Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

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Figure 6 Schematic of the cone photoreceptor outer segment, inner segment, and connecting cilium (a). (b) Base of the cone outer segment and its relationship to the plasma membrane of the connecting cilium. (c) Illustration of the continuity between cone disks and the outer segment plasma membrane. From Anderson, D. H., Fisher, S. K., and Steinberg, R. H. (1978). Mammalian cones: Disc shedding, phagocytosis, and renewal.

Investigative Ophthalmology and Visual Science 17(2): 130.

network of outer segment disks, it is homologous to other primary cilia located elsewhere in the body. For example, like other primary cilia, it possesses microtubule motor proteins that may function in the transport of outer segment components. There are numerous other proteins shared by primary cilia and photoreceptor cilia. Thus, despite the extraordinary specializations of the inner

and outer segments, the connecting link between them is an organelle that is highly conserved. This point has been made most poignantly by demonstrations in a variety of syndromic disorders, such as Senior Loken and Bardet Biedel syndromes. These disorders are characterized, in part, by retinal degeneration, as well as kidney and other disorders, caused by mutations in genes which encode ciliary proteins that subserve shared functions.

See also: Circadian Photoreception; Cone Photoreceptor Cells: Soma and Synapse; Fish Retinomotor Movements; Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; The Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: The Visual Cycle; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading

Anderson, D. H., Fisher, S. K., and Steinberg, R. H. (1978). Mammalian cones: Disc shedding, phagocytosis, and renewal. Investigative Ophthalmology and Visual Science 17(2): 117–133.

Besharse, J. C. and Horst, C. J. (1990). The photoreceptor connecting cilium: A model for the transition zone. In: Bloodgood, R. A. (ed.)

Ciliary and Flagellar Membranes, pp. 409–431. New York: Plenum Press.

Cohen, A. I. (1970). Rods and cones. In: Fjuortes (ed.) Handbook of Sensory Physiology, vol. 7, part 1B, pp. 63–110. Berlin: Springer.

Molday, R. S. (2004). Molecular organization of rod outer segments. In: Williams, D. S. (ed.) Photoreceptor Cell Biology and Inherited Retinal Degenerations. Singapore: World Scientific Publishing.

Schultze, M. (1866). Anatomie und Physiologie der Nezhaut. Archiv fu¨ r mikroskopische Anatomie 2: 175–286.

Spitznas, M. and Hogan, M. J. (1970). Outer segments of photoreceptors and the retinal pigment epithelium. Archives of Ophthalmology 84: 810–819.

Young, R. W. (1970). Visual cells. Scientific American 223: 80–91.

Glossary

Autoradiography – A technique used to localize radioactivity emitted by cells, tissues, or organisms that have been treated or injected with a radioactive isotope. Electron microscope – A microscope that uses a particle beam of electrons to illuminate a specimen and create a highly magnified image. An electron microscope has much greater resolving power than a light microscope because the wavelength of an electron is much smaller than that of visible light. Lysosome – An organelle in a cell that contains digestive enzymes.

Phagocytosis – The engulfing and internalization of particles by a cell.

Retinal pigment epithelium – A single layer of pigmented epithelial cells that borders the back of the sensory retina. The outer segments of the photoreceptor cells interdigitate with the apical processes of the retinal pigment epithelium cells.

Based upon their observations of photoreceptor disk membrane-like inclusions in the cytoplasm of rodent retinal pigmented epithelial (RPE) cells, A. Bairati and N. Orzalesi proposed in 1963 that the disk membranes of photoreceptor outer segments were in a dynamic state of turnover. A few years later, in a classic series of autoradiographic studies, Richard Young and his colleagues provided the first direct evidence that vertebrate rod photoreceptors continually replace their disk membranes. Following injection of radiolabeled amino acids into a series of frogs, a transverse band of radioactive protein became apparent at the base of rod outer segments within 24 h following injection (Figure 1). We now know that the main proteinaceous component of the phototransductive disk membranes is the visual receptor, opsin. Over the ensuing days, the band became progressively displaced toward the tip of the outer segment, and eventually became evident in inclusions within the cytoplasm of the RPE cells. Electron microscopic observations later revealed that packets of disks derived from the tips of rod outer segments were engulfed by microvillous processes on the apical surface of the RPE cells and internalized into the RPE cytoplasm. These membrane-bound

inclusions, called phagosomes, were then degraded by the RPE. Young proposed the term outer-segment renewal to refer to the overall turnover process. It encompasses a number of stages, including protein synthesis, transport to the outer segment, disk formation, disk displacement, shedding, phagocytosis and degradation, all of which result in the vectorial flow of membrane from the photoreceptor inner segment to the RPE (Figure 2).

