Connectivity of cone photoreceptor telodendria in the zebrafish retina

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second‐order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing.     

Ontogeny of cone photoreceptor mosaics in zebrafish

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a "cone mosaic." Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish expressing green fluorescent protein (GFP) in their UV-sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a "mosaic metric" statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array; i.e., the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species.     

Telodendrial contact of HRP-filled photoreceptors in the turtle retina: pathways of photoreceptor coupling

Synaptic contacts of photoreceptors in the turtle retina were studied by intracellular injection of horseradish peroxidase (HRP) and electron microscopy. Both cone and rod photoreceptors radiated basal processes (telodendria) from their terminal endings. These telodendria ran laterally in the outer plexiform layer. The telodendria of cones gave rise to many fine branches that penetrated synaptic cavities of several neighboring cones. Tips of these branches terminated near the walls of synaptic cavities. Some of the telodendrial contact formed two types of basal junction: symmetrical and punctate. The distribution of cones that made telodendrial contacts with the HRP-filled cone were quantitatively investigated. Green-sensitive cones (n = 3) made telodendrial contacts with neighboring red- and blue-sensitive cones, blue-sensitive cones (n = 4) with red- and green-sensitive cones, and red-sensitive cones (n = 9) with red- and green-sensitive cones. In contrast to these cone connections, rod telodendria did not penetrate neighboring photoreceptors. Direct synaptic contacts were not found between rods and cones. Our results clarify the variety of cone couplings in turtle retina: the three chromatic classes of cones are selectively coupled by the basal junctions at the ends of telodendrial processes.     

The development of parafoveal and mid-peripheral human retina.

The morphological development of parafoveal retina (1-1.5 mm from the foveal center) and the mid-peripheral (4 mm from the foveal center) human retina has been studied from fetal (F) 26 weeks to adulthood. At both retinal points, all layers and neuronal types are present at F26 weeks. In parafovea at F26 weeks photoreceptors have only a rudimentary inner segment and no outer segments. Short outer segments are present on both rods and cones at F36 weeks. By postnatal (P) 5-8 days the inner retina is relatively mature. Photoreceptors have elongated basal axons which cause the photoreceptor layer to become much thicker than in prenatal retina. At birth cone inner segments are untapered, but rod inner segments have already reached their adult width of 2 microns. Both rod and cone inner and outer segments are 30-50% of adult length. By 13 months both inner and outer retina are mature appearing, with the photoreceptors accounting for half the retinal thickness due to the elongation of the fibers of Henle. Cone outer segments elongate up to P5 years and rod outer segments to P13 years. At mid-peripheral or rod-ring retina outer segments are present on rods at F26 weeks and on cones at F36 weeks. At birth the inner retina is adultlike. The outer plexiform layer becomes thicker up to P45 months due to the elongation of fibers of Henle. At birth both rod and cone mid-peripheral inner segments are slightly longer and outer segments are 50% longer than in parafoveal retina. By P5 years mid-peripheral rod outer segments are slightly longer than in parafoveal retina, and this changes little thereafter. This anatomical study has found that the photoreceptors in peripheral rod-ring retina develop earlier than those in more central retina, and in turn parafoveal photoreceptors develop well in advance of foveal cones. This suggests that human neonates may utilize more peripheral retinal regions for some aspects of visual function before foveal cone vision becomes dominant.     

Ontogenetic changes in the retinal photoreceptor mosaic in a fish, the black bream, Acanthopagrus butcheri.

The morphological development of the photoreceptor mosaic was followed by light and electron microscopy in a specific region of dorsal retina of the black bream, Acanthopagrus butcheri (Sparidae, Teleostei), from hatching to eight weeks of age. The retina was differentiated when the larvae reached a total length of 3 mm (3-5 days posthatch). Single cones, arranged in tightly packed rows, were the only morphologically distinct type of photoreceptor present until the larvae were 6 mm (day 15) in standard length (SL). At this time, the rod nuclei had become differentiated and the ellipsoids of selected cones began to form subsurface cisternae along neighbouring cone membranes. In this way, double, triple, quadruple, and occasionally photoreceptor chains of up to 10 cones were formed. At 8 mm SL, there was little apparent order in the photoreceptor mosaic. However, concomitant with subsequent growth, quadruple and other multiple cone receptors disappeared, with the exception of the triple cones, which gradually reduced in both number and retinal coverage to be restricted to central retina by 15 mm SL (days 40-55). Following this stage, the arrangement of double and single cones peripheral to the region of triple cones in dorsal retina was transformed into the adult pattern of a regular mosaic of four double cones surrounding a single cone. These results demonstrate that an established photoreceptor mosaic of rows of single cones can be reorganised to form a regular square mosaic composed of single and double cones.     

