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Home , Amacrine cell, Eye, Ganglion cell layer, Inner plexiform layer, Outer plexiform layer, Receptive field

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 596-601, January 1996 Colloquium Paper

This paper was presented at a coUloquium entitled "Vision: From Photon to , " organized by John Dowling, Lubert Stryer (chair), and , held May 20-22, 1995, at the National Academy of Sciences in Irvine, CA.

Molecular biology of retinal ganglion cells MENGQING XIANG*t, HAO ZHOU*, AND JEREMY NATHANS*t#§ Departments of *Molecular Biology and Genetics, tNeuroscience, §, tHoward Hughes Medical Institute, Johns Hopkins University School of , Baltimore, MD 21205

ABSTRACT Retinal ganglion cells are the output the by emphasizing spatial . This type of spa- that encode and transmit information from the to the tially antagonistic filtering had been predicted in the 19th . Their diverse physiologic and anatomic properties have century by both Hering and Mach (4, 5) on psychophysical been intensively studied and appear to account well for a grounds, and it accounts for the illusory black dots seen in the number of psychophysical phenomena such as lateral inhibi- Hermann grid in Fig. 2. In the of old world primates, tion and chromatic opponency. In this paper, we summarize many ganglion cells also relay chromatic information by re- our current view of retinal ganglion properties and pose porting either the difference between red and green cone a number of questions regarding underlying molecular mech- inputs or the difference between blue cone input and a sum of anisms. As an example of one approach to understanding red and green (= yellow) cone inputs. For reasons that are not molecular mechanisms, we describe recent work on several obvious, most ganglion cells of the red vs. green type have both POU domain transcription factors that are expressed in chromatically and spatially opponent receptive fields, whereas subsets ofretinal ganglion cells and that appear to be involved most ganglion cells of the blue vs. red + green type have nearly in ganglion cell development. coextensive excitatory and inhibitory zones and therefore a much smaller degree of spatial opponency (6). The channeling of This paper reviews our current knowledge of retinal ganglion chromatic information into two pathways with red vs. green and cell structure and function with an emphasis on those areas in blue vs. yellow opponent organization was deduced on which molecular biological approaches may be expected to psychophysical grounds by Hering (4). It accounts for the chro- provide new insights. We begin with an overview of the matic afterimages generated by selective desensitization of one or physiological, anatomical, and psychophysical experiments another limb of the opponent processing system (Fig. that have revealed the diversity of ganglion cell properties and 2). the significance of that diversity for . Al- Hartline's original recordings showed that in some ganglion though little is currently known about the molecular basis of cells a prolonged evoked a steady response, this diversity, it is likely that many of the relevant molecules whereas in others it evoked a transient (i.e., nonlinear) re- will be identified in the near future. As an illustration of one sponse (Fig. 1). The latter type of response filters the image by area in which significant progress seems likely, we conclude emphasizing temporal changes. Beginning in the mid-1960s, with a description of recent work on transcription factors in this distinction was systematically investigated in both cat and retinal ganglion cells. monkey retinas (reviewed in refs. 6 and 7). In the cat two major ganglion cell types were identified and termed X and Y, the Physiological Properties of Retinal Ganglion Cells former responding both spatially and temporally in a linear manner and the latter responding nonlinearly (8). In primates, Ganglion cells are the output units of the . Because their a similar dichotomy was found in temporal response proper- cell bodies and are relatively accessible, they were among ties, with one class, now referred to as parvocellular or P-type the first neurons for which single unit responses ganglion cells, responding linearly, and a second class, now were determined. In 1938 Hartline (1) recorded from individ- referred to as magnocellular or M-type ganglion cells, respond- ual axons at the vitreal surface of the retina while ing nonlinearly (9). P- and M-type cells have been found to stimulating the retina with a spot of light. These seminal differ in a number of properties. In general terms, P cells are