The organization of the retina of the turtle species Mauremys caspica, found in fresh water ponds of Israel, has been examined by light microscopical techniques including examination of fresh wholemount retina, one micron blue-stained vertical sections and Golgi-stained material. The anatomical findings on Mauremys retina have been compared with those of the Pseudemys retina (Kolb, 1982) which is more commonly used for electrophysiological and neurochemical studies in the USA. The photoreceptors of Mauremys are similar in type and oil droplet content to Pseudemys photoreceptors except for the double cone in Mauremys. This cone type appears more abundant than in Pseudemys and the principal member contains a yellow oil droplet instead of an orange oil droplet. Golgi staining reveals that all the cell types that have been seen in Pseudemys are found in Mauremys with identical morphology. In addition, two amacrine cell types that were not before described for Pseudemys have been added to the classification. One of these is the tristratified dopaminergic amacrine cell described in immunocytochemical studies (Witkovsky et al., 1984; Nguyen-Legros et al., 1985; Kolb et al., 1987). We have used these anatomical studies on Pseudemys and Mauremys retina to form a catalogue of neural types for the turtle retina in general. We conclude with an attempt to combine findings from anatomy, electrophysiology, and neurochemistry to form an overview of the organization of this reptilian retina.

Monoclonal antibodies to the three isozymes of protein kinase C (PKC) (α, β, and γ) were applied to postmortem human retina. Immunostaining was done on wholemount, or cryostat-sectioned retina, and visualized after ABC/DAB procedures by light (LM) and electron (EM) microscopy.

The PCK-α antibody stained rod bipolar cells throughout the retina. EM analysis confirmed they were PKC-α-immunoreactive (IR) on their characteristic dendritic and axonal synaptology. Putative blue cone bipolar cells with wide-field axon terminals, stratifying in s5 of the inner plexiform layer (IPL), were also PKC-α-IR, and EM showed them to engage in narrow-cleft ribbon junctions in blue cone pedicles.

The PKC-β antibody stained cone bipolar cells, many amacrine cells, and most ganglion cells. Cone bipolar cells were stained all the way into the foveal center: both midget and diffuse varieties were included. The IPL was densely PKC-IR and individual neurons could not be identified on stratification patterns. EM of the outer plexiform layer (OPL) revealed that both flat and invaginating cone bipolar types were IR and that IR axon terminals were presynaptic in all strata of the IPL. The occurrence of PKC-β-IR bipolar axons in stratum 2 of the IPL suggests that OFF-center as well as ON-center types were included.

The PKC-γ antibody gave inferior staining compared with results from the other two antibodies; however, two varieties of wide-field monostratified amacrine cell and a large-bodied ganglion cell type were discernible.

PKC in one form or another appears to be a second messenger used in neurotransmission by both rod and cone systems and ON- and OFF-center systems in the human retina.

Two physiologically distinct, HRP-marked turtle retinal ganglion cells were examined for their morphology, GABAergic, glycinergic, and bipolar cell synaptic inputs, using electron-microscopic autoradiography and postembedding immunocytochemistry. One cell was a color-opponent, transient ON/OFF ganglion cell. Its center response to red was a sustained hyperpolarization, and its center response to green was a depolarization with increased spiking at onset. The HRP-injected cell most resembled G6, from previous Golgi-impregnation studies (Kolb, 1982; Kolb et al., 1988). It was a narrow-field bistratified cell, whose two broad dendritic strata peaked at approximately levels L20–25 (sublamina a) and L60 (sublamina b) of the inner plexiform layer. Bipolar cell synapses onto G6 were found evenly distributed between its distal and proximal dendritic strata, spanning L20–75. These inputs probably originated from several different bipolar cells, reflecting the complexity of the center response. GABAergic inputs were found onto both the distal and proximal strata, from near L20–L85. Only a few glycinergic inputs, confined to dendrites at L50–70, were observed.

