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

Figure 12.1 Light micrograph obtained with differential interference contrast illustrating the mosaic of rods and cones in the mouse retina. The matrix surrounding the cones has been stained with peanut lectins and appears dark. Cones make up only 3% of all the photoreceptors in the mouse retina. (From Jeon et al., 1998.)

of protein kinase C, which allows their study as a population (Haverkamp and Wässle, 2000).

Electron microscopy of the outer plexiform layer (OPL) shows typical ribbon contacts between rod spherules and rod bipolar cell apical dendrites; these form the central elements of triads, while processes originating from the axonal arborizations of horizontal cells form the lateral processes (figure 12.2). Because rod spherules are small and densely packed, histological sections cut at a perfect right angle with respect to the retinal surface are not easy to obtain. This reason, along with the fact that each spherule makes connections to only one (rarely, two) triplet of processes (see figure 12.2), explains why typical triads can be observed in only a fraction of all the spherules encountered in conventional ultrathin sections. Most commonly, only one or two dendrites are visible, facing the ribbon of each spherule. Ribbon contacts are termed invaginated and represent the only kind of synapse established by rod spherules. At the postsynaptic site of the invagination, rod bipolar dendrites carry a retinalspecific type of metabotropic glutamate receptor, mGluR6 (Ueda et al., 1997). By means of mGluR6, the hyperpolarizing light response of rods is transformed into a graded depolarization of postsynaptic rod bipolar cells. Hence, invaginating synapses are called sign-inverting. On binding to mGluR6, glutamate released onto retinal ON bipolar neurons activates a heterotrimeric G protein, Go, that ultimately closes a nonspecific cation channel (Dhingra et al., 2002). In particular, the light response of ON bipolar cells requires the strongly expressed splice variant of the G protein known as Gαo1. The pathway ultimately leading from glutamate binding to mGluR6 to the final change in membrane potential in ON bipolar cells is still somewhat obscure, although recent studies suggest the existence of multiple transduction mechanisms (Huang et al., 2003).

Figure 12.2 Electron micrograph of the mouse outer plexiform layer. A single cone pedicle of typical conical shape is surrounded by several rod spherules (Sph), each carrying one synaptic ribbon (arrows). Cone pedicles establish multiple ribbon synapses with cone bipolar (CBc) and horizontal cell dendrites (HC), as well as flat contacts (arrowhead) with cone bipolar cells. Spherules establish ribbon contacts with rod bipolar cells (RBc) and with axonal processes of horizontal cells (HC).

Rod bipolar cells have circular receptive fields (RFs) and depolarize in response to light stimuli; hence, as in all mammals, they belong to the functional type of ON-center neurons. The average width of their RF is approximately 70 μm (Berntson and Taylor, 2000), slightly larger than the dendritic diameter. This suggests there is not extensive signal spread in the OPL through gap junction connections between the terminals of the photoreceptors. It also shows that each rod bipolar cell receives contacts from all the rods within reach (around 22; Tsukamoto et al., 2001). In addition, each rod terminal establishes gap junctions with processes (telodendria) originating from cone pedicles.

Mice have typical AII amacrine cells. These neurons were first described almost 30 years ago in the retina of the cat (Kolb, 1979) and are considered a hallmark of the mammalian retinal architecture. They are narrow-field cells with a distinctive bistratified morphology (figure 12.3). Their somata, localized to the innermost part of the INL, bulge into the IPL, giving rise to one or more sturdy primary dendrites from which large varicosities (1–4 μm in diameter) termed lobular appendages branch in sublamina a. Several thinner, bushy dendrites ramify further down in the IPL in sublamina b; these dendrites are thin and long and cover a diameter of 8–12 μm.

Electron microscopy of serial sections has shown that rod bipolar cells’ axonal endings express ribbon synapses directed mostly at the AII amacrine cell’s innermost dendritic branches. In turn, each AII cell sends conventional chemical synapses at the axon terminals of OFF cone bipolar cells (see figure 12.4) by means of the lobular appendages

Figure 12.3 A typical AII amacrine cell of the mouse retina, stained with DiI, loaded on tungsten bullets and delivered with a gene gun. The sublaminae a and b of the IPL are indicated. The cell has a characteristic bistratified morphology.

(Tsukamoto et al., 2001). In addition, AII form large gap junctions with axon terminals of ON cone bipolar cells (figure 12.4).

Thus, as in all mammals, in the mouse retina too AII amacrine cells are the main postsynaptic target of rod bipolar cells (Strettoi et al., 1992).

