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

et al., 1996). These transgenic mice also display increased numbers of bipolar and some type of amacrine cells, suggesting that Bcl-2 may prevent death in these populations also (Strettoi and Volpini, 2002). However, because different antiapoptotic members of the Bcl-2 family may compensate for one another, interpretation of the alterations observed in knockout mice is complex. In summary, current evidence indicates that antiapoptotic Bcl-2 family members can interfere with programmed cell death in retinal development, but their intrinsic role in this process remains to be elucidated.

As to the BH3-only protein, no evidence of a causal involvement of BH3-only protein in retinal cell death is yet available, but rat RGCs express the BH3-only protein BIM during the period of postnatal programmed cell death, and the relative levels of BIM mRNA in RGCs have a time course correlated with that of RGC death after axotomy (Napankangas et al., 2003).

Finally, many studies indicate the involvement of individual caspases in retinal cell death. Caspases are proteases with selective cleavage sites that execute the death program. In the most characterized death core program, mature caspase-9 recruits and activates downstream caspases, such as caspase-3, resulting in controlled demise of the cell. Caspase-3 activation and the presence of DNA strand breaks due to a caspase-dependent endonuclease are the basis of two popular methods for death detection (activated caspase- 3 immunoreactivity and TUNEL, respectively), which, however, do not detect all types of cell death. Mouse embryos deficient in either caspase-3 or caspase-9 display retinal hyperplasia, disorganized cell deployment, and delayed optic fissure closure. These knockout strains have a high level of perinatal death (Kuida et al., 1996). In the few animals that are born, abnormal death kinetics after birth were limited to the inner nuclear layer (Zeiss et al., 2004). In the chick retina, acute treatment with caspase-3 inhibitors reduced to 50% cell death among RGCs in the phase of optic nerve formation (Mayordomo et al., 2003).

In summary, caspase-mediated apoptosis regulated by the Bcl-2 family has been securely established as one way of cell death in the retina, but the causal link between the different molecular players is still less clear than in other model systems. Furthermore, this death paradigm might not be the only one in the developing retina, and the different phases of cell death may not share the same death pathway. In general, as indicated by studies in isolated retinas, there is evidence for multiple posttranslational pathways of death in the developing retina (Guimaraes et al., 2003).

An important aspect of cell death is the complex realm of activators, modulators, and inhibitors of the death program. This field has attracted increasing interest in recent years, but a general picture is still lacking. Much of what is known comes from well-established models of cell death in culture.

Very schematically, an emerging picture has been identified that involves (1) extracellular signals that trigger death (NGF/p75 and in the Fas signaling system) and (2) intracellular signaling pathways that regulate activation of the death program. The latter include transcription factors and kinases. In neuronal culture model systems, some of these intracellular effectors appear implicated in the way neurotrophin promotes cell survival, but whether this occurs during retinal development is unknown. As we have seen, NGF/p75 signaling promotes cell death during embryonic retinal development, whereas Fas signaling, another established extracellular death trigger, appears to have little effect on retinal cell death, as Fas knockout mice exhibit a delayed period of cell death in retinal development but eventually have normal retinas and appear to lose the same number of retinal cells as do normal mice (Pequignot et al., 2003).

Mutations or deletions of several transcription factors have been shown to induce cell death among developing photoreceptors and RGCs. Of these, Ap3b1, encoding a subunit of the AP-3 adaptor complex, corresponds to the pearl mutation that is associated with an accelerated time course of cell death and CRB1, mutations of which are related to a thickening of the human retina that is probably dependent on alteration of natural cell death (see Linden and Reese, 2006).

Finally, several kinases have been involved in modulating neuronal death (reviewed in Putcha and Johnson, 2004). In the retina, constitutive expression of an activated form of the regulatory subunit of the phosphoinositide 3-kinase causes retinal dysplasia and an increased number of photoreceptors, attributed to reduced death of these neurons during development (Pimentel et al., 2002).

