Parallel Processing in the Mammalian Retina The Proctor Lecture

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1 Parallel Processing in the Mammalian Retina The Proctor Lecture Brian Boycott 1 and Heinz Wdssle 2 The projection from the eye to the mammalian brain is organized into parallel routes. 1 In the optic pathway retinal axons separate into several tracts that terminate in different subcortical areas, such as the suprachiasmatic nucleus, the lateral geniculate complex, the pretectum, the superior colliculus, and the accessory optic nuclei. In this way different aspects of the light signal are transfered to these centers. 2 Parallel routes also can be distinguished in the projection from the eye through the lateral geniculate nucleus to the visual cortex. Here, in primates, the parvocellular (P) and magnocellular (M) pathways are well established. 3 ' 4 Recently a further pathway in parallel to these has been found in the interlaminar regions (K-layers) of the geniculate. 5 ' 6 This may carry a blue cone signal among others. 7ab The specific tracts are made up by the axons of the 10 to 20 different types of retinal ganglion cells. 8 " 11 Their dendritic fields cover the retina homogeneously without leaving gaps, so a spot of light on the retina independent of retinal location hits at least one member of each of the ganglion cell types. Thus, the ganglion cells represent 10 to 20 niters that encode, in parallel, different aspects of the image projected onto the retina and their axons send specific messages into the different visual centers of the brain. A brightness signal, for instance, is sent to the suprachiasmatic nucleus for regulating the diurnal rhythm (see Ref. 19), a signal about retinal image movements to the accessory optic nuclei, which stabilize the eyes' positions and so on. The input from the photoreceptors to the ganglion cells is through bipolar and amacrine cells' synapses onto the ganglion cells' dendrites in the inner plexiform layer GPL)- The IPL is precisely organized: ganglion cells have dendrites at characteristic levels within the IPL. There is an overall subdivision of the IPL into ON- and OFF-layers. Dendrites of OFF-ganglion cells stratify in the outer half of the IPL, those of ON-ganglion cells in the inner half Within the ON- and OFF-layers there are further subdivisions. Dendrites of ON- and OFF-parasol cells in primates keep a very narrow level of stratification close to the center of the IPL. 14 In contrast, dendrites of ON- and OFFmidget ganglion cells stratify more diffusely and are found more toward the inner and outer regions of the IPL, respectively. l5 The two dendritic planes of the small bistratified (blue) ganglion cells stratify even further toward the outer and inner edges of the IPL. 16 This suggests that when a retinal From the 'Department of Visual Science, Institute of Ophthalmology, University of London, London, United Kingdom; and 2 Max-Planck- Institut fiir Hirnforschung, Frankfurt/Main, Germany. Proprietary interest category: N. Reprint requests: Heinz Wassle, Max-Planck-Institute fur Hirnforschung, DeutschordenstralSe 46, D Frankfurt/Main, Germany. image is neurally encoded it is different at different levels within the IPL. How are specific aspects of the light signal transferred from the outer to the inner retina? This is the role of 9 to 10 different types of bipolar cells 17 ; but many details are still missing. Considering primates, the only mammals that are known to have three cone types; one can ask which bipolar cells transfer the cone-specific chromatic signals into the IPL and at what level do their axons terminate. 71 ' It is probable that achromatic transient and sustained signals and direction-selective (DS) responses involve different sets of bipolar cells whose axons terminate at different levels within the IPL. Bipolar cells providing high spatial resolution should convey signals from one to only a few cones, whereas those bipolar cells exhibiting high contrast sensitivity should get convergent input from many cones. This multiplicity of pathways, from the outer toward the inner retina, implies that parallel processing starts immediately at the cone pedicles, the first synapse of the retina. So a key question to ask is how the synapses at an individual cone pedicle are organized to feed into all these different bipolar cells. And, of course, the answers are critically dependent on an understanding of how these synapses, through their molecular composition, create ON- or OFF- and phasic or tonic signals. Clearly the type of glutamate receptor expressed is decisive. These and related questions will be dealt with in what follows. We shall approach answers by comparing the circuitry of the retinae of primates and rats. Because rats, like other mammals, are dichromats, 18 this comparison elucidates some aspects of the circuitry subserving trichromacy in primates. Bipolar Cells of the Mammalian Retina Cajal 26 recognized rod bipolar (RB) cells as a type separate from cone bipolar cells (Fig. 1: RB). Dendrites of RB cells make invaginating contacts with rod spherules, and their axons terminate in the inner part of the IPL, close to the ganglion cell layer. 20 " 22 ' 51 There is no direct output from RB cell axon terminals onto ganglion cells; instead a narrow-field amacrine cell, the so-called All-amacrine cell, is interposed. 23 The Allcells make conventional inhibitory chemical synapses with OFF-cone bipolar and OFF-ganglion cells, and electrical synapses through gap junctions with ON-cone bipolar cells. Since the rod circuit has been reviewed several times in recent years, it will not be dealt with further here. 24 ' 25 There is only one type of RB cell, but many types of cone bipolar cells have been recognized in several mammalian species. 26 In the rabbit retina, 9 types have been described from Golgi staining. 27 Recently two of these were confirmed by intracellular dye injection and an additional type was de- Investigative Ophthalmology & Visual Science, June 1999, Vol. 40, No. 7 Copyright Association for Research in Vision and Ophthalmology 1313

2 1314 Boycott and Wassle A IOVS, June 1999, Vol. 40, No. 7 OPL GCL MONKEY IPL GCL FIGURE 1. (A) Bipolar cells of the rat retina as seen in a vertical retinal slice after intracellular injection with Lucifer Yellow or Neurobiotin. 3 '' (B) Bipolar cells from Golgi-stained rhesus macaque retina also as observed in vertical section. 36 The cells are arranged according to the stratification level of their axons in the IPL. The horizontal line dividing the IPL represents the border between the OFF (upper) and the ON (lower) sublamina. OPL, outer plexiform layer; IPL; inner plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. scribed. 2H ' 2y In the cat retina, 8 to 10 different types of cone bipolar cells have been recognized. 27 ' 30 " 33 Figure 1 is a comparison of the bipolar cells of the rat retina with those of midperipheral macaque monkey retina. The nine putative cone bipolar cell types (labeled 1 to 9) and the RB cell of the rat retina are arranged according to the stratification level of their axon terminals in the IPL. The cells were drawn after intracellular injections with Lucifer Yellow (LY) of vertical retinal slices ( jam thick). 34 The cone inputs of the nine cells have not yet been analyzed in detail, but they contact several neighboring cone pedicles, with one exception: bipolar cell 9 has a wide dendritic tree that is cone selective and might, therefore, represent a S-cone bipolar cell. The rat retina is considered to be rod dominated because only 1% of its photoreceptors are cones. 35 However, the perspective changes when one examines the absolute number of cones; the cone density is between 4000 and 5000 cones/mm 2, similar to peripheral cat, rabbit, and macaque monkey retinae. Thus, the cone bipolars of peripheral macaque monkey retina (Fig. IB) are from a region of cone density comparable to that of the rat retina (Fig. 1A). The types of bipolar cells of the monkey retina, shown schematically in Figure IB, were determined initially from Golgi-stained whole mounts. 36 There is a striking similarity between the rat and the monkey bipolar cells with respect to the shapes and stratification levels of their axons. However, there is also a clear difference; midget bipolar cells (FMB, 1MB) are only found in the monkey retina. The dendritic trees of FMB and 1MB cells are restricted to a single cone pedicle, and their axons terminate at two different levels within the IPL Following Polyak's nomenclature, bipolar cells with dendritic trees contacting several neighboring cone pedicles are called "diffuse" bipolar cells (DB1-DB6). 36 To summarize: there are approximately 10 types of cone bipolar cells in mammalian retinae whose major denning features are the shape and stratification of their axons in the IPL. Immunocytochemical Staining of Bipolar Cells The bipolar cell scheme of Figure 1 is based on intracellular injections of the rat retina and on Golgi staining for monkey retina. Both methods are selective, and stain cells unpredictably. A bipolar cell is accepted as a type only if it is found all across the retina ("mosaic") and if ever)' cone has access to one of its members ("coverage"). We, therefore, searched for immunocytochemical markers that specifically stain all bipolar cells of a given category. Figure 2 shows immunolabeling of monkey bipolar cells with an antiserum to the glutamate transporter GLT-1 (Fig. 2A) and for protein kinase C (PKC, Fig. 2B).