Many cell types have short lives and are replaced on a regular basis. However in a terminally differentiated cell, such as a photoreceptor cell, the components must be turned over to prevent the formation and build up of macromolecular byproducts that can interfere with cellular function. Accordingly, it is widely believed that the turnover of phototransductive membrane is part of a normal preemptive process to replace macromolecules before they become dysfunctional. Because phototransductive membrane is typically an extremely amplified membrane system, its turnover involves an extraordinarily high rate of synthesis and degradation of membrane proteins. Some nocturnal arthropods provide the most extreme examples, with the turnover of nearly all of their phototransductive membrane each day. Even at the more pedestrian rate of 10% per day, as found in rodent and primate rods, the turnover of disk membranes represents a major metabolic challenge for the photoreceptor and RPE cells. In each human retina, which contains approximately 100 million photoreceptor cells, an average of 9 billion opsin molecules turn over every second. Not surprisingly, therefore, the processes involved in disk membrane turnover are critical for photoreceptor cell viability, as shown with some of the first-studied rodent models of retinal degeneration. For example, in the RCS rat, phagocytosis by the RPE is defective, and in the retinal degeneration slow (rds) mouse disk membrane morphogenesis is blocked; photoreceptor cell death and blindness ensues in both these models.

Whereas the experiments using radiolabeled amino acids convincingly demonstrated the renewal of the rod outer-segment membranes, and the involvement of the RPE in the degradation phase, the case for cone outersegment renewal was less compelling. In contrast to rods, no discrete band of radioactivity was detected at the base of frog cone outer segments; instead, it appeared to be distributed diffusely throughout the cone outer segment. However, in the cones of diurnal rodents, quantitative electron microscopic autoradiographic studies showed that a

Scale bar = 10um

Figure 1 Light micrograph autoradiograph of a retina from a frog injected with radiolabeled amino acids, illustrating the band of radiolabeled protein (B) near the base of the outer segments (OS). IS, inner segments. Scale bar ¼10 mm. From Hall MO, Bok D, and Bacharach ADE (1968) Visual pigment renewal in the mature frog retina. Science 161: 787–789.

RPE

Phagocytosis and degradation

Distal displacement

OS

of disk membranes

Disk morphogenesis

CC

Transport along CC

Transport through IS

IS

Synthesis

N

9 billion opsin molecules per sec. in each human retina

S

Figure 2 Illustration of the stages involved in the turnover of phototransductive membrane in vertebrate photoreceptor cells. Modified from Williams DS (2002) Transport to the photoreceptor outer segment by myosin VIIa and kinesin II. Vision Research 42: 455–462.

concentration of radioactive glycoconjugates could be detected in the proximal portion of cone outer segments within a few hours after injection of radiolabled fucose (Figure 3). In retrospect, it is now appreciated that the banding pattern in rods is due to the confinement of newly synthesized membrane proteins to rod disks, which

Figure 3 Electron micrograph autoradiograph of a retina from a ground squirrel, shortly after injection with radiolabeled fucose, illustrating the concentration of radiolabel near the base of the cone outer segments. OS, outer segment; IS, inner segment.

mature into discrete units. In contrast, cone outer-segment disks remain interconnected, so that radiolabeled protein is free to diffuse longitudinally throughout the disk stack. Studies demonstrating the presence of disk shedding in cones, and the presence of phagosomes in RPE cells overlying regions of high cone density, such as in the human fovea and in all-cone retinas, also helped to demonstrate that the unidirectional flow of disk membrane proteins, from inner segment to outer segment, and then to the RPE, occurred in cones as well as rods.