Much effort is being devoted to understanding whether developmental death programs are reactivated during neurodegeneration in retinal pathologies, as well as to elucidate the differences between developmental and pathological death. Reviewing research on pathological cell death in the retina is beyond the scope of the present chapter, but a few aspects deserve consideration. It has been suggested that antiapoptotic “brakes” are set into action to warrant survival of normally maturing neurons. In the retina, downregulation of key pro-apoptotic factors, including pro-apoptotic Bcl-2 family members, Apaf-1, and caspase-3, correlates with maturation, while the expression of XIAP, a potent caspase inhibitor, increases in the adult retina (O’Driscoll et al., 2006), suggesting that adult neurons might survive by switching off elements of the developmental death program. In line with this view, following axotomy, RGCs activate caspase- 3 and caspase-9 (Kermer et al., 2000). Experiments testing whether caspase inhibitors prevent RGC death following axotomy, however, have had conflicting results (Kermer et al., 2000; Weishaupt et al., 2003; Spalding et al., 2005;

McKernan et al., 2006). There are also indications that the death of adult retinal cells in neuronal pathologies and related models might involve different mechanisms than those involved in developmental death. For example, Bax or Fas deletion has no effects on photoreceptor survival in a mouse model of retinitis pigmentosa (Mosinger Ogilvie et al., 1998), whereas during development, Bax deletion decreases and Fas deletion delays developmental death (Pequignot et al., 2003). Similarly, overexpression of Bcl-2 or Bcl-xL transgenes does not rescue photoreceptor cells in models of retinitis pigmentosa (Joseph and Li, 1996).

Concluding remarks

Despite considerable progress, we are still far from a clear picture of the network of interactions that control retinal development. Cell death is obviously an important component of retinal development, but many aspects of its role and regulation are still obscure. For example, we have little idea of the role of extracellular matrix or of mechanical interactions in regulating cell survival and death in the retina, and we do not know the relevance of pools of dying cells to the evolutionary potential of the system.

Cell death is observed throughout eye and retinal development, with a first peak at the time of optic vesicle formation and two later phases of death, while retinal neurons are generated and when they later differentiate. Much effort has been devoted to understanding the role of cell death in specific processes such as connection refinement and the matching of interconnected cell populations. Most of these studies have shown a modest contribution of cell death, suggesting that death is involved in these processes but is not designed to subserve them. Rather, death appears as one of the fates open to differentiating retinal cells, a fate that is realized with the activation of genetic death programs.

Much current evidence favors the view that cell survival during retinal development is due to trophic intercellular interactions, without which death occurs. This view suggests that death might be a default fate for differentiating neurons.

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Prior to vision, the developing vertebrate retina spontaneously generates a firing pattern termed retinal waves. During a retinal wave, retinal ganglion cells (RGCs) spontaneously fire correlated bursts of action potentials that propagate across the retina. Retinal waves have been characterized in a wide variety of vertebrate species (for reviews, see Wong, 1999; Firth et al., 2005). In mice, retinal waves have been detected as early as embryonic day 16 (E16) and persist until the time of eye opening at postnatal day 14 (P14).

Retinal waves coincide with a period of visual system development when there is a dramatic level of refinement of the retina’s projections to its primary targets in the brain, the superior colliculus (SC) and the lateral geniculate nucleus of the thalamus. In vivo blockade or significant alteration of retinal waves prevents normal refinement of these circuits, indicating that retinal waves are required for normal development of the visual system (reviewed in Torborg and Feller, 2005). However, the mechanisms by which retinal waves drive developmental processes are not fully understood.

In this chapter, we describe (1) the synaptic circuits that mediate retinal waves, (2) the spatial and temporal correlations of retinal waves, and (3) the role of retinal waves in establishing circuits throughout the developing visual system.

in ferret retina (Meister et al., 1991; Wong et al., 1993). MEA recordings have been used extensively to characterize spontaneous firing patterns in mice (for recent examples, see Demas et al., 2003; Cang et al., 2005; Torborg et al., 2005).

A large body of work on retinal waves has been conducted using calcium imaging (Wong et al., 1995; Feller et al., 1996), which can monitor activity over larger regions of the retina (up to 2 mm2) than is possible with MEA (less than 0.2 mm2) (Wong, 1998). In calcium imaging, intracellular concentrations of calcium are measured using fluorescent indicators (Wong, 1998). Calcium imaging indirectly measures cell depolarization by monitoring the influx of calcium triggered through voltage-gated calcium channels. Simultaneous electrophysiological recordings from individual RGCs and calcium imaging show that the bursts of action potentials and calcium transients occur simultaneously (Penn et al., 1998; Zhou, 1998; Singer et al., 2001).

Finally, single-cell physiology experiments, such as wholecell voltage-clamp and current-clamp recordings, have been used to determine the synaptic inputs and membrane potential changes of individual neurons involved in retinal waves (Feller et al., 1996; Zhou, 1998; Butts et al., 1999; Singer et al., 2001).

