Cone synapses in macaque fovea: I. Two types of non-S cones are distinguished by numbers of contacts with OFF midget bipolar cells.

L and M cones, divided into two groups by absorption spectra, have not been distinguished by structure. Here, we report what may be such a difference. We reconstructed the synaptic terminals of 16 non-S cones and the dendritic arbors of their ON and OFF midget bipolar cells from high-magnification electron micrographs of serial thin sections of a small region of macaque fovea. Each cone terminal contacted a similar number (~16) of invaginating central elements provided by its ON midget bipolar cell. By contrast, the numbers of connections between a cone terminal and its OFF midget bipolar cell were grouped into two clusters: 30-37 versus 43-50 basal contacts in the triad-associated position and 41-47 versus 61-74 Outer Densities within those basal contacts. The coefficients of variation of these distributions were all in the range of 10% or lower, characteristic of single populations. If these two clusters correspond to M- and L-cone circuits, the results reveal structural differences between M and L cones and between their corresponding OFF midget bipolar cells.

Cone synapses in macaque fovea: II. Dendrites of OFF midget bipolar cells exhibit Inner Densities similar to their Outer synaptic Densities in basal contacts with cone terminals.

As described in the companion paper, the synaptic terminal of a cone photoreceptor in macaque monkey makes an average of 35 or 46 basal contacts with the tips of the dendrites of its OFF midget bipolar cell. Each basal contact has one or more symmetrically thickened dense regions. These "Outer Densities," averaging 48 or 67 in number, harbor clusters of ionotropic glutamate receptors and are ~0.8 µm (and ~1-ms diffusion time) from active zones associated with synaptic ribbons. Here, we show similarly appearing "Inner Densities," averaging 53 or 74 in number, located more proximally on the dendrites of these OFF midget bipolar cells, ~0.4 µm inward from the tips of the dendrites and out of contact with the basal surface of the cone terminal. Compared to desmosome-like junctions, Inner Densities are closer to the terminal and are less dense and less thick. Each Inner Density is shared with another cell, the partners including diffuse bipolar cells, ON midget bipolar cells, and horizontal cells. Given the diversity of the partners, the OFF midget bipolar cells are unlikely to be in a synaptic relationship with the partners. Instead, Inner Densities are near enough to the active zones associated with synaptic ribbons to receive pulses of glutamate at concentrations effective for glutamate receptors. The role of Inner Densities is not known, but they might represent additional clusters of glutamate receptors.

Evidence that each S cone in macaque fovea drives one narrow-field and several wide-field blue-yellow ganglion cells.

A rule of retinal wiring is that many receptors converge onto fewer bipolar cells and still fewer ganglion cells. However, for each S cone in macaque fovea, there are two S-cone ON bipolar cells and two blue-yellow (BY) ganglion cells. To understand this apparent rule reversal, we reconstructed synaptic patterns of divergence and convergence and determined the basic three-tiered unit of connectivity that repeats across the retina. Each foveal S cone diverges to four S-cone ON bipolar cells but contacts them unequally, providing 1-16 ribbon synapses per cell. Next, each bipolar cell diverges to two BY ganglion cells and also contacts them unequally, providing approximately 14 and approximately 28 ribbon synapses per cell. Overall, each S cone diverges to approximately six BY ganglion cells, dominating one and contributing more modestly to the others. Conversely, of each pair of BY ganglion cells, one is dominated by a single S cone and one is diffusely driven by several. This repeating circuit extracts blue/yellow information on two different spatiotemporal scales and thus parallels the circuits for achromatic, spatial vision, in which each cone dominates one narrow-field ganglion cell (midget) and contributes some input to several wider-field ganglion cells (parasol). Finally, because BY ganglion cells have coextensive +S and -(L+M) receptive fields, and each S cone contributes different weights to different BY ganglion cells, the coextensive receptive fields must be already present in the synaptic terminal of the S cone. The S-cone terminal thus constitutes the first critical locus for BY color vision.

Macaque retina contains an S-cone OFF midget pathway.

Psychophysical results suggest that the primate visual system is equally sensitive to both the onset and offset of short-wavelength light and that these responses are carried by separate pathways. However, physiological studies of cells in the retina and lateral geniculate nucleus find far fewer OFF-center than ON-center cells whose receptive-field centers are driven by short-wavelength-sensitive (S) cones. To determine whether S cones contact ON and OFF midget bipolar cells as well as (ON) "blue-cone bipolar" cells (Mariani, 1984), we examined 118 contiguous cone terminals and their bipolar cells in electron micrographs of serial sections from macaque foveal retina. Five widely spaced cone terminals do not contact ON midget bipolar cells. These five cone terminals contact the dendrites of "blue-cone bipolar" cells instead, showing that they are the terminals of S cones. These S-cone terminals are smaller and contain more synaptic ribbons than other terminals. Like neighboring cones, each S cone contacts its own OFF midget bipolar cell via triad-associated (flat) synaptic contacts. Moreover, each S-cone OFF midget bipolar cell has a synaptic terminal in the outer half of the inner plexiform layer, where it contacts an OFF midget ganglion cell.