3 IOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture 1315 FIGURE 2. Fluorescence micrographs of vertical sections through parafoveal macaque monkey retina.(a) Immunostained for the glutamate transporter GLT-l.' f0 The labeled bipolar cells have flat dendritic tops in the OPL, and their axon terminals are confined to the outer half of the [PL. (B) This section was immiinostained for the a-isoform of protein kinase C. 22l3 ' f The labeled bipolar cells are rod bipolar cells and DB4 cells.'"' Their dendritic tops appear irregular because of the many processes invaginating into rod spherules. Their axon terminals are confined to the inner half of the I PL. ONL, outer nuclear layer; other conventions as in Figure 1. Analysis of sections and whole-mounted retinae showed that GLT-1 immunoreactivity was expressed by flat midget and DB2 bipolar cells, 39 ' 40 whereas PKC immunoreactivity was restricted to DB4 and rod bipolars. 40 Immunocytochemical markers that recognize the two types of midget bipolar,'"' 12 the DB3 bipolars, 40 and blue cone bipolar cells 43 have been found. Such staining makes it possible to measure the relative densities of the cells throughout the retina. Combining the number of cone contacts of individual diffuse bipolar cells (usually between 5 and 10) with the density estimates of diffuse bipolar cells and cones, we concluded that each cone pedicle of the primate retina contacts 10 to 15 individual bipolar cells comprising all six types of diffuse bipolar cell. In addition, each cone pedicle is in contact with at least two midget bipolar cells: one FMB and one 1MB cell. The S-cone circuit is special and has been analyzed by Kouyama and Marshak. 43 Immunocytochemical markers, which recognize specific types of bipolar cells, also were found for the rat retina. 34 Their relative densities could be measured. It is clear that, here also, each cone feeds into every type of cone bipolar cell. Cone Contacts of Bipolar Cells The synaptic terminals of cone photoreceptors, the cone pedicles, have three different kinds of synaptic specializations. First, each pedicle has gap junctions for electrical contacts with other cone pedicles and rod spherules. 44 * 47 Second, the pedicle contains invaginations, 48 each of which comprises a presynaptic ribbon and three invaginating processes: two lateral elements, which are horizontal cell dendrites, and a central element, which, in mammals, is an ON-bipolar cell denclrite. 49 This synaptic arrangement has been named a triad. 48 Although there may be more than one central element, 50 that name is retained. Third, flat (basal) contacts with putative OFF-bipolar cells have ultrastructural specializations with the cone pedicle base. 48 ' 49 ' 5152 Flat contacts can be found close to a triad (triad associated, TA) or away from triads (non-triad associated, Reconstruction of Golgi-impregnated midget bipolar cells of the primate retina by serial electron microscopy (EM; Figs. 3, 4) showed a clear dichotomy: 1MB cells made exclusively invaginating contacts, whereas FMB cells made only flat contacts. 5 ' 55 ' 1 Individual 1MB bipolar cells make up to 25 contacts with a cone pedicle (Figs. 3A, 4A) all of which are invaginating. An FMB cell, depending on retinal position, makes approximately 2 to 35 times that number of basal synapses. In central retina they are all TA, at eccentricities beyond 35 mm approximately 20% are NTA (Figs. 3B, 4B). 53 ' 35b Since the axons of 1MB and FMB cells terminate in the ON- or OFF-stratum of the IPL, respectively, it is thought that they represent an ON- and OFF-bipolar cell pair for each cone. This has been formulated as a general rule: ON-bipolar cells make invaginating contacts, whereas OFF-bipolar cells make flat contacts. 52 ' 56 ' 57 However, this correlation has been challenged after comparison with results from other vertebrate retinae. 58 Reconstructions of cone contacts of Golgi-impregnated diffuse bipolar cells by EM revealed the complexity of cone-tobipolar cell connections in midperipheral primate retina (Figs. 3C, 4Q. 5 ' f - 55l> ' Like FMB cells, bipolar cells DB1, DB2, and DB3, which have their axon terminals in the outer IPL (Fig. IB), make exclusively basal junctions with the cone pedicle. They always have TA and NTA contacts, the proportions varying according to the cell type, as does the average number of

4 1316 Boycott and Wassle IOVS, June 1999, Vol. 40, No. 7 A B 4 FIGURE 3. Apical dendrites, viewed from the photoreceptor side, of Golgi-Colonnier-stained bipolar cells in a whote-mounted rhesus macaque monkey retina (A) An 1MB cell with apical process slightly oblique to the viewer. (B) An FMB cell with well-oriented apical processes. (C) A DB2 cell with altogether 9 terminal aggregates. Scale bar, 10 jam. contacts per cone, which is between 10 and 20. There is no regularity in distribution of TA and NTA contacts, nor do some cones have all TA contacts and others all NTA. Bipolar cells DB4, DB5, and DB6 have their axon terminals in the inner part of the 1PL (Fig. IB), and, depending on the bipolar cell type, they have an average of between 4 and 8 invaginating synapses per cone pedicle. In addition, cell DB5 may have as many as 10% of its contacts as basal junctions. 60 Cell DB6 averages rather more, with 30% of the contacts basal, 61 but cell DB4 55b6 has between 30% and 60% of its contacts in a predominantly TA position. Thus, while the dichotomy "invaginating = ON, flat contacts = OFF" holds for midget bipolars and DBS is close to that pattern, DB6 does not conform so clearly and DB4 appears to FIGURE 4. Diagrams of the synaptic contacts of the three bipolar cells shown in Figure 3 55b The cone pedicles (circular outlines') were reconstructetl by EM from serial sections; their maximum diameter is approximately 8 jlim. The different symbols represent synaptic contacts; their sizes are not to scale; but their type, number, distribution, and relative position are taken from the EM reconstructions. (A) The 1MB cell made 25 invaginating contacts (A) inserted into 25 different triads; it did not make any basal synapses. (B) The FMB cell made 97 flat contacts, of which 77 were triad associated ( ) and 20 were non-triad associated (O). (Q The DB2 cell contacted altogether 9 cone pedicles and made triad-associated ( ) and non-triad-associated (O) contacts. Neither FMB or DB2 made any invaginatiiig contacts. t

5 IOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture 1317 FIGURE 5. Reconstruction of the synaptic complex of a cone pedicle of the macaque monkey retina. 50 The reconstruction was performed from horizontal serial sections, observed and photographed by EM. The width of each frame represents 10 jam. (A) The solid circumference shows the outline of the pedicle; the short lines inside represent synaptic ribbons. Two rather long ribbons are marked by arrowheads. (B) Ribbons and the horizontal cell processes forming the lateral components of the triads are shown. The long ribbons indicated in (A) are each engaged with two triads. (C) Central invaginating processes and their location with respect to the ribbons are shown. (D) Profiles of the dendritic processes immediately underneath the cone pedicle. have about half its synapses invaginating and half as basal contacts. As discussed later, the type of synapse made by a bipolar cell at a cone pedicle, flat versus invaginating, is not the decisive feature; it is rather the glutamate receptor expressed there. 62 Synaptic Organization of the Cone Pedicle of the Primate Retina The question arises as to whether there are enough synaptic sites on a cone pedicle to accommodate the dendritic tips of FMB and 1MB cells and those of all six types of diffuse bipolar cells. To address this, we have serially sectioned cone pedicles for EM and measured the number of ribbons and invaginating processes (Fig. 5). 50 The cone pedicles were from midperipheral retina (6-7 mm), corresponding to the eccentricity at which the bipolar cells from Figure IB were classified and from where the cells in Figure 3 were selected. The diagram in Figure 5A shows the outline of the pedicle and its 46 synaptic ribbons. The reconstruction of the lateral elements, formed by two horizontal cell processes flanking each ribbon, is shown in Figure 5B; 48 triads were found. The invaginating central elements and their location with respect to the ribbons also were reconstructed (Fig. 5C), and 104 were found in this particular cone pedicle. Of these, 24 were thicker, filled with organelles in the electron micrographs, and we interpret them as the dendritic tips of an invaginating midget bipolar cell. The number of 24 invaginating contacts agrees well with the contacts made by the reconstructed midget bipolar cell (Fig. 4A). Unfortunately it was not possible also to reconstruct the flat contacts from the horizontal sections through the cone pedicle. However, it has been estimated from vertical sections that there might be as many as 500 flat contacts at a cone pedicle base. 48 The drawing in Figure 5D shows a reconstruction of all processes connected to that particular cone pedicle; some 450 were found. The ribbons and triads of 14 peripheral cone pedicles were reconstructed. The minimum number of triads was 38, the maximum 48, and the average 41.8 (±3). The reconstructions of cone pedicles show that they provide enough synaptic sites to contact both types of midget bipolar cells and all six types of diffuse bipolar cells. We also have reconstructed cone pedicles from the macaque monkey fovea. They have fewer triads (21.4 ± 1.6) and consequently, as discussed in detail elsewhere, the predominantly invaginating bipolar cells DB4, DB5, and DB6 have a higher proportion of flat contacts. 50 ' 63 ' 64 The schemes presented in Figure 1 are, therefore, not only an illustration of individual types of bipolar cell. Since each type provides a complete coverage of the retina with its dendrites, and because cone pedicles provide sufficient numbers of synaptic sites, so that all dendrites actually can make functioning synapses, they fulfil the proposition that a light spot projected onto the retina hits independent of the retinal location at least one member of each of these bipolar cell types. Thus, at the first retinal synapses parallel pathways or channels are formed to transfer different aspects of the light signal into the inner retina.

