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

Retinal waves and the development of visual circuits

Retinal waves exist during a period of development when several different visual circuits are being established (for reviews, see Wong, 1999; Torborg and Feller, 2005). RGCs project to two primary targets in the brain, the SC and the dorsal lateral geniculate nucleus of the thalamus (dLGN). In these targets, RGCs respectively establish an arrangement of connections in target fields, termed a retinotopic map, that reflects the spatial arrangement of the RGCs in the retina, and eye-specific maps with inputs from the two retinas layering in neighboring but nonoverlapping regions. The precise retinotopic and eye-specific targeting of RGC axons observed in the adult mouse emerges prior to visual experience from initially diffuse and overlapping projections. In addition, during the first postnatal week, neurons in the dLGN are forming connections within the visual cortex, where they are also organized in precise retinotopic maps (Cang et al., 2005). In mice, there are no ocular dominance columns, although there is a distinct region of visual cortex where neurons receive inputs strongly driven by one eye or the other.

There is a clear role for both neural activity and molecular factors, such as the ephrins and their corresponding Eph receptors, in the establishment of these maps, though the relative importance of the two throughout the process of axon targeting and refinement is the subject of ongoing research (reviewed in O’Leary and McLaughlin, 2005). Controversy remains as to whether neural activity is instructive or permissive during these developmental events. If retinal waves play an instructive role in retinotopic map refinement, then retinotopic information must be contained within the spontaneous firing pattern (Crair et al., 2001; Eglen et al., 2003), and this information must be used to guide axonal refinement. Retinotopic information is in fact contained in the correlation structure of retinal waves: their propagating nature ensures that cells that are closer together are more temporally correlated in their firing than cells that are farther apart (Meister et al., 1991; Wong, 1993; Eglen et al., 2003). Alternatively, if retinal waves play a permissive role in retinotopic refinement, then the spontaneous firing pattern creates an environment in which molecular cues that contain retinotopic information, such as ephrins/Eph, can function. For example, in developing spinal cord, the periodicity of rhythmic activity affects the expression of axon guidance proteins (Hanson and Landmesser, 2004, 2006). Retinal waves periodically activate the cAMP/PKA pathway (Dunn et al., 2006), which may be critical for modulating protein function in individual RGCs.

Mice lacking β2-nAChRs have become a model system for establishing an instructive role for retinal waves in

visual system development. β2-nAChR−/− mice do not have stage II retinal waves (Bansal et al., 2000). MEA recordings reveal that individual β2 RGCs fire bursts of action potentials, but these bursts are not correlated with the firing of neighboring cells (McLaughlin et al., 2003) (figure 28.3A). Indeed, the average firing rate of individual RGCs is the same in wild-type and β2 mice, but both the temporal structure within bursts and the nearest-neighbor correlations are significantly reduced. β2 mice have poorly refined retinotopic maps in their retinocollicular (Grubb et al., 2003; McLaughlin et al., 2003; Chandrasekaran et al., 2005; Pfeiffenberger et al., 2006) (figure 28.3B) and thalamocortical projections (Cang et al., 2005), as well as altered eye-specific layers in the dLGN (Rossi et al., 2001; Muir-Robinson et al., 2002; Pfeiffenberger et al., 2005), indicating that the correlated firing patterns induced by retinal waves is indeed important for the establishment of visual maps.

One caveat to the findings that visual maps are altered in β2 mice is that β2 mice are global knockouts, meaning β2 is lacking in the target tissue as well as in the retina (Moretti et al., 2004; Gotti et al., 2005). However, the observed phenotypes are likely to be due to the effects of β2 on retinal waves, since (1) they can be reproduced by intraocular injections of nAChR antagonists (Cang et al., 2005; Chandrasekaran et al., 2005; Pfeiffenberger et al., 2005) and (2) the defects are constrained to the time that retinal waves are altered. If the altered visual maps observed in β2 mice were due to, say, a defect in β2-mediated plasticity, this deficit should persist throughout the life of the mouse. However, β2 mice have mostly normal stage III retinal waves, and visual responses are quite robust. These later patterns of activity drive refinement in both the SC and dLGN (MuirRobinson et al., 2002; Grubb et al., 2003; Chandrasekaran et al., 2005).

