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Kolb H, Fernandez E, Nelson R, editors. Webvision: The Organization of the Retina and Visual System [Internet]. Salt Lake City (UT): University of Utah Health Sciences Center; 1995-.

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Webvision: The Organization of the Retina and Visual System [Internet].

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Outer Plexiform Layer

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Created: ; Last Update: May 1, 2007.

A certain degree of integration of the visual message takes place at the first synapse in the retina, in the outer plexiform layer. Here cone pedicles and rod spherules are synaptic upon various bipolar cell and horizontal cell types. In addition, as mentioned in the previous section, cone pedicles pass electrical messages between each other and between rod spherules so that a small amount of rod and cone signal mixing occurs at this layer (1, 2).

Two important synaptic interactions that occur at the outer plexiform layer are:

  1. the splitting of the visual signal into two separate channels of information flow, one for detecting objects lighter than background and one for detecting objects darker that background
  2. the instillation of pathways to create simultaneous contrast of visual objects

In the first synaptic interactions, the channels of information flow are known as the basis of successive contrast, or ON and OFF pathways, respectively, whereas the second interaction puts light and dark boundaries in simultaneous contrast and forms a receptive field structure, with a center contrasted to an inhibitory surround. We shall see how these two necessary, visual information processes are created by synaptic interactions at the outer plexiform layer.

Techniques That Have Been Used to Understand Neural Pathways in the Retina

By using various anatomical techniques, the morphologies of individual neurons that make up the retina and contribute processes for synaptic interaction in the plexiform layers have been discovered and described over the years. The most important technique among these is a specific neural stain named after a famous early Italian neuroanatomist, Camillo Golgi (1885), who lived at the end of the last century (3). This staining method was used most extensively and with extraordinary success by the great Spanish anatomist, Ramon y Cajal (1892) (Fig. 1) (4).

Figure 1. Picture of Cajal.

Figure 1

Picture of Cajal.

In fact, the monumental studies of Ramon y Cajal form the basis of neuroanatomy for the vertebrate nervous system in general and the retina in particular. One of Cajal's drawings of nerve cells in the retina stained by Golgi techniques (4) is shown in Fig. 2. In this one figure from Cajal's book on the vertebrate retina, we see photoreceptors, bipolar cells, horizontal cells, and some amacrine cell types. Cajal pointed out that photoreceptors-bipolar cells-ganglion cells were involved in passing rod or cone information through the vertical pathways, and that horizontal and amacrine cells were involved in lateral interactions. He introduced the idea that synaptic connections were set up between specific cell types in the plexiform layers by virtue of nerve cells' dendrites and axons costratifying precisely to ensure that the correct presynaptic cell talked to the correct postsynaptic cell.

Figure 2. Cajal's drawing.

Figure 2

Cajal's drawing.

In 1941, Stephen Polyak authored landmark books concerning the organization of the primate retina and visual system and was the man most responsible for applying the Golgi stain to monkeys and chimpanzees (5). Even today, we continue to use the Golgi method in research on cat, monkey, and human retinas (6-12).

Some of the cells we have described since Cajal and Polyak are shown in Fig. 3. It becomes immediately obvious that our drawings of Golgi-stained cells differ from those of Cajal, in that we have drawn complete cells from a surface or wholemount view looking into the retina mounted flat. Being able to prepare wholemounts of retinas was a great advance for our understanding of the morphologies of nerve cells in the retina becauser it allowed us to see complete dendritic tree spreads of stained cell, which were often truncated in the sectioned retinas that Cajal and Polyak studied. Thus, we have been able to add significant numbers of new cell types to the original descriptions. Also, with the advent of electron microscopy, histochemical and immunocytochemical staining, and electrophysiological single-cell recording and staining, we can now direct these techniques at elucidating neural circuits in the retina in a way not available to our predecessors. All of the descriptions of cells and circuits that follow in this chapter come from experiments over the years using a combination of these techniques but always with the morphological data from Golgi staining as a basis.

Figure 3. Golgi-stained neurons of cat retina.

Figure 3

Golgi-stained neurons of cat retina.

Bipolar Cells

In human retina, 11 different bipolar cell types are revealed by Golgi staining (10, 12-14). Ten are for cones, and one type is for rods (Fig. 4).

Figure 4. Bipolar cell types in human retina.

Figure 4

Bipolar cell types in human retina.