6 1318 Boycott and Wassle IOVS, June 1999, Vol. 40, No. 7 FIGURE 6. (A) Slice preparation of a rat retina during patch-clamp recordings from a bipolar cell. 72 In this Nomarski micrograph, the cell bodies are visible, and the patch electrode, of which only the tip is in focus (arrowhead), approaches a bipolar cell. The seven-barrel puffer pipette for drug application can be seen to the left (arrow). (B) Cell body of a bipolar cell that was injected with Lucifer Yellow (LY) after the recordings. (C, D) Fluorescence micrographs showing the LY-injected bipolar cell from (B) in two focal planes. The axon-terminal of this rype-2 bipolar cell branches in the outer part of the IPL (as indicated by the white arrows in B). Scale bar, 30 p-m. Physiological and Pharmacologic Responses of Bipolar Cells By contrast with the wealth of morphologic data only limited, and sometimes conflicting, information is available for the physiological responses of mammalian bipolar cells. Intracellular recordings from RB cells of the cat retina suggest that they give OFF-responses, 65 whereas ON-responses were found in rabbits. 21 Intracellular recordings have been made from three types of cone bipolar cells of the cat retina; Cb2 : Cb5, and Cb6. 66 Cell Cb2 was an OFF-cell, and cell Cb5 was an ON-cell. Their axons terminated in the outer and inner half of the IPL, respectively. Cell type Cb6 broke the rule; it was an OFF-cell but its axon terminated in the inner half of the IPL. However, in the light of more recent work these observations in the cat retina need to be repeated. Pharmacologic and physiological studies in non-mammalian retinae have shown that ON-bipolar cells express a special type of metabotropic glutamate receptor. Binding of (±)-2- amino-4-phosphonobutyric acid (AP-4) to this receptor causes a closure of non-selective cation channels. 67 ' 6 * 1 In contrast, conventional ionotropic glutamate receptors have been found in OFF-bipolar cells. 69 Dissociated RB cells of nit and cat retinae have an AP-4- type glutamate receptor (GluR) and are, therefore, ON-bipolar cells. 70 ' 71 Dissociated cone bipolar cells of the cat retina were found to display either AP-4-type GluRs or conventional ionotropic Glulls. 71 Unfortunately, the morphologic types of these dissociated cone bipolar cells could not be denned. Recently it became possible to record from bipolar cells using the patch-clamp technique on retinal slices and to study their responses to glutamate, the photoreceptor transmitter (Fig. 6A). 7273: - C The patch electrode wasfilledwith LY, which permitted the identification of the cells from which recordings had been made (Figs. 6B, 6C, 6D). In this way, it could be decided whether the cells expressed an AP-4 type of GluR or an ionotropic GluR and whether they were ON- or OFF-bipolar cells, respectively, could be predicted. Whole cell recordings from a type-2 bipolar cell in a rat retinal slice preparation are shown in Figures 7A, 7B, and 7C. The cell was voltage clamped at a holding potential of V c = 85 mv. Application of AP-4 did not elicit any current in this cell, but application of glutamate (GLU) evoked an inward current (Fig. 7B). This inward current had a transient peak and showed a fast de-sensitization. Kainate application evoked an even stronger inward current than GLU did, and this inward current was not significantly changed by using a Co 2+ containing bathing solution; however, it was abolished by CNQX, a non-selective GluR antagonist (Fig. 7C). We interpret these findings as showing a direct action of GLU and kainate on a fast desensitizing ionotropic GluR expressed by type-2 cone bipolar cells. 72 Application of GLU in the current clamp mode caused a depolarization of these cells. Since GLU release from cones is high in darkness, these cells must be OFF-bipolar cells. Whole cell recordings from a rat type-8 bipolar cell are shown in Figures 7D and 7E. At the holding potential of V c = -53 mv, the cell exhibited a sustained inward current of 12 pa. Application of AP-4 evoked a net outward current accompanied by a reduction in membrane noise. The current was resistant to superfusion with Co 2+ and application ofglu elicited a similar current (data not shown). We interpret this as a direct action of AP-4 on a GluR that causes the closure of a non-selective cation channel. In the current clamp mode, application of GLU caused a hyperpolarization of these cells. Since GLU release from cones is high in darkness, these cells must be ON-bipolar cells. Similar responses to the application of GLU also have been reported by Hartveit. 73 There is agreement that the cone bipolar cells type 1, type 2, type 3, and type 4, all of which have their axon terminals in the outer part of the IPL (Fig. 1A) express ionotropic GluRs. In contrast bipolar cells type 6, type 7, type 8, and RB cells express an AP-4 type of GluR, and their

7 IOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture 1319 B OPL AP-4 GLU KA,. ^,^j V c = - 85mV IPL AP-4 V c = - 53mV GCL FIGURE 7. Whole cell currents recorded from identified type-2 (A) and type-8 (D) bipolar cells of rat retmae. 72 (B) The dotted horizontal line represents the steady state current. Application of AP-4 had no effect; application of glutamate induced an inward current with phasic and tonic components. (C) Same cell as in (B). Kainate (KA) induced a strong inward current, which could not be blocked by application of Co 2+ in the bath. This inward current was blocked by the glutamate receptor antagonist CNQX. (E) This type-8 cell exhibited a steady state inward current of ~12 pa (dotted horizontal line, vertical arrow). Application of AP-4 caused an outward current and a reduction of membnine noise. Horizontal scale bar represents 10 seconds in (B), (C), and (E); vertical scale bar represent 20 pa in (B) and (C) but 5 pa in (E). axons stratify in the inner part of the IPL (Fig. 1A). The cone bipolar cell type 5 has an axon that stratifies in the center of the IPL and it is a matter of discussion whether type 5 expresses an ionotropic receptor 72 or an AP-4 type of receptor. 73ab These recordings from rat retinal slices suggest a close correlation between the stratification level of bipolar cells and their physiological light responses: bipolar cells stratifying in the outer part of the IPL are OFF-bipolar cells; those with axons stratifying in the inner part of the IPL are ON-bipolar cells. Recently it became possible to elicit light responses in mammalian retinal slices. 74 ' 75 Recordings from an identified rod bipolar cell are shown in Figures 8A and 8B. When the RB cell was voltage clamped at V c = 60 mv, a light pulse of 10 msec elicited an inward current of approximately 10 pa. In the current clamp mode the cell was depolarized by approximately 6 mv. This shows that RB cells of the rat retina are indeed ON-bipolar cells, as had been suggested by their responses to AP-4. Glutamate Receptors in Mammalian Retina Molecular cloning has revealed a multiplicity of glutamate receptors and receptor subunits. Ionotropic receptors are integral membrane proteins that form an ion channel. This channel, usually a non-selective cation channel, is opened when glutamate binds to the receptor. Three major groups of ionotropic glutamate receptors can be distinguished: AMPA, kainate, and NMDA receptors. They comprise the following subunits: AMPA (GluRl, GluR2, GluR3, GluR4); kainate (GluR5, GluR6, GluR7, KA-1, KA-2); NMDA (NR1, NR2A, NR2B, NR2C, NR2D); further subunits are the orphan receptors 81 and 52. In addition multiple splice variants of the different subunits have been identified, e.g., the 10 splice variants of NRl. 76a Metabotropic receptors belong to the family of receptors that have 7 membrane-spanning domains and, when they bind with glutamate, G-proteins and second messenger systems are activated. So far 8 different metabotropic glutamate receptors have been identified (mglurl-mglur8). It is clear that a simple retinal scheme: glutamate released from photoreceptors acts on horizontal and bipolar cells and then, in turn, glutamate released from bipolar cells activates amacrine and ganglion cells, can have any degree of complexity depending on the glutamate receptors that are expressed. Work on the localization of GluRs in the retina has been recently reviewed. 62 ' 761 ' 77 Here we concentrate on the synap-