Each rod bipolar axonal ending expresses approximately 40 ribbon synapses by which it contacts several AII amacrine cells. The AII cell, collecting from several rod bipolar cells, expresses many gap junctions and diverges onto several ON bipolar terminals. The AII cell also expresses many conventional synapses by which it diverges to several OFF bipolar terminals of homogeneous type. Finally, AII amacrines are electrically coupled through small gap junctions occurring among their dendrites in sublamina b (see figure 12.4).

Therefore, as in the cat, rabbit, rat, and monkey retina, the rod-generated signal is fed into the ON and OFF pathway through AII amacrine cells that establish sign-conserving electrical connections with the axonal endings of ON cone bipolar cells while forming sign-inverting, glycinergic synapses with OFF cone bipolars. Because neither rod bipolars nor AII amacrines form direct connections to ganglion cell dendrites, the largest part of the scotopic signal reaches ganglion cells through cone bipolar cells. In fact, the final transfer to the retinal exit occurs through sign-conserving ribbon

Figure 12.4 The rod pathway of the mouse retina reconstructed from electron micrographs of serial sections. Each rod diverges to one to two rod bipolar cells (RB); in turn, 22 rods converge onto one rod bipolar cell. The rod bipolar cell provides 43 ribbon synapses to AII amacrine cells. One AII amacrine cell forms 16 gap junctions with ON cone bipolar terminals (CBON) and 19 conventional synapses with the OFF cone bipolar terminals (CBOFF). Numbers in a circle, square, and triangle represent the number of input or output synapses between a given pair of adjacent cells. (From Tsukamoto et al., 2001.)

synapses established between ON and OFF cone bipolar axonal endings and corresponding sets of ganglion cell dendrites in the ON and OFF laminae of the IPL. These synapses use glutamate as a neurotransmitter. The particular array by which rod signals exploit cone bipolar cells to gain access to ganglion cell is known as a piggyback arrangement.

AII amacrine cells can be stained selectively with antibodies against the cytoplasmic protein disabled-1 (Rice and Curran, 2000) and thus studied as a single population. There are approximately 49,000 AII amacrine cells in the retina of the C57Bl6/J mouse; their density shows a peak in the central retina. As in the rabbit, and probably as in all mammals, they constitute the largest population of amacrine cells, of which they represent around 12%.

The cell body and primary dendrite of each AII amacrines are surrounded by a ring of amacrine cell varicosities containing dopamine as well as γ-aminobutyric acid (Contini and Raviola, 2003). Dopaminergic rings are one of the sites of output of wide-field dopaminergic amacrine cells, which ramify in a narrow stratum at the INL/IPL border and also send processes to the OPL (they are also called interplexiform cells); these neurons can be visualized clearly with antibodies against tyrosine hydroxylase. There are fewer than 500 dopaminergic amacrine cells in the mouse retina;

however, their long, ramified processes cover the retina uniformly. From studies carried out in various mammals, it is known that dopaminergic amacrines receive input primarily from other amacrine cells and, to a lesser degree, from cone bipolar cells (Dowling and Ehinger, 1978; Hokoc and Mariani, 1988; Kolb et al., 1990). They are believed to modulate the light adaptation state of the retina by providing lateral inhibitory signals to AII amacrine cells. They spontaneously generate action potentials in a rhythmic fashion, and their molecular determinants have been studied in great detail by means of transgenic technology (Raviola, 2002).

In general, dopamine is a powerful modulator of gap junction permeability and a regulator of retinal sensitivity to light. Hence, dopamine is capable of influencing many components of the retinal circuitry. A well-known action is control of the conductance of gap junctions occurring between horizontal cells and between amacrine cells. In addiction, this transmitter increases the responses of ionotropic glutamate receptors on bipolar cells, and ultimately influences the center-surround balance of ganglion cells. Part of the dopamine action is exerted nonsynaptically, through a form of extrasynaptic, paracrine release. Various dopamine-dependent functions result in increased signal flow through cone circuits and a diminution in signal flow through rod circuits (Witkovsky, 2004). Variations in dopamine release are also responsible for the modulation of homologous electrical coupling between AII amacrine cells, as well as between AII amacrine cells and ON cone bipolar cells, in different conditions of illumination (Bloomfield et al., 1997). In total dark adaptation, the average size of the AII-AII network matches the size of AII cell ON-center RFs. However, as light increases, AII cells form much larger networks, comprising more than 300 amacrine cells, with a corresponding increase in RF size.

Additional rod pathways

Besides the standard mammalian circuit for night vision, two additional pathways exist that route rod-generated information to ganglion cells.

