Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Figure 11.1 Three different hypothesized biological mechanisms underlying the local spacing rule that creates the patterning in retinal mosaics. Left, Fate determination events establish a minimal spacing between like-type cells, creating a template for the patterning found in the mature mosaic. Center, Cell death eliminates a

density, relying on the periodic distribution of somata to ensure a uniformity of coverage. In species where dendritic field extent and local cell density are inversely related (e.g., Wässle et al., 1978), it is not obvious which of these strategies is responsible, since a differential expansion of the retina that dilutes peripheral cell density as the eye grows may as well be responsible for a passive, or interstitial, elongation of the dendrites. Although experimental studies in fish and chick retina have attempted to analyze these competing accounts of the regulation of dendritic coverage among various types of retinal ganglion cell classes (Hitchcock and Easter, 1986; Hitchcock, 1989; Troilo et al., 1996), studies on the mouse retina have recently provided compelling evidence for or against particular types of cellular interactions playing a role in this process for a variety of retinal nerve cell classes. They have generally ruled out a third possible explanation, namely, that field extent depends on the afferent or target cells ( Farajian et al., 2004; Lin et al., 2004; Reese et al., 2005).

This chapter focuses on three different types of retinal interneuron to consider the variation in mechanism responsible for the establishment of mosaic patterning and dendritic coverage. The discussion draws on recent studies that have all made use of the unique opportunities afforded by the mouse model, whether it is the natural variation that exists between different inbred strains, the induced variation that selective breeding strategies have allowed, the existence of mutations on those genetic backgrounds, or the genetic engineering of transgenic and knockout mice.

Horizontal cells

There is only one class of horizontal cell in the mouse retina, making it an attractive candidate for study for several

subset of an initially overproduced population of neurons, preferentially killing those in close proximity. Right, Mutual repulsion between neighboring neurons propels them to disperse tangentially until they minimize proximity to like-type neighbors.

reasons (Peichl et al., 1998). First, it is readily labeled with antibodies that do not label other cells at this depth within the retina, enabling the determination of its mosaic properties over large regions of retina (Raven and Reese, 2002). Second, it is an axon-bearing horizontal cell, permitting the ready labeling of dendritic or axonal arbors using tracers applied to the axon itself (Raven, Oh, et al., 2007). Third, it is sparsely distributed across the retina, accounting for less than 0.3% of all retinal neurons (Young, 1985), yet it extends processes to provide a coverage of the retinal surface well in excess of one (Reese et al., 2005). And fourth, the population of retinal horizontal cells exhibits a substantial variation between different strains of mice (Raven et al., 2005b), allowing analysis of the natural variation in mosaic regularity and dendritic coverage as a function of this variation in cellular density while in the absence of any differences in retinal growth. For example, the C57BL/6 and A strains of mice exhibit a nearly twofold difference in total horizontal cell number (C57BL/6J: 18,424 ± 279; A/J: 9835 ± 260, mean ± SE) while not differing in the size of their retinas.

If the patterning present in the horizontal cell mosaic were dependent on a local spacing rule of fixed distance, as has been shown for the photoreceptors in the ground squirrel’s retina (Galli-Resta et al., 1999), different strains of mice might be expected to exhibit strain-specific spacing rules to maintain mosaic regularity at a comparable level between strains (figure 11.2, top). Alternatively, such a spacing rule might not vary between strains, so that variation in density would simply lead to a variation in the regularity of the mosaic, as exhibited in the ground squirrel’s retina (GalliResta et al., 1999), and lower-density strains would show less efficient packing and less regularity than the higher-density strains (see figure 11.2, bottom). In fact, although a compari-

Figure 11.2 Different strains of mice (left and right) might exhibit different fixed spacing rules to achieve the same regularity despite variation in cell density (top). Alternatively, the identical fixed spacing rule may be employed in different strains, yielding lowerdensity strains with mosaics of lesser regularity (bottom).

son of horizontal cell mosaics between four different strains showed significant differences in average intercellular spacing, those strain differences were shown to be a byproduct of the average difference in density; that is, the variation in density, even within individual strains, was shown to be a near perfect predictor of intercellular spacing, indicating the operation of a flexible rather than fixed spacing rule (Raven et al., 2005b). Horizontal cells appear to space themselves apart from one another, independent of genotype. If anything, mosaic regularity increases slightly with the decline in density, contrary to the prediction of a fixed spacing rule operating beneath maximally permissible packing.