experiments introduced the concept of a , defined characterized by relatively slow conduction velocities, insen- by Hartline as the region of the retina that must be illuminated sitivity to small changes in luminance contrast, and high spatial in order to obtain a response in a given fiber. As shown in Fig. 1, resolution, especially near the fovea. Most P cells have a these experiments also revealed a multiplicity of response prop- chromatically opponent receptive field organization as de- erties among retinal ganglion cells, including both activation and scribed above. By contrast, M cells are characterized by inhibition: "This diversity of response among fibers from closely relatively fast conduction velocities, sensitivity to small adjacent regions of the same retina is extreme and unmistakable; changes in luminance contrast, and low spatial resolution. M it does not depend upon local conditions of stimulation or cells have achromatic center-surround receptive fields and , but appears to be an inherent property of the therefore detect luminance but not chromatic contrast. The individual ganglion cells themselves" (1). distinction drawn between cat X and Y cells in spatial response In 1953 Barlow and Kuffler (2, 3) independently discovered properties does not appear to carry over to the primate P/M that many ganglion cells have an antagonistic spatial organi- as all zation in which either an excitatory center is paired with an system P-type and most M-type ganglion cells show linear inhibitory surround or an inhibitory center is paired with an spatial summation (10). The distinct P and M systems appear excitatory surround. The center-surround organization filters Abbreviations: IPL, ; LGN, lateral geniculate nucleus. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: 805 Preclinical payment. This article must therefore be hereby marked "advertisement" in Teaching Building, 725 North Wolfe Street, Johns Hopkins Univer- accordance with 18 U.S.C. §1734 solely to indicate this fact. sity School of Medicine, Baltimore, MD 21205. 596 Downloaded by guest on September 29, 2021 Colloquium Paper: Xiang et al. Proc. Natl. Acad. Sci. USA 93 (1996) 597

FIG. 1. Light responses obtained from isolated ganglion cell axons in the frog retina (reproduced from ref. 1). The interval between the regular marks at the bottom of each trace correspond to 0.2 sec. The duration of illumination is indicated by the blackened portion of the strip near the bottom of each trace. The three cells reveal responses to the onset of illumination, the cessation of illumination, steady illumination, or various combinations of these. (Note: in trace A the apparent activity following cessation of illumination is from another cell.) to represent a critical point at which the image is divided into These are likely to be related, at least in part, to the segregation separate and parallel streams. of chromatic inputs. In one well characterized example, the blue ON/yellow OFF color opponent type of ganglion cell has Morphologic and Anatomic Properties of Retinal Ganglion been shown to be bistratified (17). One dendritic tree is located Cells at that level in the inner part of the IPL where the processes of blue cone bipolar cells terminate, and the second dendritic From the earliest histologic studies of the vertebrate retina it tree is located in the outer part of the IPL where it presumably has been apparent that each major class of cells- receives inhibitory signals from bipolar cells driven by red and photoreceptor, bipolar, horizontal, amacrine, and ganglion- green cones. contains within it morphologically distinct subtypes (11). A A third structure-function correlation can be seen in the major theme during the past century of retina research has different projections made by retinal ganglion cells, with the been the identification of functional correlates for these mor- result that distinct aspects of the retinal image are delivered to phologic differences (12). Among ganglion cells, one correla- different destinations in the brain (reviewed in refs. 6 and 7). tion that is now well established (and is perhaps not surprising) The two principal projections from the retina are to the is between the area of the dendritic field and the area of the and to the dorsal lateral geniculate nucleus (LGN) of receptive field, the former appearing to coincide with and to the , the latter projecting to the primary . determine the extent of the latter. Both dendritic field size and In amphibia and other lower the midbrain projec- cell body size differ markedly between physiologically distinct tion (the retinotectal pathway) constitutes the major output ganglion