Outer retinal junctions

In the outer retina, gap junctions couple neighboring photore-ceptors, or horizontal cells or bipolar cells (Fig. 2), but only rarely join different major cell types. Interestingly,Trexler et al. (2001) reported various ON and OFF bipolar cells that were occasionally found to be coupled to the horizontal cell network in the rabbit.

However, these connections were concluded to be rare.

4.1.1. Photoreceptor-to-photoreceptor junctions

In most vertebrate species, photoreceptors couple one another via homologous, rod-to-rod, cone-to-cone as well as heterologous rod-to-cone electrical synapses (Figs. 2and4A and B). While such coupling may reduce visual acuity, it is also beneficial for retinal signaling.

Observations using tracer coupling, electrophysiology and electron microscopy demonstrate that rod-to-rod coupling exists in fish (Owen, 1985), amphibians (Fain et al., 1976;Owen, 1985;Krizaj et al., 1998), reptiles (Schwartz, 1975a;Firsov and Green, 1998) and mammals (Tsukamoto et al., 2001;Hornstein et al., 2005). However, the cellular location of gap junctions differs slightly. For example, gap junctions are located quite distally between amphibian pho-toreceptors; they are found on radially extruding membranousfins of rod myoids in axolotls and toads (Fain et al., 1976;Owen, 1985), or even more distal inner segment regions in theXenopus(Krizaj et al., 1998). In contrast, rod-to-rod gap junctions in teleostfish, turtles and mammals are found rather proximally between telo-dendria, between the soma and the rod terminal or the rod ter-minal and the passing rod axon (Owen, 1985; Tsukamoto et al., 2001; O’Brien et al., 2012). While the possession of a single rod type is quite general across vertebrates, some anurans have two or

more rod populations with different spectral sensitivities whose coupling is restricted to the like spectral type (Gold and Dowling, 1979). The connexin subunit makeup of rod-to-rod gap junctions has only been identified in a few animals, including Cx43 in zebrafish and carp (Janssen-Bienhold et al., 1998) and Cx35/36 in the salamander (Zhang and Wu, 2004). These connexin subunits typically form channels with relatively low unitary channel con-ductances (Cx43,Bennett and Verselis, 1992; Cx36,Teubner et al., 2000), allowing for reciprocal, symmetrical but weak electrical coupling between rods (Gj: 500 pS;Zhang and Wu, 2005). Further, low conductance is ascertained by the relatively small size of focal gap junctions between photoreceptors. It is important to point out here, that the strength of coupling is not a function solely of the gap junction conductance, but also that of the cell. So for example, a 500 pS conductance would be trivially small in a horizontal cell with 10 MU input resistance, but it is a lot more significant in a photoreceptor (which has about a 1 GUinput resistance). Col-lectively, therefore, the gap junctions play a large role in the rod network. A possible corollary point is that evolution has matched the gap junction conductance to its cell type.

There is a paucity of data on the connexin makeup of reptile and mammalian rod-to-rod electrical synapses but the extent of tracer-coupling (Firsov and Green, 1998;Hornstein et al., 2005) as well as the relatively small gap junction plaques between rod receptors in ultrastructural studies (Tsukamoto et al., 2001) suggest low overall junctional conductances similar to those offish and salamander. In fact, guinea pig rod-to-rod electrical synapses have recently been presented displaying a low overall conductance of 386 pS (Li et al., 2012) similar to those observed in lower vertebrates. The low conductance of rod-to-rod electrical synapses has been thought to favor the signal passage with slow kinetics, which is a characteristic of single photon evoked rod responses (Zhang and Wu, 2005). In addition, rod homologous coupling increases the signal-to-noise ratio of rod photoreceptors. This is especially important in low light levels when rods absorb photons only sporadically, making light-evoked signals relatively weak considering the high level of dark-noise of the individual rods. It is also possible that, similar to a feedback loop of an amplifier, they moderate the high gain of the output synapse to make the best possible use of its dynamic range (Jacobs and Werblin, 1994). Hypothetically, rod-to-rod coupling fulfills yet another role in mammals, where rods and cones gen-erally form synapses with a separate set of rod and cone bipolar cells, respectively. In this scheme, rod-to-rod coupling pools signals to those scarce (<20%) rods that form‘irregular’chemical synapses with one type of OFF cone bipolar cell (Hack et al., 1999;Tsukamoto et al., 2001;Fyk-Kolodziej et al., 2003;Li et al., 2004a,b).