Cone mosaic development in the goldfish retina is independent of rod neurogenesis and differentiation

The goldfish retina displays a characteristic arrangement of cone photoreceptors that develop in a stereotyped sequence according to spectral phenotype. It has been suggested that the earliest differentiating photoreceptor in the teleost, the rod photoreceptor, might play an instructive role in development of the cone mosaic. This hypothesis was tested, first by examining the expression pattern of a cone subtype-specific marker with respect to that of rod opsin, and then by killing the cells that generate rods and examining the cone mosaic that formed in the absence of new rods. We find that, although there is potential for interactions between developing cones and immediately postmitotic rods, a role for such interactions in cone mosaic pattern formation is not likely.     

Chromatic organization of retinal photoreceptors during eye migration of Atlantic halibut (Hippoglossus hippoglossus)

The retinas of fishes often have single and double cone photoreceptors that are organized in lattice-like mosaics. In flatfishes experiencing eye migration (i.e., the metamorphic process whereby one eye migrates to the other side of the head), the hexagonal lattice of single cones present in the larva undergoes major restructuring resulting in a dominant square mosaic postmetamorphosis consisting of four double cones surrounding each single cone. The expression of different opsin types during eye migration has not been examined despite its importance in understanding photoreceptor plasticity and whether cell fate (in terms of spectral phenotype) could influence square mosaic formation. Here, we probed the retina of Atlantic halibut undergoing eye migration for opsin expression using two antibodies, AHblue and AB5407, that labeled short wavelength sensitive 2 (SWS2) opsin and longer wavelength (predominantly middle wavelength sensitive, RH2) opsins, respectively. Throughout the retina, double and triple cones labeled with AB5407 exclusively, whereas the vast majority of single cones labeled with AHblue. A minority (<5%) of single cones in the square mosaic of the centroventral retina labeled with AB5407. In regions of mosaic transition and near peripheral growth zones, some single cones co-expressed at least two opsins as they labeled with both antibodies. Short wavelength (SWS2 expressing, or S) cones formed a nonrandom mosaic gradient from central to dorsal retina in a region dominated by the larval single cone mosaic. Our results demonstrate the expression of at least two opsins throughout the postmetamorphic retina and suggest opsin switching as a mechanism to create new cone spectral phenotypes. In addition, the S cone gradient at the onset of eye migration may underlie a plastic, cell induction mechanism by which a cone's phenotype determines that of its neighbors and the formation of the square mosaic.     

Specificity of the horizontal cell‐photoreceptor connections in the zebrafish (Danio rerio) retina

Horizontal cells (HCs) are involved in establishing the center‐surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)‐ and ultraviolet (UV)‐sensitive single cones alternate with rows of red (R)‐ and green (G)‐sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small‐field (H1 and H2) and two large‐field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1‐GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI‐labeled cone HC and connected to 50–200 rods. J. Comp. Neurol. 516:442–453, 2009. © 2009 Wiley‐Liss, Inc.     