cell types. For example, in the cat, X and Y cells pathway from the retina and mediates simple visually guided correspond, respectively, to the medium (,B) and large (a) cell behaviors. In primates, the analogous pathway is devoted types, and in the monkey, P- and M-type cells correspond, principally to the control of eye and movements. Many respectively, to the small (midget) and large (parasol) cell types ganglion cells that project to the midbrain exhibit receptive (reviewed in refs. 6 and 13). For P and M cells, both dendritic fields with a high degree of selectivity-for example, to field and size increase progressively with increasing movement in a particular direction. retinal eccentricity, and this increase is matched by a corre- Ganglion cell axons navigate with extraordinary precision to sponding increase in the size of the receptive field. The contact their appropriate targets within the brain. At the optic eccentricity-dependent change in receptive field size accounts chiasm, most axons from the nasal but not the temporal half for the absence of an illusory dark spot in the one intersection of each retina cross the midline to follow the contralateral of the Hermann grid upon which the observer fixates (Fig. 2). . Central to the chiasm, ganglion cell axons in the In the human retina, receptive field sizes have been measured primate retinothalamic tract undergo further segregation. psychophysically by determining the threshold for detection of Axons from M-type ganglion cells project to the ventral two a small test flash in the presence of a superimposed circular layers of the LGN while axons from the P-type ganglion cells background of varying diameter and constant brightness (14). project to the dorsal four layers; axons derived from the When the superimposed background is confined to the exci- contralateral eye innervate the first, fourth, and sixth layers of tatory center of a center-surround receptive field it produces the LGN, while those derived from the ipsilateral eye inner- a persistent activation, thereby decreasing the sensitivity of the vate the second, third, and fifth layers; and within each layer cell to dim test flashes. When the superimposed background is of the LGN the pattern of innervation generates a precise enlarged so that it also includes the inhibitory surround, the retinotopic map that is aligned with each of the retinotopic level of persistent activation is reduced and the sensitivity of maps above and/or below it. the cell approaches that seen with the test flash alone. This psychophysical measure closely matches the eccentricity- Molecular Biological Questions dependent size of primate M-type ganglion cell dendritic fields (15) and receptive fields (16). The diversity of ganglion cell properties and the precision with A second correlation between ganglion cell structure and which these properties are programmed invite numerous ques- function relates the level at which the ganglion cell tions regarding underlying molecular mechanisms. We list arborize in the inner plexiform layer and the inputs that the cell some of these questions below. receives. By examining the morphologies of individual gan- (i) What determines the synaptic specificity of each ganglion glion cells after recording their light responses, it was discov- cell for the various classes of bipolar and amacrine cells? What ered that ganglion cells with OFF centers have dendritic arbors attractive or repulsive molecules determine the levels in the in the outer part of the inner plexiform layer (IPL), whereas IPL where ganglion cells and the various classes of bipolar and ganglion cells with ON centers have dendritic arbors in the amacrine cells ? What molecules determine the den- inner part of the IPL (reviewed in ref. 12). Further subdivisions dritic field size for each type of ganglion cell? within the IPL are evident upon close examination of ganglion, (ii) How do different ganglion cell classes differ in the types bipolar, and dendritic morphologies (Fig. 3). of they use and in the properties and Downloaded by guest on September 29, 2021 598 Colloquium Paper: Xiang et al. Proc. Natl. Acad. Sci. USA 93 (1996) regulation of their postsynaptic receptors? Do ganglion cells U.