Bipolar, Amacrine, and Ganglion Cells Interact in the Inner Plexiform Layer

The axonal endings of bipolar cells bring information from the outer plexiform layer (OPL) to the neuropil of the inner plexiform layer (IPL) (Figure 1). Here bipolar cells talk to different varieties of functionally specialized amacrine cells and to dendrites of the various ganglion cells. The neuropil is a confusing network of interconnecting profiles that, to be understood, has to be investigated at the higher magnification afforded by the electron microscope (over the light microscope) (Figure 2) and with knowledge gained from Golgi-staining for morphology and intracellular electrophysiology for function of individual cells in the network.

Figure 1

3-D block of retina with the inner plexiform layer highlighted (red).

Figure 2

Light micrograph of a vertical section of the human retina to show the inner plexiform layer (IPL).

Ultrastructure of the Neuropil of the Inner Plexiform Layer

A view of a small part of the neuropil of the inner plexiform layer is shown in the electron micrograph (Figure 3). Bipolar cell axon terminals (Figure 3, red profiles) are vesicle-filled profiles, containing irregular, long mitochondria and neurotubules. Their synapses are typified by a small synaptic ribbon pointing into a wedge with two post synaptic profiles (known as a dyad) at the apex (Figure 4, red dots in bipolar cell profiles) (1). Amacrine cell dendrites are also vesicle filled and have round mitochondria and sometimes neurofilaments as well as neurotubules. Amacrine profiles vary between being very small cross-sections through thin straight tubes and being larger varicosities budding off the dendrites.

Figure 3

Electron micrograph of the neuropil of the IPL.

Figure 4

Electron micrograph of reciprocal synapses in the IPL.

Typically, amacrine cells synapse upon other profiles (bipolar axons, amacrine cells, or ganglion cell dendrites) in the enlarged varicosities at what is known as a conventional synapse: the synapse consists of synaptic vesicles clustered at a pre- and post-membrane density (Figure 4, yellow dots in amacrine profile) (1). Ganglion cell dendrites (Figure 3, yellow profiles) are recognizable as profiles lacking synaptic vesicles but containing ribosomes, neurotubules, and filaments. Ganglion cell dendrites are seen to be postsynaptic to bipolar ribbon synapses and amacrine conventional synapses. Bipolar cells are also postsynaptic to many amacrine synapses. Often amacrine cells make what are called reciprocal synapses to the bipolar cell axons from which they receive ribbon synapses (Figure 4, yellow dots).

This reciprocal synapse is in essence a feedback synapse to the bipolar cell axon in the inner plexiform layer, with the same strategic importance as the horizontal cell dendrite making a feedback synapse at the photoreceptor ribbon synapse, in the outer plexiform layer. It will be remembered that such a local circuit in the outer plexiform layer was thought to provide the bipolar cell response with a center-surround organization. It has been suggested that another surround mechanism coming from the amacrine cells could be added at this reciprocal (feed-back) synapse in the inner plexiform layer.

Different Morphological Types of Amacrine and Ganglion Cells

It is clear that there are many different kinds of amacrine cell and ganglion cell branchings in the IPL of the human retina. From Golgi-staining studies, we know that there are at least 25 different amacrine cell types in the monkey and human retinas (2, 3). Some drawings of different amacrine cells as seen after Golgi-staining in wholemounts of human retina are shown in Figure 5.

Figure 5

Amacrine cells in human retina.

The amacrine cells are classified into different types based on morphological characteristics of dendritic tree size, such as small, medium, and large branching characteristics (i.e., tufted, varicose, linear, beaded, and radiate) and, most importantly, on the stratification of their dendrites in the IPL (2, 3). The neuropil of the IPL was arbitrarily divided into five strata by Cajal (4), because he appreciated the fact that cells branching in disparate strata could not make synaptic interactions, whereas those that costratified could. This five-strata, descriptive classification scheme has been used by morphologists since that time to classify retinal cells on their dendritic branching level. See, for example, that in Figure 6, some amacrine cells have dendrites in strata 1 and 2 (called broadly stratified types; Figure 6, cell 8) or strata 1 and 5 (called bistratified types; Figure 6, cell 1), stratum 1, or any of the five strata (called monostratified; Figure 6, cell 13) or dendrites through all strata 1 to 5 (called diffuse types; Figure 6, cells 12 and 14).

Figure 6

Stratification of amacrine cells in human retina.

In Figure 6, the amacrine cells have been stained by the Golgi procedure which, as we have seen, is very good for showing the isolated complete shape and size of a nerve cell. Newer techniques are also providing valuable information on cell shapes and sizes: techniques such as using intracellular staining through a microelectrode, and immunocytochemistry to stain cells with antibodies to their neurotransmitter or synthesizing enzymes.

