The mosaic of horizontal cells in the macaque monkey retina: with a comment on biplexiform ganglion cells.

To further characterize the H1 and H2 horizontal cell populations in macaque monkey retinae, cells were injected with the tracer Neurobiotin following intracellular recordings. Tracer coupling between cells of the same type revealed all H1 or H2 cells in small patches around the injected cell. The mosaics of their cell bodies and the tiling of the retina with their dendrites were analyzed. Morphological differences between the H1 and H2 cells observable in Neurobiotin-labeled patches made it possible to recognize H1 and H2 cells in retinae immunolabeled for the calcium-binding proteins parvalbumin and calbindin, and thus to study their relative spatial densities across the retina. These data, together with the intracellularly stained patches, show that H1 cells outnumber H2 cells at all eccentricities. There is, however, a change in the relative proportions of H1 and H2 cells with eccentricity: close to the fovea the ratio of H1 to H2 cells is approximately 4 to 1, in midperipheral retina approximately 3 to 1, and in peripheral retina approximately 2 to 1. In both the Neurobiotin-stained and the immunostained retinae, about 3-5% of the H2 cells were obviously misplaced into the ganglion cell layer. Several features of the morphology of the misplaced H2 cells suggest that they represent the so-called "biplexiform ganglion cells" previously described in Golgi studies of primate retina.

The cone synapses of cone bipolar cells of primate retina.

One each of bipolar cell types DB2 and DB4, together with a flat and an invaginating midget bipolar cell, were taken from a Golgi-stained rhesus macaque retina; then serially sectioned for EM examination of their synapses with cone pedicles. The cone input to the dendrites of the DB2 cell was exclusively at basal junctions; it had a characteristic distribution. Fifty per cent of the basal synapses were with cone pedicle membrane immediately adjacent to the dendrite of a bipolar cell invaginating to end opposite the ribbon of a cone triad (this, therefore, is called triad-associated). The remainder were one or more synapses distant from the triad-associated position (and, therefore, non-triad associated). The DB4 cell had both basal (predominantly in the triad-associated position) and ribbon-related synaptic input. But the basal to invaginating ratio differed from that of our previously published cell; 56% basal, 43% invaginating, as compared with 31% basal and 69% invaginating. Like foveal IMB cells the synapses of the mid-peripheral invaginating midget bipolar cell were exclusively invaginating; but were about 25% more numerous. The flat midget bipolar cell made exclusively basal synapses. These were 2.5 times more numerous than those of foveal flat midget bipolar cells, and 3.5 times the number of invaginating midget bipolar synapses at equivalent eccentricity. The synapses between cones and diffuse and midget bipolar cells are characteristic for each particular bipolar cell type, but the details depend on a cell's distance from the fovea (eccentricity). A rather constant number of cone pedicle synaptic ribbons 38.6 +/- 2.5 (n = 60) was found across mid-peripheral macaque and vervet monkey retinae. The smaller mean number for vervet monkey, 27.4 +/- 3.5 (n = 23), suggests there can also be generic differences in synaptic detail at cone bipolar cell synapses.

The cone synapses of DB1 diffuse, DB6 diffuse and invaginating midget, bipolar cells of a primate retina.

The distribution of synapses between cones and two types of diffuse cone bipolar cell in a rhesus monkey retina is described. The dendrites of representative Golgi-stained cells of each of the diffuse cone bipolar cell types DB1 and DB6 were serially sectioned for EM examination. Bipolar cells of the DB1 type have axons terminating in the outer half of the inner plexiform layer. The dendrites of the cell examined were postsynaptic to seven cones at 71 basal synapses; in addition, they had two ribbon synapses with one cone, and one with another. A DB6 type of bipolar cell has axons ending in the inner half of the inner plexiform layer. The dendrites of the cell examined received input from seven cones at 30 ribbon synapses; in addition there were 13 basal junctions distributed between five of the seven cones contacted. Two invaginating midget bipolar cells were found to be postsynaptic at 25 and 26 ribbon synapses of cone pedicles containing 44 and 40 ribbons respectively. These results combined with our previously published work, show that the position and number of synapses is characteristic for each category of cell. Those bipolar cells (flat) making basal synapses have more sites of synaptic contact with the cones than those bipolar cells (invaginating) with predominantly ribbon synaptic input. Over 95% of the cone junctions of the three types of diffuse bipolar cell, DB1, DB2 and DB3, are basal; and their axons always end in the a- (Off-) layer of the inner plexiform layer. All three types of diffuse invaginating cone bipolar cell, DB4, DB5 & DB6, have axons terminating in the b- (On-) layer of the inner plexiform layer; their dendrites are predominantly postsynaptic as central elements invaginating at the cone triads. However, unlike invaginating midget bipolar cells, whose dendrites are exclusively postsynaptic at ribbon synapses, between 10% (DB5) and 40% (DB4 and DB6) of the cone input to diffuse invaginating bipolar cells is through basal junctions. These data are discussed in the context of recent work on the synapses between foveal cones and their bipolar cells.