As mentioned earlier, cone pedicles have processes, called telodendria, that extend laterally in the OPL and are engaged in small gap junctions with neighboring photoreceptors (rods and cones) (Raviola and Gilula, 1975). Electrophysiological recordings have long shown that mammalian cones carry rod signals (Nelson, 1977). These appear as a slow hyperpolarization following the initial response to a brief flash of light. Through this additional pathway, rod signals can utilize the fast-tuned cone pathways to access the inner retina.

Recent recordings from mouse ganglion cells suggest a direct pathway from rods to cone bipolar cells (Soucy et al., 1998). In a mouse retina genetically modified to be “coneless,” a fast rod signal was detected in OFF ganglion cells,

suggesting the existence of direct connections between rods and OFF cone bipolar cells.

Confocal and electron microscopy have demonstrated the existence of symmetrical contacts involving rod spherules and the dendrites of OFF cone bipolar cells, which therefore collect direct input from rods (Hack et al., 1999; Tsukamoto et al., 2001). The dendrites of such cone bipolar cells express ionotropic glutamate receptors at the site of apposition to rod spherules. Hence, rod-generated signals can exit the retina through gap junctions between rods and cones, as well as through this third pathway using mixed cone-rod bipolar cells. Apparently, only 20% of all the rods are involved in this particular type of connection with OFF cone bipolar cells. However, it has been proposed that rod-generated signals can enter this pathway through rod-rod gap junctions, which are infrequent in most mammals but apparently common in the mouse retina (Tsukamoto et al., 2001).

It must be noted that, although the alternative rod pathway was first discovered in rodents, anatomical evidence for direct connections between rods and OFF cone bipolar cells has now been provided for the rabbit as well (Li et al., 2004). However, electrophysiological and pharmacological experiments have shown that, in the mouse retina, only a low proportion of OFF signals are carried in parallel to rod bipolar cells, and no ganglion cells at all in the rat retina display OFF responses attributable to direct contacts between rods and OFF cone bipolar cells (Protti et al., 2005). This observation suggests that the alternative rod pathway may be a common feature of the mammalian retina but that its relative importance and significance differ between species.

A multidisciplinary approach has demonstrated that all three rod pathways are functional in the mouse retina but operate under stimulus intensity ranges that are widely different, so that the primary rod pathway carries signals with the lowest threshold, whereas the secondary rod pathway (based on rod-cone gap junctions) is approximately 1 logarithmic unit less sensitive (Völgyi et al., 2004). Some ganglion cells receive signals preferentially from one pathway, while others exhibit convergent signals.

It is worth noting that all three rod pathways, the standard route and the two indirect ones, ultimately exploit cone bipolar axonal endings to gain access to ganglion cells. Thus, although in the beginning the neural network strictly associated with rods (composed of rods, rod bipolar cells, and AII amacrine cells) is quite minimal, ultimately the whole retinal machinery is shared by both the scotopic and the photopic pathways.

I am fond of the idea that the general use of the piggyback arrangement might be justified in evolutionary terms: conemediated vision and color vision evolved in parallel and before dim light vision (Bowmaker, 1998). Hence, the ancestral inner retina was shaped by cones. Insofar as each cone brings in several types of cone bipolar cells with various

functional properties, it is tempting to speculate that the antique vertebrate retina was constituted by several types of cone bipolar cells and cone-driven amacrine cells that diversely made connections to ganglion cells. Later in evolution rhodopsin appeared, and the ancestors of modern rods emerged. The preexisting retinal network, with the already achieved computational capabilities, was made available to the newly evolved photoreceptors tout court and without undesirable duplication: rod bipolar cells (of a single type) ensured high convergence (and thus high sensitivity) of rods in the scotopic pathway; AII amacrines, which received the bulk of rod bipolar synapses, recruited the cone pathways, connecting to ON and OFF cone bipolar cells in the IPL. Among other advantages, the piggyback architecture ensures access of the scotopic signal to parallel processing, which originally evolved in the cone system.

The notion that rod and cone pathways are not only exquisitely balanced but also deeply integrated is reinforced by, among other things, the identification of secondary effects caused by various forms of inherited photoreceptor degeneration (such as retinitis pigmentosa) on neurons of the inner retina. In this family of diseases, even though the primary genetic defect occurs in rods, which die first, cones undergo secondary degeneration. Inner retinal cells, and particularly rod bipolar cells, horizontal cells, and cone bipolar cells, display abnormal morphologies and eventually die out, while gliosis and general atrophy are observed. Such a complex chain of events, called remodeling (Jones et al., 2003; Marc et al., 2003; Gargini et al., 2007), strongly suggests that maintaining a normal morphology, as well as the long-term survival of second-order neurons, requires the presence of viable photoreceptors. This brings to light the existence of (possibly trophic) interactions normally occurring between the outer and the inner retina and acting in parallel to the synaptic-related communication.