8 1320 Boycott and Wiissle A M UJ O TIME (sec) FIGURE 8. Light responses recorded from a rod bipolar cell in a dark-adapted preparation of the rat retina (kindly provided by N. Flores and D. Protti, unpublished results), (A) A light pulse (Ganzfeld) of 15-ms duration (tipper horizontal trace) caused a depolarization of the RB cell. The voltage trace represents the average of eight individual responses. (B) In the voltage clamp mode, the light pulse induced an inward current. This shows that RB cells have depolarizing light responses and, therefore, are ON-bipolar cells. tic localization of glutamate receptors related to the photoreceptor triads in the OPL and to the bipolar ceil dyads in the 1PL. There is a specific distribution of glutamate receptors in the mammalian retina. Figures 9A, 9B, and 9C show this for the IOVS, June 1999, Vol. 40, No. 7 metabotropic receptor mglur6 (Fig- 9A), for the AMPA receptor subunit GluR2 (Fig. 9B) and for the NMDA receptor subunit NR2B (Fig. 9C). All three micrographs show a punctate imrnunofluorescence, which has been shown by EM to represent a clustering of the receptors at postsynaptic sites. By light microscopy it is possible to predict that mglur6 is restricted to synapses in the OPL (Fig. 9A); GluR2 is found both in the OPL and in the IPL (Fig. 9B), whereas NR2I3 is restricted to the 1PL (Fig. 9C). Within the IPL, GluR2 and NR2B immunoreactive puncta are in discrete bands of higher density, suggesting they are expressed preferentially at certain synapses. However, definitive synaptic localization requires EM. In a series of seminal experiments, S. Nakanishi has cloned mglur6, localized it with specific antibodies, and studied its function by gene directed (knock out) mutagenesis These experiments have shown that mglur6 is expressed at the dendritic terminals of rod bipolar cells inserted into the rod spherules (Fig. 9A) and that mglur6 represents the AP-4 receptor expressed in the ON-channel. Now Vardi et al. 62 have shown that mglur6 also is expressed in putative ON-cone bipolar cells at their invaginating, and occasionally flat, contacts with cone pedicles. However, although mglur6 seems to be the functionally predominant glutamate receptor expressed by the ON-bipolar cells, it has to be emphasized that additional glutamate receptors have been localized to them, and their functions still need to be elucidated (mglurl 80 ; ionotropic GluRs 62 ' 7781 ' 82 ). Both kainate and AMPA receptors have been localized to specific synapses in the OPL. The electron micrograph in Figure 10B shows a surprising, but consistent result: the kainate receptor GluR6/7 is localized in horizontal cell processes invaginating into cone pedicles and rod spherules; however, always, only one of the two lateral processes is labeled. The AMPA receptor subunit GluR4 is often restricted to only one horizontal cell process within the triad (Brandstatter, personal communication). In the cat retina Morigiwa and Vardi 83 also have observed that GluR2/3, GluR4, and GluR6/7 are often located in only one horizontal cell process of a triad. This suggests that the two horizontal cell processes within the FIGURE 9- Fluorescence micrographs of vertical sections through rat retinae that were immunostained for different glutamate receptors. (A) The metabotropic glutamate receptor mglur6 is restricted to the dendritic tips of RB and ON-C13 cells in the OPL (antibodies gift of S. Nakanishi; micrograph kindly provided by U. Griinert). (B) The AMPA receptor subunit GluR2 shows a punctate (synaptic) distribution in both the OPL and [PL. (C) The NMDA receptor subunit NR2B is confined to the IPL. Scale bar, 30 /am.

9 10VS, June 1999, Vol. 40, No. 7 the Proctor Lecture 1321 k "" < * $ CP \ i t * : *..* * m* GiLUR t CP f CB f B GLUR 6/7 FIGURI; 10. Electron microscopic localization of different subunits of ionotropic glutamate receptors in mouse (A) and rat (B, C) retinae using preembedding immunocytochemistry. (A) Localization of the AJMPA receptor subunit GluRl in the outer plexiform layer (kindly provided by I. Hack and J. Brandstatter, unpublished results). The plane of section is through the synaptic complex of a cone pedicle Cupper half of the micrograph, CP). The synaptic ribbons of two triads are indicated by arroivbeads. Two basal contacts (Jilted arrows') are imnuinoreactive for GluRl; one basal contact (open arrow) is unlabeled. (B) Localization of the kainate receptor subunits GluR6/7 in a cone pedicle triad (arrowhead: synaptic ribbon). H, horizontal cell dendrites; B, putative invaginating bipolar cell dendrite. Only one of the two horizontal cell dendrites expresses GliiR6/7. (C) Localization of the kainate receptor subunit KA2 to a cone bipolar axon-terniinal (CB) in the I PL. 1 *' Two postsynaptic processes (asterisks) are opposed to the presynaptic ribbon (arrowhead) at this dyad; only one is labeled. Scale bar, 0.5 in (A), 0.25 /xm in (B), and 0.15 /am in (C). photoreceptor triads express different sets of kainate/ampa receptors and possibly have different functional roles, Since the invaginating bipolar cell dendrites within the triad preferentially express the mglur6 receptor, it seems that all three members of a triad express different glutamate receptors. The electronmicrograph in Figure 10A shows that basal synapses at cone pedicles are immunoreactive for the AJVIPA receptor subunit GluRl. Previously we have shown that basal contacts express the kainate receptor subunit KA2, but not GluR6/7. 8f There is localization of GluR2/3, GluR4, and

10 1322 Boycott and Wassle IOVS, June 1999, Vol. 40, No. 7 GluR6/7 at basal contacts postsynaptic to cone pedicles in the cat retina. 83 Immunoreactivity for GluRl has been found in a subset of cat flat cone bipolar cells. 85 There are some differences in detail in these studies but all them clearly show that multiple ionotropic glutamate receptors are expressed at basal contacts of cone pedicles. The receptors are apparently only of the AMPA/kainate type since NMDA receptor subunits have not been localized to bipolar cell dendrites in the OPL (Fig. 9C). 86 Unfortunately it has not yet been shown whether different types of cone bipolar cells express different glutamate receptors. The staining of subsets of bipolar cells in the cat retina suggests there may be type-specific expression of glutamate receptor by cone bipolar cells. 85 The anatomic results demonstrating the presence of different subtypes of AMPA and kainate receptors at basal synapses of cone pedicles are in contrast to a recent physiological study in which only kainate receptors have been found to mediate the synaptic transmission between cones and OFFbipolar cells. 73c Glutamate Receptors in the IPL The punctate immunofluorescence of GluR2 and NR2B in Figures 9C and 9D suggest the two subunits are preferentially clustered at particular synaptic sites. Similarly, by light microscopy, we have studied further AMPA, kainate, and NMDA receptor subunits, which all had a clustered appearance exhibiting, however, different bands of higher and lower density of puncta within the IPL (Figs. 9B, 9C). This suggests that there is a rather precise pattern of stratification of the IPL with respect to the expression of glutamate receptor subunits. Most strikingly it was found that there seem to be no NMDA receptor subunits associated with the axon terminals of RB cells. By EM (Figs. IOC), as expected, the receptors were exclusively found in processes postsynaptic to bipolar cell ribbon synapses (dyads). As a rule, only one of the two processes at a dyad expressed a given subunit. A similar result has been reported for the cat retina by Qin and Pourcho. 85 The postsynaptic processes at cone bipolar cell dyads are usually a ganglion cell dendrite and an amacrine cell process. 49 Our result suggests that they too express different glutamate receptors. This is not to say that a ganglion cell dendrite postsynaptic to a cone bipolar cell dyad cannot express a combination of AMPA and NMDA receptors, as has been shown in other parts of the brain. It is to say, that the ganglion cell dendrite and the amacrine process are, in general, likely to express different sets of glutamate receptors. In conclusion, a multiplicity of glutamate receptors is expressed at synapses both in the OPL and in the IPL. Most still have to be linked to the different types of bipolar cells. The synapses that express different sets of glutamate receptors show a specific laminar distribution within the IPL (Figs. 9B, 9C). It is attractive to suppose that this laminar distribution of the synapses corresponds to the stratification pattern of the bipolar cell axons. The seeming exclusion of NMDA receptors from the synapses of the rod bipolar cell axons is a clear example. This would predict, that not only do the glutamate receptors expressed on the bipolar cells dendrites in the OPL shape their light responses, but also glutamate receptors expressed postynaptically in the IPL could specify the roles of the differerent types of bipolar cells. DISCUSSION In the preceding sections we have mixed anatomic results from the primate retina, physiological recordings from the rat retina, and the molecular characterization of the glutamate receptors in cat, mouse, and rat retinae, rather as if we studied the prototype of a mammalian retina. At first thought this seems to be absurd, given the fact that rat and cat retinae are rod dominated, whereas the primate retina has a highly developed fovea that is cone dominated and has three different cone types demanding specific circuits. However, as mentioned before, the cone densities of the rat retina and the cone density of now-foveal primate retina, which comprises more than 90% of that retina, are closely similar. The rod pathway through the rat retina is closely similar to that of the primate retina 25 ; the same holds for the cone bipolar pathways, as shown in Figure 1. Certain amacrine cell circuits, such as the cholinergic amacrine cells, the dopaminergic amacrine cells, and the All amacrine cells, are indistinguishable in their shapes and relative numbers. The relative percentages of the main cell classes are very similar. 87 This does not preclude differences in detail, such as only one morphologic horizontal cell type in rats 88 versus two types in primates. 89 ' 90 However, even when molecular details, such as, for example, the expressions of mglur6 and NR2A, or of different subunits of the glycine or GABA receptors are considered, there is a striking similarity between their distribution in rats and primates. One of the reasons to use the rat retina is that many of the transmitter receptors have been cloned for the rat and rat retinae are more readily available for in vitro physiology. In addition when going a step further, the rat retina is even more similar to the mouse retina, and transgenic mouse retinae are now becoming important in retinal research. The rest of the discussion will be concentrated on the primate retina and uses rat data better to understand the function of primate bipolar cells. Thus, it is safe to predict that all primate bipolar cells that have axons terminating in the outer part of the IPL and that make flat contacts (DB1, DB2, DB3 and FMB) are OFF-bipolar cells, whereas those that have axons terminating in the inner half of the IPL and that preferentially make invaginating contacts are ON-bipolar cells. However, there is no definitive answer to the question, which bipolar cells of the primate retina transfer chromatic signals from the outer to the inner retina. Clearly, the responses to light of all the different bipolar cell types have to be measured together with their chromatic modulation upon stimulation of the different cone types. These experiments have not yet been done; therefore, firm answers cannot be given, but there are some predictions and constraints with respect to bipolar cells and their chromatic selectivity to be derived from anatomy. 7b,91,92 Primate Bipolar Cells and Chromatic Signaling There is not much doubt that S-cone bipolar (BB) cells must be the channel transfering a blue ON-signal into the IPL. Their selectivity for S-cone pedicles, their invaginating contacts, and the termination of their axons in the inner IPL are all predictors for this functional role. 93a ~ c They are not restricted to the primate retina but seem to be present in the rat (Fig. 1, type 9) and in the rabbit 27 and possibly in other mammals. Axon terminals of BB cells contact the inner dendritic tier of the small field bistratified blue ON-ganglion cell. i6 ' 93: " c94 These

