Volumetric Reconstruction of a Human Retinal Pigment Epithelial Cell Reveals Specialized Membranes and Polarized Distribution of Organelles.

Purpose: Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles. Methods: A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods. Results: In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin. Conclusions: Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Origins of direction selectivity in the primate retina.

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system.

Connectomic Identification and Three-Dimensional Color Tuning of S-OFF Midget Ganglion Cells in the Primate Retina.

In the trichromatic primate retina, the "midget" retinal ganglion cell is the classical substrate for red-green color signaling, with a circuitry that enables antagonistic responses between long (L)- and medium (M)-wavelength-sensitive cone inputs. Previous physiological studies showed that some OFF midget ganglion cells may receive sparse input from short (S)-wavelength-sensitive cones, but the effect of S-cone inputs on the chromatic tuning properties of such cells has not been explored. Moreover, anatomical evidence for a synaptic pathway from S cones to OFF midget ganglion cells through OFF midget bipolar cells remains ambiguous. In this study, we address both questions for the macaque monkey retina. First, we used serial block-face electron microscopy to show that every S cone in the parafoveal retina synapses principally with a single OFF midget bipolar cell, which in turn forms a private-line connection with an OFF midget ganglion cell. Second, we used patch electrophysiology to characterize the chromatic tuning of OFF midget ganglion cells in the near peripheral retina that receive combined input from L, M, and S cones. These "S-OFF" midget cells have a characteristic S-cone spatial signature, but demonstrate heterogeneous color properties due to the variable strength of L, M, and S cone input across the receptive field. Together, these findings strongly support the hypothesis that the OFF midget pathway is the major conduit for S-OFF signals in primate retina and redefines the pathway as a chromatically complex substrate that encodes color signals beyond the classically recognized L versus M and S versus L+M cardinal mechanisms.SIGNIFICANCE STATEMENT The first step of color processing in the visual pathway of primates occurs when signals from short (S)-, middle (M)-, and long (L)-wavelength-sensitive cone types interact antagonistically within the retinal circuitry to create color-opponent pathways. The midget (L versus M or "red-green") and small bistratified (S vs L+M, or "blue-yellow") ganglion cell pathways appear to provide the physiological origin of the cardinal axes of human color vision. Here we confirm the presence of an additional S-OFF midget circuit in the macaque monkey fovea with scanning block-face electron microscopy and show physiologically that a subpopulation of S-OFF midget cells combine S, L, and M cone inputs along noncardinal directions of color space, expanding the retinal role in color coding.

Nonselective Wiring Accounts for Red-Green Opponency in Midget Ganglion Cells of the Primate Retina.

In primate retina, "red-green" color coding is initiated when signals originating in long (L) and middle (M) wavelength-sensitive cone photoreceptors interact antagonistically. The center-surround receptive field of "midget" ganglion cells provides the neural substrate for L versus M cone-opponent interaction, but the underlying circuitry remains unsettled, centering around the longstanding question of whether specialized cone wiring is present. To address this question, we measured the strength, sign, and spatial tuning of L- and M-cone input to midget receptive fields in the peripheral retina of macaque primates of either sex. Consistent with previous work, cone opponency arose when one of the cone types showed a stronger connection to the receptive field center than to the surround. We implemented a difference-of-Gaussians spatial receptive field model, incorporating known biology of the midget circuit, to test whether physiological responses we observed in real cells could be captured entirely by anatomical nonselectivity. When this model sampled nonselectively from a realistic cone mosaic, it accurately reproduced key features of a cone-opponent receptive field structure, and predicted both the variability and strength of cone opponency across the retina. The model introduced here is consistent with abundant anatomical evidence for nonselective wiring, explains both local and global properties of the midget population, and supports a role in their multiplexing of spatial and color information. It provides a neural basis for human chromatic sensitivity across the visual field, as well as the maintenance of normal color vision despite significant variability in the relative number of L and M cones across individuals.SIGNIFICANCE STATEMENT Red-green color vision is a hallmark of the human and nonhuman primate that starts in the retina with the presence of long (L)- and middle (M)-wavelength sensitive cone photoreceptor types. Understanding the underlying retinal mechanism for color opponency has focused on the broad question of whether this characteristic can emerge from nonselective wiring, or whether complex cone-type-specific wiring must be invoked. We provide experimental and modeling support for the hypothesis that nonselective connectivity is sufficient to produce the range of red-green color opponency observed in midget ganglion cells across the retina. Our nonselective model reproduces the diversity of physiological responses of midget cells while also accounting for systematic changes in color sensitivity across the visual field.

