Roles of amacrine cells
[General characteristics]
[Amacrine cell circuitry as revealed by EM]
[A2]
[AII]
[A8]
[A13]
[A17]
[A19 and A20]
[A22]
[A18]
[Starburst cell]
[References]
1. General characteristics.
Fig. 1. Drawing of the retina made by Cajal (67 K jpeg image)
Since the time of Cajal we have known that amacrine cells come in all shapes,
sizes and stratification patterns. Since those days many more morphological
subtypes have and continue to be described from further Golgi studies,
intracellular recordings and immunocytochemical staining. Thus, we presently
have a classification of amacrine cells consisting of about 40 different
morphological subtypes. Fig. 2. Picture of Camillo Golgi (59 K jpeg image)
It is useful and most easily understandable to group the amacrine cell types
into the general descriptors of narrow-field (30-150um), small-field (150-300
um), medium-field (300-500 um) and wide-field (>500 um) based on a
measurement of their dendritic field diameters (Kolb et al., 1981). Then the
next most important criterion of classification involves knowing the cells
stratification. It is generally agreed now that the IPL can be subdivided into
five equi-thickness strata or sublayers (Cajal, 1892) into which amacrine,
bipolar and ganglion cell processes can be assigned. All of these cell types
are now classified primarily on the stratum or strata of the IPL in which their
dendrites or axons are located. This is because, as mentioned in previous
chapters, the IPL of vertebrate retinas can be divided up into areas of
neuropil where specific cells are put into synaptic contacts and form circuits
only with cells earmarked for a particular functional role.
Many varieties of amacrine cell are monostratified, restricted to a single
stratum, while others are bi- or tri-stratified. When amacrine or ganglion
cell processes pass through all the strata of the IPL from distal to proximal
or vice versa, they are called diffuse cells. Superimposed upon Cajal's five
strata subdivision of the IPL, is a sublaminar division of the IPL. The first
two strata, 1-2, are known as sublamina a of the IPL while strata 3-5
are known as sublamina b by this scheme (Famiglietti and Kolb, 1976). It
will be remembered from previous chapters that sublamina a contains
bipolar axons and ganglion cell connections that lead to OFF-center ganglion
cell physiology, while sublamina b contains bipolar to ganglion cell
connections resulting in ON-center ganglion cell physiology (Nelson et al.,
1978).
This figure shows drawings of some small field amacrine cells of the
monkey retina as seen in vertical sections. Small-field cells like these can be
well visualized in section because their dendritic trees are contained within
the depth of the section. However, large field cells are not so well described
in section where their dendrites get cut off. Fig. 3. Amacrine cells of the monkey retina (98 K jpeg image)
Amacrine cells of the vertebrate retina are interneurons that interact at the
second synaptic level of the vertically direct pathways consisting of the
photoreceptor-bipolar-ganglion cell chain. They are synaptically active in the
inner plexiform layer (IPL) and serve to integrate, modulate and interpose a
temporal domain to the visual message presented to the ganglion cell. Amacrine
cells are so named because they are nerve cells thought to lack an axon (Cajal,
1892). Today we know that certain large field amacrine cells of the vertebrate
retina can have long "axon-like" processes which probably function as true
axons in the sense that they are output fibers of the cell (see later section
on dopaminergic amacrine cells). However these amacrine axons remain within the
retina and do not leave the retina in the optic nerve as do the ganglion cell
axons.
It was only when wholemount
preparations, from Golgi staining (Stell and Witkovsky, 1972; Boycott and Kolb,
1973) or immunocytochemical staining (Karten and Brecha, 1980) were attempted
that we could classify such cells. Then the full extent of their dendritic
trees which can be up to one millimeter in spread could be visualized (see
right figure) and a whole new understanding of amacrine cells became available.