Melanin and the Regulation of Mammalian Photoreceptor Topography

Melanin, or products directly associated with it, regulates the maturation of the neural retina because in hypopigmented mammals the central retina fails to develop fully. To determine whether this deficit is reflected in the distribution of photoreceptors, their topography has been studied in the retinae of normally reared pigmented and albino ferrets and animals reared under reduced light conditions. In both strains, the general distribution of rods and cones was similar to that in the cat, cone density peaking in the central retina and rod density peaking in an annulus around the area centralis. The cone population was organized in the form of an orderly mosaic whose regularity was measured at a wide range of retinal eccentricities. No differences were found in cone numbers or their mosaic distribution between pigmented and albino strains, either at the area centralis or at more peripheral regions. In both cases order within the cone mosaic was independent of cell density or retinal eccentricity. In the albinos there was a significant deficit in the number of rods at all retinal locations when compared with rod numbers in the pigmented animals. There were no differences between normally reared and dark-reared animals in this respect either within or between the strains. Therefore, the albino gene must have a selective and specific effect on the development of this cell type in the outer retina. Ganglion cells and rods are both affected by the albino gene, while cones are not. Because cones and ganglion cells are generated during the same period and rods are generated later, the albino gene cannot be acting during a particular developmental time window. Because the cone mosaic was normal in the albinos, in spite of a large rod deficit, the factors that regulate the spacing of cones cannot depend in any significant manner upon the later generation and subsequent addition of rods to the outer retina.     

Bipolar cell-photoreceptor connectivity in the zebrafish (Danio rerio) retina

Bipolar cells convey luminance, spatial, and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1′‐Dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole‐mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI‐labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly ordered mosaic: rows of alternating blue‐ (B) and ultraviolet‐sensitive (UV) single cones alternate with rows of red‐ (R) and green‐sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which—G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod, and RRod bipolar cells—accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further subdivided into ON, OFF, and ON–OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the nine common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels. J. Comp. Neurol. 520:3786–3802, 2012. © 2012 Wiley Periodicals, Inc.     

Cytodifferentiation of photoreceptors in larval goldfish: delayed maturation of rods

This study describes the differentiation of photoreceptors in larval goldfish retina. The earliest photoreceptors to differentiate were cones; 3H-fucose labeled cone but not rod outer segments in larval as well as adult goldfish. All major cone types known to be present in the adult goldfish retina (double cones, long and short single cones) were found in the larval retina by 2 days after hatching. The cones matured rapidly; within a few days they had well-developed outer segments and synaptic pedicles that were smaller, but otherwise similar to those in adults. Rods were slower to mature. Their outer segments were at first short, wide, and misshapen; only as they grew longer and narrower did they become straight and properly aligned. Rod spherules were first seen in fish older than 1 month; immature rods contained perinuclear synaptic ribbons and invaginating processes penetrated the cell body. These results suggest that the influence of rods and cones on visual function in larval goldfish may be quite different from the adult.     

Expression of the vesicular glutamate transporter vGluT2 in a subset of cones of the mouse retina

Cone photoreceptors have a continuous release of glutamate that is modulated by light. Vesicular glutamate transporters (vGluT) play an essential role for sustaining this release by loading synaptic vesicles in the cone synapse, the so-called cone pedicle. In the present study mouse retinas were immunostained for vGluT1 and vGluT2. vGluT1 was localized to all cone pedicles and rod spherules, whereas vGluT2 was found in only 10% of the cone pedicles. The vGluT2-expressing cones were characterized in more detail. They are distributed in a regular array, suggesting they are a distinct type. Their proportion does not differ between dorsal (L-cone-dominated) and ventral (S-cone-dominated) retina, and they are not the genuine blue cones of the mouse retina. During development, vGluT1 and vGluT2 expression in cones starts at around P0 and right from the beginning vGluT2 is only expressed in a subset of cones. Bipolar cells contact the vGluT2-expressing cones and other cones nonselectively. The possible functional role of vGluT2 expression in a small fraction of cones is discussed.     