Not all manipulations that alter spontaneous retinal firing patterns alter visual map formation. For example, Cx36−/− mice, which have extra action potentials between retinal waves, have normal eye-specific layers (Torborg et al., 2005). In addition, pharmacological manipulations in ferrets that alter different features of the spontaneous firing patterns do not prevent normal eye-specific segregation (Stellwagen and Shatz, 2002; Huberman et al., 2003). Hence, the nature of the instructive signals provided by retinal waves is still controversial. An understanding of the cellular basis of the different features of spontaneous activity patterns, and therefore the ability to precisely manipulate activity patterns, will be critical for resolving this issue. Future studies in which targeted manipulations alter specific features of spontaneous activity patterns should help determine what features of retinal waves drive the normal development of visual circuits.

Figure 28.3 Knockout mice lacking the β2 subunit of nAChR have disrupted retinal firing patterns and altered retinotopic projections. A, β2−/− retinas have altered firing patterns during the first postnatal week. Spike trains were recorded with a multielectrode array (MEA) from four representative cells in a P4 wild-type retina and eight representative cells in a P4 β2−/− retina. Hexagons to the left of each spike train show the position of the electrode on which that unit was recorded (black circle) relative to the other represented units (gray circles). The maximum extent of the array is 480 μm. B, β2−/− mice have defective topographic remodeling of the retinocollicular projection. Fluorescence images of DiI-labeled

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Parallel processing of neuronal signals is a fundamental feature of most if not all sensory systems in vertebrates. In the visual system, the most extensively studied example is the parallel processing of increments and decrements in luminance of visual stimulation by two separate synaptic pathways, the ON and OFF pathways. The separation of these two synaptic pathways starts at the first synapse between photoreceptors and bipolar cells in the outer retina. They remain separated to a large extent in the inner retina, the lateral geniculate nucleus (LGN), and the visual cortex. The separation of ON and OFF signals forms the basis of virtually all visual signal processing in higher centers of visual system.

Although much has been learned about the structures and function of the synaptic pathways in mature retinas, considerably less is known about how retinal synaptic pathways are formed during development and what regulatory mechanisms may guide maturation of the retinal synaptic pathways. It was earlier assumed that retinal synaptic circuitry matures early in development. It is indeed the case that many aspects of synaptic signaling in the retina reach maturity before the retina begins receiving visual stimulation. For example, by the time of eye opening in rodents, rabbits, cats, and ferrets, most of the morphological features of the retina and the expression of synthesizing enzymes, transporters, and receptors for neurotransmitters of retinal neurons resemble those in adult animals (Fisher 1979b; Greiner and Weidman, 1981; Redburn and Madtes, 1987; SassoePognetto and Wässle, 1997; Pow and Barnett, 2000; Johnson et al., 2003). However, recent studies have demonstrated that connectivity between neurons, synaptic structures and functions, and the neuronal processing of mammalian retina can be modified and refined before and after eye opening during postnatal development.

In this chapter I discuss the development of ON and OFF pathways in the retina and the possible roles of visual stimulation in the maturation of these synaptic pathways. I first highlight the basic cellular/synaptic structure and the developmental processes of ON and OFF pathways, focusing on

the developmental segregation of retinal ganglion cell (RGC) dendrites into ON and OFF pathways. I then review the modifications of maturation of ON and OFF pathways induced by alteration of synaptic activity during postnatal development of the mouse retina. Finally, I discuss the possible mechanisms regulating the developmental segregation of ON and OFF pathways in mouse retina. Although some of the evidence discussed in this chapter is from other mammalian species, the major conclusions derived from those data appear to apply well to mouse retina. This is reflected in an excellent review that addressed the similar developmental processes of ON and OFF synaptic pathways primarily based on the observations from cats and ferrets (Chalupa and Günhan, 2004).

