The types of retinal ganglion cells: current status and implications for neuronal classification.

In the retina, photoreceptors pass visual information to interneurons, which process it and pass it to retinal ganglion cells (RGCs). Axons of RGCs then travel through the optic nerve, telling the rest of the brain all it will ever know about the visual world. Research over the past several decades has made clear that most RGCs are not merely light detectors, but rather feature detectors, which send a diverse set of parallel, highly processed images of the world on to higher centers. Here, we review progress in classification of RGCs by physiological, morphological, and molecular criteria, making a particular effort to distinguish those cell types that are definitive from those for which information is partial. We focus on the mouse, in which molecular and genetic methods are most advanced. We argue that there are around 30 RGC types and that we can now account for well over half of all RGCs. We also use RGCs to examine the general problem of neuronal classification, arguing that insights and methods from the retina can guide the classification enterprise in other brain regions.

Neurite arborization and mosaic spacing in the mouse retina require DSCAM.

To establish functional circuitry, retinal neurons occupy spatial domains by arborizing their processes, which requires the self-avoidance of neurites from an individual cell, and by spacing their cell bodies, which requires positioning the soma and establishing a zone within which other cells of the same type are excluded. The mosaic patterns of distinct cell types form independently and overlap. The cues that direct these processes in the vertebrate retina are not known. Here we show that some types of retinal amacrine cells from mice with a spontaneous mutation in Down syndrome cell adhesion molecule (Dscam), a gene encoding an immunoglobulin-superfamily member adhesion molecule, have defects in the arborization of processes and in the spacing of cell bodies. In the mutant retina, cells that would normally express Dscam have hyperfasciculated processes, preventing them from creating an orderly arbor. Also, their cell bodies are randomly distributed or pulled into clumps rather than being regularly spaced mosaics. Our results indicate that mouse DSCAM mediates isoneuronal self-avoidance for arborization and heteroneuronal self-avoidance within specific cell types to prevent fasciculation and to preserve mosaic spacing. These functions are analogous to those of Drosophila DSCAM (ref. 6) and DSCAM2 (ref. 7). DSCAM may function similarly in other regions of the mammalian nervous system, and this role may extend to other members of the mammalian Dscam gene family.

The tasks of amacrine cells.

Their unique patterns of size, numbers, and stratification indicate that amacrine cells have diverse functions. These are mostly unknown, as studies using imaging and electrophysiological methods have only recently begun. However, some of the events that occur within the amacrine cell population--and some important unresolved puzzles--can be stated purely from structural reasoning.

Structural remodeling of fibrous astrocytes after axonal injury.

Reactive astrocytes are a pathological hallmark of many CNS injuries and neurodegenerations. They are characterized by hypertrophy of the soma and processes and an increase in the expression of glial fibrillary acidic protein. Because the cells obscure each other in immunostaining, little is known about the behavior of a single reactive astrocyte, nor how single astrocytes combine to form the glial scar. We have investigated the reaction of fibrous astrocytes to axonal degeneration using a transgenic mouse strain expressing enhanced green fluorescent protein in small subsets of astrocytes. Fibrous astrocytes in the optic nerve and corpus callosum initially react to injury by hypertrophy of the soma and processes. They retract their primary processes, simplifying their shape and dramatically reducing their spatial coverage. At 3 d after crush, quantitative analysis revealed nearly a twofold increase in the thickness of the primary processes, a halving of the number of primary processes leaving the soma and an eightfold reduction in the spatial coverage. In the subsequent week, they partially reextend long processes, returning to a near-normal morphology and an extensive spatial overlap. The resulting glial scar consists of an irregular array of astrocyte processes, contrasting with their original orderly arrangement. These changes are in distinct contrast to those reported for reactive protoplasmic astrocytes of the gray matter, in which the number of processes and branchings increase, but the cells continue to maintain nonoverlapping individual territories throughout their response to injury.

Physiological clustering of visual channels in the mouse retina.