A second ganglion cell type that we physiologically characterized and HRP-injected had sustained ON-center, sustained OFF-surround responses. Two examples were studied; both were bistratified in sublamina b, near L60–70 and L85–100, with branches up to near L40. They resembled G10, from previous Golgi-impregnation studies (Kolb, 1982; Kolb et al., 1988). One cell was partially reconstructed to look at the distributions of GABAergic and glycinergic amacrine cell, and bipolar cell inputs. Although synapses from bipolar cells were equally divided between the two major dendritic strata of G10, the inputs to the distal stratum were close to the soma, and the inputs to the more proximal stratum were on the peripheral dendrites. This arrangement may reflect input from two distinct types of ON-bipolar cell. GABAergic and glycinergic inputs to G10 costratified to both strata and to the distal branches; but where glycinergic inputs were found distributed throughout the arbor, GABAergic inputs appeared to be confined to peripheral dendrites. We hypothesize on the neural elements involved and the circuitry that may underlie the physiologically recorded receptive fields of these two very different ganglion cell types in the turtle retina.

The turtle retina has been shown to have a variety of different morphological ganglion cell types as well as distinct physiological ganglion cell types. The major projection of the retina to the brain in nonmammalian vertebrates is to the optic tectum. In this study, we address the question of which retinal ganglion cell types project to the optic tectum in the turtle.

Fluorescent rhodamine-labeled microspheres were used to trace the retinal ganglion cell projection to the superficial layers of the optic tectum. The fluorescent ganglion cell somata, retrogradely marked by transport from the contralateral optic tectum, were impaled with micropipettes containing rhodamine-horseradish peroxidase solution and this dye was iontophoresed into the cells under visual control.

Most of the morphological ganglion cell types described in Golgi studies (Kolb, 1982; Kolb et al., 1988) were stained. Thus, the small cell types G1, G2, G3, G5, G6, and G7; the medium-sized types G10, G11, G12, G13, and G14; and the large-sized types G15, G16, G19, G20, and G21 project to the optic tectum in the turtle. We have added a new type, G2a, which proves to have some differences from the original G2 in branching pattern. We were unable to stain the small type G4, the medium-sized types G8 and G9, and the large cell types G17 and G18; this suggests that they might not project to the superficial layers of the dorsolateral optic tectum, at least, in the turtle.

The morphology of the axon which connects the cell body and pedicle of turtle cone photoreceptors was studied by light and electron microscopy. The axon which contains numerous synaptic vesicles, some endoplasmic reticulum, and a few cisternae is basically filled with cytoplasm. The length of the axon is related to the class of cone and varies slightly with retinal location, with axons as short as 3–6 μm found in red cones, and as long as 60 μm in cones containing colorless oil droplets. By simultaneously voltage clamping the cell body and pedicle regions of single isolated cones, we measured the longitudinal axonal resistance and the cell body and pedicle membrane resistances. For each cell studied, the axonal resistance of cones with short axons was lower than the cell and pedicle membrane resistances. Thus, the cell can be considered to be an isopotential structure. However, in some cones with long axons, the axonal, cell body, and pedicle resistances were comparable. The pedicles of these cones, therefore, could act like summing points and may provide a locus for spatial signal integration. Electrical coupling between the principal and accessory members of double cones was also studied. Electron-microscopic observation of the membrane junction between the apposed inner segments of the double cones in the intact retina show narrow segments which resemble gap junctions. However, in every double cone studied in culture, passing currents into one member of the double cone did not result in measurable current flow in the adjacent cell. Thus, the two members of the double cone, isolated from the turtle retina, are not electrically coupled.

The major neurons and neural circuits that are involved in the transmission of color signals through the human retina to produce the color and spatially opponent P cell or midget ganglion cell responses are described. The older findings of single cone to midget bipolar connectivity is reviewed, and the single midget bipolar cell to midget ganglion cell connectivity as revealed by a recent serial section electron microscope study is described in detail. Our present knowledge concerning the discrimination of the blue-cone subtype from the other longer wavelength cones in the human at the outer plexiform layer is summarized, and our most recent findings concerning horizontal cell connectivity to the different spectral types of cones are discussed. Finally, a hypothetical pathway is proposed for color-opponent surrounds of midget ganglion cells using both horizontal cells at the outer plexiform layer and amacrine cell pathways at the inner plexiform layer.