The de novo synthesis of opsin-containing membranes occurs mainly in the proximal region of the inner segment (i.e., the myoid). Membranes containing newly synthesized protein are then transported from the trans-Golgi network, through the ellipsoid, to the base of the connecting cilium (Figure 2). Opsin contains motifs that appear to be important for its targeting to the outer segment. The best characterized are its C-terminal amino acids. Missense mutations that affect this region result in mistargeting of opsin and underlie forms of inherited retinal degeneration in humans. It seems likely that a molecular motor, like a dynein, which travels toward the minus ends of microtubules, is involved in this vectorial transport. In support of this notion is the finding that opsin binds to tctex-1, a light chain of dynein, in vitro. Further studies have reported roles for a number of other proteins that appear to be important for the targeting and fusion of membrane near the base of the cilium. These proteins include small GTPases, such as ADP ribosylation factor 4 (ARF4) and the RAS oncogene-related protein RAB8. It is likely that RAB8 promotes membrane fusion near the base of the cilium, and may be recruited to the post-Golgi membranes by a complex of Bardet–Biedl syndrome (BBS) proteins; mutations in BBS proteins are responsible for BBS, which includes photoreceptor degeneration

along with defects in cilia throughout the body. In photoreceptors that have a high rate of delivery of disk precursor membrane to the outer segment, the site of membrane fusion appears as a periciliary ridge complex, where the surface area of the plasma membrane has been greatly amplified by forming a series of ridges and grooves around the base of the cilium.

Transport along the cilium to the site of disk membrane morphogenesis involves molecular motors and associated proteins. Myosin VIIa, an actin-based motor, participates in this transport, although there is a clearer requirement for the kinesin-2 family of microtubule motors. In the absence of functional heterotrimeric kinesin-2, opsin delivery to the outer segment fails, and the ectopic build up of opsin causes rapid photoreceptor cell death. A homodimeric kinesin-2, KIF17, may also function in ciliary transport of outersegment proteins. A group of intraflagellar transport proteins appear to function in concert with kinesin-2 motors. These proteins, which function in anterograde transport in primary cilia in other cells, are present in the photoreceptor axoneme, and IFT88 is required for the assembly and turnover of the outer-segment disk membranes. The specific cargoes of the different motor systems along the photoreceptor cilium are not known, but it is likely that there are a variety of different routes along the cilium. For example, evidence indicates that opsin and the rim-specific protein, peripherin-rds, are transported by different mechanisms.

Historically, the process of disk formation in rods was thought to result from an invagination of the outer plasma membrane toward the centric face of the connecting cilium. However, in 1980, an alternative evagination model of disk membrane morphogenesis was proposed that could account for disk morphogenesis in both rods and cones. Over the years, experimental evidence has been generated in support of this model. According to this model, as successive evaginations of new disk membrane occur, a second membrane growth phase forms the nascent rims around the newly formed evaginations. In rod cells, rim formation occurs relatively quickly, so that only a small number of disks at the base of the stack remain continuous with the outer plasma membrane. The vast majority of rod disks are discrete units that form a stack, completely surrounded by the plasma membrane. In cone cells, however, the process of rim formation remains incomplete and a connection(s) between the disk and the outer plasma membrane is retained in many, if not all cone disks.

Although there have been some challenges to the evagination model of disk membrane morphogenesis, some key observations provide strong support. First, in contrast to mature rod disks, the evaginating membranes can be labeled with tracer molecules, showing that they are open to the extracellular milieu. Second, the nascent disks contain specific membrane proteins that are not found in mature disks, and they lack the proteins added at the stage of rim formation. This latter observation indicates that there must be a mechanism(s) to sort different groups

of outer-segment proteins prior to disk membrane morphogenesis.

Most cells are responsible for the complete turnover of their lipids and proteins. However, vertebrate photoreceptor cells are unusual in that they have recruited another cell type, the RPE cell, for the catabolic phase of disk membrane turnover. The disposal of the distal outer-segment disks requires an interaction between the photoreceptor and RPE cells. When the photoreceptors are detached from the RPE, the distal disks are not shed, indicating that disk membrane shedding and phagocytosis by the RPE are not independent events (Figure 4). In 1976, Matthew LaVail reported that rod disk shedding in rodents followed a daily rhythm, and that a peak of shedding was apparent near the

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Figure 4 Light micrograph autoradiographs of retinas from frogs injected with radiolabeled amino acids. The band of radiolabel had migrated to the distal disks, and the eyecups were removed and placed in culture for the next shedding phase. Where the retina has remained attached to the RPE (a, and to the left of arrow in (c)), radiolabeled phagosomes are evident in

the RPE. Where the retina was detached from the RPE (b, and to the right of arrow in (c)), the band of radiolabel is still evident at the distal end of each outer segment, indicating that the distal disks have not been shed. Scale bar ¼50 mm. From Williams DS and Fisher SK (1987) Prevention of rod disk shedding by detachment from the retinal pigment epithelium. Investigative Ophthalmology and Visual Science 28: 184–187.

time of light onset. Subsequent studies have shown that the shedding of phototransductive membrane occurs at dawn in other vertebrates, as well as invertebrates. However, cone photoreceptors of most vertebrate species provide an exception to this rule; the peak of cone disk shedding typically occurs shortly after dusk (or light-offset). In crepuscular and nocturnal arthropods, the morphogenesis and shedding phases occur at dusk and dawn, respectively, resulting in different-sized light-absorbing structures (known as rhabdoms) between day and night. In this manner, the turnover of phototransductive membrane is coordinated to increase the efficiency of photon absorbance by individual photoreceptors at night.

