peripheral primate retina would be informative.

Structural arrangements and connectivity with surviving cones near the cone-depleted zone also involved horizontal cells. Some cone pedicles were heavily invaginated by complex dendritic endings. We were not able to identify the cell bodies of these unusual processes but suspect that they belong to horizontal cells because they extended beyond the volume. In fact, we were not able to identify any horizontal cell bodies within the PDZ, akin to observations in the rd/rd mouse retina which has fewer horizontal cells (22) and in another study of photocoagulation of the primate fovea (32). Together, all these observations imply a local loss of horizontal cells and a remodeling of neighboring horizontal cell processes to capture novel cone partners.

The cone synapse rearrangements at the PDZ border we observed may or may not be attributed to cellular changes in response to the loss of cone neighbors. We cannot rule out the possibility that cones at the edges of the lesion were affected functionally by the photocoagulation. The photocoagulation’s heat energy may have caused damage to the neighboring photoreceptors of the ablation site (40). The rapid loss of ribbons by 2 wk postablation would suggest that cones at the PDZ border that survived may have been compromised. If so, our observations still provide insight into the ability of mature photoreceptors and MBCs to remodel, and in conditions where injury is caused by laser damage. It also raises the question of whether remodeling of cones and midget bipolars differ in regions that are partially versus wholly depleted of photoreceptors. Previous work would suggest that at least in mice, mature cone bipolar cell dendritic rearrangements are regulated on a dendrite-by-dendrite basis (26, 27). Finally, our observations here raise questions about the potential remodeling of the connectivity of photoreceptors that survive injury (or those that eventually die) and the impact on remaining retinal function.

Differential Effects on ON and OFF Midget Connections in the Inner Retina upon Cone Loss.

Our reconstructions demonstrated that despite the loss of cone contact and input, MBCs remain stratified appropriately and mostly still connect along a private line arrangement. Total axonal lengths of OFF MBCs appear to be largely maintained, at least up to 6 mo postablation. However, OFF MBCs clearly made fewer ribbon synapses with their MGC partners although the remaining synapses are still largely dyads as in controls. ON MBCs also demonstrated a significant decrease in their total axonal ribbon numbers but to a lesser extent than that of the OFF MBCs. Loss of ribbon synapses in the inner retina was found previously in mice in which bipolar cell transmission was diminished (41).

Although ON MBCs showed a decrease in overall axonal ribbon numbers, ribbon synapses associated with ON MGC targets did not seem to decrease significantly. This suggests that non-MGC ribbon synapses may have been lost in the PDZ. In contrast, OFF MBC-OFF MGC synapses were severely reduced. This differential effect on ON and OFF inner retinal midget connections is reminiscent of the different time course of changes in ON and OFF retinal ganglion cell connectivity in rodent models of ocular hypertension (42–44) and a mouse optic nerve crush model (45). As mentioned earlier, cone convergence and divergence in the OFF pathway at the PDZ border also altered more rapidly compared to the ON pathway. Thus, differential responses of ON and OFF pathways to injury and trauma may be common across many mammalian species, including primates.

Challenges to Future Cell Replacement Therapy Approaches to Foveal Circuit Repair.

Cell therapy has moved from basic research to clinical application (46), but the ultimate goal of cell therapy in reconstructing the foveal circuit to recover high-acuity vision remains to be fulfilled. Our present findings suggest that mature MBCs can undergo rewiring, implying the potential of integrating transplanted photoreceptors into the midget circuit. However, our observations also underscore significant structural challenges to the repair of foveal circuits. MBCs normally connected to the central cones are located hundreds of micrometers from the foveal pit. Thus, reconnection of these bipolar cells would necessitate either new cones to target the bipolar cells and/or the bipolar cell dendrites to extend toward the cones transplanted far away. We did not find significant extension of the dendrites of disconnected MBCs, not even toward the scotoma boundary where cones were present. Therefore, in cell replacement therapy, photoreceptors will need to be transplanted proximal to the disconnected bipolar cells, which are likely still wired appropriately to postsynaptic partners. Alternatively, factors might need to be introduced to enhance axon elongation of the transplanted photoreceptors toward the disconnected MBCs, mimicking the formation of Henle fibers during development (47). In the present study, we found that cone axons can extend an appendage process toward the PDZ and make contact with an OFF MBC, raising the possibility that foveal cones might be able to reach novel synaptic partners if axonal extensions can be promoted over long distances.

Even after successful transplantation of photoreceptors, inner retinal remodeling might hinder the full recovery of visual function. For the fovea, inner retinal midget connectivity appears relatively stable, except for the OFF connections. The differential response to injury between the ON and OFF pathway may be another factor to consider in cell replacement therapy. We observed early changes in the OFF pathway after input loss, raising the possibility that the OFF pathway might be more difficult to repair after cell therapy. This is in fact consistent with the previously reported recovery of ganglion cell light responses in degenerated rodent retinas after stem cell–derived retinal organoid transplantation, which is largely limited to ON pathway activity, and very few OFF responses (18).