Using an antibody against tyrosine hydroxylase on newborn to 30-day kitten retinas, we have been able to follow the development of the dopaminergic amacrine cells of the cat retina by light-microscopical investigations of retinal wholemounts. The Type 1 or large Toh+ amacrine cells described by others (Oyster et al., 1985; Törk & Stone, 1979) and named A18 from a Golgi study (Kolb et al., 1981), is at birth (P1) an immature neuron with a small cell body and two or three simple thick radiating dendrites stratifying in stratum 1 with many of the dendrites ending in enlarged growth cones. With increasing postnatal age, the cell body size increases from 12.5 μm diameter to reach 15.5 μm diameter at P30. The dendritic fields also increase in size and complexity. At P1, cells of the central area exhibit dendritic appendages which then develop progressively until at P13 (after eye opening) they are part of rudimentary rings and by P30 the dendritic plexus of Toh+ dendrites and rings in stratum 1, typical of the adult cells, are complete. Toh+ stained processes with growth cones that run deep in stratum 5 of the inner plexiform layer and processes passing to the outer plexiform layer first become apparent at P1 in cells of central inferior retina but not till after P13 are these processes clearly expressed. At P1, the total number of Toh+ Type 1 cells is approximately 4000 and this number remains unchanged to the adult retina. However, the retina increases in size over the Pl–P30 stage and thus the mean density of Type 1 Toh+ cells decreases from 30/mm2 at P1 to 18/mm2 at P30. The maximum density of Type 1 Toh+ cells occurs in central retina 2–4 mm superior temporal to the area centralis, corresponding to the maximum rod photoreceptor concentration.

A second type of small Toh+ amacrine cell can be visualized at P1. This Type 2 cell is characterized by a much smaller cell body than Type 1 cells (9 μm diameter), and with faintly stained dendrites located in stratum 3 of the inner plexiform layer. During later postnatal days, Type 2 cells gradually become unstainable and only few are still seen in far peripheral retina by P23. Type 2 Toh+ cells form a total population of 40,000 cells at P1 with their highest density occurring in peripheral retina. By P13, they cannot be seen in central retina and are reduced to a total population of cells staining for the antibody of 7400 cells in far peripheral retina. Their density decreases from 233/mm2 at P1 to 0/mm2 at P30. The transiently staining population of Type 2 Toh+ immunoreactive cells probably correspond to the small THI-CA-cell seen in rat retina (Nguyen–Legros et al., 1983), and the second type of dopaminergic amacrine cell seen in macaque retina (Mariani & Hokoc, 1988).

Immunocytochemistry was used to reveal a population of bipolar cells that contain γ-atrial natriuretic peptide 1–25 (γ-ANP) in turtle retina. This same antibody was also used in rat retina as a comparative control. The retinas were examined by both conventional light microscopy and confocal microscopy with double-labeling to determine whether protein kinase C-α-like immunoreactivity (PKC-α-LI) was colocalized with the γ-ANP-LI. Some thick sections of turtle retina immunostained with only the γ-ANP antibody were also examined by electron microscopy. In rat, a subpopulation of bipolar cells with axons terminating close to the ganglion cell layer was labeled. Double-labeling experiments indicated that the γ-ANP-LI and PKC-α-LI were colocalized in rat retina, and thus all the bipolar cells with γ-ANP-LI were rod bipolar cells. In turtle, the γ-ANP antibody labeled certain bipolar cells that were characterized by bistratified axon terminals arborizing on the borders of strata S2/3 and S3/4 in the inner plexiform layer (IPL). Double labeling with PKC-α antibody indicated that bipolar cells with γ-ANP-LI were not the same bipolar cell types with PKC-α-LI. Thus, γ-ANP-LI appears to be a new marker for a distinct type of bipolar cell in turtle retina. At the ultrastructural level, the γ-ANP-LI was visible throughout the cytoplasm of the bipolar cells from dendrites to axon terminals. In the outer plexiform layer (OPL), labeled dendrites contacted photoreceptor pedicles almost exclusively at narrow-cleft basal junctions, but infrequently formed the central element at a photoreceptor ribbon synapse. In the IPL, axon terminals with γ-ANP-LI made ribbon synapses onto a combination of amacrine and ganglion cells. Since narrow-cleft basal junctions and photoreceptor ribbon-related junctions are known to be associated with ON-center bipolar cells in turtle, and since the axon terminals of bipolars with γ-ANP-LI stratify primarily in the ON-strata of the IPL, we suggest that these cells are likely to be ON-center cells. It is possible that the γ-ANP may be involved in regulating the activity of Na+/K+ ATPase or in the modulation of cGMP levels.