Cone coupling has been found in most examined vertebrates (turtle:Baylor et al., 1971; turtle, monkey and rabbit:Raviola and Gilula, 1973). The molecular structure of cone-to-cone gap junc-tions are only known in a few systems, where they are formed by Cx36 subunits (birds:Kihara et al., 2009; mammals:Deans et al., 2002;Feigenspan et al., 2004;Lee et al., 2003) that allow for low trans-receptor conductance. It appears that coupling is generally present between spectrally identical receptor subtypes in reptiles (turtle:Detwiler and Hodgkin, 1979) and mammals (ground squir-rel:Li and DeVries, 2004; monkey:O’Brien et al., 2012). However, spectrally indiscriminate coupling occurs in the trichromate primate retina between red and green cones, in which case coupled cones contain spectrally different but evolutionarily closely related pho-tosensitive molecules (Hornstein et al., 2004). Similar to roderod gap junctions, cone coupling averages asynchronous noise out and sums correlated light-evoked signals (DeVries et al., 2002). In con-clusion, rod-to-rod and cone-to-cone electrical coupling seems ubiquitous in all vertebrate classes, in which both are comprised by similar connexin building blocks and play very similar roles.

Regardless of the retinal pathway by which photoreceptor sig-nals reach the output ganglion cells, rod and cone mediated sigsig-nals mix in most vertebrate species. In lower vertebrates this mixing of information is achieved by the convergence of rod and cone signals onto the same second order bipolar and horizontal cells. In contrast, mammals have two sets of bipolar cells specialized to receive in-formation from either rods or cones. However, the mixing of rod and cone signals still occurs in mammals through electrical syn-apses that connect rods to neighboring cones (Schwartz, 1975b;

Scholes, 1975;Copenhagen and Owen, 1976). An unusual feature of

mammalian horizontal cells is that its perikaryal dendrites connect only to cones, whereas its axon terminal contacts rods exclusively.

Moreover, these two cellular compartments are electrically iso-lated. Thus mixing of rod and cone signals in horizontal cells de-pends on gap junctional connections between rod and cone photoreceptors. Mixing signals at the photoreceptor level ensures that both the soma-dendritic region and the axon terminal of horizontal cells operate under both scotopic and photopic condi-tions. Most vertebrates also contain axonless horizontal cells as well that receive classical chemical synaptic inputs exclusively from Fig. 4.Tracer coupling patterns of outer retinal neurons.A. Neurobiotin injection into a single rod photoreceptor (asterisk) in theXenopusretina displays the coupling of a group of neighboring rods (arrows) and a single cone (open arrow).B. Rodecone coupling in cross-sections of theXenopusretina. A tracer-coupled cone (asterisk) adjacent to neurobiotin-injected and tracer-coupled rod outer segments. OS: outer segments, IS: inner segments, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer.C. Lucifer yellow injected luminosity-type horizontal cells in the turtle retina. Courtesy of Dr. Paul Witkovsky. An extensive array of coupled axon terminals (open arrow) as well as several coupled cell bodies (arrows) are shown. The soma of the Lucifer yellow injected horizontal cell is marked with an asterisk.D.

Neurobiotin-injected luminosity-type horizontal cells in a section from resin-embeddedXenopusretina. The injected cell is labeled with an asterisk, arrows indicate cell bodies of coupled cells while the open arrow shows a dendritic bundle.E. Neurobiotin-injected chromatic horizontal cells (arrows) in a cross section of theXenopusretina. Retinal layers are as above.F: Photomicrograph showing an extensively coupled synthitium of axon-bearing horizontal cells in the mouse retina. Arrows point to somata, whereas open arrows mark the horizontal cell axon terminals. Scale bars: 40mm inA,B,D,E; 100mm inCand 200mm inF.

cones, whereas rod signal reaches them indirectly via rodecone electrical coupling (Nelson, 1977; Smith et al., 1986). It appears that low conductance Cx36 subunits makeup the cone hemi-channels of most studied mammals (guinea pig:Lee et al., 2003;

mouse: Güldenagel et al., 2001; Feigenspan et al., 2001; Deans et al., 2002). Interestingly, a mixture of Cx34.7 and Cx35 (thefish orthologs of mammalian Cx36) was detected in the bass retina (O’Brien et al., 2004). Contrary to the apparent dominance of Cx36, the identity of rod hemi-channels is uncertain in cones, as a num-ber of reports claim that rods do not express Cx36 (Lee et al., 2003;

O’Brien et al., 2004). A more recent study nonetheless suggests that Cx36 in fact forms the rod hemichannel in the monkey retina as well (O’Brien et al., 2012). There is an unfortunate lack of data on the connexin structure of amphibian, reptile and bird retina.