Techniques used to measure retinal waves

Several physiological methods have been used to record retinal waves. Spontaneous activity in the developing retina was first detected in vitro in rabbits using extracellular recordings from single RGCs (Masland, 1977). Ten years later, spontaneous correlated bursts of action potentials were detected in vivo in fetal rat pups (Galli and Maffei, 1988). The evidence that these spontaneous bursts of action potentials propagated in the form of waves was provided by a series of experiments using a multielectrode array (MEA), which allowed simultaneous recording from tens of RGCs

Cellular mechanisms underlying retinal waves change with retinal development

Retinal waves are detected in mouse retina from a few days before birth until approximately 2 weeks after birth. During this time, the retina itself undergoes a dramatic amount of development (Morgan and Wong, 2006). At E16, the mouse retina consists of a ganglion cell layer that is two to three cell bodies thick and contains both RGCs and cholinergic and GABAergic amacrine cells. Around birth, the first chemical synapses are detectable by electron microscopy (Olney, 1968). These synapses have the morphological character-

istics of classic chemical synapses, such as those between amacrine cells and RGCs. At P10, ribbon synapses are first detected in electron micrographs of the inner plexiform layer (IPL), representing the first functional synapses between bipolar cells and RGCs (Fisher, 1979).

The mechanisms underlying retinal waves go through three functional stages as the retina develops (figure 28.1). The stages are defined by the combination of receptor antagonists that block waves (reviewed in Firth et al., 2005; Torborg and Feller, 2005). Stage I retinal waves consist of simultaneous increases in [Ca2+]i in small clusters of cells that are blocked by gap junction antagonists, and activity that propagates over substantially larger regions that is blocked by nicotinic acetylcholine receptor (nAChR) antagonists. At birth (P0), when chemical synapses are present in the IPL, stage I waves end and stage II waves begin. Stage II waves are blocked by nAChR antagonists and by gap junction antagonists. Stage II waves end and stage III waves begin at P10, when bipolar cells are forming glutamatergic synapses with RGCs. Stage III waves are blocked by ionotropic

Figure 28.1 Three functional stages of retinal wave–generating circuits. A, Schematic of circuits that mediate retinal waves. (Modified from Catsicas and Mobbs, 1995.) B, Summary of development of the synaptic circuitry that mediates waves in mice. Each color corresponds to a different wave-generating circuit. Yellow corresponds to non-nAChR circuitry, which mediates the nonpropagating events in embryonic mice. There is pharmacological evidence that stage I waves in other species are mediated by gap junctions, but this has not been directly demonstrated in mouse retina. In addition, it is not known which gap junction–coupled networks mediate stage I waves. Red corresponds to stage II circuits, which require activation of nAChRs. Stage II waves are initiated and propagate through a network of starburst amacrine cells. Blue corresponds to stage III circuits, which require activation of ionotropic glutamate receptors. The source of stage III wave initiation and the location of the horizontal coupling that drives coordinated release of glutamate during this stage are not yet known. See color plate 17. (Modified from Bansal et al., 2000.)

glutamate receptor antagonists and gap junction antagonists, but not by nAChR antagonists.

The circuitry that underlies stage II retinal waves has been extensively studied and therefore is the best understood. Here we focus primarily on the properties of stage II waves, both their spatial and temporal characteristics and the cellular mechanisms underlying them.

Spatiotemporal properties of stage II retinal waves

A complete description of retinal waves must link the known circuitry that mediates the waves with their global spatial and temporal properties. The spatiotemporal properties of retinal waves have been divided into three parts—wave initiation, propagation, and termination (figure 28.2A). Calcium imaging and MEA recording have both been used to describe these properties of stage II retinal waves in mice. Extensive studies have also been conducted in ferrets (Feller et al., 1997; Butts et al., 1999), rabbits (Zhou and Zhao, 2000; Zhou, 2001a), and turtles (Sernagor et al., 2000, 2003) but are not presented here.

How do retinal waves start? Stage II retinal waves begin in random locations in the retina, with all locations having equal probability for wave initiation. Hence, there is no particular region of the retina that functions as a pacemaker for retinal waves. We proposed a computational model that assumes a randomly distributed population of cells with a finite probability of spontaneously depolarizing in which the cells are connected to each other by recurrent excitation (Feller et al., 1997; Butts et al., 1999). When the activity level surpasses a threshold level of depolarization, a retinal wave occurs. After this event, all cells participating in the wave enter a refractory period during which they cannot initiate or participate in subsequent waves. Hence, wave initiation results from a combination of spontaneous depolarization in individual cells and network interactions. Recent evidence supporting this model is presented later in the chapter.