Cell density ratios in a foveal patch in macaque retina.

We examine the assumptions that the fovea contains equal numbers of inner (invaginating or ON) and outer (flat or OFF) midget bipolar cells and equal numbers of inner and outer diffuse bipolar cells. Based on reconstruction from electron photomicrographs of serial thin sections through the fovea of a macaque monkey, we reject both assumptions. First, every foveal L and M cone is presynaptic to one inner and one outer midget bipolar cell; however, S cones are presynaptic to one outer but no inner midget bipolar cell. Second, we measure the density of all foveal cells in the same patch of fovea, affording accurate cell density ratios. For each foveal cone pedicle, at a density of 26,500 mm(-2), there is close to one (0.88) outer diffuse bipolar cell but only 0.40 inner diffuse bipolar cells. This asymmetry may be related to differences in resolution and sensitivity for light increments and decrements. We also find one (1.01) Müller cell, one (1.01) amacrine cell in the inner nuclear layer, and close to one (0.83) horizontal cell for each cone pedicle. In addition, for each S cone, there are two inner S-cone bipolar cells and two small bistratified ganglion cells. In total, there are 3.4 cone bipolar cells per cone but only 2.6 ganglion cells per cone. The latter ratio is enough to accommodate one midget ganglion cell for each midget bipolar cell.

Inner S-cone bipolar cells provide all of the central elements for S cones in macaque retina.

Synaptic terminals of cones (pedicles) are presynaptic to numerous processes that arise from the dendrites of many types of bipolar cell. One kind of process, a central element, reaches deeply into invaginations of the cone pedicle just below an active zone associated with a synaptic ribbon. By reconstruction from serial electron micrographs, we show that L- and M-cone pedicles in macaque fovea are presynaptic to approximately 20 central elements that arise from two types of inner (invaginating) bipolar cell, midget and diffuse. In contrast, S-cone pedicles, with more synaptic ribbons, active zones/ribbon, and central elements/active zone, are presynaptic to approximately 33 central elements. Moreover, all of these arise from one type of bipolar cell, previously described by others, here termed an inner S-cone bipolar cell. Each provides approximately 16 central elements. Thirty-three is twice 16; correspondingly, these bipolar cells are twice as numerous as S cones. (Specifically, each S cone is presynaptic to four inner S-cone bipolar cells; in turn, each bipolar cell provides central elements to two S cones.) These bipolar cells are presynaptic to an equal number of small-field bistratified ganglion cells, giving cell numbers in 2G:2B:1S ratios. Each ganglion cell receives input from two or more inner S-cone bipolar cells and thereby collects signals from three or more S cones. This convergence, along with chromatic aberration of short-wavelength light, suggests that S-cone contributions to this ganglion cell's coextensive blue-ON/yellow-OFF receptive field are larger than opponent L/M-cone contributions via outer diffuse bipolar cells and that opponent L/M-cone signals are conveyed mainly by inner S-cone bipolar cells.

Two ribbon synaptic units in rod photoreceptors of macaque, human, and cat.

The rod photoreceptor's synaptic terminal (or spherule) uses an elaborate synaptic structure to signal absorption of one or more photons to its postsynaptic targets. This structure includes one or two synaptic ribbons inside the terminal and a pouch-like "invagination" outside the terminal, into which enter a widely variable number of incoming fibers and postsynaptic targets-central elements supplied by rod bipolar cells and lateral elements supplied by horizontal cells. Nonetheless, our three-dimensional reconstructions of this synaptic structure in foveal retina of macaque monkey and peripheral retina of human and cat reveal several features that are highly conserved across species and with eccentricity: 1). every spherule has one invagination; 2). with rare exceptions, every spherule has two ribbon synaptic units with these features: a). on the presynaptic side, each ribbon synaptic unit has a ribbon or part of a ribbon and one trough-shaped arciform density that demarcates its active zone; b). on the postsynaptic side, each ribbon synaptic unit has two apposed lateral elements and one or more central elements; 3). the volume of the extracellular space in the single invagination is small, approximately 0.1 microm(3); and 4). the largest distance from active zone to receptor regions on bipolar cells is small, less than approximately 1.5 microm. With such small dimensions, release of one quantum of transmitter can pulse glutamate to a concentration comparable to the EC(50) of the metabotropic glutamate receptors on the central elements associated with both synaptic units. We speculate that two ribbon synaptic units are required to sustain the high quantal release rate needed to signal a single photon.