In the present study, we focused on the structural changes along the midget pathway after acute photoreceptor cell loss. Future investigations using electrophysiological recordings will be necessary to probe the functional consequences of the remodeling induced by cone loss. As for the molecular mechanisms that trigger remodeling or confer resilience to the midget system, single-cell profiling transcriptomics of foveal cones and ON and OFF MBCs after cone ablation will be informative. How the midget pathways are perturbed over time in retinal diseases such as retinitis pigmentosa or age-related macular degeneration, also remains to be investigated. While our current findings underscore repair challenges unique to the fovea, they also offer hope because by and large, the disconnected MBCs do not remodel extensively or rapidly after acute cone loss, preserving their basic features and demonstrating some plasticity that could be useful for future repair.

Materials and Methods

In Vivo Targeted Ablation of Foveal Photoreceptors.

The retinas of two adult (4-5-year-old males) M. fascicularis monkeys underwent laser photocoagulation to ablate photoreceptors, a procedure that commonly occurs in the transplantation studies (17, 48, 49). The retinas did not receive any transplants. Animals were sacrificed and fixation of the eyecups were performed by the RIKEN laboratory under approval by the RIKEN Animal Use oversight committee. The procedure typically lasts 30-40 mins under anesthesia with ketamine hydrochloride (40 to 80 mg/kg) and xylazine (5 to 10 mg/kg). A 577 nm OPSL PASCAL laser (Topcon Medical Laser System) was used to carry out the photocoagulation. Fifteen spots (5 vertical × 3 horizontal; 90 to 110 mW) were applied in rapid succession to obtain an ablation area of approximately 200 × 500 µm. Optical coherence tomography (OCT) was carried out 2 wk later to evaluate whether the laser intensity was sufficient to photocoagulate the outer nuclear layer (ONL) while leaving the inner nuclear layer (INL) intact. The effectiveness of the photocoagulation was later corroborated by TEM. Photocoagulation was performed at different time points: 2 wk, 2 mo, and 6 mo before retinal fixation.

SBFSEM Sample Preparation and Cellular Reconstruction.

Three eyecups with retinas that received laser photocoagulation (right eye of #1, both eyes of #2) were fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH7.4, for 3 h at room temperature and then overnight at 4 °C. The tissue was washed 3 × 5 min in 0.1 M cacodylate buffer, pH7.4, and shipped to the University of Washington. Export permission was authorized by the Japan Ministry of Economy, Trade and Industry, and the import permit for Infectious Biological Agents was issued from Centers for Disease Control and Prevention (CDC). The eyecups were shipped in a temperature-controlled container. Upon arrival, the retinas were dissected from the eyecups, and sample pieces were then removed and processed for SBFSEM as before(35). Retinal pieces were incubated in 1.5% potassium ferrocyanide and 2% osmium tetroxide (OsO4) in 0.1 M cacodylate buffer (0.66% lead in 0.03 M aspartic acid, pH 5.5) for 1 h. After washing, the tissue was placed in a freshly made thiocarbohydrazide solution (0.1 g TCH in 10 ml dH2O heated to 60 °C for 1 h) for 20 min at room temperature (RT). After rinsing at RT, the tissue was incubated in 2% OsO4 for 30 min, rinsed again, and stained en bloc in 1% uranyl acetate overnight at 40 °C, then washed, and stained with Walton’s lead aspartate for 30 min. After a final wash, the retinal pieces were dehydrated in a graded ice-cold alcohol series and finally embedded in Epon resin. For one sample (from #1R), semithin sections (0.5 to 1 μm thick) were cut and stained with toluidine blue, until a region without cone pedicles appeared. The block was then trimmed for SBFSEM (Thermo Fisher VolumeScope). Serial EM sections were cut at 50 to 55 nm thickness and imaged at an x-y resolution of 5.0 to 5.5 nm. Montages of 5 × 3 tiles, each tile about 40 × 40 μm, were obtained with an overlap of about 10%. Image stacks were concatenated and aligned using TrackEM2 (Image J). The 3D volume was 110 (x) × 185 (y) x106 (z) µm. Retinal cells were reconstructed using TrakEM2 and displayed in 3D using AMIRA (Thermo-Fisher).

Supplementary Material

Appendix 01 (PDF)

This work was supported by NIH grant EY033447 (Wong, P.I.), EY01730 (Vision core grant, Neitz, M., P.I.), Japan Society for Promotion of Science Overseas Fellowship (Akiba, R.), and AMED grant number JP20bm0204002 (Mandai, M.). We thank Dr. Felice Dunn for her insightful reading of the manuscript.

Author contributions

R.A., H.U., H.-Y.T., M.T., M.M., and R.O.W. designed research; R.A., S.L.B., S.K., H.U., H.-Y.T., and R.O.W. performed research; R.A., S.L.B., S.K., and R.O.W. analyzed data; and R.A., S.L.B., H.U., H.-Y.T., T.B., M.T., M.M., and R.O.W. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

Some study data available (Due to the size of the dataset (>2 Tb), we provide the original dataset by sending the raw images on an external hard-drive upon request.). All study data are included in the article and/or SI Appendix.

Supporting Information

Reviewers: D.K., Washington University in St Louis; and W.L., NIH.
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