Horizontal cells

Cell bodies of horizontal cells form the outermost tier of the INL; their processes connect within the borders of the OPL. Each horizontal cell is postsynaptic to a large cohort of photoreceptors and has the important task of averaging their signals, feeding them back onto photoreceptor synaptic terminals, and at the same time feeding them forward onto the dendrites of bipolar cells. Horizontal cells are connected to each other by large gap junctions that provide electrical coupling. The strength of the coupling varies with the retinal adaptation to light and is modulated by, among other substances, dopamine released by amacrine cells. Horizontal cells therefore play a key role in the mechanism of neural adaptation, because through their feedback they adjust cone sensitivity and shape the RFs of bipolar and ganglion cells.

Figure 12.5 Confocal image of a whole mount mouse retina in which horizontal cells are revealed with antibodies against calbindin D (red signal), while their axonal complexes are labeled with antineurofilament antibodies (green staining). See color plate 4.

Although most vertebrates have at least two types of horizontal cells, rodents (and thus mice) are a noticeable exception in that they carry only a single variety (Peichl and Gonzalez-Soriano, 1994), the one with a long, thin axon that ramifies into an elaborate and rich axonal arborization. Although dendritic branches emerging from the cell somata are postsynaptic to cones, axonal arborizations connect to rod spherules exclusively. Electrophysiology has shown that cone input from the dendrites does not reach the axonal arborization, connected to rods (Suzuky and Pinto, 1986). Therefore, a single soma provides metabolic support to two sets of processes with completely different connections and virtually isolated. In the mouse retina, the whole plexus of horizontal cells can be stained by antibodies against the calcium-binding protein calbindin D. Antibodies against the heavy subunit of neurofilament proteins instead reveal only horizontal cell axonal arborizations (figure 12.5). There are approximately 18,000 horizontal cells in the retina of the C57Bl6/J mouse.

Cone pathways to ganglion cells

Retinal parallel processing begins at the first synapse between photoreceptors and different types of bipolar cells. These carry glutamate receptors of heterogeneous molecular composition that thereby give rise to a variety of parallel channels that run vertically across the retina (Wässle, 2004).

Cones respond to light stimuli with a graded hyperpolarization and release glutamate at multiple synaptic sites endowed in each pedicle. Glutamate release is higher in the dark and is reduced by light shed onto cones. Unlike rod spherules, which only make connections to the terminal

process of one horizontal cell and to one to two dendrites of rod bipolar cells, each cone pedicle has numerous postsynaptic partners (see figure 12.2). As in all vertebrates, two types of bipolar cell contacts are typically found: flat (or basal) contacts and invaginating (or ribbon) contacts (Dowling and Boycott, 1966). The dendritic tips of invaginating bipolar cells are flanked by two lateral horizontal cell dendrites in the typical triad configuration, much like that described earlier for rod spherules. The dendritic terminals of flat bipolar cells make numerous contacts at the pedicle membrane facing the OPL (see figure 12.2). Basal contacts mediate sign-conserving synapses with cone bipolar cells, which therefore respond to light with graded hyperpolarizations (OFF cone bipolar cells); ribbon contacts, instead, mediate sign-inverting synapses with another group of cone bipolar cells that respond to light with graded depolarizations (ON cone bipolar cells). The molecular basis for the functional effects of these synaptic contacts is well known: cone bipolar cells establishing flat contacts carry ionotropic glutamate receptors on their dendritic tips, whereas those engaged in invaginating contacts instead mainly express mGluR6, exactly like rod bipolar cells. According to a general rule in retinal architecture that has no known exceptions, OFF bipolar cells have axonal endings that ramify in the outer third of the IPL, sublamina a; conversely, cone bipolar cells that respond to light with a depolarization end in the deepest part of the IPL, or sublamina b. The ON-OFF dichotomy, established at the first synaptic station in the retina, is maintained throughout the visual system.