What determines the speed of retinal wave propagation? Stage II retinal waves propagate at a speed of 150 μm/s in mice. This speed is two to three times faster than speeds predicted for the extracellular diffusion of excitatory substances, such as those observed in spreading depression (Martins-Ferreira et al., 1974, 2000; Somjen, 2001), but an order of magnitude slower than epileptic waves that are induced in cortical circuits by blocking all inhibitory synaptic transmission (see, e.g., Prince and Connors, 1986). From these measurements, it has been hypothesized that retinal wave speed is determined by fast chemical synapses, with one step of propagation “slowed” by some diffusive component, such as activation of a G protein–coupled receptor or diffuse release of neurotransmitter.

Stage II retinal waves propagate over a finite region of the retina, stopping at well-defined but shifting boundaries. We

Figure 28.2 Calcium imaging reveals spatial and temporal properties of stage I and stage II waves. A, Time evolution of a single stage II retinal wave visualized with fluorescence imaging of the calcium indicator fura-2. Decreases in fura-2 fluorescence associated with the increased calcium evoked by waves are shown at successive 0.5 s intervals. The final frame represents the total area of tissue covered by a single wave. B, Retinal waves of embryonic day 17 (E17) and postnatal day 2 (P2) retinas. Each frame summa-

have shown that these wave boundaries are determined by a “refractory period,” defined as a finite period of time lasting 30–40 s after activation of an area of the retina, during which it cannot participate in subsequent waves (Feller et al., 1997). In addition to controlling the distance over which waves propagate, the refractory period acts to define the frequency with which a local region of the retina participates in waves (Feller et al., 1997; Butts et al., 1999).

Mechanisms of stage II retinal waves

A breakthrough in understanding wave-generating mechanisms was the discovery that stage II retinal waves are medi-

rizes 90 s of activity in control ACSF (top row) and in 100 μM d-tubocurarine, a general nAChR antagonist (bottom row). Gray background represents the total retinal surface labeled with fura2AM. Each color corresponds to individual domains, with a colorcoded time bar below each frame to indicate the time of occurrence of each wave. Scale bar = 100 μm. See color plate 18. (Modified from Bansal et al., 2000.)

ated by a cholinergic circuit. Studies in turtle (Sernagor and Grzywacz, 1999) and in ferret (Feller et al., 1996) showed that curare, a general nAChR antagonist, blocks stage II retinal waves (figure 28.2B).

The retina contains many different nAChR subunits. Nicotinic AChRs found in the CNS are either homomultimers consisting entirely of α7 subunits or heteromultimers containing a combination of α and β subunits (Sargent, 1993; McGehee and Role, 1995; Role and Berg, 1996). In heterologous expression systems, α3 subunits form functional nAChRs only in the presence of either the β2 or the β4 subunit (Role and Berg, 1996; Gotti et al., 2005). Which nAChR subunits mediate retinal waves? Antagonists

selective for nAChRs containing particular subunits or subunit combinations exist, but these drugs become nonselective at high concentrations.

To identify the functional receptors that mediate retinal waves, a screen of different knockout mice lacking specific nAChR subunits was performed (Bansal et al., 2000). Mice lacking β4-containing nAChRs have normal retinal waves, whereas mice lacking α3-containing nAChRs have altered retinal waves. Mice lacking β2-containing nAChRs do not have stage II retinal waves. Hence, although many classes of nAChRs exist in the retina (Moretti et al., 2004), only β2-containing nAChRs are critical for mediating retinal waves.

Whole-cell voltage-clamp recordings from RGCs show that RGCs receive cholinergic and GABAergic inputs during retinal waves (Feller et al., 1996; Zheng et al., 2004). During development, activation of GABAA receptors is excitatory (Zhang et al., 2006) and therefore provides some of the depolarization associated with waves (Stellwagen et al., 1999; Wong et al., 2000). However, blockade of GABAA receptors does not alter the frequency of retinal waves and therefore is not thought to play a critical role in the generation of stage II retinal waves (Stellwagen et al., 1999).