The signaling mechanisms involved in the phagocytosis of disk membranes are not well understood; however, the requirement for a number of molecules has been determined. The c-mer proto-oncogene (Mertk) gene encodes a receptor tyrosine kinase, which is localized to the apical membrane of the RPE. In the absence of a functional form of this receptor, the ingestion of disk membranes does not occur, as is found in the retina of the RCS rat which carries a mutation in the Mertk gene. A role for the avb5 integrin receptor and its ligand, milk fat globule EGF factor 8 (MFG-E8), has also been detected. This receptor appears to be required for the diurnal rhythm of shedding and phagocytosis since, in b5 integrin knockout mice, disk membranes are still phagocytosed, but there is no peak of disk shedding at light onset.

Following internalization, the phagosomes are transported out of the apical region of the RPE where they subsequently fuse with lysosomes, and are degraded enzymatically. In mice lacking myosin VIIa, phagosome transport in the cytoplasm is impaired, thus indicating a role for this actin-based motor in the transport process. Microtubule motors also appear to be required in facilitating phagosome–lysosome fusion. A major enzyme in the degradation of opsin is cathepsin D, which is highly concentrated in the RPE lysosomes. Lysosomal enzymes have also been detected in RPE melanosomes, which may

have a minor degradative role. The degradation of disk membrane proteins and lipids represents the end of the catabolic phase of the turnover process.

See also: Injury and Repair: Light Damage; Photoreceptor Development: Early Steps/Fate; The Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration.

Further Reading

Besharse, J. C. (1986). Photosensitive membrane turnover: Differentiated membrane domains and cell–cell interaction. In: Adler, R. and Farber, D. B. (eds.) The Retina, Part I, pp. 297–352. New York: Academic Press.

Deretic, D. (2006). A role for rhodopsin in a signal transduction cascade that regulates membrane trafficking and photoreceptor polarity.

Vision Research 46: 4427–4433.

Insinna, C. and Besharse, J. C. (2008). Intraflagellar transport and the sensory outer segment of vertebrate photoreceptors.

Developmental Dynamics 237: 1982–1992.

LaVail, M. M. (1976). Rod outer segment disk shedding in rat retina: Relationship to cyclic lighting. Science 194: 1071–1074.

Papermaster, D. S., Schneider, B. G., and Besharse, J. C. (1985). Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas. Investigative Ophthalmology and Visual Science 26: 1386–1404.

Steinberg, R. H., Fisher, S. K., and Anderson, D. H. (1980). Disc morphogenesis in vertebrate photoreceptors. Journal of Comparative Neurology 190: 501–508.

Williams, D. S. (ed.) (2004). Photoreceptor Cell Biology and Inherited Retinal Degenerations, chs. 1, 3–6, and 13–15. Singapore: World Scientific Publishing.

Young, R. W. (1967). The renewal of photoreceptor cell outer segments.

Journal of Cell Biology 33: 61–72.

Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. Journal of Cell Biology 42: 392–403.

Glossary

Endocytosis – Active incorporation of external membrane into the cell, used to recover vesicular membrane that has fused with the external membrane at a chemical synapse.

Invagination – A permanent infolding of a cell’s external membrane, associated in photoreceptors with their synaptic ribbons, and containing fine dendritic processes of bipolar and horizontal cells. Mesopic – The 3-log unit range of background illuminance in which rod and cone signals temporally sum in cones and the cone bipolar pathway. Poikilotherm – A species such as fish, turtles, and frogs whose body temperature varies with the environment.

Refractory period – A period immediately after an event, for example, the release of a vesicle of neurotransmitter, during which the next event cannot occur.

Rhodopsin – The rod pigment molecule that absorbs photons and starts the visual transduction cascade.