Human retina, like most mammalian retinas, is rod dominated outside of the fovea. Therefore, rod bipolar cells form the numerically superior part of the bipolar population in human retinas.

The rod bipolar is typically a stout bipolar, with a cell body situated middle to high in the inner nuclear layer and producing a tuft of dendrites entering the OPL and reaching up to different levels between cone pedicles to reach the stacked rod spherules (Fig. 5 and Fig. 6).

Figure 5. Rod bipolar cell at low magnification.

Figure 5

Rod bipolar cell at low magnification.

Figure 6. EM and drawing of a rod triad (monkey).

Figure 6

EM and drawing of a rod triad (monkey). rb, rod bipolar dendrite; HC, horizontal cell axon terminals.

The rod bipolar dendritic terminals end one to a rod spherule as the central invaginating dendrite (shown by electron microscopy in Fig. 5 and Fig. 6) (7). In the central retina, rod bipolar dendritic trees are small (15 μm across), and 15–20 rods are contacted. In the peripheral retina, the dendritic tree is 30 μm across and contacts 40–50 rods.

Ten different types of cone bipolar are present in human retina. Seven of them are concerned with converging information from many cones. They are known as diffuse-cone bipolar types (DBs). Three cone bipolar types are concerned only with single-cone contacts in a one-to-one relationship. These are known as midget bipolars, and blue-cone-specific types.

Some of the diffuse-cone bipolars are very wide field (giant) in dendritic spread (70–100 μm) and connect with as many as 15–20 cones (8). Little is known concerning the wide-field cells and and their role in retinal processing. These cells need further exploration. Commonly, the smaller diffuse bipolar cells collect information from 5 to 7 cones in the central retina and from 12 to 14 cones in the peripheral retina. The midget bipolar cells contact single cones, but there are two different varieties of them per cone. Thus, cones of the fovea have output to two midget bipolar cells, and of course, some still output to the diffuse bipolar cells as well. The two types of midget bipolar differ in their contact with the cone pedicle (Fig. 7).

Figure 7. Midget bipolar cell contacts.

Figure 7

Midget bipolar cell contacts.

The invaginating midget bipolar type (IMB) connects with the cone pedicle as central invaginating dendrites (Fig. 7, red profiles) at ribbon synapses in the cone pedicles (7). Flat midget bipolar cells (FMBs) contact the cone pedicle by means of semi-invaginating, wide-cleft basal junctions (Fig. 7, green profiles). Often, FMB dendrites make two contacts with the cone pedicle on either side of the central invaginating dendrite from the other midget bipolar cell (7).

A cone bipolar cell that is thought to be specific for the short-wavelength cones, or blue cones (S-cones), has been described in monkey (10, 15) and in human retina (12). This blue S-cone bipolar (Fig. 8) typically contacts one cone heavily, by several dendrites converging on that particular cone pedicle as central elements at the ribbons; thus, it is essentially another type of midget bipolar cell, but it differs from regular IMBs and FMBs in having also two or more wispy dendrites contacting either another cone pedicle or ending blindly in the OPL (see the later chapter on S-cone pathways). There is also a type of giant, bistratified cone bipolar cell in primate retina (Fig. 8, GBB) that has been proposed to be involved in the blue-cone system, because its axonal branching level in the IPL corresponds exactly to the dendritic branching levels of the bistratified blue/yellow ganglion cell of the S-cone pathways through the retina (see the later chapter on the S-cone pathways) (16).

Figure 8. S-cone bipolar cell types of primate retina.

Figure 8

S-cone bipolar cell types of primate retina.

The remaining diffuse bipolar types in primate retinas (Fig. 8) are analogous to cone bipolar cells of other mammalian retinas in contacting clusters of cone pedicles, as mentioned above. Similar to the midget bipolar cells, though, these diffuse types also differ in their types of synaptic contacts with cone pedicles, being either flat-contacting types (making basal junctions with cone pedicles) or invaginating and ribbon-contacting types, or even being types that mix these types of contacts (17) (see also Kolb and Nelson (18) for a review on photoreceptor to bipolar contacts in the vertebrate retina).

Horizontal Cells

All mammalian retinas have two types of horizontal cell (HC) as the laterally interconnecting neurons in the outer plexiform layer (14). The cat has been studied extensively as a model mammalian retina, and the two types, known as A-type and B-type, are illustrated in Fig. 9 (19). The A- and B-type HCs are very similar in appearance in the rabbit retina as well.

Figure 9. Horizontal cells in cat retina.