- exhibit physiological alterations in synaptic efficacy and, if so, U. by which mechanisms? (iii) What are the identities of the guidance molecules that lead ganglion cell axons across the retinal surface to the optic Eu nerve, determine which axons cross the midline at the , direct different axons to the midbrain or thalamus (as well as to other destinations), and produce the precise ar- Eu rangement of synaptic contacts within the midbrain and LGN? (iv) What genetic regulatory circuits distinguish types and how are these set up during develop- Eu ment? How are the numbers of different ganglion cell types determined, and what are the mechanisms by which these differ between species? How are the numbers and morphol- ogies of each type of ganglion cell programmed to vary as a function of retinal eccentricity? I I Transcription Factors in Retinal Ganglion Cells Many of the questions posed above are under active investi- gation. As an illustration of one area in which some progress has been made, we discuss below current work on the identi- fication and characterization of transcription factors that are likely to be involved in controlling ganglion cell development. The specification of a final differentiated cellular phenotype consists, in large part, of the selective transcriptional activation of particular genes. Work on myoblast differentiation in the mouse (18) and on early embryonic development in Drosophila (19) suggests that this is accomplished by a combinatorial network of interacting transcription factors. These act both to stably set the cell along a particular pathway of differentiation a and to activate a battery of downstream genes, the products of which are the structural proteins, enzymes, etc., that function- ally distinguish one cell type from another. A number of transcription factors have been localized to the retina; most are also present in a variety of neural, and in some cases nonneural, tissues. Pax6 is the best characterized of these factors. It contains both a PAX domain and a homeodomain and is expressed in all or nearly all ocular tissues including the , , and retina (20). In mice, homozygous Pax6 mutants lack and nasal primordia (21). In the heterozygous condition, mutations in the murine Pax6 gene cause a small eye phenotype, and mutations in the human PAX6 gene cause aniridia (22, 23). SOHo-1, a homeodomain gene identified in FIG. 2. Psychophysical demonstrations of chromatic and spatial chickens, is expressed in all layers of the developing retina as signal processing in the retina. (Upper) Spatial opponent processing well as in other sensory organs including the otocyst and dorsal demonstrated by the Hermann grid. Viewing the figure at one-half root and facial ganglia (24). Several homeodomain genes that arm's length produces the illusion of gray dots at the intersections are highly homologous to the Drosophila NK-2 formed by four black corners. The effect can be understood with gene-Nkx2.2, reference to excitatory center-inhibitory surround receptive fields. TTF1, and DLb-are expressed in the developing retina and in More light falls on the inhibitory annulus of a ganglion cell that has its a complex pattern in other regions of the developing central receptive field centered over the image of an intersection compared to (CNS) (25, 26). Isll, which contains both a LIM a ganglion cell that has its receptive field centered in the white space domain and a homeodomain, is expressed in endocrine organs, between two adjacent black squares. Therefore, the former cell will be in the brain and spinal cord, and in the retina in subsets of cells inhibited to a greater extent than the latter, with the result that the in the inner nuclear and ganglion cell layers (27). ChxlO, a white area at the intersection will appear relatively dimmer. When the homeodomain gene, is expressed in retinal neuroblasts but not figure is viewed at one-half arm's length, illusory gray dots are seen at in the developing ; in the adult retina it is all intersections except for the one upon which the observer fixates, an effect that arises from the smaller receptive field sizes in the central confined to the (28). Two transcription retina. (Lower) Color opponent processing demonstrated by the factors that do not contain homeodomains have been charac- induction of chromatic afterimages. To achieve the full effect, the terized in the retina. NRL, a member of the basic region viewer should fixate on the central black dot for ten seconds while leucine zipper family, is expressed only in the retina, where it the figure is illuminated by intense white light (e.g., ). If the is present in most or all neurons (29). Mash-1, a member of the observer then views a white piece of paper, an afterimage is seen in basic region helix-loop-helix family, is expressed in many which each square appears as its opponent color. The effect occurs regions of the developing CNS; in the developing retina, it is because within the retinal region illuminated by each colored square present in neuroblasts and is absent from the ganglion cell those cones and/or cone