Anatomy predicts that mammalian retinas should have in excess of 12 physiological channels, each encoding a specific aspect of the visual scene. Although several channels have been correlated with morphological cell types, the number of morphological types generally exceeds the known physiological types. Here, we attempted to sort the ganglion cells of the mouse retina purely on a physiological basis. The null hypothesis was that the outputs of the ganglion cells form a continuum or should be divided into only a few types. We recorded the spiking output of 471 retinal ganglion cells on a multielectrode array while presenting 4 classes of visual stimuli. Five parameters were chosen to describe each cell's response characteristics, including relative amplitude of the ON and OFF responses, response latency, response transience, direction selectivity, and the receptive field surround. We compared the results of four clustering routines and judged the results using the relevant validation indices. The optimal partition was the 12-cluster solution of the Fuzzy Gustafson-Kessel algorithm. This classification contained three visual channels that carried predominately OFF responses, six that carried ON responses, and three that carried both ON and OFF information. They differed in other parameters as well. Other evidence suggests that the true number of cell types in the mouse retina may be somewhat larger than 12, and a definitive typology will probably require broader stimulus sets and characterization of more response parameters. Nonetheless, the present results do allow us to reject the null hypothesis: it appears that in addition to well-known cell types (such as the ON-OFF direction selectivity cells) numerous other cell classes can be identified in the mouse retina based solely on their responses to a standard set of simple visual stimuli.

Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin.

The rod and cone cells of the mammalian retina are the principal photoreceptors for image-forming vision. They transmit information by means of a chain of intermediate cells to the retinal ganglion cells, which in turn send signals from the retina to the brain. Loss of photoreceptor cells, as happens in a number of human diseases, leads to irreversible blindness. In a mouse model (rd/rd) of photoreceptor degeneration, we used a viral vector to express in a large number of retinal ganglion cells the light sensitive protein melanopsin, normally present in only a specialized subset of the cells. Whole-cell patch-clamp recording showed photoresponses in these cells even after degeneration of the photoreceptors and additional pharmacological or Cd(2+) block of synaptic function. Interestingly, similar responses were observed across a wide variety of diverse types of ganglion cell of the retina. The newly melanopsin-expressing ganglion cells provided an enhancement of visual function in rd/rd mice: the pupillary light reflex (PLR) returned almost to normal; the mice showed behavioral avoidance of light in an open-field test, and they could discriminate a light stimulus from a dark one in a two-choice visual discrimination alley. Recovery of the PLR was stable for at least 11 months. It has recently been shown that ectopic retinal expression of a light sensitive bacterial protein, channelrhodopsin-2, can restore neuronal responsiveness and simple visual abilities in rd/rd mice. For therapy in human photodegenerations, channelrhodopsin-2 and melanopsin have different advantages and disadvantages; both proteins (or modifications of them) should be candidates.

The unsolved mystery of vision.

Vision looms large in neuroscience--it is the subject of a gigantic literature and four Nobel prizes--but there is a growing realization that there are problems with the textbook explanation of how mammalian vision works. Here we will summarize the evidence behind this disquiet. In effect, we shall present a portrait of a field that is 'stuck'. Our initial focus, because it is our area of expertise, is on evidence that the early steps of mammalian vision are more diverse and more interesting than is usually imagined, so that our understanding of the later stages is in trouble right from the start. But we will also summarize problems, raised by others, with the later stages themselves.

Image processing: how the retina detects the direction of image motion.

In the retina, the beautifully symmetrical 'starburst' amacrine cells interact with each other in a way that creates asymmetrical responses to moving images at their dendritic tips. This computation, occurring in a retinal interneuron, is a foundation of the directional signals transmitted by the retina to the brain.

Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice.

Using a variety of double and triple labeling techniques, we have reevaluated the death of retinal neurons in a mouse model of hereditary glaucoma. Cell-specific markers and total neuron counts revealed no cell loss in any retinal neurons other than the ganglion cells. Within the limits of our ability to define cell types, no group of ganglion cells was especially vulnerable or resistant to degeneration. Retrograde labeling and neurofilament staining showed that axonal atrophy, dendritic remodeling, and somal shrinkage (at least of the largest cell types) precedes ganglion cell death in this glaucoma model. Regions of cell death or survival radiated from the optic nerve head in fan-shaped sectors. Collectively, the data suggest axon damage at the optic nerve head as an early lesion, and damage to axon bundles would cause this pattern of degeneration. However, the architecture of the mouse eye seems to preclude a commonly postulated source of mechanical damage within the nerve head.

Remodeling of cone photoreceptor cells after rod degeneration in rd mice.

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell--though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.

Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts.

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.

Sensory systems: fine-tuning the visual scene.

The visual system adjusts its properties for efficient representation of the objects present in the environment at the time. A new report suggests that complex processing of this sort can begin as early as the retina itself, but some important issues remain unresolved.

Contextual tuning of direction-selective retinal ganglion cells.

A direction-selective (DS) retinal ganglion cell responds well to a small object moving within its receptive field center, but less well when there is also a moving stimulus in the surrounding area; this has been described as tuning for local motion. We show here an additional selectivity, such that the surround has less effect if there is a discontinuity--that is, a difference in spatial phase, spatial frequency or velocity--between the center stimulus and that present in the surround.