The horizontal cells of artiodactyl retinae: a comparison with Cajal's descriptions.

The morphology of horizontal cells in ox, sheep, and pig retinae as observed after Lucifer Yellow injections are described and compared with the descriptions of Golgi-stained cells by Ramón y Cajal (1893). Horizontal cells in the retinae of less domesticated species, wild pig, fallow and sika deer, mouflon, and aurochs were also examined. All these retinae have two types of horizontal cell; their morphologies are in common, although with some familial differences. Their basic appearance is as Cajal described; except in one important respect, a single axon-like process could not be identified on the external horizontal cells. It is concluded that external horizontal cells of artiodactyls correspond to the axonless (A-type) cells of other mammals. Cajal's internal horizontal cells have a single axon which contacts rods. This type corresponds to the B-type cells of other mammalian retinae. Artiodactyl A- and B-type horizontal cells differ from those of many other mammals in that the B-type dendritic tree is robust and the A-type dendritic tree is delicate. Historically, this morphological difference between orders of mammals has led to some confusion. The comparisons presented here suggest that the morphological types of primate horizontal cells can be integrated into a general mammalian classification.

Blue-cone horizontal cells in the retinae of horses and other equidae.

The morphology of horizontal cells chiefly of the horse, but also of asses, mules, and a zebra, has been examined by Lucifer yellow injections into lightly fixed retinae and by immunocytochemistry. In common with other mammals, equids have a B-type horizontal cell, i.e., a cell with dendrites synapsing with cones and possessing a single axon synapsing with rods. Most mammalian retinae have a further type of horizontal cell, the A-type, also synapsing with cones but without an axon. The second type of horizontal cell in equids also has no axon; otherwise, it is most unusual. Compared with other mammalian A-type cells, it has a vary large dendritic field, both absolutely and relative to the dendritic fields of B-type cells. The dendrites are fine and sparsely branching. Their most striking feature is that they bear a low density of irregularly spaced synaptic terminal aggregates, suggesting their cone contacts are selective. Immunolabelling of S (blue)-cones in horse retina showed that they comprise, depending on retinal location, 10-25% of the cone population. For a single horse A-type cell, it is shown that 44 of its 45 terminal aggregates are congruent with the pedicles of S-cones. Immunostaining with a calbindin antibody demonstrated that each type of horizontal cell forms an independent regular mosaic. The density ratio of B- to A-type cells varied between 5 and 10. This is the first demonstration in a mammalian retina of a horizontal cell type with a direct input exclusively from S-cones.

The rod pathway of the macaque monkey retina: identification of AII-amacrine cells with antibodies against calretinin.

AII-amacrine cells were characterized from Golgi-stained sections and wholemounts of the macaque monkey retina. Similar to other mammalian retinae, they are narrow-field, bistratified amacrine cells with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smoother dendritic tree in the inner half. AII cells of the monkey retina were stained immunocytochemically with antibodies against the calcium-binding protein calretinin. Their retinal mosaic was elaborated, and their density distribution across the retina was measured. Convergence within the rod pathway was calculated. Electron microscopy of calretinin-immunolabelled sections was used to study the synaptic connections of the AII cells. They receive a major input from rod bipolar cells, and their output is largely onto cone bipolar cells. Thus, the rod pathway of the primate retina follows the general mammalian scheme as it is known from the cat, the rabbit, and the rat retina. The spatial sampling properties of macaque AII-amacrine cells are discussed and related to human scotopic visual acuity.

Synapses between cones and diffuse bipolar cells of a primate retina.

