Orin S. Packer

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.

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.

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 α-Y cell of other mammals, yet two key α-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 ∼15 μm. 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.

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.

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.

Photopigment transmittance imaging of the primate photoreceptor mosaic

We introduce a new technique for classifying many photoreceptors simultaneously in fresh, excised primate retina on the basis of their absorptance spectra. Primate retina is removed from the pigment epithelium and illuminated under a microscope from the same direction as in the intact eye. To facilitate the guiding of light into the receptor outer segments, the optical axes of the photoreceptors are oriented parallel to the optical axis of the microscope. Photoreceptor outer-segment tips are imaged on a charge-coupled device array, which provides radiometric measurements of the light passing through each photoreceptor. These images are acquired sequentially at three wavelengths chosen to maximize the absorptance differences among the three cone photopigments. After the photopigment is bleached, a second set of three images is acquired. The ratios of the images before and after bleaching at each wavelength are photopigment transmittance maps of the retina. These are combined into a single trichromatic image showing the distribution of photopigment if the retina could be viewed directly in white light without bleaching. We have found patches of receptors in peripheral macaque retina where the measured absorptance at the wavelength of maximum absorptance is consistent with the predicted axial absorptance of th photopigment. The cones in these patches cluster into two groups corresponding to the middle wavelength- sensitive (n = 53, mean absorptance = 0.28) and the long wavelength- sensitive (n = 63, mean absorptance = 0.30) cones. The mean absorptances of 273 macaque and 183 human rods were 0.51 and 0.41, respectively.

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.

Characterization and use of a digital light projector for vision research.

For creating stimuli in the laboratory, digital light projection (DLP) technology has the potential to overcome the low output luminance, lack of pixel independence, and limited chromaticity gamut of the cathode ray tube (CRT). We built a DLP-based stimulator for projecting patterns on the in vitro primate retina. The DLP produces high light levels and has good contrast. Spatial performance was similar to that of a CRT. Temporal performance was limited by the refresh rate (63 Hz). The chromatic gamut was modestly larger than that of a CRT although the primary spectra varied to a small degree with light output and numerical aperture.

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.

A whole mount method for sequential analysis of photoreceptor and ganglion cell topography in a single retina

Photoreceptors (PR) in human and monkey retina are visible in whole mounts cleared with glycerol or dimethyl sulfoxide and viewed with Nomarski differential interference contrast microscopy. These preparations substantially decrease the large tissue volume changes associated with dehydration and sectioning and reveal many details of PR organization and cytology with great clarity. Tissue may be subsequently stained to reveal ganglion cells so that the topography of both cell types may be studied in the same retina.