Ribbon – A presynaptic structure that collects vesicles of neurotransmitter for release. Scotopic – The range of background illuminance

from starlight to moonlight in which rods absorb one photon or less per integration time ( 200 ms). Spherule – The rod terminal, normally in the shape of a sphere, that contains synaptic ribbons. Telodendria – Fine axonal processes extending laterally from the base of the cone terminal, which contact neighboring rod and cone terminals.

Introduction

Photoreceptors are the vertebrate retina’s primary site for transduction of light into a neural signal. The rod photoreceptor is responsible for vision at night. Rods are essential for vision over the scotopic range (starlight through moonlight) into the mesopic range (twilight) where their signals mix with cone signals. In starlight they must transduce single-photon signals, but in twilight they function more like sensitive cones to temporally integrate photon signals and to adapt before finally saturating in bright

daylight. The rod’s exquisite combination of signalprocessing mechanisms makes it a marvel of signal processing for a dedicated purpose.

Structure

Morphology and Topology

The rod is a specialized neuron consisting of an outer segment, inner segment, soma, axon, and axon terminal (Figure 1(a)). The biochemical pathways responsible for transducing light into an electrical signal are contained in the outer segment. The electrical signal passes to the inner segment where it is transformed by voltage-gated channels. The inner segment of the vertebrate rod lies just above the external limiting membrane. The rod soma lies in a variable location in the outer nuclear layer, connected to the inner segment above and the terminal below by the axon. For rods of most species, the soma is larger in diameter ( 3–5 mm) than the outer segment. It contains the nucleus which holds the cell’s DNA, necessary for development and to maintain the cell’s biochemical machinery. Many mammalian species have 20-fold more rods than cones, so the outer nuclear layer consists mainly of several layers of rod somas. Rods of most species fill the space between the cones, and are closely spaced ( 100 000–200 000 mm–2) to capture as many photons as possible.

Axon and Terminal

The rod axon, which carries the electrical signal from the soma to the axon terminal, varies in length depending on the species and the eccentricity (distance from central retina) of the rod. In para-foveal rods near the center of the primate eye, rod axons extend along with cone axons up to 200–400 mm laterally to allow the cone outer segments to be packed tightly together in the fovea and the axon terminals to be given adequate space outside the fovea to make their synaptic connections. In other mammals, the rod axon is shorter; in a cat it is vertical and typically extends 50 mm through 6–10 layers of rod somas in the outer nuclear layer, and in guinea pig the axon is shorter, typically 10–25 mm, depending on the number of layers of rod somas. Typically, rod axons are 0.5 mm in diameter, interspersed between rod somas and cone axons of 1–2 mm diameter (Figure 1(a)). The mammalian rod axon terminal is typically 2–3 mm in diameter, and is spherical; hence, it is

commonly called a spherule. The spherules of adjacent rods are located at different heights, allowing them to fit just above the cone terminals where they can be reached by cone telodendria and fine dendritic processes of bipolar cells.

Synapse

The rod makes chemical synaptic contacts onto one type of rod bipolar cell, one or more types of OFF-cone bipolar cell, and one type of horizontal cell at its axon terminal (Figure 1(b)). The synapse releases glutamate via small packets of membrane called synaptic vesicles, which are small ( 30 nm) organelles, created by endocytosis from the cell’s external membrane and filled with glutamate by transporter proteins. The vesicles diffuse

freely around the cytoplasm while they are being filled. The presynaptic machinery in the terminal contains one or two dense structures called ribbons, because in cross section they are thin and extend vertically away from the cell’s membrane, easily seen in electron micrographs or by confocal visualization of specific presynaptic proteins (ribeye, kinesin). The ribbon is thought to be a specialization to allow high release rates, for it collects several rows of vesicles and tethers them for release. The mechanism for vesicle release is complex and contains several dozen proteins, and its details are not yet understood, but it is known to be initiated by calcium ions binding to a receptor protein which causes the vesicle to fuse with the external membrane and thus release its contents into the extracellular space. The ribbon is thought to gather vesicles ready to be docked to provide a larger readily

releasable pool. As the rod is depolarized for long periods at night, its ribbon mechanism is specialized to release vesicles at a high rate continuously.

Invagination

The rod’s ribbon synapse is located in a way similar to the cone’s ribbon, in an extension of extracellular space into the bottom surface of the spherule called an invagination, where very fine processes of horizontal and bipolar cells extend to form postsynaptic specializations. Each invagination contains two ribbons, each of which is presynaptic to two horizontal cell dendrites from different horizontal cells. In addition, each ribbon is presynaptic to one rod bipolar cell, for a divergence from a rod to two rod bipolars. In addition, the rod terminal in some species contacts one or more types of OFF bipolar cells. The function of the invagination is unknown, but it has been suggested to limit diffusion of the neurotransmitter released by the cone or by horizontal cells for negative feedback.