A synaptic signature for ON- and OFF-center parasol ganglion cells of the primate retina.

In the primate retina, parasol ganglion cells contribute to the primary visual pathway via the magnocellular division of the lateral geniculate nucleus, display ON and OFF concentric receptive field structure, nonlinear spatial summation, and high achromatic temporal-contrast sensitivity. Parasol cells may be homologous to the alpha-Y cells of nonprimate mammals where evidence suggests that N-methyl-D-aspartate (NMDA) receptor-mediated synaptic excitation as well as glycinergic disinhibition play critical roles in contrast sensitivity, acting asymmetrically in OFF- but not ON-pathways. Here, light-evoked synaptic currents were recorded in the macaque monkey retina in vitro to examine the circuitry underlying parasol cell receptive field properties. Synaptic excitation in both ON and OFF types was mediated by NMDA as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors. The NMDA-mediated current-voltage relationship suggested high Mg2+ affinity such that at physiological potentials, NMDA receptors contributed ∼20% of the total excitatory conductance evoked by moderate stimulus contrasts and temporal frequencies. Postsynaptic inhibition in both ON and OFF cells was dominated by a large glycinergic "crossover" conductance, with a relatively small contribution from GABAergic feedforward inhibition. However, crossover inhibition was largely rectified, greatly diminished at low stimulus contrasts, and did not contribute, via disinhibition, to contrast sensitivity. In addition, attenuation of GABAergic and glycinergic synaptic inhibition left center-surround and Y-type receptive field structure and high temporal sensitivity fundamentally intact and clearly derived from modulation of excitatory bipolar cell output. Thus, the characteristic spatial and temporal-contrast sensitivity of the primate parasol cell arises presynaptically and is governed primarily by modulation of the large AMPA/kainate receptor-mediated excitatory conductance. Moreover, the negative feedback responsible for the receptive field surround must derive from a nonGABAergic mechanism.

Distinct synaptic mechanisms create parallel S-ON and S-OFF color opponent pathways in the primate retina.

Anatomical and physiological approaches are beginning to reveal the synaptic origins of parallel ON- and OFF-pathway retinal circuits for the transmission of short (S-) wavelength sensitive cone signals in the primate retina. Anatomical data suggest that synaptic output from S-cones is largely segregated; central elements of synaptic triads arise almost exclusively from the "blue-cone" bipolar cell, a presumed ON bipolar, whereas triad-associated contacts derive primarily from the "flat" midget bipolar cell, a hyperpolarizing, OFF bipolar. Similarly, horizontal cell connectivity is also segregated, with only the H2 cell-type receiving numerous contacts from S-cones. Negative feedback from long (L-) and middle (M-) wavelength sensitive cones via the H2 horizontal cells elicits an antagonistic surround in S-cones demonstrating that S versus L + M or "blue-yellow" opponency is first established in the S-cone. However, the S-cone output utilizes distinct synaptic mechanisms to create color opponency at the ganglion cell level. The blue-cone bipolar cell is presynaptic to the small bistratified, "blue-ON" ganglion cell. S versus L + M cone opponency arises postsynaptically by converging S-ON and LM-OFF excitatory bipolar inputs to the ganglion cell's bistratified dendritic tree. The common L + M cone surrounds of the parallel S-ON and LM-OFF cone bipolar inputs appear to cancel resulting in "blue-yellow" antagonism without center-surround spatial opponency. By contrast, in midget ganglion cells, opponency arises by the differential weighting of cone inputs to the receptive field center versus surround. In the macula, the "private-line" connection from a midget ganglion cell to a single cone predicts that S versus L + M opponency is transmitted from the S-cone to the S-OFF midget bipolar and ganglion cell. Beyond the macula, OFF-midget ganglion cell dendritic trees enlarge and collect additional input from multiple L and M cones. Thus S-OFF opponency via the midget pathway would be expected to become more complex in the near retinal periphery as L and/or M and S cone inputs sum to the receptive field center. An important goal for further investigation will be to explore the hypothesis that distinct bistratified S-ON versus midget S-OFF retinal circuits are the substrates for human psychophysical detection mechanisms attributed to S-ON versus S-OFF perceptual channels.

Horizontal cell feedback without cone type-selective inhibition mediates "red-green" color opponency in midget ganglion cells of the primate retina.