The photoreceptor synapses of three representative cells of the six types of diffuse bipolar cell of the rhesus macaque monkey's retina are described at 3.5-4.0 mm eccentricity. Bipolar cell DB3 was found to be postsynaptic to 11 cones at 155 basal synapses; about 70% of these were triad associated. Bipolar cell DB4 as postsynaptic to eight cones at 52 ribbon synapses; in addition it was found also to make an average of two or three basal (non-ribbon) synapses per cone (total 23). The DB5 bipolar cell type had 57 invaginating synapses with seven cones. It too had basal synapses, but only two with each of three cones. The diffuse invaginating bipolar cell described by Mariani (1981) is identified as a member of the DB5 category. Dendrites of cone bipolar cell types which have axons ending in the a-layer of the inner plexiform layer make only basal synapses with the cone pedicle. Those so far investigated are the flat midget bipolar cell and the DB2 and DB3 flat diffuse bipolar cells. All bipolar cells whose axons terminate in the b-layer of the inner plexiform layer are postsynaptic at the ribbon synapses of the cone pedicles. They now appear to fall into two groups. Those whose dendrites are exclusively postsynaptic at the ribbons; these are the blue cone and invaginating midget bipolar cells. And the diffuse bipolar cell DB4, that has both ribbon and basal synapses in a ratio of about 2.3:1. It is uncertain into which category cell DB5 should be placed; its basal synapses are so few the cell could be anomalous. It now seems that at least one primate bipolar cell type may be like those of other vertebrates in having, as defined ultrastructurally, two different kinds of synaptic connection with its cones. The results are discussed in the context of a brief review of the photoreceptor synapses of other mammalian bipolar cells.

Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location.

Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used for in situ and in vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable by in situ immunocytochemistry. However, B-type cells could be seen to express all three subunits in vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical characterization and spatial distribution of midget bipolar cells in the macaque monkey retina.

Midget bipolar cells form the first distinct step in the parvocellular (P-) pathway of the primate visual system, and are the major determinant of the receptive field properties of colour selective midget ganglion cells. This paper describes the sampling properties of the midget bipolar cell population and relates this to the processing of chromatic information in the P-pathway. Immunocytochemical markers were used to label midget bipolar cells so that their spatial density could be compared with that of cones and ganglion cells. Sections through macaque monkey retinae were immunostained with antibodies against cholecystokinin (CCK), and recoverin. In CCK-labelled sections, in addition to blue cone bipolar cells, numerous thin bipolar cell dendrites, which could be associated with individual cone pedicles are stained. CCK-immunoreactive midget bipolar cells are found throughout the retina. A different population of midget bipolar cells is revealed in recoverin-labelled sections. Based on a comparison with midget bipolar cells in Golgi-stained retinae we propose that ON-midget (invaginating) bipolars are immunoreactive for CCK and confirm that OFF-midget (flat) bipolar cells are immunoreactive for recoverin [Milam, Dacey and Dizhoor (1993) Visual Neuroscience, 10, 1-12]. The density of recoverin labelled midget bipolars matches the cone density to an eccentricity of about 10 mm; from there outwards it drops to 60% of the cone density. This suggests convergence of several cones to individual midget bipolar cells in peripheral retina. We conclude that midget bipolar cells are present throughout the entire primate retina, and could, in peripheral as well as in central retina, provide chromatically specific input to the P-pathway.

Cone synapses of a flat diffuse cone bipolar cell in the primate retina.

A Golgi-stained flat diffuse cone bipolar cell from a vervet monkey's retina (Cercopithecus aethiops), contacting six cones, was serially sectioned for electron microscopy (EM) to determine the types of synapses it made with the cone pedicles. All the synapses were basal (flat) contacts. Their distribution and ultrastructural type were similar at each pedicle. Approximately half the synapses were definable as triad-associated and the rest were elsewhere on the cone pedicle base. Their ultrastructure is the same regardless of those positions. About 25 synapses were made with each cone. Thus this type (DB2 of Boycott & Wässle, 1991) of flat diffuse cone bipolar cell is in contact with six cones through about 150 synapses. At the eccentricity studied each cone pedicle probably makes 90-100 basal synapses with between three and four DB2 bipolar cells. This is between two and three times the number that are made with all the types of invaginating bipolar cells. A brief review of cone photoreceptor synapses with bipolar cells shows that, for those so far examined in the primate retina, the dichotomy into two types of bipolar cell invaginating (ribbon-related), with axons ending in the b-layer of the inner plexiform layer (IPL) (hence presumptive On-bipolars) and flat (basal synapses), with axons ending in the a-layer of the inner plexiform layer (hence presumptive Off-bipolars) is the rule. But other vertebrate retinae, including that of the cat, also have bipolar cells which vary from this pattern.