Biophysical Properties

The inner segment and axon terminal of rods are similar to those of cones in that they contain several membranebound ion channels, including Kv, KCn, BK, and L-type Ca2+. The K+ channels (Kv, KCn, and BK) of the inner segment and soma are activated by depolarization, providing an outward current to balance the inward dark current through the light-modulated cyclic guanosine monophosphate (cGMP)-gated channels of the outer segment. These channels provide an adaptational influence, opposing the dark signal, and indirectly opposing the light signal by deactivating with hyperpolarization. The KCn channels in the soma, axon, and terminal underlying the hyperpolarization-activated current (Ih) provide a delayed depolarization when activated by hyperpolarization. The rod terminal’s L-type Ca2+ channels (Cav1.4) uniquely include a 1F subunits and may have special gating properties in conjunction with CaBP4, a calmodulin-like binding protein that shifts the Cav1.4 gating activation curve to provide higher gain. In addition, the terminal contains a calcium-sensitive chloride current (ICl(Ca)) which provides signal enhancement because the chloride gradient is depolarizing in rods. However, the resulting chloride efflux is thought to downmodulate the calcium current. Further, the rod terminal contains a calcium-sensitive potassium current (BK) which limits depolarization and calcium entry, and calcium-induced calcium release (CICR) from internal stores which amplifies the internal calcium signal. The rod terminal contains a high-affinity calcium system which includes buffers, plasma membrane calcium ATPase pumps (PMCAs), ryanodine receptors, and inositol triphosphate (IP3) receptors to modulate calcium release from internal stores. One possible reason for these specializations

is that the calcium channels that trigger vesicle release must be modulated by a tiny single-photon signal and thus the system must have high gain. The rod terminal contains several transporters with special functions. Some of them transport glutamate, including at least two isoforms in the membrane of vesicles to load them with glutamate. A glutamate transporter sitting in the external membrane of the rod terminal is important for uptake of glutamate from the extracellular space.

Gap Junctions

The rod spherules in most vertebrates are electrically coupled by gap junctions to the neighboring cones. The gap junctions are made between the rod spherule and cone telodendria which are basal processes emanating from the cone terminals. This coupling allows rod signals to enter cones at twilight and to be carried through cone pathways to ganglion cells. In addition, rods in some, possibly all, mammals are directly coupled by small gap junctions which may reduce voltage noise in the rod terminal that originates in transduction and membrane ion channels.

Function

The Single-Photon Signal and Noise

The rod is faced with a difficult task. Vertebrate species active in night, such as most mammals, can see in starlight backgrounds when photons are rare and a rod receives a photon only every 20 min. Over the 3-log unit range of scotopic backgrounds, a rod receives one photon or less per integration time (200 ms) so that its signal is binary. To generate a continuous visual image at such low backgrounds requires that signals from many rods be summed. A typical mammalian ganglion cell sums signals from several thousand rods, and can detect the signal from just one photon in that huge field. This extraordinary feat is a challenge because the single-photon signal is tiny, about 1 mV in amplitude. To detect such a tiny signal in a huge field of rods would be straightforward if all the rods were silent in the dark; but the mechanisms of transduction and synaptic transmission are noisy. The noise from the rods converging to a ganglion cell, if linearly summed with the single-photon signal, would completely mask it, preventing its transmission through the visual pathway. Therefore, the rod pathway has evolved mechanisms to remove the dark continuous noise before it is summed with photon signals.

Sources of Noise

The mammalian rod generates dark continuous noise of thermal origin in its transduction cascade with an amplitude of 10–30% of the peak single-photon signal.

In addition, the rod generates thermal isomerizations of its pigment molecule rhodopsin, called dark thermal events, at a rate equivalent to starlight on a cloudy night. The signals generated by dark thermal events are identical to the single-photon signal, so they appear as real photons from the visual environment and are sometimes referred to as dark light. Another source of noise is variability in the single-photon signal’s amplitude. Further, the rod synapse generates robust noise from fluctuation in vesicle release, which masks single-photon events in a way similar to the dark continuous noise. These sources of noise would not be a problem for detecting a singlephoton signal from one rod, but if the signals of more than 10 rods were linearly summed, the noise would mask the single-photon signal. To mitigate the problem, synaptic convergence in the rod pathway is accomplished in several stages. The first stage of convergence, from rods to the rod bipolar, is limited to between 20:1 and 100:1 depending on the species, but even this limited amount of convergence would mask the rod signal without special synaptic mechanisms.