Traditionally, the stratification and morphology of the axonal arborizations of bipolar cells have been used as main distinguishing criteria, more than the shape of their dendritic arbors or the size of the soma. Unfortunately, the molecular determinants of cone bipolar cells are very similar, and it is difficult to make a distinction among them using cell-type- specific antibodies, a method that has been largely used to visualize retinal neurons. Hence, population studies of types of cone bipolar cells are rare for the mouse retina. Antibodies against the neurokinin receptor 3 label a large population of OFF cone bipolar cells whose axonal arbors span the

whole thickness of sublamina a. These cells number approximately 90,000 per retina in the C57Bl6/J mouse (Pignatelli and Strettoi, 2004), but it is not easy to tell whether they form a homogeneous or a mixed population. In addition, a subset of them can be stained with recoverin antibodies (Haverkamp and Wässle, 2000), but the intensity of the staining and the number of stained cells vary in degenerating retinas, making it difficult to assign a functional meaning to the immunolabeling. It is clear that at least a fraction of them coincide with the type called CBb1 by Tsukamoto et al. (2001), which receives multiple contacts from the lobular appendages of AII amacrine cells in the sublamina OFF of the IPL (see figure 12.4).

A transgenic mouse line (357) has been created that expresses GFP in all members of a single type of ON cone bipolar cell and coincides with that termed CB4a by Pignatelli and Strettoi (2004) and with type 7 of Ghosh et al. (2004). One type of monostratified ON ganglion cell and the inner dendrites of one bistratified ganglion cell tightly cofasciculate with axon terminals of the line 357 bipolar cells and are likely to receive synaptic input from them (Lin and Masland, 2005).

Superimposed on the ON/OFF dichotomy are four types of OFF and five types of ON cone bipolar cells (figure 12.6). We are just beginning to understand their distinguishing features and their functional roles (Euler and Wässle, 1995; Hartveit, 1997; Euler and Masland, 2000; Berntson and Taylor, 2000; Freed, 2000). For instance, different types of ON cone bipolar cells express at least two different connexins at their gap junctions with AII amacrine cells (Han and Massey, 2005; Lin et al., 2005). It is not unlikely that two types of gap junction have distinctive physiological or regulatory properties (Mills and Massey, 2000), optimized to meet the functions of particular subsets of cone bipolar cells. Hence, the visual signal could be differently processed by types of ON cone bipolar cells expressing different electric junctions.

Some of the bipolar cells select certain types of cones, such as the blue cone bipolar cells, and thus transfer a chromatic signal into the IPL. In the mouse, the type labeled 9 in the

Figure 12.6 The various types of bipolar cells of the mouse retina, classified after labeling with fluorescent dyes delivered with a gene gun. AII amacrine cells are represented in light gray in the background. Nine types of cone bipolar cells (CB) and one type of

rod bipolar cell (RB) have been identified. (From Pignatelli and Strettoi, 2004.) A classification produced by Ghosh et al. at the same time and based on intracellular injection is consistent with the types illustrated here.

classification of Ghosh et al. (2004) and CB5 in the classification of Pignatelli and Strettoi (2004) has sparsely branching and wide dendrites in the OPL; the axonal arborization is narrow and unistratified in the innermost tier of the IPL (see figure 12.6). This ON cell is strongly reminiscent of the blue cone bipolar cell described in other species (Boycott and Wässle, 1991; Euler and Wässle, 1995). Recently, blue cone bipolar cells have been labeled in a transgenic mouse expressing clomeleon, a chloride-sensitive fluorescent protein under the control of the Thy1 promoter. It was shown that blue cone bipolar cells constitute only 1%–2% of the bipolar cell population, and their dendrites selectively contact cones that express short-wavelength opsin (S cones) (Haverkamp et al., 2005).

Unlike the blue ON cone bipolar, most bipolar cells contact all the cones, usually five to ten, within their dendritic field; despite the nonselectivity in their synaptic input, these bipolar cells differ in intrinsic properties. For instance, OFF bipolar cells can be further subdivided according to the specific expression of AMPA or kainate receptors on their dendrites (DeVries, 2000). The physiological consequences of this molecular diversity are different temporal resolution and possibly different threshold sensitivity. It has been observed that the different types of OFF and ON cone bipolar cells can provide separate channels for high-fre- quency and low-frequency information.

Recent data support the notion that bipolar cells are capable of discriminating between the sustained and transient components of the light stimuli (Cohen and Sterling, 1992; Roska and Werblin, 2001; Werblin et al., 2001). This possibility would arise from the ordered and layered organization of the retina: each type of cone bipolar cell should be able to provide a characteristic stimulation of a selected type of ganglion cell that cofasciculates at the same level of the IPL. Inhibition, on the contrary, would take place more diffusely, by means of amacrine cells (Masland, 2001). The presence of a variety of cone bipolar cell types is thought to reflect a variety of parallel functions; although their different roles are only beginning to be understood, all the available evidence indicates that the various types transmit different representations of the visual scenery to the inner retina. The power of computation of the cone pathway is exploited by the rod pathway as well, which is “grafted” onto the cone system by means of the connections of AII amacrine cells.