Parasol (P alpha) ganglion-cells of the primate fovea: immunocytochemical staining with antibodies against GABAA-receptors.

Retinae of macaque monkeys were immuno-stained with antibodies against GABAA-receptors. In peripheral retina most ganglion cells were immunoreactive. In central retina, around the fovea, staining in the ganglion cell layer was selective and only 5-8% of all ganglion cells were labelled: these had the largest cell bodies and their dendrites occupied a broad stratum in the middle of the inner plexiform layer. From comparison with Golgi-stained ganglion cells it is concluded that the entire population of parasol (P alpha)-cells at the fovea was labelled. The mosaic and sampling properties of parasol cells were determined by combining dendritic field measurements of Golgi-stained cells with their density when immuno-stained. There is convergence of 30-50 cones onto each foveal parasol ganglion cell. The dendritic fields of both ON- and OFF-parasol cells provide complete retinal coverage. The Nyquist limits of their mosaics are 4 min of arc.

Synaptic contacts of a two-cone flat bipolar cell in a primate retina.

The Golgi-stained dendrites of a two-cone bipolar cell of a vervet monkey were serially sectioned for electron microscopy and shown to make basal synapses with two neighboring cones. The synapses were all on the cone pedicle membrane adjacent to the bipolar processes invaginating to form the triads. This is the characteristic position for flat midget bipolar cells. The numbers of triads in nine adjacent cone pedicles were not significantly different from those in the cones contacted by the bipolar. Based on this measure the two-cone bipolar does not contact a special population of cones. The advantages and disadvantages of using vertical or horizontal sections for the determination by electron microscopy of the connectivity of the dendrites of Golgi-stained flat bipolar cells are discussed.

Morphological Classification of Bipolar Cells of the Primate Retina.

Bipolar cells were studied in Golgi - Colonnier-stained whole mounts of macaque monkey retinae. A piece of retina, at 6 - 7 mm eccentricity, was particularly well stained for the analysis of the different bipolar cell types. Many midget bipolar cells were encountered and the dichotomy into flat and invaginating midget bipolars was confirmed. Six types of diffuse cone bipolar cell are distinguished. They differ in their dendritic branching pattern, in the number of cones contacted-usually between five and ten-and in the shape and branching level of their axons. The size, shape and stratification of the axons were found to be the most reliable distinguishing features for classifying diffuse cone bipolar cells. The stratification of the axons in the inner plexiform layer (IPL), whether closer to the amacrine or ganglion cells, was used to name diffuse cone bipolar cells in the order DB1 to DB6. Blue cone and rod bipolar cells were confirmed as distinct types. Axon terminals of diffuse cone bipolars were found to tile their sublamina of the IPL in a territorial manner. From this the density of each type could be estimated, and it is shown that a single cone is likely to be in contact with as many as 15 individual diffuse bipolar cells, as well as two midget bipolars. The diffuse bipolar cells observed contact all the cone pedicles in their dendritic fields. It is, therefore, unlikely that they carry a chromatic signal into the inner retina. The presence of many midget bipolar cells, which make contact with one cone pedicle only, suggests that midget bipolars provide chromatic input to ganglion cells in peripheral retina as well as in the fovea. The data show that the P- and M-cell pathways of the primate visual system are, to a significant extent, already anatomically discrete at the photoreceptor synapse.

Cone bipolar cells and cone synapses in the primate retina.

Primate retinal bipolar cells synapsing with two adjacent cones (2C bipolars) are further described. Their synaptic contacts are either as the central (invaginating) component of the cone triads or as basal (flat) contacts on the membrane of the cone pedicle base. Correspondingly, their axons end either in the b (inner half or in the a (outer) half of the inner plexiform layer. The shape and size of the axon terminals of 2C bipolars are indistinguishable from those of adjacent midget bipolars. Therefore 2C bipolars, like midget bipolars, probably synapse with midget ganglion cells. Two C bipolars have not been identified as connected to foveal cones. But they are not restricted to the retinal periphery, as has previously been supposed, since they occur, mixed with midget (single cone) bipolars, throughout all parts of the retina from about 2.5 mm to at least 10.0 mm from the fovea. It is likely that 2C bipolars are a variant of the midget bipolars; and that they contact some members of the same population of cones, instead of the midgets. This paper briefly reviews, and raises some new, problems concerning our current understanding of the synaptic connectivity patterns of the midget, 2C, and diffuse cone bipolar cells.