Synaptic Transfer Function: High Gain

and Temporal Filtering

The rod ribbon synapse is specialized to transmit the rod signal at scotopic backgrounds. It is thought to transmit a binary single-photon signal because, over the range of starlight to moonlight, single-photon events predominate, and when measured with brighter flashes, the variance of the rod bipolar response saturates at the single-photon level. The rod’s presynaptic release function (vesicle release as a function of voltage) has very high gain, possibly as high as 1 mV e–1-fold change, allowing the tiny single-photon signal to modulate a large fraction of the dark release rate. Indeed, the rod’s calcium channels, high-affinity buffering, internal calcium stores, and PMCA are thought to participate in the high-gain release, to maximize modulation of vesicle release by the 1 mV hyperpolarization of the singlephoton signal. In addition, the postsynaptic half of the rod synapse in the rod bipolar dendrite includes several mechanisms specialized for its unique single-photon function (Figure 1(c)). It contains a second-messenger cascade that inverts the signal. A photon signal causes a drop in the rod’s glutamate release, which reduces glutamate binding to the postsynaptic mGluR6 receptor to deactivate the second-messenger cascade, opening the postsynaptic ion channels and depolarizing the rod bipolar. The secondmessenger cascade includes several steps of temporal filtering, which remove the high-frequency components of the dark continuous noise. In some species (e.g., salamander), the synaptic filter in the rod synapse is 10-fold slower than the corresponding cone synapse. This matches the slower rod transduction response and allows more complete removal of the noise. In mammals, the light-modulated

ion channel in the rod bipolar dendrite is blocked after a short delay by calcium entering the channel. The effect is to generate a transient that limits the duration of the light response in the rod bipolar.

Rate of Vesicle Release

The random release of vesicles by the rod ribbon is a major source of noise for the rod pathway. Although the details of the ribbon’s release mechanism are not known, it is stochastic (noisy), similar to a modulated Poisson distribution, for which the standard deviation is equal to the square root of the mean. The rod’s ribbon synapse is similar to other ribbon synapses in the cone and bipolar cell, but its challenge is simpler and yet more extreme. The rod’s binary signal simplifies the requirements for the synapse; it must only transmit two discriminable levels, signifying the presence or absence of a photon. However, to transmit a discriminable level, the rod must drop its calcium level, in response to a 1 mV hyperpolarization, by a fraction adequate to modulate its random release by more than one standard deviation over the single-photon signal’s rise time (100 ms). The rod’s rate of vesicle release is thought to be 100 s–1, equivalent to 10 vesicles in 100 ms, with a standard deviation of 3 vesicles, which would be adequate if the release were modulated by more than 30%, but the 1 mV single-photon signal is thought to modulate the rod’s calcium channels by only 20%. This implies either (1) a higher release rate, for example, 250 ves/s, (2) a higher voltage gain for calcium-channel gating, (3) an unknown mechanism to remove vesicle fluctuation noise, or (4) an unknown mechanism for amplifying the single-photon signal so that it can modulate a greater fraction of release. There is some evidence of a refractory period that could generate more regular release. Protons released along with a cone photoreceptor’s vesicles bind to the local calcium channels which may generate a short refractory period, allowing release to be more regular. If release in this case could be more regular than a Poisson distribution, it raises the question of why synaptic release is typically found to be Poisson-like.

Synaptic Transfer Function: Nonlinear

Threshold

The second-messenger cascade in the rod bipolar dendritic tip contains a nonlinear threshold to process the single-photon signal embodied in the binding of glutamate to the mGluR6 receptor. The rod bipolar’s dendritic ion channels do not open unless the rod signal rises above the nonlinear threshold. This mechanism removes much of the dark continuous noise remaining after the cascade’s temporal filter. When the peak amplitude of the single-photon signal falls below the nonlinear threshold,