Fig.4. Amacrine cells as seen in wholemount
(78 K jpeg image)
A new technique of intracellular staining by a photochemical method has been developed In Richard Masland's group as an alternative to the unreliable Golgi technique (MacNeil and Masland, 1998). Amacrine cells of the rabbit retina are labelled with the nuclear stain DAPI and then selected single nuclei are irradiated by a narrow beam of light to drive DAPI to the oxidation of non-fluorescent dihydrorhodamine 123 to the fluorescent rhodamine 123. The complete cell body and the dendritic tree is thus revealed under viewing in the fluorescence or confocal microscopes. By this method, 30 or so different varieties of amacrine cell can be photographed and drawn in full detail in the rabbit retina. 22 varieties of amacrine cell have been seen in Golgi preparations in cat and primate retinas so either some have been missed that were seen in rabbit, or else they are not as well deveoped in these less complex mammalian retinas. In any event the narrow field and medium field types revealed by MacNeil and Maslands work (1998) are shown in Figures 4b and c below. A further 5 different wide field monostratified types were also encountered in rabbit retina by this method (not illustrated). They correspond closely to the wide field types seen in monkey, cat and human (Fig. 4, above) (Mariani, 1990; Kolb et al., 1981, Kolb et al. 1992).
2. Amacrine cell circuitry as revealed by electron microscopy.
Kidd (1962) and later Dowling and Boycott (1966) were the first to
identify the three types of profile that contribute to the IPL by electron
microscopy. The electron micrograph below shows the cytological criteria on
which we now recognize bipolar, amacrine and ganglion cell profiles in the
neuropil. Thus bipolar cell axonal endings are recognized
by being filled with synaptic vesicles and having a ribbon-shaped density (red spots) pointing to two postsynaptic profiles (amacrine and ganglion). Amacrine profiles are also filled with synaptic vesicles but make
synapses characterized by membrane densities at which the vesicles are particularly
clustered (yellow spots). Ganglion cell profiles are recognized as
being only postsynaptic to either bipolar axons or amacrine processes,
containing no vesicles but instead a content of neurotubules, ribosomes and
glycogen granules.
Fig. 5. Electron micrograph of several profiles at the IPL level (117 K jpeg image)
We have learned much concerning the synaptic relationships of certain narrow-field amacrine cells as well as bipolar and small ganglion cell types such as midget ganglion cells of the primate retina, from reconstructions of serial-section electron micrographs. The circuitry of the AII amacrine cell in the cat retina was first appreciated by this means (Famiglietti and Kolb, 1975; Kolb, 1979). However, with the advent of intracellular dye injection of electron-dense materials (horseradish peroxidase, HRP, or the photoreduction of Lucifer yellow) after physiological recordings or the development of electron dense immunostains for electron microscopy, neurocircuitry was made easier for us. We could look at amacrine cells and their synaptic inputs by study of fewer sections and it was not as critical to photograph every single section in a series. The amacrine cell of interest would always be clearly marked black, and easily found in the synaptic neuropil. It is from this technique that we have learned most about amacrine cells and their circuitry in the mammalian retina. The remainder of this chapter will describe the morphology, circuitry and intracellular responses of the amacrine cells that are most completely understood at present.
3. A2: narrow-field, cone pathway amacrine cell.
A2 is a narrow-field amacrine with a 20-60 um wide dendritic tree composed of
multibranched, beaded and appendage-bearing dendrites mostly confined to
stratum 2 of the IPL.
Fig. 6. Golgi drawings of A2 amacrine cells (59 K jpeg image)
Intracellular recordings from A2 cells (formerly called A4) indicate that these cells give true slow potential hyperpolarizing response to light (OFF-center) at all positions of the slit in their receptive fields and they have no sign of an inhibitory surround (Kolb and Nelson, 1984).
A2 cells receive bipolar input from OFF-center types of cone bipolar cell of sublamina a and make reciprocal synapses to these bipolar axons. A2 amacrine cells then synapse upon OFF-center ganglion cell dendrites of sublamina a. The A2 cell makes an inhibitory synapses upon these ganglion cells, because it is thought to be a GABA-ergic cell type (Pourcho and Goebel, 1983).