pathways that were most strongly stimulated layer (30). A number of more ubiquitous transcription factors were selectively desensitized. The desensitization must occur within have also been found in the retina but are unlikely to play a role the retina because the appears to move space as eye afterimage in the in moves. Consistent with a retinal origin, if the figure is viewed with only distinguishing cell types. one eye the afterimage will be confined to that eye. The observed With respect to the generation and differentiation of retinal afterimage reveal two systems for chromatic analysis: red vs. ganglion cells, four POU domain transcription factors are green and blue vs. red + green (= yellow). likely to be important, based on their expression in subsets of Downloaded by guest on September 29, 2021 Colloquium Paper: Xiang et al. Proc. Natl. Acad. Sci. USA 93 (1996) 599

FIG. 3. Ganglion and amacrine cells in the dog retina (from ref. 11). A, B, and C, amacrine cells; a-i, ganglion cells. ganglion cells in a variety of vertebrate retinas. The POU retina because of their low level of immunoreactivity with domain family of transcription factors is defined by the pres- currently available antibodies. ence of a bipartite DNA binding domain consisting of a The Brn3 proteins are also expressed in the developing POU-specific domain of -70 amino acids and a POU-specific dorsal root and trigeminal ganglia (33, 35), reminiscent of the homeodomain of '60 amino acids, separated by a 10- to expression pattern of the chicken homeobox gene SOHo-1 30-amino acid linker. More than 10 distinct POU domain (24). In adult mice, each of the anti-Brn3 antibodies stains a family members have been identified thus far in vertebrates, subset of cells within these ganglia (37). Anti-Brn3a antibodies including both ubiquitously expressed factors such as Oct-1 label most of the neurons; anti-Brn3b antibodies label <50% and tissue-specific factors such as the pituitary-specific factor of the neurons; and anti-Brn3c antibodies label only occasional Pit-1 (reviewed in ref. 31). Three of the four POU domain neurons. The expression of these transcription factors in both factors implicated in ganglion cell development-Brn3a, the somatosensory and visual systems is intriguing, given that Brn3b, and Brn3c-are highly homologous members of the class IV POU domain subfamily. The fourth, RPF-1, is a newly discovered member of the class VI POU domain subfamily. Os The first member of the Brn3 subfamily was identified in Is developing rat brain cDNA (32). Subsequent experiments led to the identification and characterization of the three Brn3 ONL -4i..

genes in mice (33-35) and in humans (36, 37). The Brn3 O PL .. proteins are closely related to Unc86, a protein involved in the INL development of sensory neurons in Caenorhabditis elegans (38, 39). In the adult mouse, each Brn3 gene is expressed in a small IPL number of midbrain nuclei, in the dorsal root and trigeminal ganglia, and in the retina. Within the retina, expression is GCL, confined to subsets of cells within the ganglion cell layer (Fig. 4). In cat and retinas, all of the Brn3-expressing cells appear to be ganglion cells rather than displaced amacrine cells Os-N. - (which constitute an appreciable fraction of the cells in the ganglion cell layer) as determined by double immunostaining ONL with AB5, an antibody previously shown to label only ganglion cells (40). In all retinas examined thus far, a characteristic and OPL reproducible heterogeneity is observed in the intensity of INL ganglion cell immunolabeling. In the developing mouse retina, the Brn3 proteins are found in the ganglion cell layer beginning IPL between embryonic days 12 and 15, the time at which this layer first separates from the underlying layer of dividing neuro- blasts (M.X. and J.N., unpublished). In cat and macaque retinas, it has been possible to correlate the pattern of expression of the Brn3 genes with the known morphologic and anatomic classes of ganglion cells (37). In the Iss cat, Brn3a is found at high levels in small (y) ganglion cells and at lower levels in medium (,3) and large (a) cells; Brn3b is ONL found at high levels in all ganglion cells; and Brn3c is found OPL ' only in small ganglion cells. A similar pattern is seen in the INL mouse retina where Brn3a and Brn3b are present in '40% of cells in the ganglion cell layer and largely colocalize; anti-Brn3c IPL immunoreactivity is present in '15% of cells in the ganglion cell layer and these constitute a subset of the cells that contain GCL Brn3a and Brn3b. In the macaque retina, immunostaining also reveals colo- calization of Brn3a and Brn3b. The density