The distinctive red-green dimension of human and nonhuman primate color perception arose relatively recently in the primate lineage with the appearance of separate long (L) and middle (M) wavelength-sensitive cone photoreceptor types. "Midget" ganglion cells of the retina use center-surround receptive field structure to combine L and M cone signals antagonistically and thereby establish a "red-green, color-opponent" visual pathway. However, the synaptic origin of red-green opponency is unknown, and conflicting evidence for either random or L versus M cone-selective inhibitory circuits has divergent implications for the developmental and evolutionary origins of trichromatic color vision. Here we directly measure the synaptic conductances evoked by selective L or M cone stimulation in the midget ganglion cell dendritic tree and show that L versus M cone opponency arises presynaptic to the midget cell and is transmitted entirely by modulation of an excitatory conductance. L and M cone synaptic inhibition is feedforward and thus occurs in phase with excitation for both cone types. Block of GABAergic and glycinergic receptors does not attenuate or modify L versus M cone antagonism, discounting both presynaptic and postsynaptic inhibition as sources of cone opponency. In sharp contrast, enrichment of retinal pH-buffering capacity, to attenuate negative feedback from horizontal cells that sum L and M cone inputs linearly and without selectivity, completely abolished both the midget cell surround and all chromatic opponency. Thus, red-green opponency appears to arise via outer retinal horizontal cell feedback that is not cone type selective without recourse to any inner retinal L versus M cone inhibitory pathways.

Blue-yellow opponency in primate S cone photoreceptors.

The neural coding of human color vision begins in the retina. The outputs of long (L)-, middle (M)-, and short (S)-wavelength-sensitive cone photoreceptors combine antagonistically to produce "red-green" and "blue-yellow" spectrally opponent signals (Hering, 1878; Hurvich and Jameson, 1957). Spectral opponency is well established in primate retinal ganglion cells (Reid and Shapley, 1992; Dacey and Lee, 1994; Dacey et al., 1996), but the retinal circuitry creating the opponency remains uncertain. Here we find, from whole-cell recordings of photoreceptors in macaque monkey, that "blue-yellow" opponency is already present in the center-surround receptive fields of S cones. The inward current evoked by blue light derives from phototransduction within the outer segment of the S cone. The outward current evoked by yellow light is caused by feedback from horizontal cells that are driven by surrounding L and M cones. Stimulation of the surround modulates calcium conductance in the center S cone.

Parallel ON and OFF cone bipolar inputs establish spatially coextensive receptive field structure of blue-yellow ganglion cells in primate retina.

In the primate retina the small bistratified, "blue-yellow" color-opponent ganglion cell receives parallel ON-depolarizing and OFF-hyperpolarizing inputs from short (S)-wavelength sensitive and combined long (L)- and middle (M)-wavelength sensitive cone photoreceptors, respectively. However, the synaptic pathways that create S versus LM cone-opponent receptive field structure remain controversial. Here, we show in the macaque monkey retina in vitro that at photopic light levels, when an identified rod input is excluded, the small bistratified cell displays a spatially coextensive receptive field in which the S-ON-input is in spatial, temporal, and chromatic balance with the LM-OFF-input. ON pathway block with l-AP-4, the mGluR6 receptor agonist, abolished the S-ON response but spared the LM-OFF response. The isolated LM component showed a center-surround receptive field structure consistent with an input from OFF-center, ON-surround "diffuse" cone bipolar cells. Increasing retinal buffering capacity with HEPES attenuated the LM-ON surround component, consistent with a non-GABAergic outer retina feedback mechanism for the bipolar surround. The GABAa/c receptor antagonist picrotoxin and the glycine receptor antagonist strychnine did not affect chromatic balance or the basic coextensive receptive field structure, suggesting that the LM-OFF field is not generated by an inner retinal inhibitory pathway. We conclude that the opponent S-ON and LM-OFF responses originate from the excitatory receptive field centers of S-ON and LM-OFF cone bipolar cells, and that the LM-OFF- and ON-surrounds of these parallel bipolar inputs largely cancel, explaining the small, spatially coextensive but spectrally antagonistic receptive field structure of the blue-ON ganglion cell.

The smooth monostratified ganglion cell: evidence for spatial diversity in the Y-cell pathway to the lateral geniculate nucleus and superior colliculus in the macaque monkey.