A possible role is in disinhibition of the ganglion cells' center responses. Alternatively, A2 cells, despite being small-field types, might have a role in the generation of antagonistic surrounds of ganglion cells (Kolb and Nelson, 1993). A2 cells receive a great many amacrine inputs to their dendritic trees which could be from wider field cells than they are are themselves so giving them a much larger receptive field size than their actual dendritic tree size would indicate.
Fig. 7. Summary diagram of A2 amacrine cells (59 K jpeg image)
4. AII: a bistratified rod amacrine cell.
Fig. 8. AII amacrine cells stained with different methods (59 K jpeg image)
Above are shown four examples of the best studied amacrine of all in the vertebrate retina: the AII "rod amacrine" of the mammalian retina. These cells have been recorded from by microelectrodes and dyes have been iontophoresed into the cell after the intracellular recordings (Nelson, 1982). The AII cell, was first described from Golgi staining and electron microscopic examination (Famiglietti and Kolb, 1975; Kolb and Famiglietti, 1974).
AII is a narrow field amacrine (dendritic tree diameter typically 30-70 um) with a bistratified morphology: the mitral shaped cell body gives off a single, stout apical dendrite and a cluster of lobular appendages (round blobs just below the cell body, above) arise from the main dendrite in sublamina a of the IPL. The finer "arboreal dendrites" (Vaney et al., 1991) penetrate down into sublamina b to end close to the ganglion cell layer. In the human retina, such an AII amacrine cell is seen in a surface view of a wholemount.
Fig. 9. AII amacrine cells (59 K jpeg image)
In cat and rabbit retinas where AIIs have been recorded from, the AII cell is a rod-dominated depolarizing (ON-center) cell (Bloomfield, 1992; Dacheux and Raviola, 1986; Nelson, 1982). Thus, in the center of its receptive field the cell gives a transient depolarizing response with a pronounced sustained plateau (ON-center) and a long drawn out hyperpolarization after light off. By 140 um to either side of the center, the response to a light flash is now an inverted response indicating a hyperpolarizing surround (OFF-surround) (Nelson, 1982).
Electron microscopy has shown that the AII, is primarily postsynaptic to rod bipolar axon terminals in lower sublamina b of the IPL (30% of its input, Strettoi et al., 1992). Its major output is upon ganglion cells that have dendrites only in sublamina a, i.e. AII cell lobular appendages synapse upon OFF-center a and b ganglion cells (Kolb and Nelson, 1993).
Fig. 11. Electron micrographs of AII amacrine synapses (59 K jpeg image)
The AII also passes rod-driven information through the ON-center cb5 cone bipolar to ON-center a and b ganglion cells by means of gap junctions (black spots on AII primary dendrites to pink cb axon). A little OFF-center cone bipolar input is provided to the AII lobular appendages by cb1 and cb2 OFF-center cone bipolar cells in sublamina a (19% of input, Strettoi et al., 1992) (yellow cb profiles).
Thus, AII cells do carry some cone pathway
components to their ON-center responses, which could come from excitatory input
from ON-center cb5 at the gap junctions, or from the direct cb1 or cb2 synapses
which would have to be inhibitory, in this case. AII amacrine cells are also
coupled across the retina in a weak electrical syncytium by virtue of their gap
junctions between their arboreal dendrites in sublamina b (gj, lower
right) (Famiglietti and Kolb, 1975; Nelson, 1982; Vaney, 1994a).