11 JOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture 1323 ganglion cells give OFF-responses when L- and M-cones are stimulated, and this input is probably derived from an OFFdiffuse bipolar cell synapsing onto the outer tier of the bistratified dendritic tree The axon terminals of DB1, DB2, and FMB cells overlap with the outer tier of the dendrites of the small bistratified ganglion cells. So which of these three bipolar cells provides the input? Were it to be derived from DB1 or DB2 cells, then cone selectivity becomes an issue. 93bc>96 Are the cone contacts of DB1 or DB2 cells restricted to L- and M-cones, or do they, non-selectively, contact all three cone types? Because the midget bipolar cells contact a single cone they must transfer the chromatic signature of this cone to the inner retina. As we have shown, there is a 1:1 correspondence between both 1MB and FMB cells and the cones of central and midperipheral retina. 42 Thus, the complete cone mosaic is represented in detail in both the ON- and the OFF-sublamina of the IPL. The situation is different in far peripheral retina. Here midget bipolar cells contact several neighboring cone pedicles non-selectively. 42 This does not necessarily exclude cone selectivity because M- and L-cones have a clumped distribution However, at the border of such patches a mixed cone input would occur, and so the chromatic signal might become degraded. In the fovea it has been shown by EM that ON- and OFF-midget ganglion cells (P-cells) receive their major excitatory input from a single 1MB or FMB cell. 91 ' 92 ' 98 ' 99 Here the center response of the receptive field of a P-cell would be dominated by whichever (L- or M-) cone the MB cell happened to contact. Two models of the surround response can be considered. To be strictly color-selective, it should receive input from only one cone type. 100 " 102 Horizontal cells, because of their non-selective cone connections, cannot provide such a surround opponency " 105 Small field amacrine cells recently have been shown to receive input from both L- and M-cones, 106 and hence, like horizontal cells, it is considered they cannot provide a pure surround. This favors the second model of a mixed cone input to the surround of the P-cells The situation becomes more complicated in peripheral retina, where there are more cones than there are ganglion cells. 109 Midget ganglion cells in midperipheral retina have extended dendritic trees ; thus, several midget bipolar cells must converge to create the receptive field center of a peripheral midget ganglion cell. 112 If peripheral midget ganglion cells have "pure cone" centers, their dendrites must make selective contacts with MB-axon terminals; however, if peripheral midget ganglion cells have "mixed cone" centers, they can contact all MB-axon terminals within their reach. Recent physiological recordings 95 suggest peripheral midget ganglion cells have mixed L- and M-cone inputs to their receptive field centers; this is consistent with non-selective wiring. However, L- and M-cones are clustered, so there may be an occasional peripheral midget ganglion cell with "a pure cone center." This is even more likely since it has been found that there are rather substantial differences in the L/M cone ratio in the human retina." 3 These few cells and their chromatic signal might be sufficient to account for the residual color sensitivity of the peripheral visual field. Mullen and Kingdom 114 recently measured the peripheral color sensitivity in human observers and compared their results with the model of non-selective wiring using a "Hit and Miss" analysis. They showed that the losses of red-green color sensitivity across the human visual field can be accounted for by a non-selective wiring of the peripheral midget ganglion cells. Cone Selectivity of Diffuse Bipolar Cells There are two possible mechanisms by which diffuse bipolar cells could gain chromatically specific light responses. The first model postulates cone selective contacts. One type of diffuse bipolar cell might avoid L-cones and contact only M-cones; another type might avoid M-cones and contact solely L-cones. There is a precedent for such connectivity in the bipolar cells offish retinae The second model is more sophisticated and proposes that diffuse bipolar cells contact all cones within their dendritic fields, but express specific glutamate receptors: at synapses with L-cones a kainate/ampa receptor and at synapses with M-cones a mglur6 receptor, or vice versa. Again, there is a precedent in the fish retina where specific glutamate receptors on single bipolar cells can subserve separate, functionally defined, synaptic inputs For horizontal cells of the fish retina, it has been found that the transmitter released from S-cones decreases the membrane conductances of Hlhorizontal cells, whereas the synapses made by L- and M-cones are of a classical excitatory type. The question arises whether similar mechanisms are present in the mammalian retina, and more specifically in the primate retina. 7b It is possible to estimate the number of cones contacted by monkey diffuse bipolar cells (Fig. 3C) by observation of the dendritic trees and the cone mosaic in Golgi-stained whole mounts of the retina. 36 Diffuse bipolar cells contacted between 5 and 10 neighboring cones. The L- and M-cones of the monkey retina are present in equal numbers and randomly distributed. 97 The chances that a diffuse bipolar cell contacting 7 cones has a pure L- or M-cone input are less than 1%. Hence an individual diffuse bipolar cell most probably contacts both L- and M-cones. Even if there might be an occasional DB-cell with a pure cone input, because the diffuse bipolar cells converge onto ganglion cell dendrites, it is very likely that such a chromatic signal would be lost. The situation is different in the case of S-cone input. Given the low number of S-cones, most diffuse bipolar cells would miss them. 96 The second model postulating cone-specific expression of glutamate receptors cannot be excluded at present. It is unlikely for the following reason. Electrophysiological recordings from dissociated cone bipolar cells of cat retina 71 and from cone bipolar cells in slices of rat or ground squirrel retinae 72 ' 731 '" 0 did not reveal two opponent glutamate responses within the same bipolar cell (see Fig. 7a). Electron microscopic reconstructions of the cone contacts of diffuse bipolar cells of the primate retina 54 ' 60 ' 61 did not reveal any signs of cone selectivity as postulated by this model. Nor have two types of diffuse bipolar cells, one making flat contacts with L-cones and invaginating contacts with M-cones, or vice versa, been observed. The Functional Role of Diffuse Bipolar Cells Comparable to other mammalian retinae, primate diffuse bipolar cells transfer a luminosity signal to the IPL. Those terminating in the outer half of the IPL are most likely OFF-bipolar cells, whereas those terminating in the inner half of the IPL are ON-bipolar cells. But why are there several types of bipolar cell