In light of the similarities of the various types of cone bipolar cells, the use of transgenic mice that express fluorescent markers in one or more populations of bipolar and ganglion (or amacrine) cells appears to be the most promising method for understanding their specific pattern of connections. The small size of these neurons and their relatively high density make it difficult to use single-cell injection as a major tool to study bipolar cell circuitry. Because of the resolution limits of confocal microscopy, however, the assess-

ment of true synaptic contacts remains to be identified at the electron microscopy level.

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Just as for other CNS loci, the major mode of neuronal communication in the retina is chemically mediated synaptic transmission. However, it has been long known from serial reconstructions of electron micrographs that some neighboring retinal neurons form gap junctions between their closely opposed plasma membranes, suggesting a role for electrical transmission as well. In fact, coupling between horizontal cells was described more than 40 years ago by Yamada and Ishikawa (1965), some 5 years before Goodenough and Revel (1970) coined the term “gap junction.”

Gap junctions, the morphological substrate of electrical synapses, are composed of two hemichannels or connexons that link across the extracellular space to form an intercellular pathway for diffusion of molecules up to about 1,000 daltons. Hemichannels are hexameric structures composed of six subunit transmembrane proteins called connexins. Twenty different connexin genes have been characterized in the mouse, and a number of connexin proteins are widely expressed in murine retinal neurons, including connexin 36 (Cx36), connexin 45 (Cx45), and connexin 57 (Cx57) (Söhl and Willecke, 2003; Söhl et al., 2005; Kamasawa et al., 2006). In fact, recent studies suggest that gap junctions are found in almost all of the approximate 60 subtypes of neuron in the retina, indicating that electrical synaptic transmission plays a significant role in retinal signal processing. The degree of coupling between retinal neurons does not appear to be a static process but shows high plasticity regulated by neuromodulators such as dopamine and nitric oxide, whose release is dependent on the adaptational state of the retina (Lasater and Dowling, 1985; Witkovsky and Dearry, 1991; DeVries and Schwartz, 1992; Hampson et al., 1992; Bloomfield et al., 1997; Xin and Bloomfield, 1999a, 1999b).

Still, the role of electrical transmission in the retina and brain has long been underestimated. Over the past decade, owing to recent technical advances in cell labeling techniques, particularly the advent of the gap junction permeable biotinylated tracers, electrophysiology, and molecular cloning, studies of electrical synaptic transmission in the retina have proliferated. The use of mouse mutants in which

selective gap junctions are disrupted by targeting connexin genes has become a particularly important tool to characterize the function of these electrical synapses. In this chapter we review the distribution of gap junctions in the mouse retina and recent work detailing their significant and varied functional roles in visual processing.

Photoreceptor gap junctions

As shown in a variety of mammalian retinas (Raviola and Gilula, 1973; Kolb and Jones, 1985; Tsukamoto et al., 1992), direct gap junctional coupling occurs between the axon terminals of neighboring cone photoreceptors and also between rods and cones in the mouse (Tsukamoto et al., 2001). β-gal and PLAP reporters in heterozygous Cx36 knockout (KO) mouse lines were detected within cell bodies in the outer nuclear layer (ONL) and in processes extending distally to the region occupied by photoreceptor inner segments (Deans et al., 2002) (figure 13.1A). The widespread labeling of photoreceptors suggested that Cx36 was at least expressed by rods, which constitute 97% of all photoreceptors in the mouse (Jeon et al., 1998). However, a study of two Cx36 transgenic mutants indicated that Cx36 was expressed only in cone photoreceptors and thus subserved both the homologous cone-cone and heterologous rod-cone coupling (Feigenspan et al., 2004). The Cx36 expression limited only to murine cone photoreceptors was consistent with the distribution described in the guinea pig retina (Lee et al., 2003).

Electrical coupling between cones has been shown to increase the signal-to-noise ratio of their visually evoked responses (DeVries et al., 2002). Since the intrinsic noise produced in neighboring cones is independent, whereas their visual signals are partially shared, electrical coupling averages out the noisy fluctuations in voltage more than the response signals. The conductance of cone-to-cone gap junctions does result in a small blur, but this is lower than that of the eye’s optics. Thus, the signal fidelity gained by electrical coupling between cones outweighs any compromise in visual acuity.