Cellular and synaptic structure of retinal

ON and OFF pathways

The detailed organization of the mouse retinal synaptic pathways is described in other chapters; here I highlight only the cellular and synaptic structure of the ON and OFF pathways. The separation of ON and OFF pathways originates at the first synapse between photoreceptors (rods and cones) and bipolar cells in the outer retina (figure 29.1). In all vertebrate retinas, light stimulation hyperpolarizes the membrane potentials of photoreceptors and decreases the synaptic release of glutamate from these cells. Glutamate released from photoreceptors activates an ionotropic glutamate receptor on cone OFF bipolar cells and depolarizes their membrane potentials. On cone ON bipolar cells and on all rod bipolar cells, glutamate activates a metabotropic glutamate receptor and hyperpolarizes the membrane potentials of these cells (see chapters 12 and 14, this volume, for details). This sign-reversing and nonreversing action of glutamate on the ON and OFF bipolar cells separates the increment and decrement luminance signals into ON and OFF pathways.

Different from ON and OFF bipolar cells, which have different types of glutamate receptors at their postsynaptic

Figure 29.1 Schematic drawing of the principal anatomical components, synaptic connections, and representative light responses of ON and OFF pathways of mammalian retina. Photoreceptors (rods and cones) synapse with bipolar and horizontal cells in the OPL. ON and OFF signals are generated in the OPL by the activation of metabotropic and ionotropic glutamate receptors on ON and OFF bipolar cells, respectively. For the cone bipolar cells, whereas all ON bipolar cells synapse with RGCs in sublamina b, all OFF bipolar cells make synapses with RGCs in sublamina a of IPL. A subpopulation of RGCs receives synaptic inputs from both

sites to transmit visual signals, all RGCs use ionotropic glutamate receptors at their synapses as their primary synaptic receptors to conduct glutamatergic synaptic inputs from bipolar cells. The separation of ON and OFF pathways at the level of synaptic inputs to RGCs relies on the RGC dendritic distribution and selective synaptic connections with ON and OFF bipolar cells in distinct sublaminae of the inner plexus layer (IPL). There are more than a dozen morphologically distinctive subtypes of RGCs in adult mouse retina (Rockhill et al., 2002; Sun et al., 2002; Diao et al., 2004; Kong et al., 2005; Coombs et al., 2006). Despite the enormous diversity in structural and functional properties among different subtypes of RGCs, all ON RGCs ramify their dendrites only in sublamina b (ON layer) of the IPL and synapse with cone ON bipolar cells. In contrast, all OFF RGCs ramify their dendrites only in sublamina a (OFF layer) of the IPL and synapse with cone OFF bipolar cells (Famiglietti and Kolb, 1976; Nelson et al., 1978). Thus, the ON and OFF pathways are maintained functionally and structurally separated. A subset of RGCs, the ON-OFF RGCs, ramify their dendrites in both sublaminae, synapse with both ON and OFF bipolar cells, and signal both the onset and termination of light (Amthor et al., 1984).

ON and OFF bipolar cells. Bipolar cells that receive rod inputs synapse with AII amacrine cells, which in turn make electrical synapses with cone OFF bipolar cells and chemical synapses with cone ON bipolar cells. Light responses in outer retinal neurons, such as photoreceptors and bipolar and horizontal cells, are graded potentials. In the inner retina, transient signals and spikes originate on RGCs and amacrine cells. AC, amacrine cell; AII, AII amacrine cell; GC, ganglion cell; HC, horizontal cell; Off CBC, cone OFF bipolar cell; On CBC, cone ON bipolar cell; PhR, photoreceptor; RBC, rod bipolar cell. (Adapted from Xu and Tian, 2004.)

The rod bipolar cells, on the other hand, do not directly synapse with RGCs. Instead, they synapse with a specific group of amacrine cells, the AII amacrine cells, and depolarize these cells when light is on. The latter then depolarize cone ON bipolar cells through gap junction connections and hyperpolarize cone OFF bipolar cells and OFF RGCs by releasing the inhibitory neurotransmitter glycine onto these cells (Bloomfield and Dacheux, 2001). Therefore, the separation of rod-driven ON and OFF signals starts at the synaptic connections between AII amacrine cells and cone bipolar cells.