The only source of ACh in the retina is a class of interneurons called starburst amacrine cells (SACs) (Vaney, 1990; Zhou, 2001b), though there is some evidence of transient high expression of ACh in horizontal cells during development (Zhou, 2001b). The properties of SACs and the SAC network are sufficient to explain most characteristics of stage II retinal waves. Direct recordings from SACs indicate they receive synaptic input during waves (Butts et al., 1999; Zheng et al., 2004). In addition, SACs form nAChRmediated synapses with each other and with RGCs (Zhou, 1998; Zheng et al., 2004, 2006). Paired recordings from SACs show that neighboring SACs monosynaptically release both ACh and GABA onto one another and that each SAC receives input from around 20 other SACs (Zheng et al., 2004, 2006). Current-clamp recordings from SACs show that when all synaptic transmission is blocked, SACs undergo spontaneous depolarizations, followed by long, Ca2+- dependent after-hyperpolarizations (AHPs) (Zheng et al., 2006). The model for how these properties of SACs combine to create stage II retinal waves is as follows (Zheng et al., 2006): (1) Individual SACs spontaneously depolarize and release small amounts of ACh onto other SACs. (2) When a critical mass of neighboring SACs spontaneously depolarize concurrently, they release enough ACh onto other neighboring SACs, causing them to depolarize, and the newly recruited SACs in turn excite other SACs, causing a wave to propagate across the retina. (3) The large calcium influx caused by the depolarization during the wave elicits a large AHP that decays over 30 s. This AHP is the basis of the 30 s refractory period. (4) SACs also synapse onto RGCs, so that

when waves of depolarization pass through the SAC network, RGCs receive cholinergic input from the SACs and GABAergic input from SACs or other amacrine cells. ACh and GABA both excite RGCs, causing the RGCs to fire the bursts of action potentials that characterize retinal waves.

Interestingly, in contrast to RGCs, which express nAChRs in the adult retina, expression of nAChRs in SACs decreases with postnatal development (Zheng et al., 2004). Hence, downregulation of nAChRs on SACs may be responsible for the end of stage II retinal waves.

The hypothesis that stage II waves are initiated and propagate through a network of spontaneously active SACs is quite compelling, but there is much left to be explained. First, it is important to note that these experiments have all been done in rabbit retina and have not yet been reproduced in the mouse, though the circuitry is likely to be similar. Second, the channel that underlies the spontaneous depolarization of SACs, and what causes it to be activated, are not yet known. Third, the AHP is mediated by a potassium conductance that is calcium dependent and strongly modulated by cAMP levels; its identity is also unknown. Fourth, it is not known whether these conductances are unique to SACs. In a study of dissociated rat retinal neurons, both cholinergic and noncholinergic amacrine cells exhibited spontaneous, cell-autonomous depolarizations (Firth and Feller, 2006), though these findings have not been reproduced in the intact retina. By verifying this model of stage II retinal wave initiation and propagation in the mouse, targeted gene knockouts will allow the testing of specific hypotheses.

Gap junctions and stage II retinal waves

The role of gap junctions in stage II retinal waves is unclear. Gap junctions coordinate neuronal firing in many brain areas, including the adult retina (Connors and Long, 2004; Sohl et al., 2005), and extensive gap junction coupling exists in the neonatal retina (Penn et al., 1994; Catsicas et al., 1998). Gap junction antagonists have been used extensively to study the role of coupling in retinal waves, but the results of these pharmacological studies are inconsistent: in some studies gap junction antagonists block retinal waves (Hansen et al., 2005), in others they do not (Stacy et al., 2005). Part of the difficulty may arise from the fact that gap junction antagonists have nonspecific effects such as blockade of voltage-gated calcium channels (Vessey et al., 2004). Another limitation to using general pharmacological agents to block gap junctions is that since most cells in the retina are gap junction coupled (Sohl et al., 2005), general gap junction antagonists do not identify specific coupled networks that may mediate retinal waves.

Recent evidence that gap junction coupling can mediate retinal wave propagation under certain conditions came

from a study using a transgenic mouse in which SACs located in a large segment of the retina did not produce acetylcholine (ACh) (Stacy et al., 2005). At P3, ACh-knockout regions of the retina did not exhibit retinal waves. However, by P5, waves were recorded in ACh-knockout regions, and these waves were blocked by gap junction receptor antagonists, while retinal waves in wild-type littermates were not. Hence, expression of gap junctions can compensate for an absence of normal synaptic connections.