Retinal ganglion cell density and cortical magnification factor in the primate.

The question of whether the large area occupied by the primate fovea in the visual cortex (V1) is the result of a selective amplification of the central visual field, or whether it merely reflects the ganglion cell density of the retina, has been a subject of debate for many years. Measurements of the ganglion cell densities are made difficult by lateral displacements of cells around the fovea and the occurrence of amacrine cells in the ganglion cell layer. We have now identified and counted these amacrine cells by GABA immunocytochemistry and by retrograde degeneration of ganglion cells. By reconstructing the fovea from vertical and horizontal serial sections, we were able to measure the densities of cones, cone pedicles and ganglion cells within the same retina. We found 3-4 ganglion cells for every foveal cone. This ratio decreased to one ganglion cell per cone at an eccentricity of 15-20 deg (3-4 mm) and in peripheral retina there are more cones than ganglion cells. The ganglion cell density changes by a factor of 1000-4000 between peripheral and central retina. A comparable gradient has been reported for the representation of the peripheral and central visual field in V1. We suggest that ganglion cell density can fully account for the cortical magnification factor and there is no need to postulate a selective amplification of the foveal representation.

Cortical magnification factor and the ganglion cell density of the primate retina.

It has long been contentious whether the large representation of the fovea in the primate visual cortex (V1) indicates a selective magnification of this part of the retina, or whether it merely reflects the density of retinal ganglion cells. The measurement of the retinal ganglion-cell density is complicated by lateral displacements of cells around the fovea and the presence of displaced amacrine cells in the ganglion cell layer. We have now identified displaced amacrine cells by GABA immunohistochemistry and by retrograde degeneration of ganglion cells. By reconstructing the fovea from serial sections, we were able to compare the densities of cones, cone pedicles and ganglion cells; in this way we found that there are more than three ganglion cells per foveal cone. Between the central and the peripheral retina, the ganglion cell density changes by a factor of 1,000-2,000, which is within the range of estimates of the cortical magnification factor. There is therefore no need to postulate a selective magnification of the fovea in the geniculate and/or the visual cortex.

Horizontal Cells in the Monkey Retina: Cone connections and dendritic network.

Horizontal cells of the macaque monkey retina were quantified and the number of cones converging onto an individual horizontal cell as well as the number of horizontal cells contacting a single cone were determined. This was done by combining data from individual horizontal cells stained by the Golgi method with the results of immunocytochemical staining described in the preceding paper (Röhrenbeck et al., 1989). The observation (Boycott et al., 1987) that all horizontal cells contact all cones in their dendritic field irrespective of cone type was confirmed. The particular cones contacted by the terminal aggregates of each horizontal cell were found. The dendritic fields of H1 and H2 cells increase with increasing eccentricity; close to the fovea H1 cells are smaller than H2 cells, at 6 mm eccentricity they are about the same size and in peripheral retina H1 cells are much larger than H2 cells. The density gradients of the two cell types balance their denritic field changes so that throughout the retina each and every cone synapses with 3 - 5 horizontal cells of each type. Horizontal cells of both cat (Wässle et al., 1978) and monkey retina follow the general rule that all cones in the dendritic fields are contacted, their perikarya form a regular mosaic and the boundaries of their dendritic fields are marked by the perikarya of their homologous neighbours.

Neurofibrillar long-range amacrine cells in mammalian retinae.

A distinct population of wide-field, unistratified amacrine cells are shown to be selectively stained by using neurofibrillar methods in rabbit and cat retinae. Their cell bodies may be located in the inner nuclear, inner plexiform or ganglion cell layers and they branch predominantly in stratum 2 of the inner plexiform layer. Characteristically, each cell has two or more long-range distal processes which extend for 2-3 mm beyond a more symmetrical, proximal dendritic field of 0.6-0.8 mm diameter. Although the neurofibrillar long-range amacrines account for less than 1 amacrine in 500, they achieve effective coverage of the retina by both the proximal and distal dendrites.