of immunostained FIG. 4. Anti-Brn3a, anti-Brn3b, and anti-Brn3c immunoreactivity in the mouse retina [reproduced with permission from ref. 37 (copy- cells in the ganglion cell layer falls steeply in going from the right 1995, Society for Neuroscience)]. Immunoperoxidase staining center to the periphery of the retina, a distribution that roughly with affinity-purified polyclonal antibodies specific for each of the matches the overall distribution of retinal ganglion cells in the Brn3 transcription factors (Left) and 4',6-diamidino-2-phenylindole primate retina (Fig. 5). Immunolabeling of macaque retinae (DAPI) staining of the same sections (Right). (A and B) Anti-Brn3a. following retrograde tracing from the lateral geniculate nu- (C and D) Anti-Brn3b. (E and F) Anti-Brn3c. Immunoreactive nuclei cleus shows high levels of Brn3a in a minority of P-type are found almost exclusively within the ganglion cell layer; the rare ganglion cells and low levels in all of the remaining P- and immunostained cells found in the inner nuclear layer are presumed to represent displaced ganglion cells. Note that the purple horseradish M-type ganglion cells. In the same retinae, high levels of Brn3b reaction were seen in peroxidase product partially quenches DAPI fluorescence nearly all P-type ganglion cells and low levels were when the two are present in the same nucleus. OS, outer segment;IS, seen in nearly all M-type ganglion cells (Fig. 6). Brn3c- inner segment; ONL, ; OPL, ; containing cells have not yet been mapped in the macaque INL, inner nuclear layer; GCL, ganglion cell layer. Downloaded by guest on September 29, 2021 600 Colloquium Paper: Xiang et al. Proc. Natl. Acad. Sci. USA 93 (1996) of cells in the ganglion cell layer contain high levels of RPF-1. In contrast to the eccentricity-dependent decrease in overall cell density in the ganglion cell layer, the density of cells that contain high levels of RPF-1 changes little with retinal eccen- tricity. The most direct evidence that any of the POU domain transcription factors play a role in ganglion cell development comes from recent experiments in which the Brn3b gene has been inactivated by homologous recombination in embryonic stem cells (M.X., J.N., L. Gan, and W. Klein, unpublished FIG. 5. Distribution of anti-Brn3a immunoreactivity among gan- data). Mice that are homozygous for the mutant allele are glion cells in the central (A) and peripheral (B) macaque retina viable but show specific defects in retinal structure. While [reproduced with permission from ref. 37 (copyright 1995, Society for Brn3b knockout retinae resemble those of the wild type in Neuroscience)]. overall structure, they have 70% fewer ganglion cells. Other neurons within the retina and brain appear to be minimally or both systems divide a complex sensory input into parallel not at all affected. streams (41). These data suggest a homology in the develop- An intriguing aspect ofthe Brn3 and RPF-1 immunolabeling ment of these two sensory systems, based on a partial overlap patterns is the characteristic heterogeneity in nuclear labeling of transcriptional regulators. intensity. This heterogeneity in levels of transcription factors RPF-1, the fourth POU domain sequence implicated in suggests that stable differentiated states may be determined ganglion cell development, was identified in human and mouse not only by the presence or absence of different transcription genomic DNA and subsequently found in the human retina factors but by the maintenance of these factors at particular where it is expressed in subsets of ganglion and amacrine cells intermediate levels. A graded mechanism of this general type (H.Z. and J.N., unpublished data). As described above for the has been shown to mediate anterior-posterior fate determi- pattern of immunostaining for the Brn3 factors, immunostain- nation in the Drosophila embryo, in which case concentration ing for RPF-1 shows a characteristic heterogeneity of nuclear gradients of a small set of maternally derived regulatory staining intensity. In the cat retina, the highest levels of RPF-1 proteins determine the level of expression of a larger set of are found in medium (13) and small (-y) ganglion cells; the large target genes at different positions in the embryo (19). (a) ganglion cells contain little or no RPF-1. In the macaque, The authors thank Dr. Stewart Hendry for helpful comments on the many ganglion cells contain low levels of RPF-1 and a minority manuscript. This work was supported by the National Eye Institute (National Institutes of Health) and the Howard Hughes Medical Institute. 1. Hartline, H. 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