12 1324 Boycott and Wassle IOVS, June 1999, Vol. 40, No. 7 to operate this physiological dichotomy? Their precise functional role still has to be elucidated. The most likely explanation is they provide input to different ganglion cell types. Those terminating close to the center of the IPL, such as DB2, DB3, DB4, and DB5, probably provide input to the dendrites of parasol ganglion cells.' l Parasol cell light responses are phasic and have a high contrast sensitivity. 3 ' 4122l23al24b Bipolar cells DB2 and DB5 are the most likely candiates for providing input to OFF- and ON-parasol ganglion cells, respectively, because they have a high coverage factor 36 and make relatively more synapses with cone pedicles than other diffuse bipolar cells. 60 This is consistent with the notion of high contrast sensitivity. Whether they give phasic light responses still has to be shown. Of course, it also is possible that other DB-cells, particularly DB3 and DB4 contribute inputs to parasol cells. In the rat retina it has been shown that bipolar cells 5 and 6 co-stratify with the dendrites of the displaced cholinergic amacrine cells (Fig. 1A) and thus may provide direct and indirect input to directional selective ganglion cells. l23b DB4 and DB5 cells could be the homologous types of the primate retina and also could provide input to directional selective ganglion cells. Dacey and Lee 93a have shown that the "blue ON" ganglion cells have a bistratified dendritic tree, with the outer tier of dendrites close to the amacrine cell layer. There they receive an OFF-input from diffuse bipolar cells. 93bc ' 95 In general, there seems to be a tendency in mammalian retinae for phasic ganglion cells to stratify toward the center of the IPL, whereas tonic ganglion cells have their dendrites further from the center. This holds for cat a (phasic) and j3 (tonic) ganglion cells, 124 " and it also has been found for monkey parasol (phasic) and midget (tonic) ganglion cells. 16 One might predict that the diffuse bipolar cells terminating in the respective strata also have phasic or tonic light responses. A more general idea is that the different bipolar cells are subdivided according to their temporal bandwith. 17 The basis for this could be the expression of different GluR subunits and combinations of subunits at their synapses with the cone pedicles (Figs. 9, 10). It is well known that such receptors can have different sensitivities to glutamate, different desensitization characteristics, or specific modulatory sites. 76a Alternatively, the change in the temporal properties of the bipolar cells might occur in the IPL through different feedback circuits (reciprocal synapses) or through intrinsic differences in the time course of transmitter release (Ca 2+ channels). The number of ganglion cell types in any mammalian retina exceeds the number of bipolar cells types. So how do the ganglion cells get their specific light responses? As shown in Figures 9 and IOC there is a multiplicity of different glutamate receptors postsynaptic at bipolar cell dyads. Let us suppose two different types of ganglion cell receive input from the same type of bipolar cell. One ganglion cell may express an NMDA receptor at its synapses, the other a kainate receptor. Clearly, the physiological signals in the two ganglion cells will differ Thus, parallel processing of the visual image in the retina, which follows the anatomic routes defined by the different bipolar cells, can be multiplexed by the different glutamate receptors expressed in amacrine and ganglion cell dendrites. Evolution of Primate Color Vision Why has trichromacy evolved in primates and not in other mammals? Cone pigments must have mutated from time to time given die perfect adaptation of mammalian cone pigments to their habitats. 18 For a mutation in the cone pigment to be advantageous to an animal die brain must have direct access to diat information. For instance, were the pigment of all cones to shift to a wavelength more appropriate to the animal's habitat, the diffuse bipolar cells might give stronger signals and diis information could be used by die brain. The situation is different, however, if a split of cone pigments into L- and M-pigments comparable to die mutation in primates occurs. The split might result in equal numbers of L- and M-cones randomly distributed across die retina. In mammals, other than primates, bipolar cells pool die signals of several neighboring cones and, in turn, ganglion cells pool die signals from several converging bipolar cells. In such a highly convergent system die chromatic information introduced into the cone mosaic by die L/M mutation would be lost within the retina and dius would never reach the brain. We would argue that die situation 30 million years ago was different in Old World monkeys. During its evolution the primate eye and retina has been optimized for highest spatial resolution. This has required a high cone density and a low cone-to-ganglion cell convergence in die "acuity padiway." The anatomic limits for this optimization are reached when each cone is connected tiirough a midget bipolar cell to a midget ganglion cell. Thus, a "private line" to die brain is established. 37 We suggest that only after this one-to-one connection in die central retina had evolved, did a subsequent mutation in die L-cone pigment create approximately equal numbers of L- and M-cones at random spatial locations ' 126 The midget system of the central retina was able to transmit this chromatic information to die brain where it could be used, for example, to detect red fruit among green leaves Gradually, die selective advantages of trichromatic vision must have lead to a proliferation of color processing pathways in cortical and perhaps even subcortical centers. The wellknown plasticity of the brain and of die subcortical visual pathways 129 could well have allowed such changes. This "midget theory" of the evolution of tricliromacy in Old World monkeys has its basis in die general pattern of mammalian retinal wiring. It is not necessary to postulate, in addition, specific mutations to change the cone selectivity of bipolar cells, the expression of glutamate receptors, or the selectivity of ganglion cells. The idea that trichromacy "piggy-backs" on die high acuity system also postulates that midget ganglion cells perform a "double duty" in visual signaling, an idea that has been promoted for some years. 126, Acknowledgments Over more than 30 years we have been helped by more friends and colleagues than can be listed here. They are mostly to be found as coauthors on our papers. For this review J. H. Brandsditter, M.-H. Chun, T. Euler, E. Fletcher, N. Flores, U. Griinert, J. M. Hopkins, I. Hack, P. Koulen, P. Martin, and D. Protti made important contributions. The authors thank F. Boij for valuable technical assistance and I. Odenthal for typing the manuscript. Authors' Note For convenience, primates is used to mean Old World monkeys, trichromatic New World monkeys, and humans. Other primates are specified as necessary.

13 JOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture 1325 References 1. Stone J. Parallel Processing in the Visual System: The Classification of Retinal Ganglion Cells and Its Impact on the Neurobiology of Vision. New York: Plenum Press; Roclieck RW, Brening RK, Watanabe M. The origin of parallel visual pathways. In: Shapley R, Lam DM-K, eds. Proceedings of the Retinal Research Foundation Symposia, Vol. 5, Contrast Sensitivity: From Receptors to Clinic. Cambridge, MA: MIT Press; 1993: Kaplan E, Lee BB, Shapley RN. New views of primate retinal function. In: Osborne NN, Chader G, eds. Progress in Retinal Research. Oxford: Pergamon Press; 1990: Lee BB. Receptive field structure in the primate retina. Vision Res. 1996;36: Casagrande VA. A third parallel visual pathway to primate area VI. Trends Neurosci. 1994;17:3O5-31O. 6. Hendry SHC, Yoshioka T. A neurochemically distinct third channel in the macaque dorsal lateral geniculate nucleus. Science. 1994;264: a.Martin PR, White AJR, Goodchild AK, et al. Evidence that blue-on cells are part of the third geniculocortical pathway in primates. EurJ Neurosci. 1997;9: b.Martin PR. Colour processing in the primate retina: recent progress. / Physiol. 1998;513: Roclieck RW, Watanabe M. Survey of the morphology' of macaque retinal ganglion cells that project to the pretectum, superior colliculus, and parvicellular laminae of the lateral geniculate nucleus./ Comp Neurol : Pu M, Berson DM, Pan T. Structure and function of retinal ganglion cells innervating the cat's geniculate wing: an in vitro study. / Neurosci. 1994; 14: Stein JJ, Berson DM. On the distribution of gamma cells in the cat retina. Vis Neurosci. 1995;12: Berson DM, Isayama T, Pu M. The eta ganglion cell type of cat retina. J Comp Neurol (in press). 12. Nelson R, Famiglietti EV Jr, Kolb H. Intracellular staining reveals different levels of stratification for on- and off-center ganglion cells in cat retina. /Neurophysiol. 1978;4l: Peichl L, Wassle H. Morphological identification of on- and offcentre brisk transient (Y) cells in the cat retina. Proc R Soc Lond B. 1981;212: Jacoby R, Stafford D, Kouyama N, et al. Synaptic inputs to ON parasol ganglion cells in the primate retina. / Neurosci. 1996; 16: Watanabe M, Rodieck RW. Parasol and midget ganglion cells of the primate retina. / Comp Neurol. 1989;289: Dacey DM. Physiology, moq^hology and spatial densities of identified ganglion cell types in primate retina. In: Higher-Order Processing in the Visual System, Ciba Foundation Symposium 184. Chichester: Wiley; 1994: Sterling P, Smith RG, Rao R, et al. Functional architecture of mammalian outer retina and bipolar cells. In: Djamgoz MBA, Archer SN, Vallerga S, eds. Neurobiology and Clinical Aspects of the Outer Retina. London: Chapman & Hall; 1995: Jacobs GH. The distribution and nature of colour vision among the mammals. Biol Rev. 1993;68:4l Moore RY, Speh JC, Card JP. The retinohypothalamic tract originates from a distinct subset of retinal ganglion cells. / Comp Neurol, 1995;352: Boycott BB, Kolb H. The connections between bipolar cells and photoreceptors in the retina of the domestic at. J Comp Neurol. 1973;148:91-H Dacheux RF, Raviola E. The rod pathway in the rabbit retina: a depolarizing bipolar and amacrine cell. / Neurosci. 1986;6: Greferath U, Griinert U, Wassle H. Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity. / Comp Neurol. 1990;301: Kolb H, Famiglietti EV. Rod and cone pathways in the inner plexiform layer of cat retina. Science. 1974;l86: Wassle H, Yamashita M, Greferath U, et al. The rod bipolar cell of the mammalian retina. Vis Neurosci. 1991;7: Wassle H, Griinert U, Chun M-H, et al. The rod pathway of the macaque monkey retina: identification of AU-amacrine cells with antibodies against calretinin./ Comp Neurol, 1995;36l : Cajal, SR y. La retine des vertebres. La Cellule. 1893;9: Famiglietti EV. Functional architecture of cone bipolar cells in mammalian retina. Vision Res. 1981;21: Mills SL, Massey SC. Morphology of bipolar cells labeled by DAP1 in the rabbit retina./ Comp Neurol. 1992;321:133-l Merighi A, Raviola E, Dacheux RF. Connections of two types of flat cone bipolars in the rabbit retina./ Comp Neurol. 1996;371: Kolb H, Nelson R, Mariani A. Amacrine cells, bipolar cells and ganglion cells of the cat retina: a Golgi study. Vision Res. 1981; 21: McGuire BA, StevensJK, Sterling P. Microcircuitry of bipolar cells in the cat retina. / Neurosci, 1984;4: Cohen ED, Sterling P. Demonstration of cell types among cone bipolar neurons of cat retina. Phil Trans R Soc Lond B. 1990; 330: Cohen ED, Sterling P. Convergence and divergence of cones onto bipolar cells in the central area of the cat retina. Phil Trans R Soc Lond B. 1990;330: Euler T, Wassle H. Immunocytochemical identification of cone bipolar cells in the rat retina./ Comp Neurol. 1995;36l : Szel A, Rohlich P, van Veen T. Short-wave sensitive cones in the rodent retina. Exp Eye Res. 1993;57:5O3-5O Boycott BB, Wa'ssle H. Morphological classification of bipolar cells in the macaque monkey retina. Eur J Neurosci ;3: Polyak SL. We Retina. Chicago, IL: Chicago University Press; Boycott BB, Dowling JE. Organization of the primate retina: light microscopy. Pbilos Trans R Soc Lond B Biol Sci. 196S>;255: Rauen T, Kanner BI. Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae. Neurosci Lett. 1994; 169: Griinert U, Martin PR, Wassle H. Immunocytochemical analysis of bipolar cells in the macaque monkey retina. / Comp Neurol, 1994;348: Milam AH, Dacey DM, Dizhoor AM. Recoverin immunoreactivity in mammalian cone bipolar cells. Vis Neurosci. 1993; 10: Wassle H, Griinert U, Martin PR, et al. Immunocytochemical characterization and spatial distribution of midget bipolar cells in the macaque monkey retina. Vision Res. 1994;34: Kouyama N, Marshak DW. Bipolar cells specific for blue cones in the macaque retina. / Neurosci. 1992;12: Cohen AJ. Some electron microscopic observations on interreceptor contacts in the human and macaque retinae, f Anat, 1965;99: Baylor DA, Fuortes MGF, O'Bryan PM. Receptive fields of single cones in the retina of the turtle. / Physiol ;214: Raviola E, Gilula NV. Gap junctions between photoreceptor cells in the vertebrate retina. Proc Nat/ Acaa'Sci USA. 1973;70:l Tsukamoto Y, Masarachia P, Schein SJ, et al. Gap junctions between the pedicles of macaque foveal cones. Vision Res. 1992; 32: Missotten L. The infrastructure of the Human Retina. Brussels: Arscia Uitgaven; Dowling JE, Boycott BB. Organization of the primate retina: electron microscopy. Proc R Soc Lond B Biol Sci. 1966; 166: Chun M-H, Griinert U, Martin PR, et al. The synaptic complex of cones in the fovea and in the periphery of the macaque monkey retina. Vision Res. 1996;36: Kolb H. Organization of the outer plexiform layer of the primate retina: electron microscopy of Golgi-impregnated cells. Phil Trans R Soc Lond B. 1970;258: Raviola E, Gilula NV. Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. / Cell Biol, 1975;65:

14 1326 Boycott and Wassle IOVS, June 1999, Vol. 40, No Boycott BB, Hopkins JM. Cone bipolar cells and cone synapses in the primate retina. Vis Neurosci. 1991;7: Boycott BB, Hopkins JM. Cone synapses of a flat diffuse cone bipolar cell in the primate retina. J Neurocytoi. 1993;22: a.Kolb H, Boycott BB, DowlingJE. A second type of midget bipolar cell in the primate retina. Appendix. Phil Trans R Soc London B. 1969;255: b. Hopkins JM, Boycott BB. The cone synapses of cone bipolar cells of primate retina. / Neurocytoi. 1997;26: Stell WK, Ishicla AT, Lightfoot DO. Structural basis for on- and off-center responses in retinal bipolar cells. Science. 1977; 198: Famiglietti EVJr, Kaneko A, Tachibana M. Neuronal architecture of on and off pathways to ganglion cells in carp retina. Science. 1977;198: Kolb H, Nelson R. The organization of photoreceptor to bipolar synapses in the outer plexiform layer. In: Djamgoz MBA, Archer SN, Vallerga S, eels. The Outer Retina. London: Chapman & Hall; 1995: Hopkins JM, Boycott BB. Synaptic contacts of a two-cone flat bipolar cell in a primate retina. Vis. Neurosci, 1992;8: Hopkins JM, Boycott BB. Synapses between cones and diffuse bipolar cells of a primate retina. / Neurocytoi. 1995;24: Hopkins JM, Boycott BB. The cone synapses of DB1 diffuse, DB6 diffuse and invaginating midget bipolar cells of a primate retina. J Neurocytoi. 1996;25:391-4O Vardi N, Morigiwa K, Wang T-L, et al. Neurochemistry of the mammalian cone 'synaptic complex.' Vision Res. 1998;38: Calkins DJ, Tsukamoto Y, Sterling P. Foveal cones form basal as well as invaginating junctions with ON-diffuse bipolar cells. Vision Res. 1996;36: Wiissle H. Parallel pathways from the outer to the inner retina in primates. In: Gegenfurtner KR, Sharpe LT, eds. Colour Vision: From Genes to Perception. Cambridge, UK: Cambridge University Press; (in press). 65. Nelson R, Kolb H, Famiglietti EVJr, et al. Neural responses in the rod and cone systems of the cat retina: intracellular records and procion stains. Invest Ophthalmol, 1976;15: Nelson R, Kolb H. Synaptic patterns and response properties of bipolar and ganglion cells in the cat retina. Vision Res. 1983;23: Slaughter MM, Miller RF. 2-Amino-4-phosphonobutyric acid: a new pharmacological tool for retina research. Science ;211: Shiells RA, Falk G, Naghshineh S. Action of glutamate and aspartate analogues on rod horizontal and bipolar cells. Nature. 1981; 294: Attwell D, Mobbs P, Tessier-Lavigne M, et al. Neurotransmitter induced currents in retinal bipolar cells of the axolotl, Ambystoma mexicanum. JPhysiol Lond. 1987;387:125-l6l. 70. Yamashita M, Wiissle H. Responses of rod bipolar cells isolated from the rat retina to the glutamate agonist 2-amino-4-phosphonobutyric acid (APB). / Neurosci ;11: Villa de la P, Kurahashi T, Kaneko A. L-Glutamate-induced responses and cgmp-activatecl channels in three subtypes of retinal bipolar cells dissociated from the cat. / Neurosci. 1995;15: Euler T, Schneider H, Wassle H. Glutamate responses of bipolar cells in a slice preparation of the rat retina. / Neurosci. 1996;l6: a.Hartveit E. Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation. / Neurophysiol. 1996;76: b.Hartveit E. Functional organization of cone bipolar cells in the rat retina. J Neurophysiol. 1997;77:17l cDeVries SH, Schwartz EA. Kainate receptors mediate synaptic transmission between cones and "OFF" bipolar cells in a mammalian retina. Nature. 1999;397:157-l Cohen ED. Interactions of inhibition and excitation in the lightevoked currents of X type retinal ganglion cells. / Neurophysiol. 1998;80: Euler T, Masland RH. Light responses recorded from bipolar cells in rat retinal slices. Soc Neurosci Abstr. 1998;24:S135. Abstract nr a.Hollmann M, Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci. 1994;17: b.Brandstatter JH, Koulen P, Wassle H. Diversity of glutamate receptors in the mammalian retina. Vision Res. 1998;38: Lo W, Molloy R, Hughes TE. Ionotropic glutamate receptors in the retina: moving from molecules to circuits. Vision Res. 1998; 38: Nomura A, Shigemoto R, Nakamura Y, et al. Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell. 1994;77:36l Masu M, Iwakabe H, Tagawa Y, et al. Specific deficit of the ON response in visual transmission by targeted disruption of the mglur6 gene. Cell. 1995;80: Koulen, P, Kuhn R, Wassle H, et al. Group I metabotropic glutamate receptors mglurla and mglur5a: localization in both synaptic layers of the rat retina. J Neurosci. 1997;17: Hughes TE. Are there ionotropic glutamate receptors on the rod bipolar cell of the mouse retina? Vis Neurosci. 1997;l4: Wenzel A, Benke D, Mohler H, et al../v-methyl-d-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells. Neuroscience. 1997;78: Morigiwa K, Vardi N. Differential expression of ionotropic glutamate receptor subunits in the outer retina. / Comp Neurol, 1999;405: Brandstatter JH, Koulen P, Wassle H. Selective synaptic distribution of kainate receptor subunits in the two plexiform layers of the rat retina. / Neurosci. 1997;17: Qin P, Pourcho RG. Synaptic relationships of AMPA-selective glutamate receptor subunits in the cat retina. Vis Neurosci. 1999; 16: Hartveit E, Brandstatter JH, Sassoe-Pognetto M, et al. Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina. / Comp Neurol. 1994;348: Jeon C-J, Strettoi E, Masland RH. The major cell populations of the mouse retina. J Neurosci. 1998; 18: Peichl L, Gonzalez-Soriano J. Morphological types of horizontal cell in rodent retinae: a comparison of rat, mouse, gerbil, and guinea pig. Vis Neurosci. 1994;11: Kolb H, Mariani A, Gallego A. A second type of horizontal cell in the monkey retina. / Comp Neurol. 1980; 189: Boycott BB, Hopkins JM, Sperling HG. Cone connections of the horizontal cells of the rhesus monkey's retina. Proc R Soc Lond Biol. 1987;229: Kolb H. Anatomical pathways for color vision in the human retina. Vis Neurosci. 1991;7:6l Kolb H. The architecture of functional neural circuits in the vertebrate retina. Invest Ophthalmol Vis Sci. 1994;35: a.Dacey DM, Lee BB. The 'blue-on' opponent pathway in primate retina originates from a distinct bistratified ganglion cell type. Nature. 1994;367: b.Calkins DJ, Tsukamoto Y, Sterling P. Microcircuitry and mosaic of a blue-yellow ganglion cell in the primate retina. / Neurosci. 1998;18: c.Ghosh KK, Martin PR, Griinert U. Morphological analysis of the blue cone pathway in the retina of a new world monkey, the Callithrix jacchus. J Comp Neurol. 1997: Dacey DM. Morphology of a small-field bistratified ganglion cell type in the macaque and human retina. Vis Neurosci. 1993;1O: Dacey DM. Circuitry for color coding in the primate retina. Proc NatlAcadSci USA. 1996;93: Calkins DJ. Synaptic organization of cone pathways in the primate retina. In: Gegenfurtner KR, Sharpe LT, eds. Colour Vision: From Genes to Perception. Cambridge, UK: Cambridge University Press; (in press).