The many roles of starburst amacrine cells.

Starburst amacrine cells release two classical neurotransmitters, ACh and GABA. In a tour de force of paired-cell recording, Zheng et al. now show that the starburst cells are mutually excitatory during early development but mutually inhibitory in adult animals. The change occurs by remodeling of both the cholinergic and the GABAergic synapses between starburst cells. The finding gives a precise mechanistic basis for the developmental waves of activity in the retina.

Expression of mRNA for glutamate receptor subunits distinguishes the major classes of retinal neurons, but is less specific for individual cell types.

PURPOSE: To investigate the expression of ionotropic glutamate receptor subunits by retinal neurons, to assess the extent to which different functional types of retinal neurons are characterized by the expression of the receptor subtypes. METHODS: Rod photoreceptor cells and bipolar cells were identified in retina dissociates. Amacrine cells were identified in dissociates from transgenic mice or by staining with an antibody against the extracellular carbohydrate epitope CD15. Ganglion cells were identified by retrograde axonal transport of FITC-dextran or by green fluorescent protein (GFP) fluorescence in a transgenic strain. We examined the receptors simultaneously using non-quantitative single-cell reverse transcriptase polymerase chain reaction for GluR1-R4 (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors), GluR5-R7, and KA1 and 2 (kainate receptors), delta1 and delta2 subunits, and the N-methyl-D-aspartate (NMDA) receptor subunits NR1, 2a-d, and 3a. RESULTS: The expression of glutamate receptors on bipolar cells and rod photoreceptors was limited: Neither expressed functional NMDA receptors, and rods were also negative for AMPA receptors. The sample of ganglion cells included examples of many ganglion cell types; these were distinguished morphologically using quantitative parameters defined in a previous cluster analysis. All types of ionotropic glutamate receptors were found to be expressed on ganglion cells. The iGluR subunits GluR4, KA2, delta1, and NR1 were expressed on almost all ganglion cells examined. CONCLUSIONS: Despite the heterogeneity of ganglion cell types, differences among them in this PCR-based method were minor. Thus, retinal interneurons are characterized by expression of distinctive glutamate receptor types, but functional differences among ganglion cells seem to be reflected instead in the amounts as well as spatial distributions of a widely expressed group of receptors.

Vision: Two Speeds in the Retina.

The central (foveal) retina takes about 30 milliseconds longer to signal to the brain than the peripheral retina. In the natural world, a 30 millisecond delay could have real consequences. Why did evolution do it this way?

Different functional types of bipolar cells use different gap-junctional proteins.

Rod signals are transmitted to ON retinal ganglion cells by means of gap junctions between AII amacrine cells and ON bipolars. The AII amacrine cells are known to express connexin36 (Cx36), but previous studies of Cx36 in ON cone bipolars have been ambiguous. Here, we studied bipolar cells in a transgenic mouse line that expresses high levels of green fluorescent protein (GFP) in one type of ON cone bipolar cell. We found strong Cx36 immunostaining in the axon terminals of the GFP-labeled type 357 bipolar cells in both vertical sections and whole mounts of the retina. This finding was confirmed by single-cell immunostaining and single-cell reverse transcription-PCR (RT-PCR). As reported previously (Maxeiner et al., 2005), Cx45 was found in some ON bipolar cells, but RT-PCR showed Cx36 and not Cx45 to be expressed by the type 357 bipolar cells. Some of the remaining GFP-negative bipolar cells expressed Cx45 but not Cx36. It appears that different types of ON cone bipolar cells express different connexins at their gap junctions with AII amacrine cells.

Populations of wide-field amacrine cells in the mouse retina.

We surveyed wide-field amacrine cells in the mouse, using a large series of retinas from a transgenic strain that expresses the green fluorescent protein (GFP) in isolated retinal cells. Wide-field cells were present in surprising diversity and number. They formed groups that could be defined by arbor depth, arbor size, and soma size. By conventional criteria, these populations of cells make up 11 amacrine cell "types." Five additional types have been reported by others in the mouse. Roughly two-thirds of the wide-field amacrine cells are axon-bearing cells, which have separate dendritic and axonal arbors. The axonal arbor of a single cell sometimes covers the majority of the retinal surface. The axon-bearing cells appear to be centrifugally conducting neurons similar to those studied electrophysiologically in some other species. Although they are classified as independent morphological types, it seems likely that their physiological functions represent variations on a single organizational plan. These cells are present at every level of the inner plexiform layer, which suggests that they affect most of the mouse retina's final outputs to the brain and, by implication, almost all visual function.