These are some different amacrine cell types stained by injection of dyes through a microelectrode inserted into the cell body. The cells that look like exploding fireworks are called "starburst" cells and are known to use acetylcholine as their neurotransmitter (Figure 7) (5-7).

Figure 7

Starbust amacrine cells.

Another type of amacrine cell, thought to contain serotonin as a neurotransmitter, has been stained by the same technique (Figure 8) (8).

Figure 8

Serotonin-containing amacrine cells.

Using similar morphological techniques of Golgi staining, immunocytochemisry, and intracellular staining, the ganglion cells of the retina have been classified into various different types (Figure 9, Figure 10). As many as 25 different types exist in mammalian and human retinas, a few of which are shown in both sectioned view (Figure 9) and wholemount view (Figure 10). As with the amacrine cells, ganglion cells are classified onto the different types by cell body size, dendritic tree spread, branching patterns (e.g., radiate or tufted), and branching level in the five strata of the IPL. In both cat and human retinas, ganglion cells can be from small to large, diffuse, bistratified, and various monostratified, large-field types with different stratification levels in the inner plexiform layer.

Figure 9

Sectioned view of ganglion cells.

Figure 10

Wholemount view of Golgi-stained ganglion cells in primate retina.

Both amacrine and ganglion cells increase in dendritic tree span with eccentricity from the fovea. The very smallest dendritic fields for all cells types being possessed by cells of the fovea or area centralis are shown in Figure 11 for the common ganglion cells of the cat retina (alpha and beta types). The same ganglion cell types are ten times the size in dendritic tree spread in peripheral retina (9, 10). For this reason, classification of amacrines and ganglion cells has to always take eccentricity from the fovea into account and compare cells in similar areas of retina to be sure that they are indeed different cell types.

Figure 11

Cat ganglion cells.

In the human retina, the commonest ganglion cell types are the parasol and the midget ganglion cells, shown in Figure 12 (3, 11-13). They are also known as P cells (midget ganglion cells), because of their projection to the parvocellular layers, and M cells (parasol ganglion cells), because of their projection to the magnocellular layers of the lateral geniculate nucleus (14). We shall return to these ganglion cell types in a later chapter.

Figure 12

Primate ganglion cells.

Stratification of Amacrine and Ganglion Cells in Relationship to Bipolar Cell Axons

One may ask why amacrine cells and ganglion cells should be classified on stratification level of their dendrites, particularly if they look the same in other respects, such as field size, cell body size, and dendritic morphology. It was already clear to Cajal (4) 100 years ago that dendrites of amacrine and ganglion cells and axons of bipolar cells were arranged to costratify in some meaningful manner. The meaning of costratifications and orderly arrangements of branching levels, other than the obvious of making sure certain cell types made synapses with certain other cell types, escaped him though.

We had to wait for 70 years before the answer was found in the physiological responses of the different nerve cells of the retina. In the 1970s, it became possible to make intracellular recordings from the neurons of the retina with dye-filled glass microelectrodes. Physiologists were able to record both the response of the impaled cell to light and to fill the cell with a fluorescent dye, such as Lucifer, to see what type of cell it was (Figure 13, left) (see the animation of the ionotophoresis of Lucifer dye into a ganglion cell via a microelectrode and see the animation of the physiological recording procedure).

Figure 13

Experimental procedure for impaling ganglion cell in the retina.

Movie 1

An animation of the iontophoresis of Lucifer dye into a ganglion cell via a microlectrode.

Movie 2

An animation of the physiological recording procedure.

In the 1950s and 1960s, it became apparent from ganglion cell recordings in the mammalian retina that the visual message leaving the retina was in the form of ganglion cell axon spike discharges that occurred either when a spot of light stimulated the retina (ON discharge to light) or when the spot of light was turned off (OFF discharge). Ganglion cells were responding to either one or the other of the change of state of light. The one group was stimulated by the light brighter than background and the other by light darker than background (15, 16). Next came intracellular recordings from the five basic cell types in a mudpuppy retina, which revealed that cells preceding ganglion cells also fell into the two physiological types, i.e., bipolar and amacrine cells could give ON or OFF discharges (17, 18). Knowing now that bipolar cells could be ON- or OFF-center to a spot of light (they could have the opposite response in a surround concentrically around their central receptive field; mentioned in the chapter on the outer plexiform layer and dealt with in detail in later chapters on circuitry for rod and cone signals), it was reasonable to suggest that OFF-center bipolar chains excited OFF ganglion cells, and that ON-center bipolar cell chains excited ON ganglion cells. The typical bipolar cell types that might form the bipolar chains driving ganglion cells of the primate retina are shown in Figure 14.