Identification of pedicles of putative blue-sensitive cones in the human retina

Cone photoreceptor pedicles from midperipheral regions of the human retina (6 mm from the foveal center) have been studied by light and electron microscopy. Three areas of cone pedicle mosaic were serially thin-sectioned, in the tangential plane, from the inner border of the outer plexiform layer to the emergence of the cone axons from the cone pedicles. Semithin sections were then collected from the cone axon level through the cone cell bodies to the cone inner segment level. Two hundred twenty-one cone pedicles were followed by this means to their respective inner segments. Eight percent of the cone pedicles were from cones with inner segment characteristics of the blue cones. All 221 cone pedicles were reconstructed by tracing images from electron micrographs. The cone pedicle locations, surface areas, telodendrial projections, and synaptic ribbons could then be measured by morphometry and analyzed by statistical methods. Some selected cone pedicles were reconstructed by computer graphics methods. The cone pedicles identified as belonging to the blue cone type could be distinguished from the surrounding longer wavelength types on the following morphological criteria: 1) they were smaller (50% the area of the surrounding pedicles), 2) they contained shorter synaptic ribbons, 3) they exhibited essentially no telodendrial contact to neighboring cone pedicles, 4) they were positioned slightly more vitread in the outer plexiform layer than neighboring pedicles, and 5) their irregular occurrence in the cone mosaic coincided with the distribution criteria established in our previous paper (Ahnelt et al: J. Comp. Neurol. 255:18-34, '87) for putative blue sensitive cones in midperipheral human retina.     

Cat cones have rod input: A comparison of the response properties of cones and horizontal cell bodies in the retina of the cat

The responses of horizontal cell bodies and cones in the retina of the cat have been studied by means of intracellular recording and Procion dye injection In an isolated, arterially perfused eyecup preparation. Comparison of the hyperpolarizing responses of these units to red and blue stimuli of different intensities indicated that all morphological varieties of horizontal cells and, additionally, cones themselves, had mixed rod and cone input. The rod input into horizontal cell bodies is thus explained on the basis of cone physiology. The half-saturating intensity of 441 nm stimuli for the rod input into cones and horizontal cells was about 400 quanta/μm₂/sec and about 160,000 quanta/μm₂/sec for the cone input. Little of this difference can be related to the different quantum catching abilities of rods and cones. The spatial properties of horizontal cell bodies and cones have been characterized using stimuli consisting of long slits in conjunction with a continuous cable model. Space constants for horizontal cells ranged from 210 μm to 410pm, whereas those for cones ranged from 50μm, or possibly less, to 180 μm. It is argued that horizontal cell bodies of the cat retina form electrical networks, and that the sizes of the receptive fields generated in these networks may be limited by the diameters of the primary and secondary dendrites of horizontal cells. The rod and cone fields of horizontal cell bodies were found to be nearly coextensive in space, arguing against the notion that substantial rod input came from distant, rod-dominated terminal arborizations.     

Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina

Background

The multiplex, lattice mosaic of cone photoreceptors in the adult fish retina is a compelling example of a highly ordered epithelial cell pattern, with single cell width rows and columns of cones and precisely defined neighbor relationships among different cone types. Cellular mechanisms patterning this multiplex mosaic are not understood. Physical models can provide new insights into fundamental mechanisms of biological patterning. In earlier work, we developed a mathematical model of photoreceptor cell packing in the zebrafish retina, which predicted that anisotropic mechanical tension in the retinal epithelium orients planar polarized adhesive interfaces to align the columns as cone photoreceptors are generated at the retinal margin during post-embryonic growth.

Methods

With cell-specific fluorescent reporters and in vivo imaging of the growing retinal margin in transparent juvenile zebrafish we provide the first view of how cell packing, spatial arrangement, and cell identity are coordinated to build the lattice mosaic. With targeted laser ablation we probed the tissue mechanics of the retinal epithelium.

Results

Within the lattice mosaic, planar polarized Crumbs adhesion proteins pack cones into a single cell width column; between columns, N-cadherin-mediated adherens junctions stabilize Müller glial apical processes. The concentration of activated pMyosin II at these punctate adherens junctions suggests that these glial bands are under tension, forming a physical barrier between cone columns and contributing to mechanical stress anisotropies in the epithelial sheet. Unexpectedly, we discovered that the appearance of such parallel bands of Müller glial apical processes precedes the packing of cones into single cell width columns, hinting at a possible role for glia in the initial organization of the lattice mosaic. Targeted laser ablation of Müller glia directly demonstrates that these glial processes support anisotropic mechanical tension in the planar dimension of the retinal epithelium.

Conclusions

These findings uncovered a novel structural feature of Müller glia associated with alignment of photoreceptors into a lattice mosaic in the zebrafish retina. This is the first demonstration, to our knowledge, of planar, anisotropic mechanical forces mediated by glial cells.