However, the similar connexin makeup of such phylogenetically distant animal groups such asfishes and mammals suggests that all vertebrate rod-to-cone gap junctions are formed by similar connexins. Of course, one can argue that convergent evolution lead independently to a very similar connexin makeup infish and mammalian rod-to-cone gap junctions to serve similar functions and intermediary lineages do not necessarily share the same fea-ture. Descriptions of various species agree that rod-to-cone elec-trical synapses are not controlled by the ambient light level (monkey: Schneeweis and Schnapf, 1999; mouse and fish:

Ribelayga et al., 2008) but they rather follow the retinal circadian rhythm via dopamine controlled mechanisms in both fish and mammals (Ribelayga et al., 2008). During the day, the level of secreted dopamine increases by which a D2/D4receptor initiated and cAMP/PKA driven intracellular cascade decrease the con-ductance of rod-to-cone gap junctions (Xenopus: Krizaj et al., 1998). This quite similar dopamine mediated control of rod-to-cone gap junction gating infish, amphibians and mammals rein-force the above-mentioned hypothesis concerning the similar connexin building blocks of rod-to-cone electrical synapses.

Therefore, it is highly likely that rod-to-cone gap junctions are not just ubiquitous but their molecular structure is conserved across vertebrates as well.

4.1.2. Horizontal-to-horizontal cell junctions

Horizontal cells are second order inhibitory interneurons whose dendrites and axons extend laterally to contact cone pedicles and rod spherules, respectively (Van Haesendonck and Missotten, 1979;

Wässle and Riemann, 1978). In these synapses, horizontal cells provide inhibition onto photoreceptors, which is a crucial element in forming the receptivefield center-surround mechanism for down-stream retinal neurons. Various vertebrate species display one or more types of horizontal cell in their retina according to the level of photoreceptor diversity in the corresponding species. In essence, each photoreceptor population has its own horizontal cell type that feeds one spectrally tuned photoreceptor with inhibition while others are active (Wu,1992;Stell and Lightfoot, 1975). Irrespective of the number of subtypes, each horizontal cell population appears to form type-specific, extensively coupled syncytia (Figs. 2 and 4CeF), a universal feature of all vertebrate horizontal cells (rabbit e Bloomfield et al., 1995; mouseeJanssen-Bienhold et al., 2009;Xin and Bloomfield, 1999; dogfishe Kaneko, 1971; turtleePerlman and Ammermüller, 1994; frogseMascetti and Ogden, 1989; sala-mandereLasansky, 1980; chickeCooper and McLaughlin, 1981).

Interestingly enough, axon-bearing horizontal cells form two inde-pendent coupled arrays, one connecting the soma/dendritic regions of neighbor horizontal cells and another, spatially somewhat more restricted, array of horizontal cell axon terminals. Homologous coupling of horizontal cells serves to average background luminance mediated signals across the electrically connected horizontal cell array. This averaged signal provides an equalized inhibition over

large retinal areas (the luminous background signal) against which the local photoreceptor signal is distinguished. This is the basic cir-cuit for contrast detection in the photoreceptor layer, and it is reprized in the bipolar cells. Such signal averaging mechanism re-quires high conductance electrical synapses among horizontal cells.

The relatively easy passage of current and tracers through horizontal cell gap junctions has been indicated by a cohort of findings including: (i) the extensive tracer coupling, (ii) the extended receptive field size (Bloomfield et al., 1995; Xin and Bloomfield, 1999) and(iii)the passage offluorescent dyes like Procion Yellow and Lucifer Yellow (Kaneko, 1971;Dacheux and Raviola, 1982). The molecular structure of the low resistance horizontal cell connections were found to be slightly variable across species as Cx57 mediates coupling in mouse (Janssen-Bienhold et al., 2009;Palacois-Prado et al., 2009), Cx50/Cx57 in rabbit (Huang et al., 2005;O’Brien et al., 2006;Cha et al., 2012), Cx35/36 in carp (Liu et al., 2009) and both Cx52.6 and Cx55.5 in zebrafish (Zoidl et al., 2004). However, analyses of DNA coding sequences revealed that most of these connexins (with the lone exception of the carp Cx35/36) display remarkably high sequence homology (see alsoFig. 1), and represent connexin orthologs rather than different species-specific connexins. Among all vertebrates, perhapsfish display the highest variety of connexin subunits utilized to form horizontal-to-horizontal cell gap junctions.

This diversity in connexin makeup likely corresponds to the large diversity of horizontal cell subtypes described in the retina of vari-ousfish species (Stell and Lightfoot, 1975;Connaughton and Nelson, 2010;Schieber et al., 2012). It is tempting to speculate that each horizontal cell type expresses different connexin subunits to form homologous junctions. Indeed,Dermietzel et al. (2000)suggested that Cx55.5 is expressed by only a subset of zebrafish horizontal cells, whereas the rest of the horizontal cells expressed other connexins.