C

axon terminal

axon

s

Figure 13.1 A, β-Gal reporter in transgenic mouse retina indicates that Cx36 is expressed by photoreceptors in the ONL and by bipolar cells and amacrine cells in the INL. Small arrowheads indicate photoreceptor somata; large arrowheads indicate somata of an AII amacrine cell and bipolar cells. B, Transverse view of immunolabeling of the wild-type mouse retina for Cx36. Labeling is confined to the plexiform layers, consistent with the known locations of gap junctions between retinal neurons. C, Immunolabeling for Cx36 is absent in the Cx36 KO mouse retina. See color plate 5. (A, Adopted from Deans et al., 2002, with permission. B and C, Adopted from Deans et al., 2001, with permission.)

In contrast, the coupling between rods and cones is believed to form a secondary rod pathway in which scotopic signals can be communicated directly to cones and then relayed to ganglion cells via the cone bipolar cells. Evidence for the functionality of this pathway include demonstrations of rod signals within cone photoreceptors (Nelson, 1977; Schneeweis and Schnapf, 1995) and the survival of rod signals at the ganglion cell level after blockade of the principal rod pathway subserved by rod bipolar and AII amacrine cells (Strettoi et al., 1990, 1992; DeVries and Baylor, 1995). In addition, studies of the Cx36 KO mouse retina showed that disruption of rod-cone coupling resulted in a significant loss of rod signaling to ganglion cells (Deans et al., 2002). Consistent with human psychophysical evidence (Sharpe and Stockman, 1999), these studies further indicated that the primary rod pathway conveys the most sensitive rod signals to the ganglion cells, whereas the secondary pathway conveys higher threshold scotopic signals (Deans et al., 2002; Völgyi et al., 2004).

In contrast to larger mammals, rods are homologously coupled to each other via gap junctions in the mouse retina (Tsukamoto et al., 2001). Interestingly, approximately 20% of mouse rods form a chemical synapse with a mixed rod-cone bipolar cell, thus creating a third rod pathway for scotopic OFF signal transmission to the inner retina (Soucy

Figure 13.2 Tracer-coupled group of horizontal cells in the mouse retina following injection of single horizontal cell soma with Neurobiotin. The cluster of darkly labeled somata (s) are surrounded by axon terminals and connecting axons. Scale bar = 50 μm.

et al., 1998; Tsukamoto et al., 2001). It has been proposed that rod-rod coupling pools the scotopic signals at the photoreceptor level for conveyance to the ganglion cells via the third pathway. This third rod pathway may thus be useful at dusk and dawn, when relatively greater numbers of photons are available than during starlight and the pooled signal may thereby more efficiently encode faintly backlit objects. Physiological evidence for this third pathway was recently provided by Völgyi et al. (2004), who described ganglion cells with scotopic sensitivities that were lower than those of signals conveyed by the primary and secondary rod pathways. Further, signals presumably transmitted via the third rod pathway survived in the Cx36 KO mouse retina, suggesting that pooling of scotopic signals via rod-rod coupling was still intact. This lends further support to the notion described earlier that Cx36 is not expressed at the gap junctions between rod photoreceptors in the mouse.

Horizontal cell gap junctions

Most vertebrate retinas contain two subtypes of horizontal cell, one that is axonless and a second that maintains an axon that typically extends for a few hundreds microns before ending in an elaborate terminal arbor (Fisher and Boycott, 1974; Kolb, 1974; Boycott et al., 1978). Only the axonbearing subtype of horizontal cell is found in the mouse retina (He et al., 2000). Neighboring horizontal cells in mammalian retinas are extensively coupled via gap junctions (Kolb, 1974; Raviola and Gilula, 1975; Vaney, 1991; Bloomfield et al., 1995; He et al., 2000) (figure 13.2). The axonless

and axon-bearing horizontal cells show only homologous coupling resulting in separate electrical syncytia. Likewise, the somatic and axon terminal endings of the axon bearing horizontal cells are segregated into homologously coupled networks.

The horizontal cell gap junctions form an efficient pathway for intercellular electrical communication whereby the receptive fields (RFs) of individual horizontal cells dwarf the size of their dendritic arbors (Tomita, 1965; Naka and Rushton, 1967; Bloomfield and Miller, 1982). Coupled with feedforward and/or feedback chemical synaptic transmission, the enlarged RFs of horizontal cells are thought to mediate the surround RFs of bipolar cells necessary for contrast discrimination (Naka and Nye, 1971; Naka and Witkovsky, 1971; Bloomfield et al., 1995).