Figure 9

Horizontal cells in cat retina.

The A-type HC is a large, sturdy cell with radiating dendrites covering a dendritic field of 150–250 μm, depending on area of retina (all neurons of the retina are small in dendritic expanse in the central or foveal retina and increase in dendritic tree size with eccentricity from the central area). The B-type HC in the mammalian retina differs from the A-type in having a smaller, bushier (in general) dendritic tree (75–150 μm in diameter) and bearing an axon that travels 300 μm or more before ending in a huge, expansive axon terminal tree. The dendrites of both A-type and B-type HCs end in cone pedicles, whereas the axon terminals of the B-type HCs end in rod spherules (Fig. 9) (19).

Primates, although obviously mammals, have been thought of as somewhat different in their HC make-up compared with cats. Originally, there was thought to be only one HC type, called an HI, that looked like a miniature version of the cat B-type HC (5). In 1980, we described a second type of HC in the rhesus monkey retina and called it the HII type (20). Most recently, we have been able to distinguish a third type, the HIII type, of HC in the human retina (21). Light micrographs and drawings of Golgi-stained examples of these three types are illustrated in Fig. 10 and Fig. 11.

Figure 10. Light micrograph of human horizontal cells.

Figure 10

Light micrograph of human horizontal cells.

Figure 11. Drawings of monkey horizontal cells.

Figure 11

Drawings of monkey horizontal cells.

HI is the classic HC of primate retina (5). It is a small-field cell (15-μm-diameter dendritic tree in the fovea, 80–100-μm in the periphery) with stout dendrites, giving rise to distinct clusters of round or donut-shaped terminals contacting cones as lateral elements of the ribbon synapses (Fig. 12).

Figure 12. HI horizontal cell of primate retina.

Figure 12

HI horizontal cell of primate retina.

In peripheral retina, the HI cells have much bigger dendritic trees, and their radiating dendrites contact as many as 18 cones. The HI cell has a single, thick axon that passes laterally in the outer plexiform layer to terminate more than 1 mm away in a thickened axon terminal stalk that bears a fan-shaped profusion of lollipop-like terminals. HI axon terminals end in rod spherules as lateral elements of the ribbon synapses (Fig. 13) (7).

Figure 13. HI horizontal cell terminals.

Figure 13

HI horizontal cell terminals.

HIII cells are similar in appearance to HI cells, but everywhere in the retina, HIII cells are one-third bigger in dendritic tree size and typically, particularly in the peripheral retina, asymmetrical in shape (one or two dendrites are much longer than others). The clusters of terminals contact cones in the same manner as the HI cell terminals, and because of their bigger field size, they contact more cone pedicles (9–12 in the foveal retina, 20–25 in the peripheral retina). The axon of the HIII has not been conclusively followed to a terminal yet, so we know nothing of the nature of the photoreceptor type it contacts, although we suspect it is a mixture of rods and cones.

HII cells are more spidery and intricate in dendritic field characteristics than either of the other types (20). Their terminals are not clearly seen as clusters approaching cone pedicles, but they are known to end in cone pedicles (20, 22, 23). HII cells also bear an axon, but this is quite different from that of the other two HC types. It is short (100–200 μm), curled instead of straight, and has contacts to cone pedicles by means of small, wispy terminals.

Recent findings from electron microscopic studies of Golgi-stained HCs of the human retina show that there is some color-specific wiring going on for the three cell types (Fig. 14) (22, 23).

Figure 14. Three cell types of horizontal cells in human retina.

Figure 14

Three cell types of horizontal cells in human retina.

Thus, HIs contact medium- and long-wavelength cones primarily, but with a small number of contacts to any short-wavelength cones in the dendritic field. HII cells contact short-wavelength cones, directing major dendrites to these cones in their dendritic fields where they occur and contacting with lesser numbers of terminals of other types of non-short-wavelength cone. The HII cells axon contacts short-wavelength cones only. HIII cells have large dendritic terminals in medium- and long-wavelength cones, seemingly avoiding short-wavelength cones in their dendritic tree (23). Thus, a wiring diagram can be made (Fig. 15) that summarizes our present understanding of the spectral connections of the three HC cell types of the primate retina.

Figure 15. Summary of the spectral connections of the three HC types of the primate retina.

Figure 15

Summary of the spectral connections of the three HC types of the primate retina.