Apart from Cx50, these previously mentioned connexin subtypes form channels with relatively moderate unitary conductance (McMahon, 1994;Lu and McMahon, 1996;Srinivas et al., 1999a,b;

Zoidl et al., 2004;Palacios-Prado et al., 2009). However, many of them aggregate in huge plaque-like structures (Witkovsky et al., 1983;Janssen-Bienhold et al., 2009), thus providing afirm basis for the peculiarly high overall trans-junctional conductance (Lu and McMahon, 1996) that can be as high as 30 nS inin vitroexpression systems (Palacios-Prado et al., 2009) or can, in theory, reach themS range (O’Brien et al., 2006). The extensive coupling of horizontal cells can be dynamically modified by light levels and by circadian rhythms (Kohler et al., 1990;Kurz-Isler et al., 1992). Accordingly, horizontal cell coupling is more extensive in dim light than in darkness or bright light (Baldridge and Ball, 1991; Xin and Bloomfield, 1999), hence a gradual increase of the background light first enhances trans-junctional conductance, but further brightening initiates an opposite, conductance-decreasing mecha-nism. These light induced changes in horizontal cell coupling are mediated by altered dopamine levels (Piccolino et al., 1987). Dop-amine, by binding to D1receptors on the horizontal cell membrane, evokes an increase in intracellular cAMP level (Lasater, 1987) fol-lowed by the activation of PKA, which in turn triggers phosphor-ylation of connexins and the reduction of gap junctional conductance (McMahon et al., 1989). The observed large number of phosphorylation sites present in the C-terminal of horizontal cell connexins (Zoidl et al., 2004) is plausibly the site of intracellular regulation through phosphorylation. Besides dopamine, nitric oxide (NO) also affects horizontal cell coupling, acting through the alter-ation of the intracellular cGMP level (DeVries and Schwartz, 1989;

McMahon, 1994). In contrast to dopamine, however, NO only re-duces the opening frequency, not the average open time (McMahon, 1994), and may also suppress endogenous dopamine release (Bugnon et al., 1994). In addition to dopamine and NO, retinoic acid (RA) appears to act as a light-dependent modulator (Weiler et al.,

1998,1999,2000). It acts through a dopamine-independent mech-anism (Weiler et al., 1999) but the retinal RA receptors and the intracellular cascade involved have not yet been identified.

4.1.3. Bipolar-to-bipolar cell junctions

Bipolar cells are vertically oriented glutamatergic interneurons (with very few exceptions in some non-mammalian species) that relay signals from photoreceptors to ganglion and amacrine cells in the inner retina. While there is mounting evidence that lower vertebrate bipolar cells maintain electrical coupling with one another (Van Haesendonck and Missotten, 1983;Marc et al., 1988a;

Arai et al., 2010), their mammalian functional counterparts are less studied due to their small size and hence relative inaccessibility to intracellular recording methods. The rationale for electrical cou-pling of bipolar cells is still unclear. It may decrease the dispersion of input signals permitting bipolar cells to respond uniformly to light (Umino et al., 1994) and ultimately leading to a better signal-to-noise ratio, similar to that seen between photoreceptors (Jacobs and Werblin, 1994). Nevertheless, data on bipolar-to-bipolar cell coupling is not sufficient to assign a definite function to them, thus a comparison between vertebrate groups is premature.

Yet there are a few consistent and conserved features across examined species including:(i)electrical coupling is not general but rather is specific to only certain bipolar cell types;(ii)similar to those of photoreceptors, gap junctions connecting bipolar cells are small and focal and(iii)bipolar-to-bipolar gap junctions can occur between bipolar cell dendrites and/or axon terminals (Fig. 2;fishe Marc et al., 1988a;Arai et al., 2010; mammalseRaviola and Gilula, 1975;Kolb, 1979;Cohen and Sterling, 1990;Vaney, 1994). Although these junctions can be heterologous (Mills, 1999), coupled bipolar cells seem to follow the segregation of ON/OFF signal streams in mammals (Raviola and Gilula, 1975; Kolb, 1979; Saito and Kujiraoka, 1988;Cohen and Sterling, 1990). The connexin makeup of these peculiar gap junctions has only been described in a few instances. For example, Cx36 has been localized to mouse OFF bi-polar cell dendrites (Feigenspan et al., 2004) and goldfish Mb1 bi-polar cell axon terminals (Arai et al., 2010). In addition, Cx45 has recently been detected in juxtaposition with dendritic crossings of similar type OFF bipolar cells, whereas Cx45 subunits seem to form heterologous gap junctions between axon terminals of OFF bipolar cells in the mouse retina (Hilgen et al., 2011). It is even less detailed how these gap junctions are regulated, albeit it seems that NO rather than dopamine modulates bipolar-to-bipolar electrical and tracer coupling (Sakai and Ball, 1994).