Figure 14

Bipolar cell types in human retina (from Golgi staining).

These bipolar cells, as described in the previous section on the outer plexiform layer, make different types of synapses with cone pedicles and rod spherules. Some types make basal contacts on the surface of the cone pedicles, and others make invaginating contact to end close to the synaptic ribbons in both rod spherules and cone pedicles.

It is now thought that bipolar cells respond to light just like the photoreceptor, with a slow hyperpolarization when the bipolar dendrite is in the basal junction position. In contrast, the other type of contact, invaginating to the ribbon synapse, causes the bipolar to respond to light with an inverted sign compared with the photoreceptor. It gives a slow depolarizing response. Thus, the nature of the postsynaptic membrane channels on the cone bipolar cell dendrite is the important designator of the response sign the bipolar will have. The hyperpolarizing bipolar types are the start of OFF-center channels, and the depolarizing types are the start of ON-center channels through the retina.

The bipolar cells shown in Figure 14 will be noticed to have different axonal ending levels in the inner plexiform layer, e.g., DB1 has an axon ending in the stratum 1 neuropil close to the amacrine cell layer of the inner nucelar layer, while imb has an axon ending at the opposite side of the inner plexiform layer in straum 5 against the ganglion cell bodies. It seems obvious now, but it took a lot of painstaking serial section recontruction to show (19, 20) that DB1 and imb made contacts only with ganglion cells that had dendritic stratification in the same neuropil as the respective bipolar axonal terminals. At the level of information transfer between bipolar cells and ganglion cells in the IPL, only excitatory channels are present (21), so the type of signal transmitted to the ganglion cell, as either ON- or OFF-center, is essentially determined by the bipolar cells contacting it (Figure 15).

Figure 15

Organization of ON- and OFF-center ganglion cells.

To keep the ON and OFF channels separate through the ganglion cells to the brain, the inner plexiform layer is divided into two functionally discrete sublaminae, called a (the two strata below the amacrine cell bodies) and b (the other three strata stretching to the ganglion cell bodies) (22). Interactions are only allowed between basal-contacting cone bipolar types and one set of ganglion cells in sublamina a, whereas invaginating-contacting cone bipolar cells can interact only with another set of ganglion cells branching in sublamina b (see above). Gouras (1971) (23) was the first to suggest that this specificity of bipolar-to-ganglion-cell contacts underlay ON-center and OFF-center midget ganglion cell responses in monkey. Later, Nelson et al. (24) conclusively proved this hypothesis by means of intracellular recording and marking experiments in ganglion cells of cat (Figure 16) (see the animation of the physiological recording of ON and OFF beta ganglion cells).

Figure 16

Intracellular recordings of ON-center and OFF-center ganglion cells.

Movie 3

An animation of the physiological recording of ON and OFF beta ganglion cells.

Figure 16 shows the intracellular recordings made from two different ganglion cells of Nelson and coauthors' study (24). One cell proves to be ON-center, giving a burst of spikes riding on a depolarization of the membrane as soon as the light flash goes on. In contrast, the other cell is OFF-center, giving a hyperpolarization to the membrane when the light flash is on, but a burst of spikes riding on the depolarization when the light flash is over. The ON-center ganglion cell has dendrites restricted to branching in sublamina b and has synaptic input from invaginating types of bipolar cell. The OFF-center cell has dendrites reaching higher to branch only in sublamina a and gets synapses from basal junction contacting types of bipolar cell.

There is a definite functional architecture to the inner plexiform layer of the mammalian retina and, in fact, to all vertebrate retinas. Stratification of the neuropil is formed by specific levels of branching of bipolar, amacrine, and ganglion cells so that specialized circuits of interactions are set up. In addition, there has, during the course of evolution, been an imposition of a broad division of the two halves of the inner plexiform layer into the top half, allowing only interactions for the OFF-center ganglion cell pathways and in the bottom half, only for the ON-center ganglion cell pathways. Bipolar and ganglion cells are chiefly responsible for the bisublaminar organization, but amacrine cells, particularly if they are of the sustained physiological types, are also drawn in. However, many amacrine cells are diffuse or bistratified and serve to connect the ON- and OFF-center neuropils, whereas still others stratify in such a manner at the border between the two functional neuropils, to receive both ON and OFF inputs to drive them. Such amacrine cells may be in the majority in the vertebrate retina and are thought to be involved more in temporal facets of retinal performance. They may be important for fast transfer of information, i.e., for speeding up signals The role of some of these amacrine cells will become clearer in later chapters.

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