ON and OFF Center Pathways and Center Surround Organization of the Retina

We know that a photoreceptor neurotransmitter (which is glutamate, see Dowling (24) and Massey (25) for reviews) is released in the dark in the vertebrate retina (26). Thus, the photoreceptor, whether it be rod or cone, is in a depolarized state in the dark. On light stimulation, the photoreceptor responds with a hyperpolarization; transmitter release ceases, but the postsynaptic bipolar cells respond with either hyperpolarization or depolarization of their membranes. The hyperpolarizing type of bipolar cell is called an OFF-center cell, whereas the depolarizing bipolar cell is called an ON-center cell (27, 28).

The origin of these two important ON-center and OFF-center channels is determined by the types of synaptic contacts that these bipolars make with cone pedicles or rod spherules. Thus, the type of bipolar cell making invaginating contacts (several cone bipolar types, among them the IMB type and the rod bipolar type) responds to light with an inverted sign compared with the photoreceptor. They give depolarizing responses to light and are thought to be stimulated via metabotropic glutamate receptors, specifically mGluR6, and signal via a G protein cascade (29, 30) (see chapters on rod and cone pathways). These depolarizing bipolar cells have an APB-sensitive glutamate receptor (Fig. 16 and Fig. 17) (31).

Figure 16. Origin of ON-center and OFF-center channels.

Figure 16

Origin of ON-center and OFF-center channels.

Figure 17. Drawing of the organization of the photoreceptor synapse showing the different glutamate receptors that are presently known to be on the various postsynaptic dendrites.

Figure 17

Drawing of the organization of the photoreceptor synapse showing the different glutamate receptors that are presently known to be on the various postsynaptic dendrites.

On the other hand, the type of bipolar cell that makes contact with the photoreceptor at a basal junction responds to light just like the photoreceptor by hyperpolarizing (see above). The hyperpolarizing types are driven via ionotropic (iGluR) AMPA-kainate glutamate channels in their synapses with photoreceptors (33). Hyperpolarizing (basal junction-contacting) bipolar cells are the start of OFF-center channels, and the depolarizing (invaginating, ribbon-related) bipolar types are the start of ON-center channels throughout the whole retina and visual system. In a later chapter, we find out that only excitatory channels are present at the level of information transfer between cone bipolar cells and ganglion cells in the inner plexiform layer. Therefore, the status of the signal transmitted by the ganglion cell to the brain is essentially determined by the nature of the cone bipolar contacting it, which in turn is determined by its photoreceptor synapse.

In submammalian species, intracellular recordings from the cones have indicated that they receive a feedback inhibitory message from horizontal cells (see a movie of the intracellular recordings of a cone and bipolar cells) (33). In fish retinas, there is morphological evidence that the horizontal cell lateral elements at the triad synapse produce spinules at which feedback signaling occurs to the cone (see Djamgoz and Kolb (34) for a review). In mammalian cones, there is no unequivocal morphological evidence for this feedback synapse, but some kind of feedback is expected at this site (see above).

Movie 1. A movie of the intracellular recordings of a cone and bipolar cells.

Movie 1

A movie of the intracellular recordings of a cone and bipolar cells.

Horizontal cell lateral elements, i.e., horizontal cell dendrites, have vesicles within them that could be directed at the cone membrane. This arrangement of horizontal cell dendrites on either side of the ribbon synapse in the triad, with the bipolar cell dendrites forming the central or basal junction position, is very important (Fig. 18). A small local circuit is formed here, that influences the flow of information throughout the whole retina.

Figure 18. Electron micrograph of synaptic contacts in a cone pedicle triad synapse.

Figure 18

Electron micrograph of synaptic contacts in a cone pedicle triad synapse. Possible feedback synapses in horizontal cells are shown.

We know that a photoreceptor neurotransmitter is released in the dark in the vertebrate retina. Thus, the photoreceptor, whether it be rod or cone, is in a depolarized state in the dark. On light stimulation, the photoreceptor reacts with a hyperpolarization; transmitter release ceases, but the postsynaptic bipolar cells respond with either hyperpolarization or depolarization of their membranes. The horizontal cells respond with a hyperpolarization as well, but a feedback synapse from the horizontal cell to the photoreceptor is also thought to occur. The feedback signal sums information from a network of horizontal cells connected over a wide spatial area of the outer plexiform layer. Thus, this large spatial area influences the photoreceptor and the bipolar cell to include a response coming from a surround region of retina. This local circuit provides the bipolar cell with a center-surround organization.

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