Development of retinal synaptic circuitry and the segregation of ON and OFF pathways

The development of mammalian retinal synaptic circuitry is commonly described as a two-step process. The first step includes the commitment of major cell types and the establishment of an initial synaptic circuitry. The second step includes remodeling of the fine structure of cell-cell connections to form specific synaptic pathways. In rodents, most of the cellular and molecular machinery required for synaptic transmission between retinal neurons develops during the

period shortly before and after birth (figure 29.2A). RGCs are the first neurons to differentiate, followed by cones, amacrine, and horizontal cells before birth, and then rods, bipolar cells, and Müller cells after birth (Marquardt and Gruss, 2002; Xu and Tian, 2004). Synaptogenesis follows a somewhat similar order as neurogenesis (Maslim and Stone, 1986; Nishimura and Rakic, 1987). Morphologically identified conventional synapses between amacrine and RGCs in the IPL appear during the first postnatal week. Then cones and rods establish synaptic connectivity with horizontal cells in the outer plexus layer (OPL). The last element to establish synaptic connectivity during development is the bipolar cells, which provide postsynaptic dendrites to photoreceptors and horizontal cells in the OPL and presynaptic axons to amacrine cells and RGCs in the IPL. In mouse retina, bipolar cells start to form synapses in the OPL and IPL early in the second postnatal week (Fisher, 1979b). At this time a synaptic link is completed that is needed to elicit light responses in RGCs (Maslim and Stone, 1986; Nishimura and Rakic, 1987). Consistent with the time course of synaptogesis characterized by morphology, functional synaptic transmission carried by vesicular γ-aminobutyric acid (GABA) and glycine release from amacrine cells was found preceding vesicular glutamate transmission from bipolar cells in developing mouse retina, and the spontaneous glutamatergic synaptic inputs from bipolar cells in mouse RGCs were recorded as early as P7 ( Johnson et al., 2003).

Synaptogenesis continues for several weeks after establishment of the initial synaptic connections from photoreceptor

to RGCs (figure 29.2B). In mouse, the density of conventional synapses in the IPL increases rapidly from 85 synapses/ 1,000 μm3 at the ages of P3–P10 to 223 synapses/1,000 μm3 around the time of eye opening (P11–P15), which is very close to the adult level. The density of ribbon synapses between bipolar cells and RGCs in the IPL, on the other hand, is low around the time of eye opening (45 synapses/1,000 μm3) and increases about 2.5-fold 3 weeks after eye opening to reach the adult level (Fisher, 1979b). Consistently, functional features of synaptic maturation, measured as the frequency of vesicle-mediated spontaneous synaptic transmitter release, increase continuously with synaptogenesis (see figure 29.2B). The rates of AMPA receptor– mediated spontaneous excitatory postsynaptic currents and GABA/glycine receptor–mediated spontaneous inhibitory postsynaptic currents remain constant for a few days after eye opening and then surge fourfold around 2 weeks after eye opening, reaching a plateau by P60 (Tian and Copenhagen, 2001).

In addition to the developmental increase in the number of synapses and the frequency of spontaneous synaptic transmitter release, the initially established retinal synaptic circuitry is profoundly refined morphologically and functionally during postnatal development to form specific synaptic pathways, such as ON and OFF pathways. Early in postnatal development, the dendrites of RGCs ramify diffusely throughout the IPL in mammalian retinas (Maslim and Stone, 1988; Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995, 1999; Bansal et al., 2000; Wang et al., 2001;

Figure 29.2 Neurogenesis and synaptogenesis in developing retina. A, Neurogenesis in rodent retina begins before birth and is largely completed shortly after birth. There are roughly two waves in retinal neurogenesis. The differentiation of RGCs, horizontal cells, amacrine cells, and cones starts early during prenatal development and is mostly completed before birth. The differentiation of rods and bipolar cells, however, starts shortly before birth and continues for 1–2 weeks after birth. (Modified from Young, 1985.) B, Synaptogenesis of mouse retina starts before eye opening and continues for several weeks after eye opening. The density of both

ribbon and conventional synapse in IPL reaches a peak at age P21. (Modified from Fisher, 1979a.) The frequency of RGC spontaneous synaptic inputs increases with age and peaks around 2 weeks after eye opening. (Modified from Tian and Copenhagen, 2001.) The curves show the relative cell populations, synaptic densities, and frequencies of spontaneous synaptic inputs as functions of time. Numbers indicate prenatal and postnatal days of murine development. sEPSC, spontaneous excitatory postsynaptic current; sIPSC, spontaneous inhibitory postsynaptic current.