One way to circumvent the limitations of gap junction antagonists is to study retinal waves in transgenic mice with genes for specific connexins knocked out and reporter genes knocked in. Connexins are the proteins that make up gap junctions. Three connexins have been identified in neurons in the adult retina: Cx36, Cx45, and Cx57 (Sohl et al., 2005). Using a mouse in which the Cx36 gene is replaced by a β-galactosidase reporter (Deans et al., 2001, 2002), we have demonstrated that Cx36 is expressed during retinal development in RGCs, glycinergic AII amacrine cells, and cone bipolar cells (Hansen et al., 2005). Cx36−/− mice have mostly normal stage II retinal waves, though 15% of Cx36−/− RGCs tonically fire action potentials in the normally quiet periods between waves (Hansen et al., 2005; Torborg et al., 2005). Studies of retinal waves in other connexin knockout mice have not yet been completed. Perturbation of gap junctions has more dramatic effects during stage I and stage III retinal waves, as described in the next section.

Properties of stage I and stage III retinal waves

Stage I retinal waves, lasting from E16 to P0 in mice, are characterized by small clusters of synchronous increases in intracellular calcium that are insensitive to nAChR and GABAA receptor antagonists, as well as larger, nAChRmediated waves (Bansal et al., 2000) (see figure 28.2B). Waves seen in E17 retinas do not always respect refractory boundaries defined by previous waves but do propagate with a similar speed to that of stage II retinal waves. This early activity is blocked by gap junction receptor antagonists (Zhou and Zhao, 2000; Stacy et al., 2005). Hence, the retina follows a similar developmental pattern observed in both the developing spinal cord and cortex in which early network connectivity is mediated by gap junctions, and this electrical coupling is reduced as chemical synapses mature (Roerig and Feller, 2000). Interestingly, mice lacking the nAChR α3 subunit are able to generate retinal waves through an extension of the stage I wave-generating mechanism (Bansal et al., 2000), perhaps similarly to the mice lacking ACh in a segment of the retina (Stacy et al., 2005). Hence, the mechanisms mediating stage I waves can be extended in the absence of normal spontaneously active retinal circuits.

The circuitry underlying stage III waves has not been identified, but it is probable that the SAC network is not the source of correlated activity. Stage III waves are not blocked by nAChR antagonists (Bansal et al., 2000). During stage III waves, SACs no longer undergo spontaneous depolarizations (Zheng et al., 2006). Furthermore, SACs do not express nAChRs, and the GABA they release onto one another is inhibitory (Zheng et al., 2004). These changes in the properties of SACs eliminate all recurrent excitation in the SAC network.

Blockade of GABAA/C and glycine receptors leads to an increase in stage III wave frequency (Zhou, 2001a; Syed et al., 2004), indicating that endogenous GABA release regulates wave initiation but is not critical for the generation of retinal waves. These changes correspond to the same point in development when activation of GABAA receptors becomes inhibitory (Fischer et al., 1998; Zhang et al., 2006).

The role of gap junctions in stage III retinal waves is not fully elucidated. In Cx36−/− mice, RGCs have a significant increase in asynchronous firing of action potentials in the normally silent periods between retinal waves (Torborg et al., 2005). These extra action potentials are blocked by glutamate receptor antagonists (Hansen et al., 2005), indicating that Cx36-coupled networks normally suppress interwave release of glutamate. Cx36 is highly expressed in glycinergic AII amacrine cells, which make gap junction connections with other AII amacrine cells and cone bipolar cells. In addition, AII amacrine cells have a glycinergic input onto OFF cone bipolar cells (Pourcho and Goebel, 1985; Strettoi et al., 1992; Deans et al., 2002). Since both glycine and glutamate modulate the spontaneous firing of RGCs, one hypothesis is that Cx36 is critical for coordinating the release of these two transmitters.

Where are stage III waves initiated, and how do they propagate? Stage III glutamate receptor–mediated waves propagate at approximately twice the velocity and depolarize individual ganglion cells at approximately twice the frequency as stage II waves (Bansal et al., 2000; Muir-Robinson et al., 2002), consistent with stage III waves being mediated by mechanisms distinct from those mediating stage II waves. Stage III waves are blocked by ionotropic glutamate receptor antagonists (Bansal et al., 2000). Synapses containing ionotropic glutamate receptors are found between photoreceptors and OFF bipolar cells, between bipolar cells and amacrine cells, and between bipolar cells and RGCs. Additionally, a class of amacrine cells expressing vesicular glutamate transporter III at terminals presynaptic to RGCs and other amacrine cells has recently been identified (Haverkamp and Wässle, 2004; Johnson et al., 2004). Which of these components of the various glutamatergic circuits mediates stage III waves remains to be determined.