     

Retinal photoreceptor arrangement,SWS1 and LWS opsin sequence,and electroretinography in the South American marsupial Thylamys elegans (Waterhouse, 1839)

We studied the retinal photoreceptors in the mouse opossum Thylamys elegans, a nocturnal South American marsupial. A variety of photoreceptor properties and color vision capabilities have been documented in Australian marsupials, and we were interested to establish what similarities and differences this American marsupial showed. Thylamys opsin gene sequencing revealed two cone opsins, a longwave‐sensitive (LWS) opsin and a shortwave‐sensitive (SWS1) opsin with deduced peak sensitivities at 560 nm and 360 nm (ultraviolet), respectively. Immunocytochemistry located these opsins to separate cone populations, a majority of LWS cones (density range 1,600–5,600/mm²) and a minority of SWS1 cones (density range 100–690/mm²). With rod densities of 440,000–590,000/mm², the cones constituted 0.4–1.2% of the photoreceptors. This is a suitable adaptation to nocturnal vision. Cone densities peaked in a horizontally elongated region ventral to the optic nerve head. In ventral—but not dorsal—retina, roughly 40% of the LWS opsin‐expressing cones occurred as close pairs (double cones), and one member of each double cone contained a colorless oil droplet. The corneal electroretinogram (ERG) showed a high scotopic sensitivity with a rod peak sensitivity at 505 nm. At mesopic light levels, the spectral ERG revealed the contributions of a UV‐sensitive SWS1 cone mechanism and an LWS cone mechanism with peak sensitivities at 365 nm and 555 nm, respectively, confirming the tuning predictions from the cone opsin sequences. The two spectral cone types provide the basis for dichromatic color vision, or trichromacy if the rods contribute to color processing at mesopic light levels. J. Comp. Neurol. 518:1589–1602, 2010. © 2009 Wiley‐Liss, Inc.     

Early afferent signaling in the outer plexiform layer regulates development of horizontal cell morphology

The pedicles of cone photoreceptors, labeled with an antibody to mouse cone arrestin (blue), stratify in the outer plexiform layer, where they form synapses with the dendrites of horizontal and bipolar cells. Those synaptic sites are evidenced by the co‐localization of the synaptic ribbon protein, piccolo (red), with the cone arrestin labeling. The remaining red profiles in the outer plexiform layer indicate the sites of the rod spherules. An antibody to cytochrome oxidase (green) labels the mitochondrion‐rich inner segments of all photoreceptors and yields punctate peri‐nuclear labeling within the outer nuclear layer. Many of these features of the outer retina are altered in the Cacna1f‐mutant retina, expressing a defective calcium channel subunit that prevents normal neurotransmission in the outer plexiform layer. J. Comp. Neurol. 506:745–758, 2007. © 2007 Wiley‐Liss, Inc.     

Spectral sensitivities of seven morphological types of photoreceptors in the retina of the turtle, Geoclemys reevesii

Spectral sensitivities of photoreceptors in the turtle (Geoclemys) retina were studied by intracellular recording, and each cell was filled with Lucifer yellow (LY). Photoreceptors were classified into seven morphological types: rod, four types of single cones, and two members of a double cone. Single cones contained one of four different oil droplets: red, pale-green, orange, and clear. Double cones consisted of two apposed cones; principal members contained yellow oil droplets, while accessory members contained no oil droplet. Spectral sensitivities recorded from these seven types of photoreceptors were classified into one type of rod and three chromatic types of cones. Rods (n = 19) showed peak sensitivity at 520 nm. Single cones containing either a red (n = 51) or a pale-green (n = 9) oil droplet were red-sensitive (lambda max at 620 nm). Single cones containing an orange oil droplet (n = 14) were green-sensitive (lambda max at 540 nm). Single cones containing a clear oil droplet (n = 3) were blue-sensitive (lambda max at 460 nm). Both members of the double cone, principal (n = 22) and accessory (n = 15), were red-sensitive (lambda max at 620 nm). No diffusion of LY was detected between the apposed members of double cones. Red-sensitive cones, therefore, consisted of four different morphological types of cones, and they occupy about 70% of the photoreceptor mosaic in the turtle retina.