Diao et al., 2004), where they could synapse with both ON and OFF bipolar cells. With subsequent maturation, RGC dendrites are seen to be much more narrowly stratified, with most or all of the arbors restricted to sublamina a or b. This laminar refinement predicts there is an age-dependent decrease in the number of RGCs receiving synaptic inputs from both ON and OFF bipolar cells. Indeed, analysis of RGC dendritic arborization in mouse retina shows that whereas 53% of RGCs in P10-aged animals ramify in both sublaminae a and b of IPL, only 29% of mouse RGCs in P30-aged animals ramify in both sublaminae a and b (Tian and Copenhagen, 2003).

The developmental stratification of RGC dendritic arbors is reflected physiologically as an age-dependent decrease in the number of RGCs that respond with spikes at the onset and termination of a light. This maturational decline in the percentages of ON-OFF responding RGCs has been observed electrophysiologically in mouse, cat, and ferret

retinas (Bisti et al., 1998; Wang et al., 2001; Tian and Copenhagen, 2003). These results serve to illuminate the observation that, because the ON and OFF sublaminae of the IPL are so well regulated, the retina is one of the best places in the nervous system to directly link structural characteristics with functional responsiveness at the cellular level.

As a technical note, it should be mentioned that the morphological characterization of RGC dendritic patterns has been facilitated significantly by the availability of mouse lines in which GFP or YFP is driven by the Thy1 promoter (Feng et al., 2000). In line H of the Thy1-YFP-expressing mice, several dozen YFP-labeled RGCs are randomly distributed throughout the retina (figure 29.3A) with minimal overlap of their dendritic fields (figure 29.3B). Confocal microscopy of these individual cells in whole mounted retinas provides a precise measurement of the fine structure of the arborization patterns of RGC dendrites and the distribution in the IPL (figure 29.3C–F).

Figure 29.3 Dendritic ramification patterns of YFP expressing RGCs in the IPL can be determined using confocal microscopy from Thy1-YFP transgenic mouse retina. A, View from vitreal side of a flat-mounted retina harvested from a Thy1-YFP-expressing mouse. B, Enlarged view of the area inside the box in A. Axons from individual RGCs cross the retina from each soma to the optic nerve head. C, Four frames taken from a representative stack of confocal images of a bistratified RGC showing the soma and axon, the dendrites ramified in sublamina b (blue), the dendrites ramified in sublamina a (green) of the RGC, and immunolabeling of dopami-

nergic amacrine cells (red). D, A stacked image of the same cell as shown in panel C. E, The 90° rotation view of the cell in D. Three dashed lines indicate the inner border of the IPL, the boundary of sublaminae a and b, and the outer border of the IPL. F, Normalized pixel intensity of the dendrites of each frame (open circles) plotted as a function of IPL depth of the cell in panel C. The data were fitted with two Gaussian distributions (green and blue lines). Doublearrow lines indicate widths. Single arrows indicate the locations of the two peaks of dendritic density. See color plate 19.

How is the RGC dendritic stratification regulated in developing retina? It is postulated that RGCs achieve their mature stratified patterns by removing “misplaced” dendrites from diffuse ramification patterns. This pruning of RGC dendrites is one of the best examples of the maturational reorganization of neuronal processes (Wong and Ghosh, 2002) and has been found in cat (Dann et al., 1988; Maslim and Stone, 1988; Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995), ferret (Bodnarenko et al., 1999), rabbit (Wong, 1990), rat (Yamasaki and Ramoa, 1993), and mouse (Bansal et al., 2000; Diao et al., 2004). Although the exact underlying synaptic and molecular mechanisms regulating the RGC dendritic stratification are not clear, accumulating evidence suggests that this developmental refinement crucially depends on synaptic activities, including both spontaneous and visually evoked activities, before and after eye opening.