the photon signal is lost, resulting in a false-negative, reducing the quantum efficiency of vision. When the peak amplitude of the continuous dark noise rises above the nonlinear threshold, a false-positive event is generated, which may mask individual real photon events, confounding their detection. A high nonlinear threshold will reduce the false-positive rate, but cannot reduce the rate of dark thermal events because they are identical to real photon events. Therefore, the dark thermal rate will mask a low false-positive rate, obviating the need to reduce the false-positive rate to zero. The optimal level for the nonlinear threshold thus is a compromise between false-negative and false-positive rates, and depends on the level of continuous noise, the variability of the singlephoton signal, and the thermal event rate. The optimal level for the threshold is suggested by some studies to be in the range of 0.4–0.8 times the peak amplitude of the single-photon signal, which will cause a false-negative rate of 30–50%. In some studies, the optimal level was suggested to be 1.3 times the peak amplitude of the singlephoton signal, thus losing most of the photon signals. The discrepancy between these studies emphasizes that not all the details of the rod synapse are understood. However, it is clear that when signals from 20 rods are summed in a rod bipolar cell, the nonlinear threshold reduces the noise level enough to allow a single-photon signal to be detected downstream in the visual pathway.

Electrical Coupling in Starlight

The electrical coupling between rods not only reduces their voltage noise by averaging their signals, but also reduces the amplitude of the single-photon signal. The amplitude of the single-photon signal is reduced proportionate to the equivalent number of rods, but the noise amplitude is reduced only by the square root of the equivalent number of rods coupled, which produces a net reduction in signal-to-noise ratio at each rod synapse. This type of lateral electrical coupling would not affect performance if the rod bipolar were to sum rod signals linearly, for example, without the nonlinear threshold, but in that case the noise would swamp single-photon signals as described above. The nonlinear synaptic threshold of the rod synapse depends, for its noise-reduction effect, on the amplitude of the single-photon signal, implying that electrical coupling between rods reduces the signal-to-noise ratio of the single-photon signal transmitted to the rod bipolar cell. This paradoxical result suggests that the gap junctions may be modulated by light or by the circadian clock. Alternately, the distribution of each single-photon signal to several nearby rod terminals by electrical coupling may allow vesicle fluctuation noise from rod ribbons to be averaged downstream. The amount of improvement in this case would depend on the gain and degree of nonlinearity and the amount of noise in rod vesicle release.

Electrical Coupling in Twilight

At mesopic backgrounds, rods receive 1–1000 photons per integration time, so they temporally integrate photon signals and function more like cones except that their response gain is 30-to100-fold higher than the cone signal. As the rod synapse is specialized for single-photon signals and the rod bipolar pathway is specialized for high-gain spatial summation, the robust signals in rods at twilight need an alternate pathway. The electrical coupling between rods and cones allows twilight signals to pass from rods into neighboring cones and thence into cone pathways, where, as the background level increases, the rod component drops as rods saturate or adapt and the rod-cone coupling is downmodulated. In most mammals, the rod–cone coupling is not selective for cone type, so different cone spectral types have a similar rod contribution. This is convenient because opponent color signals computed by subtraction in retinal circuits can then remove the rod contribution. The mixing of the cone’s signal with the higher-gain rod signal represents a form of adaptation because it extends by 2–3 log units the background range over which the cone ribbon synapse can transmit a signal. Rods of some species (frog, turtle, and fish) are much larger in diameter than mammalian rods and therefore, when warm, generate more dark thermal events and are more likely to receive multiple photon events at scotopic backgrounds; so in this case rod–rod coupling imitates mesopic cone–cone coupling and is advantageous. However, at low temperatures these poikilotherms (cold-blooded species) have a low dark thermal event rate and can respond to stimuli that evoke single-photon signals.

Negative Feedback

The rod ribbon synapse is similar to the cone ribbon synapse in that two horizontal cell dendritic processes are postsynaptic to each ribbon. The horizontal cell is the type B axon terminal (HBat; H1 in primate) which contacts only rods and is known to carry exclusively rod signals. The HBat is therefore considered to be electrically isolated from its cone-driven soma and dendritic tree. The function of this horizontal cell appendage is thought to be feedback to rods, but little evidence exists for negative feedback at the rod terminal. In salamander, the buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfo- nic acid (HEPES) when applied in the perfusion bath in vitro blocks the shift in the calcium current originating in horizontal cells, suggesting that rod horizontal cell feedback functions in a way similar to cone horizontal cell feedback. The HBat collects from several hundred to several thousand rods, making it a good candidate to collect an average rod signal in starlight to control, through negative feedback, the rods’ release of neurotransmitter. One likely possibility is that feedback to