How this flexible spacing rule is established during development is unclear. The number of horizontal cells is well below the number of clonal columns derived from single progenitors at the outset of neurogenesis (Williams and Goldowitz, 1992), but because the horizontal cells are one of the earliest generated retinal neurons (Sidman, 1961; Blanks and Bok, 1977; Hinds and Hinds, 1979), a lateral inhibitory mechanism could be envisioned to produce a local periodic distribution with a density that is subsequently diluted by clonal expansion (Reese et al., 1999). There is, however, no evidence bearing directly on the early patterning or spacing achieved immediately following the determination of the horizontal cell fate.

With respect to naturally occurring cell death, the horizontal cells stand out as being the best retinal exception to what had been regarded as a general rule, that nerve cell types are overproduced (Linden and Reese, 2006). There is no published evidence that horizontal cells undergo naturally oc-

curring cell death (Young, 1984; Mayordomo, 2001), and no evidence that horizontal cell numbers are overproduced during normal development, at least postnatally (Raven et al., 2005a). Transgenic mice that overexpress the antiapoptotic gene Bcl-2 have, however, been shown to exhibit 20% more horizontal cells, raising the possibility that there is some prenatal cell death that could in principle improve mosaic regularity from an initially less orderly mosaic. There is as well some indication that the neurotrophin nerve growth factor (NGF) signals through the tyrosine kinase A (trkA) receptor in the chick retina to maintain horizontal cell numbers during development (Karlsson et al., 1998).

Following birth, when all but the most peripherally situated horizontal cells have migrated to the future outer plexiform layer, after which no detectable horizontal cell loss occurs, in both the densest and sparsest populated retinas of these various strains, the regularity and packing efficiency of the horizontal cell mosaic increase significantly until postnatal day 5. Thereafter these indices of mosaic patterning remain largely unchanged into maturity (Raven et al., 2005a; see also Scheibe et al., 1995). During the period between birth and postnatal day 5, horizontal cells disperse tangentially for short distances within the retina, as evidenced through the use of X-inactivation transgenic mosaic mice to reveal clonal boundaries (Reese et al., 1999). Unlike with other means of identifying clonally related cells, in X-inac- tivation mosaic mice a known proportion of progenitor cells is labeled, permitting direct calculation of the proportion of dispersing cells among any cell class (Reese and Tan, 1998). For the horizontal cells, virtually all must engage in this tangential dispersion (Reese et al., 1999). Because this dispersion can only change the positioning of horizontal cell neuroblasts relative to their sites of birth and determination, and relative to any spatial relationships that were established by hypothesized cell death occurring prenatally, this dispersion must change the spatial patterning established by those other mechanisms. Since its occurrence coincides with an increase in regularity and packing, a relationship between the two would be implicated: tangential dispersion increases the patterning already present at birth (Raven et al., 2005a).

The difference in mosaic regularity or packing between the mosaic of horizontal cells on the day of birth and random (density-matched and soma-constrained) distributions is still significant, indicating that something else contributes to the patterning before birth. It could be that some degree of naturally occurring cell death leads to the patterning present at birth; it may be that fate determination events set a coarse grain to the mosaic that is subsequently refined by tangential dispersion; or it may be that tangential dispersion of horizontal cells also occurs between the time of fate determination and postnatal day 1, when the dispersing horizontal cells may be situated within the amacrine cell layer, masquerading as dispersed amacrine cells that are already present on

the day of birth, before they translocate back to the future horizontal cell layer (Reese et al., 1999; Edqvist and Hallbook, 2004).

The final patterning of the horizontal cell mosaic is also independent of the afferents: transgenic mice carrying an attenuated diphtheria toxin gene under the control of a human L cone regulatory sequence lose nearly all of their cones prior to synaptogenesis with the horizontal cells (Soucy et al., 1998). Despite this loss of cone afferents, mosaic regularity shows no change relative to that seen in control littermates (Raven and Reese, 2003). These results are consistent with the finding that the patterning within retinal mosaics is largely independent of the patterning present in other mosaics, regardless of whether those mosaics are synaptic partners (Rockhill et al., 2000).

Like their intercellular spacing, the dendritic outgrowth of horizontal cells is related to local horizontal cell density (figure 11.3); higher-density strains have smaller dendritic fields than do lower-density strains, and the degree of dendritic growth appears to be modulated precisely to maintain dendritic overlap as a constant, being about six (Reese et al., 2005). This would suggest that the extent of dendritic growth is controlled by homotypic interactions between neighboring horizontal cells rather than by any cell-intrinsic program determining dendritic field size. Of course, each strain could have allelic variants of genes that drive dendritic growth. To discriminate between these possibilities, a Lim1 mosaic-con- ditional knockout mouse has been used to modulate the density of horizontal cells. Lim1 is a transcription factor that is expressed exclusively within horizontal cells during development and plays a critical role in specifying their migratory

Figure 11.3 Horizontal cell spacing and dendritic field growth are each regulated by local horizontal cell density. Horizontal cells in the mouse retina are positioned to minimize proximity with neighboring horizontal cells. Mouse strains with lower average densities of horizontal cells (e.g., the A strain, right) extend their dendritic fields farther than those with higher average densities (e.g., C57BL/6, left) to maintain a constant dendritic coverage of roughly six cells.

endpoint: horizontal cells lacking Lim1 become mispositioned in the amacrine cell layer and stratify in the inner plexiform layer (Poché et al., 2007). Those remaining Lim1positive horizontal cells, settling in their normal stratum and arborizing normally within the outer plexiform layer, show a nearly twofold increase in dendritic field area when the local density in this stratum is reduced by about 50% (Poché et al., in press), demonstrating that their field size is clearly dependent on homotypic interactions. This effect on the overall size of the dendritic field is to be contrasted with the patterning of the dendritic field, which is controlled primarily by afferent rather than homotypic interactions (Raven, Oh, et al., 2007).

Cholinergic amacrine cells

The cholinergic amacrine cells in the mouse retina are distributed to two strata within the ganglion cell and inner nuclear layers, extending their characteristic starburst dendritic arbors into an inner and an outer stratum within the inner plexiform layer, as in other mammalian retinas. The two populations are spatially independent of one another (Galli-Resta et al., 1997, 2002), as in all other species examined (Diggle, 1986; Rockhill et al., 2000). They have densities comparable to the population of horizontal cells, but unlike the horizontal cells, they extend their dendritic arbors far more extensively, yielding a dendritic overlap of about 30 (Farajian et al., 2004).

The mosaics of cholinergic amacrine cells, when compared with the horizontal cells (Raven et al., 2005b), are never as regularly distributed yet are still more regular than simulated random distributions (Whitney et al., 2008). Like the horizontal cell mosaics, the cholinergic mosaics show a decline in average intercellular spacing as density increases, although this relationship is perturbed for the mosaic in the ganglion cell layer (Whitney et al., 2008). Tangential dispersion of cholinergic amacrine cells is, as for the horizontal cells, nearly universal (Reese et al., 1999), and the intercellular spacing of cholinergic amacrine cells may be constrained solely by soma diameter prior to these cells settling within their mosaic layers, at least in the developing rat’s retina (Galli-Resta et al., 1997). Although the presence of such immediate near-neighbor pairings during radial migration suggests these cells must move apart from one another as they arrive in the mosaic layer, this population of cells is also modulated by naturally occurring cell death (Galli-Resta and Novelli, 2000), with roughly 20% being overproduced. This naturally occurring cell death can be prevented by blocking ATP signaling via P2X receptors, and these denser mosaics contain more frequent near-neighbor pairings, but their regularity and packing have not yet been adequately described (Resta et al., 2005). Yet a comparison of mosaic regularity before and after naturally occurring cell

death does not reveal any increase in regularity for this cell type at the latter time point, suggesting that cell death does not appreciably modulate the patterning associated with this population (Galli-Resta and Novelli, 2000). The presence of such closer near-neighbor pairings during the radial migratory phase of cholinergic amacrine cells (Galli-Resta et al., 1997) also suggests that the spacing present within the mosaic layers is not a passive consequence of a spacing established at the time of fate determination. But their presence is by no means an unambiguous demonstration that the patterning present at the time of fate determination or during migration is entirely random. For this cell class, there is currently no good estimate of the extent to which fate determination events contribute to the patterning achieved in maturity.

There is, however, additional evidence that neighboring cholinergic amacrine cells can modulate their intercellular spacing conspicuously during early postnatal development. Disruption of microtubule stability within the processes of cholinergic (and also horizontal) cells immediately after birth leads to a dramatic, and transient, redistribution of their somata within the mosaic layers (Galli-Resta et al., 2002). These results, in conjunction with the independent evidence for naturally occurring tangential dispersion, support the notion that neighboring cholinergic amacrine cells space themselves apart within the plane of the mosaic, mediated by some form of homotypic interaction via the dendrites. The interaction is unrelated to synaptic associations with retinal ganglion cells, since experimental or genetic manipulations that eliminate or double the latter population have no effect on the patterning of cholinergic cells (Galli-Resta, 2000). One result inconsistent with that former study is the finding that excitotoxic ablation of roughly 40% of the cholinergic amacrine cells on postnatal day 3 has no effect on the positioning of remaining cholinergic amacrine cells (Farajian et al., 2004), although it remains a possibility that this age is too late to alter the cell-cell interactions governing relative positioning.

The processes of cholinergic amacrine cells overlap one another extensively, generating dendritic coverage factors that range from 20 to 70, depending on the species. That same latter study demonstrated that the excitotoxic ablation of 40% of the cholinergic amacrine cells did not appear to harm those cells that survived into maturity; those cells went on to differentiate a normal starburst morphology, growing their dendritic fields sevenfold thereafter, well in excess of the extent of overall retinal growth. Interestingly, the fields achieved by those surviving cholinergic amacrine cells were virtually indistinguishable from those in the control condition, including their overall dendritic field area (Farajian et al., 2004). That is, cholinergic amacrine cells would appear to grow their dendrites to establish a dendritic field extent that is independent of the density of either the cholinergic somata or their processes (figure 11.4). These conclusions are

Figure 11.4 Cholinergic amacrine cells extend their dendritic fields to achieve a degree of dendritic overlap on the order of 30 (left). Early partial depletion of the mosaic, before appreciable dendritic growth has taken place, does not alter the subsequent dendritic growth of remaining cells (right).

supported by other results comparing the density and field area of cholinergic amacrine cells in the C57BL/6 and A strains of mice: despite there being 25% fewer cholinergic amacrine cells in the ganglion cell layer in the A strain, dendritic field size is no different (Keeley et al., 2008). The cholinergic amacrine cells would appear to operate a cellintrinsic growth strategy, defining a field of a particular size rather than titrating growth precisely to yield a constant degree of dendritic overlap.

Similar conclusions have recently been drawn for the dendritic growth of two types of retinal ganglion cells using Brn3b and Math5 knockout mice, cells that normally establish a degree of dendritic coverage closer to one but which do not appear to modulate their dendritic growth in the absence of normal neighbor relationships (Lin et al., 2004). That result would appear to contradict the evidence that experimental manipulations altering neighbor relations in turn alter dendritic growth among ganglion cells (Perry and Linden, 1982; Eysel et al., 1985; Kirby and Chalupa, 1986; Leventhal et al., 1988). Although this discrepancy may simply indicate an intrinsic upper limit on the size of the dendritic field that can still be biased or restricted by neighbor relationships, it may alternatively indicate that different ganglion cell classes define their dendritic growth via intrinsic constraints versus extrinsic cellular interactions.

Dopaminergic amacrine cells

The dopaminergic amacrine cells are one of the sparsest classes of neuron found in the retina, accounting for less than one-hundredth of a percent of the total complement of retinal neurons (Versaux-Botteri et al., 1984). In the mouse retina, they are all situated in the inner nuclear layer immediately adjacent to the inner plexiform layer, unlike in some

other species, where a sizable proportion is positioned in the ganglion cell layer (Peichl, 1991; Eglen et al., 2003b). Remarkably, their minuscule number is tightly regulated, so that there is minimal variation in the total number present in mice from the same strain (Masland et al., 1993), yet different strains of mice reveal a nearly fourfold variation in the size of this population (Raven, Whitney, et al., 2007). For example, the A strain contains 276 ± 9.5 dopaminergic amacrine cells, whereas the ALR strain contains 962 ± 11.9 cells. Indeed, an analysis of 25 recombinant inbred strains of mice (the AxB/BxA strain set) derived from the A and C57BL/6 strains (strains showing a twofold difference in dopaminergic amacrine cell number) indicates a putative locus for the control of dopaminergic amacrine cell number on chromosome 7 (Raven, Whitney, et al., 2007). These cells give rise to sparse and frequently asymmetric dendritic fields, plus extensive if also sparse axonal processes that may spread for millimeters within the plexiform layers, primarily within the substratum of the inner plexiform layer immediately adjacent to the inner nuclear layer (Versaux-Botteri et al., 1984; Oyster et al., 1985; Savy et al., 1989; Dacey, 1990; Zhang et al., 2004).

The somal patterning of the dopaminergic amacrine cells is close to that expected for a random distribution in nearly all species examined. The issue has been most thoroughly examined in the retina of the mouse, where the distribution of somata is found to be nonrandom using a variety of indices, although not much more regular than random (Raven et al., 2003). Modeling studies showed that a fixed spacing rule was effective at simulating a limited sample of four different fields of dopaminergic amacrine cells, although multiple spacing rules were found to be effective for some of those same sampled fields. In short, the distribution of dopaminergic amacrine cells loosely abides by a rule that reduces the frequency of pairings closer than 100 μm relative to what would be expected for a random distribution of cells at comparable density. Although such a spacing rule is large relative to those observed for most other cell types, this distance is still meager relative to the density of dopaminergic amacrine cells, the latter being far below the maximal packing limit for spheres of this size (Raven et al., 2003). The low-density mosaics generated by such a fixed spacing rule are, consequently, hardly regular.

The mechanism underlying this spacing rule is unlikely to be related to tangential dispersion, since minimal numbers of dopaminergic amacrine cells exhibit any dispersion from their clonal columns (Reese et al., 1999), rarely greater than a distance comparable to soma diameter (Raven et al., 2003). In the fish retina, the patterning of the dopaminergic amacrine cells is readily simulated by a lateral inhibitory fate determination model (Cameron and Carney, 2004), and additional observations in the fish retina are inconsistent with either dispersion or cell death accounts (Tyler

et al., 2005). In the mouse, however, the Bcl-2-overexpress- ing transgenic retina has been particularly informative, for this cell type shows a nearly 10-fold increase in total number (Strettoi and Volpini, 2002), where their distribution is indiscriminable from random simulations matched in density and constrained by soma size (Raven et al., 2003). Unlike the wild-type (C57BL/6) retina, where side-by-side dopaminergic amacrine cell pairings are never detected, their frequency in the Bcl-2-overexpressing retina is exactly to be predicted from random distributions matched for density.

The transgene is driven by a neuron-specific enolase promoter that should ensure that the construct is expressed only after neuroblasts have undergone their final mitosis and begun differentiating. Accordingly, these results have been interpreted to indicate a role for this antiapoptotic gene in regulating dopaminergic amacrine cell survival (Strettoi and Volpini, 2002), although there exists no independent evidence that dopaminergic amacrine cells undergo naturally occurring cell death (Linden and Reese, 2006). These results are, however, consistent with other studies showing a role for BDNF-trkB receptor signaling in the control of dopaminergic amacrine cell number (Cellerino et al., 1998). These results, then, suggest that dopaminergic amacrine cells are normally overproduced and that the determinants of survival or death are at least partially influenced by proximity to other dopaminergic amacrine cells, perhaps through a competition for trophic factors provided by targets or other neighboring cells. Of course, if proximity were the sole rule for reducing this population by approximately 90%, far more regular mosaics could be sculpted from random distributions of cells with this degree of cell loss (Eglen and Willshaw, 2002). Clearly, other factors must play a role in the decision to survive or die within this population of cells.

The results from these Bcl-2-overexpressing mice would imply that there are no constraints on the production of dopaminergic amacrine cells residing immediately next to one another. Unless lateral inhibitory or other fatedetermining mechanisms are somehow altered in these transgenic retinas, these results are perhaps the strongest available for ruling out any role for fate-determining events in creating the minimal distance spacing rule responsible for the patterning present in the dopaminergic amacrine cell mosaic, modest as that patterning may be for this cell type.

Little is known about the control of process outgrowth for the dopaminergic amacrine cells. In the rat retina, the dendritic fields are conspicuously asymmetric, and there may be a tendency for closer neighboring cells to extend their dendrites away from one another (Savy et al., 1989), suggesting that their growth is controlled by homotypic interactions. Given the nearly fourfold variation in dopaminergic amacrine cell number across different strains of mouse (Raven,

Whitney, et al., 2007), it would be interesting to see whether dendritic field growth scaled accordingly, as it does for the horizontal cells. But because this cell type plays a neuromodulatory role rather than a role in image-forming processes, one might predict no change in field size between the strains but rather a modulation in the release or reception of this neurotransmitter within the retina.

Outstanding issues

The studies just reviewed have implicated the differentiating processes as being the means by which horizontal and cholinergic amacrine cells may space themselves out during development, but exactly how they do so has yet to be determined. Do these cells define local domains as they first begin differentiating, when they have barely an overlap of one, shifting their somal position toward the center of this domain (Cook and Chalupa, 2000)? Might these cells establish contacts with homotypic neighbors that mediate tensile forces across the network, leading to uniform spacing (Galli-Resta, 2002)? Or are the somata truly repelled by the presence of close neighbors, engaging in active cellular migration (Reese et al., 1995)? Furthermore, what is the relationship between the interactions governing positioning and those governing constraints on dendritic overlap, for the horizontal cells? A simple contact inhibition at the tips of growing dendrites would be sufficient for establishing a dendritic coverage of one (and may be all that is required to trigger tangential dispersion), but mouse horizontal cells ultimately generate a coverage of around six. This degree of coverage approximates dendritic growth extending to neighboring somata, suggesting that the processes of each horizontal cell are inhibited from further growth on contacting those cell bodies. Given these differences in generating coverages of one versus six, one can envision symmetric versus asymmetric interactions that might mediate such contact-mediated events, but these arenas are largely unexplored for the moment (but see Tanabe et al., 2006).

The dopaminergic amacrine cell mosaic is itself puzzling, for even if it is sculpted from an initially denser mosaic, it is difficult to envision how fate determination mechanisms initially generate such a random distribution of overproduced neurons at such low density. Even more remarkable is the consistency with which developmental mechanisms yield a final number that is so reliably established among members of the same strain. Although dopaminergic amacrine cell number may depend on trophic relationships governed by the size of other populations, including the retinal ganglion cells, for which there is good evidence for allelic variants modulating neuronal number (Williams et al., 1998), it remains puzzling how such a dependency could produce precision in cellular number in the absence of any precision in cellular patterning.

Conclusion

Much of our understanding of the organizing principles of retinal mosaics had been established in other mammalian species long before the mouse came into vogue as a model for studying the retina and visual system. The use of the mouse as a model system has benefited our understanding of the development of this mosaic architecture, its coverage, and its connectivity, although so far implementation of the mouse model has largely clarified the role of cellular interactions in these processes rather than any details of the molecular mechanisms governing those interactions. Researchers may be well positioned to move in this direction as increasing numbers of genetically modified mouse resources become available. Other species traditionally associated with the field of experimental embryology (e.g., chick, frog, fish), permitting as they do the ready transfection or transplantation of progenitors to alter gene expression in known lineages of cells in order to dissect the cell-autonomous and environmental signaling controlling these interactions, have been the models of choice for addressing events that occur in utero in the mouse retina, but novel genetically engineered mice and experimental approaches are quickly overcoming such limitations (e.g., Badea et al., 2003; Matsuda and Cepko, 2007). To the extent that the processes involved in building the mosaic architecture of the retina prove to be related to more general processes associated with retinal growth, which in mammals does not proceed by an annular accumulation of cells characteristic of other vertebrates, the use of those other species may have only limited potential for understanding these systems features of the mammalian retina, further driving the application of new approaches for genetic manipulation within the developing mouse retina.

acknowledgment Supported by grant no. EY-11087 from the National Institutes of Health.