Synaptic activity and the developmental segregation of ON and OFF pathways

Both the spontaneous activity present early in postnatal development and visually evoked activity occurring later in postnatal development have been reported to influence the developmental refinement of RGC dendrites. In early developing retina, before an animal can respond to visual stimulation, RGCs fire periodic bursts of action potentials that are highly correlated and propagate across the RGC layer in a wavelike fashion (Wong, 1999). These spontaneous burst activities—namely, retina waves—are mediated by mainly excitatory neurotransmission, with a developmental shift from cholinergic to glutamatergic in mammalian retina (Wong, 1999; Wong et al., 2000). The retina wave had been implicated to direct the RGC axonal projections to their thalamus targets in LGN (see chapter 28, this volume, for details). Recent studies suggest that these spontaneous activities might also regulate RGC dendritic maturation, dendritic filopodial movement, and the maintenance or elimination of existing processes (Wong and Wong, 2000; Wong and Ghosh, 2002). Genetic deletion of the β subunit of nicotinic acetylcholine receptors diminished the retina wave mediated by acetylcholine receptors and slowed the stratification and segregation of RGC dendrites in the IPL during early (before P8) postnatal development. The retinas of the mutant mice had significantly narrower IPL, and RGC dendrites were not stratified or only weakly stratified into two distinct sublaminae. Between P8 and eye opening (P14), when retinal waves are mediated by ionotropic glutamate receptor– mediated transmission, the IPL of the mutant mice approximately doubled in size and RGC dendrites segregated into four or five distinguishable strata like those of wild-type animals, suggesting that both early cholinergic and later glutamatergic synaptic transmission contribute to the de-

velopmental refinement of RGC dendrites (Bansal et al., 2000).

The critical roles of glutamatergic synaptic inputs from bipolar cells to the developmental stratification and segregation of RGC dendrites into ON and OFF pathways were also demonstrated in another set of experiments. During the time period of normal dendritic stratification of RGCs, intraocular injection of APB, an agonist for class III metabotropic glutamate receptors exclusively expressed on the rod bipolar and cone ON bipolar cells, hyperpolarized these bipolar cells and blocked the glutamate release from these neurons, resulting in an arrest of the developmental stratification and segregation of RGC dendrites into ON or OFF layer of the IPL. About 40% of the RGCs in APB-treated adult retina have their dendrites multistratified in both sublaminae a and b of IPL, a significantly higher percentage than that seen in untreated age-matched controls (Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995, 1999), and this effect is irreversible with prolonged APB treatment (Deplano et al., 2004). In contrast, APB treatment did not alter the RGC density or somata and dendritic field size, demonstrating that excitatory synaptic inputs from bipolar cells have a highly selective impact on RGC dendritic stratification.

The role of visually evoked activity in the developmental segregation of RGC dendrites into ON and OFF pathways is shown by the finding that light deprivation retards the maturational conversion of RGCs ramified in both sublaminae a and b of the IPL into cells ramified only in sublamina a or b of the IPL. Tian and Copenhagen (2003) compared the lamination patterns of RGCs in cyclic light–reared mice to those in dark-reared mice. At P30, 53% of the RGCs ramified in both sublaminae a and b of the IPL in the darkreared mice versus 29% in cyclic light–reared mice. This difference was highly significant. However, this percentage was very close to the P10-aged mice raised in cyclic light (53%). These anatomical findings predicted that many more RGCs in P30 dark-reared mice should be ON-OFF- responsive RGCs in comparison with age-matched controls. Multielectrode array recordings of light responses from RGCs verified this prediction. In the P27–P30-aged mice raised in constant darkness, the percentage of ON-OFF- responsive RGCs was more than fourfold higher than in age-matched controls raised in cyclic light but comparable to the percentage of ON-OFF-responsive RGCs in P10– P12-aged mice. These results demonstrated that light stimulation is critical for the developmental segregation of RGC dendrites into ON and OFF pathways.

Visual stimulation also influences other morphological and functional attributes of RGCs. In dark-reared mice, the density of conventional synapses in IPL is greater than in mice reared under cyclic light conditions (Fisher, 1979a). Light deprivation also blocked the age-dependent increase