In the primate visual system approximately 20 morphologically distinct pathways originate from retinal ganglion cells and project in parallel to the lateral geniculate nucleus (LGN) and/or the superior colliculus. Understanding of the properties of these pathways and the significance of such extreme early pathway diversity for later visual processing is limited. In a companion study we found that the magnocellular LGN-projecting parasol ganglion cells also projected to the superior colliculus and showed Y-cell receptive field structure supporting the hypothesis that the parasol cells are analogous to the well studied alpha-Y cell of the cat's retina. We here identify a novel ganglion cell class, the smooth monostratified cells, that share many properties with the parasol cells. Smooth cells were retrogradely stained from tracer injections made into either the LGN or superior colliculus and formed inner-ON and outer-OFF populations with narrowly monostratified dendritic trees that surprisingly appeared to perfectly costratify with the dendrites of parasol cells. Also like parasol cells, smooth cells summed input from L- and M-cones, lacked measurable S-cone input, showed high spike discharge rates, high contrast and temporal sensitivity, and a Y-cell type nonlinear spatial summation. Smooth cells were distinguished from parasol cells however by smaller cell body and axon diameters but approximately 2 times larger dendritic tree and receptive field diameters that formed a regular but lower density mosaic organization. We suggest that the smooth and parasol populations may sample a common presynaptic circuitry but give rise to distinct, parallel achromatic spatial channels in the primate retinogeniculate pathway.

Y-cell receptive field and collicular projection of parasol ganglion cells in macaque monkey retina.

The distinctive parasol ganglion cell of the primate retina transmits a transient, spectrally nonopponent signal to the magnocellular layers of the lateral geniculate nucleus. Parasol cells show well-recognized parallels with the alpha-Y cell of other mammals, yet two key alpha-Y cell properties, a collateral projection to the superior colliculus and nonlinear spatial summation, have not been clearly established for parasol cells. Here, we show by retrograde photodynamic staining that parasol cells project to the superior colliculus. Photostained dendritic trees formed characteristic spatial mosaics and afforded unequivocal identification of the parasol cells among diverse collicular-projecting cell types. Loose-patch recordings were used to demonstrate for all parasol cells a distinct Y-cell receptive field "signature" marked by a nonlinear mechanism that responded to contrast-reversing gratings at twice the stimulus temporal frequency [second Fourier harmonic (F2)] independent of stimulus spatial phase. The F2 component showed high contrast gain and temporal sensitivity and appeared to originate from a region coextensive with that of the linear receptive field center. The F2 spatial frequency response peaked well beyond the resolution limit of the linear receptive field center, showing a Gaussian center radius of approximately 15 microm. Blocking inner retinal inhibition elevated the F2 response, suggesting that amacrine circuitry does not generate this nonlinearity. Our data are consistent with a pooled-subunit model of the parasol Y-cell receptive field in which summation from an array of transient, partially rectifying cone bipolar cells accounts for both linear and nonlinear components of the receptive field.

Synergistic center-surround receptive field model of monkey H1 horizontal cells.

Horizontal cells typical of the vertebrate retina are strongly coupled by gap junctions. The resulting horizontal cell network has extremely large receptive fields that extend well beyond the boundaries of a single dendritic tree. This network has been modeled as a syncytium of cytoplasm bounded by cell membrane (Lamb 1976; Naka & Rushton, 1967). Horizontal cells in the primate retina are also coupled by gap junctions, but their receptive fields are relatively small and in some cases may approximate the span of the dendritic tree of an individual cell (Packer & Dacey, 2002). The receptive field of the macaque H1 horizontal cell type has been modeled as the sum of two spatial components: a strong but small diameter excitatory center, and a weak but broad excitatory surround. Here we explore the hypothesis that the receptive field center of H1 cells derives from direct cone synaptic input and that the synergistic surround derives from gap-junctional coupling among H1 cell neighbors. We measured the receptive field structure of H1 cells in the presence of carbenoxolone, a gap junction blocker, to determine the effects of uncoupling center and surround components and compared these data to a neural simulation of the H1 network in which gap-junctional conductance could be manipulated. Carbenoxolone reduced the surround component and eliminated irregularities in spatial structure thought to be associated with the surround. The effects of carbenoxolone could be mimicked by manipulating gap-junctional conductance in an H1 cell network simulation. These results provide strong support for the two-component model of H1 receptive field structure. In addition, carbenoxolone eliminated a slow depolarization following light onset thought to be mediated by cone-H1 feedback (Kamermans & Spekreijse, 1999). Low concentrations of cobalt, a calcium channel blocker that spares gap junctions, had an effect similar to that of carbenoxolone but did not affect receptive field structure. These results are consistent with a calcium-mediated mechanism of feedback from H1 cells to cones that is independent of the synergistic two-component model of receptive field organization.

The classical receptive field surround of primate parasol ganglion cells is mediated primarily by a non-GABAergic pathway.

Although the center-surround receptive field is a fundamental property of retinal ganglion cells, the circuitry that mediates surround inhibition remains controversial. We examined the contribution of horizontal cells and amacrine cells to the surround of parasol ganglion cells of macaque and baboon retina by measuring receptive field structure before and during the application of drugs that have been shown previously to affect surrounds in a range of mammalian and nonmammalian species. Carbenoxolone and cobalt, thought to attenuate feedback from horizontal cells to cones, severely reduced the surround. Tetrodotoxin, which blocks sodium spiking in amacrine cells, and picrotoxin, which blocks the inhibitory action of GABA, only slightly reduced the surround. These data are consistent with the hypothesis that the surrounds of light-adapted parasol ganglion cells are generated primarily by non-GABAergic horizontal cell feedback in the outer retina, with a small contribution from GABAergic amacrine cells of the inner retina.

L and M cone contributions to the midget and parasol ganglion cell receptive fields of macaque monkey retina.

Analysis of cone inputs to primate parvocellular ganglion cells suggests that red-green spectral opponency results when connections segregate input from long wavelength (L) or middle wavelength (M) sensitive cones to receptive field centers and surrounds. However, selective circuitry is not an obvious retinal feature. Rather, cone receptive field surrounds and H1 horizontal cells get mixed L and M cone input, likely indiscriminately sampled from the randomly arranged cones of the photoreceptor mosaic. Red-green spectral opponency is consistent with random connections in central retina where the mixed cone ganglion cell surround is opposed by a single cone input to the receptive field center, but not in peripheral retina where centers get multiple cone inputs. The selective and random connection hypotheses might be reconciled if cone type selective circuitry existed in inner retina. If so, the segregation of L and M cone inputs to receptive field centers and surrounds would increase from horizontal to ganglion cell, and opponency would remain strong in peripheral retina. We measured the relative strengths of L and M cone inputs to H1 horizontal cells and parasol and midget ganglion cells by recording intracellular physiological responses from morphologically identified neurons in an in vitro preparation of the macaque monkey retina. The relative strength of L and M cone inputs to H1 and ganglion cells at the same locations matched closely. Peripheral midget cells were nonopponent. These results suggest that peripheral H1 and ganglion cells inherit their L and M cone inputs from the photoreceptor mosaic unmodified by selective circuitry.

Colour coding in the primate retina: diverse cell types and cone-specific circuitry.

How is the trichromatic cone mosaic of Old World primates sampled by retinal circuits to create wavelength opponency? Red-green (L versus M cone) opponency appears to be mediated largely by the segregation of L versus M cone signals to the centre versus the surround of the midget ganglion cell receptive field, implying a complex cone type-specific wiring, the basis of which remains mysterious. Blue-yellow (S versus L+M cone) opponency is mediated by a growing family of low-density ganglion types that receive either excitatory or inhibitory input from S cones. Thus, the retinal circuits that underlie colour signalling in primates may be both more complex and more diverse then previously appreciated.

Receptive field structure of H1 horizontal cells in macaque monkey retina.

The ganglion cells of primate retina have center-surround receptive fields. A strong candidate for mediating linear surround circuitry is negative feedback from the H1 horizontal cell onto the cone pedicle. We measured the spatial properties of H1 cell receptive fields in the in vitro macaque monkey retina using sinusoidal gratings, spots, and annuli. Spatial tuning curves ranged in shape from smoothly low pass to prominently notched. The tuning curves of approximately 80% of cells could be well described by a sum of two exponentials, giving a prominent central peak superimposed on a broad shallow skirt. The mean diameter of the combined receptive field decreased with eccentricity from 309 micro m at 11 mm to 122 micro m at 4 mm. We propose that the strong narrow field reflects direct synaptic input from the cones overlying the dendritic tree whereas the weak wide field reflects coupled inputs from neighboring H1 cells. Those cells not well fit by a sum of exponentials had tuning curves with additional peaks at higher spatial frequencies that were likely due to undersampling in the cone-H1 network. Unlike other vertebrates, the macaque H1 network is less strongly coupled, has smaller receptive fields, and shows no functional plasticity. Macaque H1 receptive fields are surprisingly small, suggesting a great reduction in electrical coupling. Because the center of the H1 receptive field gets only a small percentage of its total response from the coupled field, the smallest receptive fields are similar in diameter to the dendritic trees. They are probably small enough to form the surrounds of foveal midget cells. The H1 network is compatible with a mixed-surround model of spectral opponency.