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ENRICA STRETTOI

The fundamental plan of the mouse retina follows the blueprint common to all mammalians: rods largely predominate, as they represent 97% of the photoreceptor population (Carter-Dawson and LaVail, 1979; Jeon et al., 1998). In the common C57Bl6/J strain of mouse, there are approximately 6.4 million rods and 180,000 cones (figure 12.1). The first are presynaptic to a single type of bipolar cell, the rod bipolar cell; each cone, instead, is connected to a cohort of different cone bipolar cells, all together forming parallel, vertical channels across the retina and variably connecting to ganglion cells. Additional, mixed rod-cone pathways also exist. The signal is modulated in the outer retina by a single type of horizontal cell and, in the inner retina, by more than 20 different types of amacrine cells that are reciprocally connected to bipolar cells and are presynaptic to ganglion cell dendrites. Ganglion cells also occur in a variety of types and with different functional features.

The work of many laboratories and the use of state-of-the art neuroanatomical techniques have led to recognition of the cellular complexity of the retina, and the mouse visual system has been the object of many studies in recent years (because of the use of this species for transgenic and knockout experiments). Nevertheless, many functional issues remain unresolved. Yet the concept of a retinal fundamental circuit, constituted by a discrete number of neuronal types (about 50) and repeated across the retinal surface, with no single cell type playing a dominant role, is applicable to the retina of all mammals, including the mouse (Masland, 2001).

A quantitative analysis of the mouse inner nuclear layer (INL) demonstrated that its cells have a dome-shaped distribution, slightly more peaked than that of the photoreceptors, with a maximum density around 300 μm from the optic nerve head. The topography of cells in the ganglion cell layer is more peaked still, with a density relatively higher in the nasal quadrant of the retina ( Jeon et al., 1998). All three distributions in the mouse, though, are flatter than those found in rat, rabbit, cat, or monkey (Hughes, 1975; Mitrofanis et al., 1988; Martin and Grünert, 1992; Strettoi and Masland, 1995); thus, the mouse retina is more homogeneous across its surface than is the retina of other mammals.

The relative fractions of various cell types in the INL is known: bipolar cells make up 41% of all cells in the layer, amacrines 39%, Müller cells 16%, and horizontal cells 3% (Jeon et al., 1998). Thus, although it is generally assumed that the retinas of lower mammals are more rich in amacrine cells, these numbers demonstrate that the bipolar-to- amacrine ratio is close to one in the retina of the mouse, exactly like that of the rabbit and very close to that of the monkey. Actually, the retina of higher mammals, such as macaque monkeys and humans, is made more complex by the presence of the fovea, with a dedicated circuit of midget neurons that is completely absent from the rodent retina. However, the fovea represents only 1% of all the retinal surface; hence, the fundamental plan of the retina is highly conserved among mammals.

The relative proportion of rod versus cone bipolar cells in the mouse retina has also been estimated. In the C57Bl6/J mouse, rod bipolar cells number approximately 208,000, while cone bipolar cells are twice as numerous (Strettoi and Volpini, 2002). This confirms another general rule of retinal architecture, in that even in a strongly rod-dominated retina (and rodents are among the mammals with the highest rod : cone ratio), cone bipolar cells outnumber rod bipolar cells (Strettoi and Masland, 1995). This is partly due to the fact that each cone diverges on several cone bipolar cells of different types, whereas multiple rods converge on individual rod bipolars, thus increasing the sensitivity of the scotopic pathway.

With the main features of the mouse retina now summarized, this chapter briefly reviews the architecture of the rod pathways and that of cone pathways to ganglion cells.

The rod pathway

Mice have classic rod bipolar cells. These well-known neurons have ovoid cell bodies (around 10 μm in diameter), usually located in the outer half of the INL, and bushy dendritic arborizations. The axons are thin and straight, while the axon terminals are large, each composed of three to five bulblike varicosities that reach the deepest part of sublamina b of the inner plexiform layer (IPL). Typically, rod bipolar cells can be stained with antibodies against the alpha isoform