There is abundant evidence that the gap junctions connecting horizontal cells are dynamically regulated by the neuromodulator dopamine as a mediator of light adaptation (Witkovsky and Dearry, 1991). Application of dopamine or cAMP reduces the coupling between the somatic endings of mouse horizontal cells, whereas the coupling is significantly increased by D1 receptor antagonists (He et al., 2000). Interestingly, these agents appear to have no effect on the coupling between horizontal cell axon terminals, suggesting that the subunit composition of their interconnecting gap junctions is different from that of the somatic junctions (He et al., 2000).

Mouse horizontal cells do not express either Cx26 or Cx36 (Deans and Paul, 2001). They do express Cx57, and tracer coupling is abolished in the Cx57 KO mouse retina, indicating that this connexin protein is critical for horizontal cell electrical coupling (Hombach et al., 2004). Indeed, horizontal cells in the Cx57 KO mouse retina show a significant reduction in their RF size compared with that seen in wild-type animals (Shelley et al., 2006). Coupling appears to be lost for both somatic and axon terminal endings, indicating that Cx57 is crucial for both sets of gap junctions. However, as mentioned earlier, the results of He et al. (2000) showing different pharmacological sensitivities of these junctions suggest that whereas they both express Cx57, they may be heteromeric, with different overall connexin composition.

Recently, Kamermans et al. (2001) posited that the feedback circuit from horizontal cells to cone photoreceptors in the fish retina may rely on ephaptic transmission via Cx26 hemichannels. In this scheme, electrical charge moving across hemichannels communicates with the extracellular space and modifies the activity of nearby cone photoreceptor axon terminals. Although this mechanism has not been confirmed in the mammal, it is possible that Cx57 could also form functional hemichannels on mouse horizontal cells for which Cx26 protein has not been detected (Deans and Paul, 2001).

Bipolar cell and amacrine cell gap junctions

Bipolar cells can be coupled either heterologously to amacrine cells or homologously to other bipolar cell neighbors. The different mosaics and coverage factors for the subtypes of bipolar cells in the mouse suggest that only a few may display the dendritic overlap necessary for homologous coupling (Mills and Massey, 1992; Massey and Mills, 1996; Lin et al., 2005). Indeed, the only evidence for homologous coupling of murine bipolar cell comes from a study of transgenic mice in which Cx36 expression was found on the dendrites of three subtypes of OFF bipolar cells just below the cone pedicles (Feigenspan et al., 2004). Interestingly, Cx45 expression has been found in all four types of OFF cone bipolar cells, suggesting that a subset may express more than one connexin (Maxeiner et al., 2005), although they may be spatially segregated to the outer and inner plexiform layers.

In contrast, a number of studies have been made of the heterologous gap junctions formed between ON cone bipolar cells and AII amacrine cells. To date, only the coupling of the AII subtype of amacrine cell has been studied in the mouse retina. The junctions formed by AII amacrine cells are discussed in detail in the next section.

Gap junctions in the proximal rod pathways

As mentioned, there are three rod pathways in the mammalian retina (Bloomfield and Dacheux, 2003). The role of rod-cone and rod-rod coupling in the secondary and tertiary rod pathways, respectively, was discussed earlier. The principal rod pathway for ON signaling is rods → rod bipolar cells → AII amacrine cells → ON cone bipolar cells → ON ganglion cells. There are two sets of gap junctions found in this pathway: homologous AII-AII cell junctions and heterologous AII-ON cone bipolar cell junctions. Cx36 is abundantly expressed by AII amacrine cells (Feigenspan et al., 2001; Deans et al., 2002) (see figure 13.1) and AII cell-AII cell coupling is lost in the Cx36 KO mouse retina (Deans et al., 2002) (see figure 13.3) indicating that Cx36 comprises the homologous junctions between these cells. Based on a computational model, Smith and Vardi (1995) speculated that AII cell-AII cell coupling serves to sum synchronous signals and subtract asynchronous noise, thereby preserving the fidelity of the high-sensitivity signals carried by the primary rod pathway. This function is similar to that described earlier for cone photoreceptor coupling in the outer retina. Consistent with this idea, the intensity-response profiles of the most sensitive ganglion cells in the mouse retina show a rightward shift in the Cx36 KO retina due to an approximate one log unit loss of sensitivity (Völgyi et al., 2004) (figure 13.4). This results in equal sensitivities for the rod signals carried by the primary and secondary rod