Fig. 12. Schematic drawing of the wiring pattern of the AII amacrine cell (59 K jpeg image)
The dopaminergic amacrine cell provides a considerable number of synapses to the AII cell, either directly upon its cell body or upon its lobular appendages (A, red arrowheads) (Voigt and Wässle, 1987; Kolb et al, 1991). Dopamine cells are thought to have a function in the inner retina to uncouple AII amacrine cells from both their contacts with the depolarizing cone bipolar and the AII amacrine coupled network (Daw et al., 1990; Vaney, 1994a). As much as 51% of the input to AII amacrine cells is from various other amacrine cells though, and most of these inputs occur in the central part of the cells dendritic tree in strata 3-4 (Strettoi et al., 1992). AII amacrine cells are glycine-immunoreactive (Pourcho and Goebel, 1985; Crooks and Kolb, 1992) and contain the calcium binding proteins parvalbumin, calbindin and calretinin (Wässle et al., 1995).
Fig. 13. Parvalbumin staining of AII amacrine cells in hamster retina (78 K jpeg image)
The AII amacrine cells are the major carriers of rod signals to the ganglion cells in the retina. As such they play a role in speeding up the slow potential rod messages for presentation to ganglion cells (Nelson, 1982; Smith, 1994). Their distribution in the retina suggests that they tile the complete retina (Vaney, 1990). AII amacrine cells peak in density at 1.5 mm from the foveal center in monkey and at the area centralis in cat (Vaney, 1984). In addition, because of their high density across all parts of the retina and their synaptic involvement with millions of rod bipolar cells, they may contribute in a major way to the pattern ERG (Zrenner, 1990).
5. A8: a bistratified cone amacrine cell.
A8 is a bistratified, narrow-field amacrine cell which is easy to
confuse with AII in wholemount, stained retina. It actually looks like an
upside-down AII cell. A8 has short, wispy processes coming from the apical
dendrite to ramify in sublamina a of the IPL whereas heavy beaded
dendrites penetrate down to sublamina b, to run in strata 4 and 5. This
cell type may correspond to the DAPI-3 of the rabbit retina, described by Vaney
(1990) and Bloomfield (1992).
Fig. 14. Golgi and HRP appearance of A8 amacrine cells (39 K jpeg image)
The A8 cell has been intracellularly recorded and studied by electron
microscopy after iontophoresis of horseradish peroxidase. In Fig. 13 we shown two of the most important synapses of this cell type.
Fig.15. Electron micrograph of A8 synapses in the IPL (59 K jpeg image)
Fig. 16. Summary diagram of the wiring pattern of the A8 amacrine cell (78 K jpeg image)
Click here to see an animation of the wiring pattern of the A8 amacrine cells
(78 K quicktime movie)
The intracellular response of A8 indicates a hyperpolarization to light at the receptive field center with a rather transient OFF-response to light. The transientness could reflect the amacrine synapses (38% of input) occurring over all parts of its dendritic tree (above figures red arrowheads). (0, response is at largest magnitude).
Both rod driven and cone driven signals
contribute to the response. Its receptive field extent can be mapped with a
slit of light. As the slit is stepped some distance to either side of the
center of the receptive field the response of the cell inverts and a
depolarizing or ON-surround appears by 700 um from the central position (top
and bottom trace) (Kolb and Nelson, 1996).
Fig. 17. Summary diagram of the A8 amacrine cell (59 K jpeg image)
Some part of the amacrine input to A8, particularly that upon its cell body and proximal dendrites in stratum 1 of the IPL, may be from dopaminergic amacrine cells (A18) (Kolb et al., 1991). So it is probable that this cell type is also under the control of the dopaminergic amacrine cells, like the AII amacrine cell (see above). Thus, the dopamine cell may control A8's spatial characteristics through gating its gap junctions in light and dark. A8 is intensely glycine-immunoreactive (Pourcho and Goebel, 1985; Crooks and Kolb, 1992). We have suggested that A8 cells also function in the disinhibition of ganglion cell receptive field centers (Kolb and Nelson, 1996).
[General characteristics]
[Amacrine cell circuitry as revealed by EM]
[A2]
[AII]
[A8]
[A13]
[A17]
[A19 and A20]
[A22]
[A18]
[Starburst cell]
[References]