15 IOVS, June 1999, Vol. 40, No. 7 The Proctor Lecture Mollon JD, Bowmaker JK. The spatial arrangement of cones in the primate fovea. Nature. 1992;360: Kolb H, DeKorver L Midget ganglion cells of the parafovea of the human retina: a study by electron microscopy and serial-section reconstructions./ Comp Neurol. 1991;303:6l Calkins DJ, Schein SJ, Tsukamoto Y, et al. M and L cones in macaque fovea connect to midget ganglion cells by different numbers of excitatory synapses. Nature. 1994;371: Reid RC, Shapley RM. Spatial structure of cone inputs to receptive fields in primate lateral geniculate nucleus. Nature. 1992; 356: Smith VC, PokornyJ, Lee BB, et al. Responses of ganglion cells of the macaque retina on changing the relative phase of heterochromatically modulated light. / Pbysiol. 1992;458: Kremers J, Lee BB, Yeh T. Receptive field dimensions of primate retinal ganglion cells. In: Drum B, ed. Color Vision Deficiencies XII. Dordrecht: Kluver Academic Publishers; 1995: Wassle H, Boycott BB, Rohrenbeck J. Horizontal cells in the monkey retina: cone connections and dendritic network. Eur J Neurosci. 1989;l: Dacheux RF, Raviola E. Physiology of HI horizontal cells in the primate retina. Proc R Soc Lond B Biol Sci. 1990;239: Dacey DM, Lee BB, Stafford DK, et al. Horizontal cells of the primate retina: cone specificity without spectral opponency. Science. 1996;271: Calkins DJ, Sterling P. Absence of spectrally specific lateral inputs to midget ganglion cells in primate retina. Nature. 1996;381:6l3-6l Paulus W, Kroger-Paulus A. A new concept of retinal colour coding. Vision Res. 1983;23: Lennie P, Haake PW, Williams DR. The design of chromatically opponent receptive fields. In: Landy MS, Movshon JA, eds. Computational Models of Visual Processing. London: MIT Press; 1992: Wassle H, Griinert U, Rohrenbeck J, et al. Retinal ganglion cell density and cortical magnification factor in the primate. Vision Res. 1990;30: H Dacey DM, Petersen MR. Dendritic field size and morphology of midget and parasol ganglion cells of the human retina. Proc Natl AcadSciUSA. 1992;89: Dacey DM. The mosaic of midget ganglion cells in the human retina. / Neurosci. 1993b;13: Croner LJ, Kaplan E. Receptive fields of P and M ganglion cells across the primate retina. Vision Res. 1995;35: Roorda A, Williams DR. The arrangement of the three cone classes in the living human eye. Nature. 1999;397: Mullen KT, Kingdom FAA. Losses in peripheral colour sensitivity predicted from "hit and miss" post-receptoral cone connections. Vision Res. 1996;36: Scholes JH. Colour receptors, and their synaptic connexions, in the retina of a cyprinid fish. Philos Trans R Soc Lond B Biol Sci. 1975;270:6l Ishida AT, Stell WK, Lightfoot DO. Rod and cone inputs to bipolar cells in goldfish retina./ Comp Neurol. 1980;191: Nawy S, Copenhagen DR. Multiple classes of glutamate receptor on depolarizing bipolar cells in retina. Nature. 1987;325: Nawy S, Copenhagen DR. Intracellular cesium separates two glutamate conductances in retinal bipolar cells of goldfish. Vision Res. 1990;30: Calkins DJ, Schein SJ, Sterling P. Cone inputs to three types of non-midget ganglion cell in macaque fovea.[arvo Abstract]. Invest Ophtbalmol Vis Sci. 1995;36(4):S4. Abstract nr Jacoby RA, Marshak DW. Diffuse bipolar cell inputs to parasol ganglion cells in macaque retina. [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1995;36(4):S4. Abstract nr Jacoby RA, Marshak DW. Inputs to parasol ganglion cells in macaque retina. [ARVO Abstract]. Invest Ophtalmol Vis Sci, 1996;37(3):S95O. Abstract nr Lee BB, Martin PR, Valberg A. Nonlinear summation of M- and L-cone inputs to phasic retinal ganglion cells of the macaque. J Neurosci, 1989;9:l433-l a.Lee BB, Pokorny J, Smith VC, et al. Responses to pulses and sinusoids in macaque ganglion cells. Vision Res. 1994;34: b.Brandstatter JH, Greferath U, Euler T, et al. Co-stratification of GABA A receptors with the directionally selective circuitry of the rat retina. Vis Neurosci. 1995;12: a.Wassle H, Boycott BB. Functional architecture of the mammalian retina. Physiol Rev ;71: b.Lee BB, Wehrhahn C, Westheimer G, et al. The spatial precision of macaque ganglion cell responses in relation to vernier acuity of human observers. Vision Res. 1995;35: Mittman S, Taylor WR, Copenhagen DR. Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells. / Physiol, 1990;428: Mollon JD, Jordan G. Eine evolutionare Interpretation des menschlichen Farbensehens. Die Farbe. 1988;35/36: Osorio D, Vorobyev M. Colour vision as an adaptation of frugivory in primates. Proc R Soc Lond B. 1996;263: Regan BC, Vienot F, Charles-Dominique PC, et al. The colour signals that fruits present to primates. [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1996;37(3):S648. Abstract nr Shatz CJ. Emergence of order in visual system development. Proc Natl Acad Sci USA. 1996;93: Ingling CR Jr, Martinez-Uriegas E. The relationship between spectral sensitivity and spatial sensitivity for the primate r-g X-channel. Vision Res. 1983;23: Ingling CRJr, Martinez-Uriegas E. The spatio-chromatic signal of the r-g channel. In: Mollon JD, Sharpe LT, eds. Colour Vision: Physiology and Psychophysics. London: Academic Press; 1983: Lennie P. Recent developments in the physiology of color vision. Trends Neurosci. 1984;7: Merigan WH. Chromatic and achromatic vision of macaques: role of the P pathway. / Neurosci. 1989;9: Negishi K, Kato S, Teranishi T. Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas. Neurosci Lett. 1988;94: