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

Glossary

Amacrine cells – Neurons with cell bodies located in the proximal part of the inner nuclear layer, and neuronal processes ramifying in the inner plexiform layer.

Circadian rhythms – Changes in biological processes that occur on a daily basis, are driven by autonomous circadian clocks, and provide selective advantage to organisms by allowing them to anticipate temporal changes in their environment. Interplexiform cells – Neurons with cell bodies in the amacrine cell layer; they are distinguished from amacrine cells by having neuronal processes that project in both the inner and the outer plexiform layers.

Neuromodulators – Chemical transmitters that mediate slow, long-lasting modulatory effects on neurons and neuronal circuits through synaptic or extrasynaptic receptors; they usually have no effect on neuronal membrane potential alone, but modulate the response to other neurotransmitters.

Photopic vision – The vision in bright light that is mediated by cone photoreceptors and cone bipolar cell pathways.

Localization of Dopamine Neurons in

the Retina

Dopamine is a member of the catecholamine family of neurotransmitters, which also includes norepinephrine (also known as noradrenaline) and epinephrine (also known as adrenaline). It is the primary catecholamine in the retina; only trace amounts of epinephrine and norepinephrine are found in this tissue. It was first detected in the retina in the 1960s and has been the subject of intensive study ever since.

Dopamine is released from a unique population of neurons with cell bodies in the innermost region of the inner nuclear layer (INL). These neurons have been visualized by formaldehyde-induced histofluorescence and by immunohistochemistry, with antibodies against tyrosine hydroxylase (see Figure 1), the rate-limiting enzyme in dopamine biosynthesis. The dopamine neurons have long axons that ramify in sublamina 1 of the

inner plexiform layer (IPL), at the border of the INL. The axons contain varicosities (presumptive sites of dopamine release) along their entire length. Although the number of dopamine neurons is small (approximately 500 in mouse retina), the extensive length and branching of the axons result in considerable overlap of processes throughout the retina. Some of the axons form rings around the perikarya of AII amacrine cells, which transmit rod pathway signals in the inner retina. Some processes, likely dendritic, descend further into the IPL. In many species, the processes of the dopamine neurons project toward the outer plexiform layer (OPL; see arrow in Figure 1(a)), designating them as interplexiform cells.

Regulation of Dopamine Neuronal Activity

Retinal dopamine neurons spontaneously fire action potentials, stimulating the release of dopamine throughout the cell. Light increases the firing of action potentials and stimulates dopamine synthesis, release, and metabolism. Both transient and sustained light-evoked firing patterns have been observed.

Although the processes of dopamine neurons ramify in the outer portion of the IPL, where OFF bipolar cells make synapses, most evidence suggests that the light responses of dopamine neurons are driven by the ON pathway. Light-evoked dopamine release from monkey and frog retinas is inhibited by L-(þ)-2-amino-4-phosphonobutyric acid (L-AP4), which pharmacologically blocks synapses between photoreceptors and ON bipolar cells. Light-evoked firing of most, but not all, dopamine cells in mouse retina is also blocked by L-AP4. In addition, light-evoked dopamine metabolism, commonly observed in the vertebrate retina, is absent in the no b-wave (nob) mouse; this mouse has no functional ON pathway due to a mutation in the nyctalopin (nyx) gene, but has an intact OFF pathway. Retinal dopamine neurons are excited by glutamate, a bipolar cell transmitter, and inhibited by the amacrine cell transmitters gamma aminobutyric acid (GABA) and glycine. Once released, dopamine diffuses to act on extrasynaptic receptors found on multiple cells types throughout the retina.

Dopamine Neuronal Activity Is Coupled to Dopamine Synthesis and Metabolism

The light-evoked increase in dopamine neuronal activity is not associated with a depletion of the neuromodulator,

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(b)

Figure 1 Dopamine amacrine/interplexiform cells of the rat retina. (a) A vertical section through the retina showing a dopamine cell body and processes labeled with an antibody to tyrosine hydroxylase. Note the extensive labeling of processes in sublamina 1 of the ipl and processes ascending to the opl (arrow). onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

(b) Whole mount preparation of rat retina showing tyrosine hydroxylase immunoreactive cell bodies and processes from a horizontal view. Note the long, branching processes of the dopamine neurons and the extensive coverage of the ipl with these processes. Reproduced from Figure 1 in Witkovsky, P. (2004). Dopamine and retinal function. Documenta Ophthalmologica 108, 17–40. ã Kluwer Academic Publishers. With kind permission of Springer Science and Business Media.

and is sometimes correlated with an increase in the steady-state levels of dopamine and it’s primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). Several mechanisms appear to account for these observations. The dopamine neurons contain plasma membrane dopamine transporters, which recapture a fraction of the dopamine released into the extracellular space. Following reuptake, the dopamine can be repackaged into vesicles for subsequent release or metabolized by monoamine oxidase to form DOPAC. A light-evoked increase of DOPAC levels is a common feature among many vertebrate retinas. More important in maintaining a readily releasable pool of dopamine is a light-evoked increase of dopamine biosynthesis.

This occurs through phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. While tyrosine hydroxylase can be phosphorylated by several protein kinases, the cyclic adenosine monophosphate (cAMP)-dependent kinase (protein kinase A (PKA)) appears to play a prominant role in the lightevoked regulation of tyrosine hydroxylase. The phosphorylation of the enzyme by PKA results in a conformational change that decreases the Km for tetrahydrobiopterin (BH4), the co-factor of tyrosine hydroxylase. This increases the activity of the enzyme at the subsaturating concentrations of BH4 found in retinal dopamine cells.

Circadian Control of Dopamine Release

and Metabolism

In addition to being regulated by light, retinal dopamine release and metabolism, in many species, are controlled by circadian clocks that generate daily rhythms that persist in constant (24 h d–1) darkness. In these instances, dopamine release and DOPAC levels increase during the subjective day and decrease during the subjective night. In mouse retina, dopamine neurons express circadian clock genes and, therefore, may possess an autonomous circadian clock. However, evidence obtained in several species, including mouse, suggests that the circadian rhythms of dopamine release and metabolism are dependent on another neuromodulator, melatonin, which is synthesized in photoreceptor cells and released in a circadian fashion during the subjective night. Retinal dopamine neurons express melatonin receptors, the activation of which inhibits dopamine release. In turn, dopamine released in the daytime suppresses melatonin formation. Since dopamine receptors are widely distributed in the retina, dopamine plays important roles in the circadian organization of the retina.

Dopamine Receptors

Dopamine receptors are guanine nucleotide-binding protein (G-protein)-coupled receptors. There are five subtypes, organized into two families based on structural and pharmacological similarities: the D1-like and D2-like families (Table 1). The individual subtypes are named based on the chronological order in which they were cloned. The D1 family includes the D1 and D5 receptors, while the D2 family consists of D2, D3, and D4 receptors. The D1 and D5 (also known as D1B) receptors are derived from genes without introns; thus, there are no splice variants of the D1-like receptors. They show very similar pharmacologies with respect to selective agonists and antagonists; however, dopamine has a much higher affinity for the D5 receptor than for the D1 subtype. The D2, D3, and

D4 receptor genes contain introns, and splice variants of the D2 receptor have been characterized. They show similar pharmacologies; however, some selective agonists and antagonists have been identified. A common feature of all dopamine receptors is that they couple to G proteins that regulate cAMP. The D1 family receptors stimulate cAMP formation, while the D2 family members inhibit it. Dopamine receptors have also been reported to affect phospholipase C, Ca2þ currents, Kþ currents, and the protein kinases AKT, glycogen synthase kinase-3b (GSK3b), and extracellular signal-regulated kinases (ERK1/2).

Nearly all retinal cell types appear to express dopamine receptors, consistent with dopamine’s role as a paracrine neuromodulator that acts on extrasynaptic receptors. Of the D1 family members, dopamine D1 receptor immunoreactivity is found on processes in both the OPL and IPL. The receptors are expressed by horizontal cells, subtypes of cone bipolar cells, and some amacrine cells. There is evidence for the expression of functional D1-like receptors in ganglion cells of goldfish. D5 receptors are expressed by mammalian retinal pigment epithelial (RPE) cells. Of the D2 family members, dopamine D2 receptors are highly expressed by retinal dopamine neurons, and function as autoreceptors that inhibit dopamine release. D2 receptors are also expressed by other unidentified amacrine cells and possibly by some bipolar and ganglion cells. D2 receptors have also been shown to be expressed and functional in guinea pig Mu¨ller cells. Dopamine D4 receptors are highly expressed by photoreceptor cells in mammals. The photoreceptors of nonmammalian vertebrates also have D2-like receptors; in addition, these are likely to be D4 receptors based on pharmacological criteria, but no molecular proof of this has been provided yet. D4 messenger ribonucleic acid (mRNA) expression has also been observed in the IPL and ganglion cell layer; however, due to the lack of specific antibodies, the types of cells in these layers have not been identified. Interestingly, neither mRNA nor protein for the dopamine D3 receptor has been found in retina.

Functions of Dopamine in the Retina

The widespread distribution of dopamine receptors coupled with the ability of dopamine to diffuse throughout the retina suggests that this neuromodulator has many functions in regulating retinal physiology, particularly in serving as a chemical signal promoting light adaptive functions. This section describes the effects of dopamine on different cell types within the retina, beginning with the distal retina, and gives examples on how dopamine impacts retinal network adaptation, circadian rhythmicity, and ocular growth.

Retinal Pigment Epithelium

Dopamine D5 receptors stimulate cAMP formation, which inhibits rod outer segment phagocytosis by RPE cells. Cultured bovine RPE cells express D5 receptors and dopamine inhibits phagocytosis by these cells. Thus, dopamine may function as a component of the regulatory mechanism that controls rod outer segment turnover.

Photoreceptor Cells

Dopamine D2/D4 receptors on photoreceptor cells have numerous effects due to their ability to inhibit cAMP formation and decrease intracellular Ca2þ concentrations. Dopamine receptor activation inhibits the synthesis of melatonin in photoreceptor cells by inhibiting cAMP formation. It regulates the amplitude of diurnal rhythms of phosphorylation of phosducin by cAMPand Ca2þ- dependent protein kinases. Dopamine has been reported to increase the rate of dephosphorylation of rhodopsin, and probably affects the phosphorylation state of many photoreceptor proteins. Dopamine has been reported to inhibit the hyperpolarization-activated current (IH) in rods, as well as the activity of Na+/K+-adenosine triphosphate (ATP)ase, which balances the dark current. It decreases the intracellular Ca2+ concentration of photoreceptor cells and also affects rod–cone coupling, but the effects may vary by species. These effects on currents, Ca2+, and coupling may contribute to a reduction of rod synaptic transfer to bipolar and horizontal cells during the daytime.

Horizontal Cells

Horizontal cells mediate lateral inhibition and synaptic feedback to photoreceptor cells. Different horizontal cell subtypes couple together through gap junctions to form networks. In retinas of both mammalian and nonmammalian vertebrates, the activation of D1-like receptors uncouples the horizontal cells, narrowing their receptive fields. The activation of dopamine D1 receptors in darkadapted retinas depolarizes horizontal cells and reduces

responses to flickering lights. The depolarization is due to the cAMP-dependent enhancement of glutamate-gated currents through a-amino-3-hydroxyl-5-methyl-4-isoxazole- propionate (AMPA)/kainate glutamate receptors in the horizontal cell membrane; other voltage-gated channels may also be involved.

Bipolar Cells

Although dopamine receptor immunoreactivity has been observed in subtypes of mammalian cone bipolar cells, its function is yet to be investigated. In salamander retina, glutamate responses in OFF cone bipolar cells are enhanced by dopamine, similar to the enhancement observed in horizontal cells. In addition, dopamine has been shown to modulate gamma-aminobutyric acid (GABA)-mediated inhibition of Ca2þ influx and neurotransmitter release from bipolar cell terminals. GABAergic amacrine cells synapse onto bipolar cell terminals to provide feedback inhibition of glutamate release via GABA-C receptors. Dopamine, acting on D1 receptors, reverses this inhibition and increases glutamate release.

Amacrine Cells

AII amacrine cells are critical mediators of the rod pathway. They receive excitatory input from rod depolarizing bipolar cells and, thus, depolarize in response to light. They transmit hyperpolarizing light signals to OFF cone bipolar cells through inhibitory glycinergic synapses and depolarizing light signals to ON cone bipolar cells through gap junctions. In addition, AII amacrine cells form homotypic gap junctions with neighboring AII amacrines. The axons of the dopamine neurons surround the perikarya of AII amacrine cells. Dopamine and D1 receptor agonists promote the uncoupling of gap junctions in AII amacrine cells by cAMP-dependent phosphorylation of connexin proteins. The gap junctions between amacrine cells are more sensitive to the uncoupling action of dopamine than those between AII amacrine cells and ON cone bipolar cells. In addition to secreting dopamine, the dopamine amacrine/interplexiform cells also synthesize and release the inhibitory neurotransmitter GABA. The dopamine amacrine/interplexiform cells synapse onto the AII amacrines, where their processes form rings around the perikarya of the AII cells. The active zones of the synapses have GABA-A receptors. Thus, activation of the dopamine amacrine/interplexiform cells is likely to decrease the light response of AII amacrine cells through GABA. Interestingly, D1 dopamine receptors have not been observed in these active zones, suggesting that dopamine diffuses to distal receptors to promote AII–AII cell uncoupling.

Dopamine stimulates acetylcholine release in the retina through D1-like receptors; most acetylcholine in mammalian retina is released from starburst amacrine

cells, which function in the circuitry regulating directional selectivity to moving stimuli.

Most of the synapses made by dopamine amacrine/ interplexiform cells are onto other amacrine cells. Thus, it is likely that dopamine affects many other functions within the inner retinal circuitry, but the details and functional consequences for visual processing are less clear.

Ganglion Cells

The effects of dopamine on ganglion cells are complex and it is difficult to discern which responses result from direct actions on ganglion cells and which responses reflect effects on retinal circuitry. In the mammalian retina, dopamine appears to decrease the sensitivity to light and strengthen the center surround of ganglion cells. These effects are mediated by dopamine D1-like receptors, and D1 receptor antagonists have opposite effects. A D1 receptor blocker also reduces the response of ON–OFF directionally sensitive ganglion cells to the leading edge of a moving light stimulus. In addition, dopamine strengthens cone pathway input to ganglion cells and reduces rod pathway input.

A small subpopulation of ganglion cells is referred to as intrinsically photosensitive retinal ganglion cells (ipRGCs). These ganglion cells project to the suprachiasmatic nucleus (SCN) of the hypothalamus, the site of the master circadian clock in mammals; to the olivary pretectal nucleus, a relay nucleus in the pupillary light reflex circuit; and to other nuclei involved in nonimage forming vision. These ipRGCs mediate photic entrainment of the circadian clock and are essential for the normal pupillary light reflex. They contain a photopigment, melanopsin, and are directly responsive to light, showing a depolarizing response to illumination. The ipRGCs also receive input from rods and cones through retinal circuitry. In mammals, dopamine amacrine/ interplexiform cells are a component of this circuitry and synapse directly onto the ipRGCs. In addition, dopamine drives a circadian rhythm of melanopsin expression in the ipRGCs, at least in rats.

Mu¨ller Glial Cells

Mu¨ller cells are giant glia that span the width of the neural retina from the outer limiting membrane to the inner limiting membrane. They play important roles in retinal physiology by regulating the extracellular ionic milieu, especially the Kþ concentration. Mu¨ller cells are also an important source of trophic factors within the retina. Mammalian Mu¨ller cells express dopamine D2 receptor immunoreactivity. The application of dopamine or a D2 receptor agonist decreases Kþ conductance through an inwardly rectifying Kþ channel. These findings indicate that dopamine may influence the extracellular Kþ clearance, thereby affecting neuronal excitability and visual processing in the retinal circuitry.

Role of Dopamine in Photopic Visual Processing

Overall, the effects of dopamine on individual retinal cell types are consistent with an important role for this neuromodulator in visual processing in light-adapted retinas. Dopamine facilitates cone input to the inner retina and diminishes rod input. Extensive rod–cone coupling at night may shunt cone currents to rods, accounting for the apparent lack of cone input at night in the absence of dopamine. The uncoupling of the rod–cone network in the daytime in response to dopamine isolates the cones, strengthening their input signals to the inner retina. The effect of dopamine on horizontal cell coupling narrows receptive fields and makes the center surround organization more compact, enhancing spatial contrast sensitivity. The enhancement of glutamate-gated currents on OFF cone bipolar cells would also be expected to strengthen cone input. In addition, the uncoupling of the AII amacrine cell network by dopamine, together with the inhibitory effect of co-released GABA on AII amacrine cell light responses, should further suppress information flow through the rod pathway. Collectively, activation of the dopamine amacrine/interplexiform cell appears to fine-tune the retinal circuitry for high-resolution, high-contrast, lowsensitivity visual processing under photopic conditions.

Dopamine and Circadian Organization of the

Retina

Many cells types in the vertebrate retina express circadian clock genes, including the dopamine amacrine/ interplexiform cells. Dopamine can entrain or phase-shift the circadian clock in amphibian photoreceptor cells that regulates melatonin biosynthesis. Dopamine affects the circadian expression of a clock gene reporter (period 2:: luciferase) in the inner retina of mice. In addition, circadian- clock-driven dopamine release drives circadian rhythms of rod–cone coupling in mice and fish, and photoreceptor protein phosphorylation in mouse photoreceptors. With the widespread distribution of dopamine receptors in retina, the ability of dopamine to diffuse throughout the retina, the expression of clock genes in the dopamine amacrine/interplexiform cells, and the large increase in dopaminergic activity at dawn, dopamine may play important, conserved roles in the circadian organization of the retina by entraining and coordinating multiple circadian oscillators.

Dopamine, Retinal Development, Ocular growth, and Myopia

In chick embryo retinal cell cultures, dopamine has been reported to inhibit growth cone motility. In addition, the signaling mechanisms utilized by dopamine receptors appear to change with the developmental stage. Dopamine may play a role in stabilizing synapses during retinal

development. However, many of these studies were done with in vitro systems, and may not necessarily reflect the development of the retina in vivo.

Dopamine may also be involved in ocular development and establishing emmetropia (perfect, focused vision). At birth, most animals are hyperopic, with images focused posterior to the photoreceptors. During the postnatal period, the eye elongates, increasing its own focal length. Under normal conditions, the eye will elongate only to the point that the image is focused on the photoreceptor cells, yielding the condition referred to as emmetropia. However, if the eye grows excessively long, the image will focus in front of the retina, causing myopia (nearsightedness). The growth of the eye in the axial dimension is regulated by the retina through visually guided feedback mechanisms that coordinate the growth of the eye with its optics. If the retinal image is degraded, as in the case of congentital infant cataract, the eye grows excessively long, resulting in myopia. This process is referred to as form-deprivation myopia and has been studied extensively in chicken hatchlings. When a diffuser goggle is placed over the eye of a newly hatched chick, the eye will grow excessively long, resulting in a large myopic refractive error within 1–2 weeks. Interestingly, allowing short periods of vision each day without the diffusers prevents the development of myopia.

Although some controversy exists, dopamine appears to contribute to the retinal circuitry involved in the feedback mechanism controlling emmetropization. Retinal dopamine levels and metabolism are reduced in eyes of chicks exposed to form deprivation. The administration of apomorphine, a dopamine receptor agonist, prevents the development of form-deprivation myopia. Moreover, dopamine receptor antagonists block the ability of short periods of unobstructed vision to prevent the development of myopia.

Summary

Dopamine release is driven by light and regulated by circadian clocks. The neuromodulator regulates multiple functions within the retina primarily by extrasynaptic mechanisms. Dopamine receptors are widely distributed in the retina, and dopamine has the capacity to diffuse from sites of release to distant receptors. A primary function of dopamine is to regulate network adaptation within the retina to optimize vision during the daytime.

Acknowledgements

The research in the author’s laboratory is supported by NIH grants EY004864 and EY006360.

See also: Circadian Metabolism in the Chick Retina; Circadian Photoreception; Circadian Regulation of Ion

Channels in Photoreceptors; Information Processing: Amacrine Cells; Morphology of Interneurons: Amacrine Cells; Morphology of Interneurons: Interplexiform Cells; Neurotransmitters and Receptors: Melatonin Receptors; The Circadian Clock in the Retina Regulates Rod and Cone Pathways.

Further Reading

Djamgoz, M. B. A., Hankins, M. W., Hirano, J., and Archer, S. N. (1997). Neurobiology of retinal dopamine in relation to degenerative states of the tissue. Vision Research 37: 3509–3529.

Green, C. B. and Besharse, J. C. (2004). Retinal circadian clocks and control of retinal physiology. Journal of Biological Rhythms 19: 91–102.

Nguyen-Legros, J., Versaux-Botteri, C., and Vernier, P. (1999). Dopamine receptor localization in the mammalian retina. Molecular Neurobiology 19: 181–204.

Ruan, G. X., Allen, G. C., Yamazaki, S., and McMahon, D. G. (2008). An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA. PLoS Biology 6: e249.

Tosini, G., Pozdeyev, N., Sakamoto, K., and Iuvone, P. M. (2008). The circadian clock in the mammalian retina. BioEssays 30: 624–633.

Witkovsky, P. (2004). Dopamine and retinal function. Documenta Ophthalmologica 108: 17–40.

Zhang, D. Q., Zhou, T. R., and McMahon, D. Q. (2007). Functional heterogeneity of retinal dopaminergic neurons underlying their multiple roles in vision. Journal of Neuroscience 27: 692–699.

Glossary

Circadian rhythm – The term from the Latin circa which means around and diem which denotes day that is an approximate daily (24-h) periodicity in physiological processes of many organisms. Diurnal – Activities that are repeated every 24 h, but are not necessarily under the control of a biological clock.

Orthologs – Homologous sequences which are similar to each other because they originated from a common ancestor.

Paracrine – The term from the Latin para, which means near, is a form of cell signaling in which the target cell is located within the same tissues as the cell that releases the chemical signal. Pinealocytes – The main cells of the pineal gland that produce and secrete melatonin into the circulation.

Xenopus – The term from the Latin strange foot, which is a genus of frog native to Africa, and commonly used in research as a model organism.

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine hormone synthesized by pinealocytes and retinal photoreceptors. The rate of melatonin synthesis, in most species studied, is highest at nighttime, and is considered to be a chemical signal of darkness that entrains circadian rhythms. This hormone synthesized in the pineal gland is secreted immediately into the circulation and acts as an endocrine hormone on distant target sites throughout the body. Melatonin produced in the retina, however, is thought to have a local, or paracrine role. It is thought that melatonin is synthesized and released by the photoreceptors at night, and diffuses throughout the retina to bind to melatonin receptors located on a variety of retinal cells. Since melatonin is a very lipophilic molecule, it diffuses freely through plasma membranes.

The three major subtypes of melatonin receptors are members of the superfamily of guanine nucleotide binding (G-protein)-coupled receptors. Most studies have shown that melatonin receptor activation is coupled to an inhibition of adenylate cyclase activity, although many reports

demonstrate that other signaling mechanisms are conveyed by the melatonin signal. Melatonin receptors have been identified in many different retinal cells, including amacrine cells, horizontal cells, ganglion cells, photoreceptors, and the adjacent retinal pigment epithelium (RPE).

Sites of Retinal Melatonin Synthesis

Melatonin Synthesis by Photoreceptors

The photoreceptors appear to be the sites of melatonin synthesis in the retina. They express all of the enzymes involved in melatonin synthesis. Melatonin is synthesized from tryptophan in a series of four enzymatic steps: (1) tryptophan is converted into 5-hydroxytryptophan by tryptophan hydroxylase (TPH); (2) the 5-hydroxytryptophan is then converted into 5-hydroxytryptamine (serotonin) by aromatic amino acid decarboxylase; (3) serotonin is then converted into N-acetylserotonin by arylalkylamine N-acetyltransferase (AANAT); and (4) N-acetylserotonin is converted into melatonin (N-acetyl-5-methoxytryptamine) by hydroxyindole-O-methyltransferase (HIOMT). The enzyme activity and messenger RNA (mRNA) encoding TPH and AANAT exhibit circadian rhythms of expression, with highest levels occurring at night.

There is strong evidence that identifies the photoreceptors as the sites of retinal melatonin synthesis. Melatonin immunoreactivity is localized in the outer nuclear layer (ONL) of the retina which contains the cell soma of the photoreceptors. HIOMT and AANAT protein and mRNA are localized to photoreceptor cytoplasm, and a cyclic rhythm of AANAT activity persists following chemical lesion of the inner retina. The mRNA encoding TPH is localized to photoreceptors, and the photoreceptor layer of the amphibian retina continues to produce melatonin rhythmically in darkness after isolation from the inner retina.

In addition to the photoreceptors, some neurons of the inner retina may have the capacity to produce a small amount of melatonin. Melatonin immunoreactivity is observed in the inner retina, and a low level of AANAT mRNA has been detected in the inner nuclear layer (INL) and ganglion cell layer (GCL). The INL contains the cell soma of amacrine, horizontal, bipolar, and Mu¨ller cells. Since some cells in these layers also have melatonin receptors, the synthesis of melatonin by inner retinal neurons may be involved in the circadian activity of cells of the inner retina.

Phylogenetic Relationships between Photoreceptors and Pinealocytes

The ability of photoreceptors and pinealocytes to synthesize melatonin appears to be the consequence of an ancestral relationship between the retina and the pineal gland. Some primitive animals possessed three eyes which may have produced melatonin and were also capable of phototransduction. Pinealocytes of some lower vertebrates are morphologically very similar to retinal photoreceptors, and they synthesize melatonin as well as many proteins that are characteristic of retinal photoreceptors. Furthermore, the photoreceptors of the nonmammalian pineal gland are directly photosensitive, and during the embryologic development of the mammalian pineal gland, the pinealocytes undergo a transient photoreceptor-like differentiation.

It is suggested that the middle, or third eye, eventually evolved into an endocrine organ specialized for the secretion of melatonin into the circulation, in which the melatonin-producing pineal photoreceptors eventually lost their phototransduction capabilities, and the melatonin-producing cells of the lateral eyes evolved into photoreceptors specialized for phototransduction, but maintained their ability to synthesize melatonin. Genes expressing melatonin receptors in peripheral tissues may have also become expressed in ocular tissues, which enabled local paracrine signaling by melatonin in the retina.

Classification of Melatonin Receptors

Melatonin receptor expression has been identified in the retinas of several species. The major types of melatonin receptors that have been cloned are members of the superfamily of G-protein-coupled receptors. Melatonin receptors have been classified as Mel1a, Mel1b, and Mel1c subtypes. These three subtypes are expressed in tissues, including the retinas, of lower vertebrates such as amphibians, fish, and birds. Mammalian melatonin receptors are classified according to their homology to the nonmammalian receptors, and according to their pharmacological properties. In mammals, the Mel1a and Mel1b receptor subtypes are designated as the MT1 and MT2 receptor subtypes, respectively. The mammalian ortholog of the Mel1c subtype has been designated as GPR50, and does not bind melatonin. The G-alpha proteins coupled to melatonin receptors are inhibitory (Gi) to the activation of adenylate cyclase and cyclic adenosine monophosphate (cAMP) production in most tissues studied. However, receptor coupling to other G-alpha proteins (Gia2, Gia2, Giaq, Gias, Giaz, and Gia16), and hence other signaling pathways, have been reported. Nuclear melatonin receptors, which are members of the RAR-related RZR/ROR

orphan nuclear receptor superfamily, have been reported to exist in some tissues, and a melatonin-binding site on the enzyme quinine reductase 2 has been identified and is referred to as the mammalian MT3 melatonin receptor.

Most G-protein-coupled receptors interact with each other to form homodimers or heterodimers. The mammalian MT1 and MT2 melatonin receptors can exist as homodimers and as heterodimers. Dimerization of G-protein-coupled receptors has important functional consequences in regard to receptor affinity, trafficking, and signaling. The relative expression levels of melatonin receptor subtypes in the retina may have a significant impact on the function of melatonin in the target cells.

Sites of Melatonin Receptors in the Retina

Melatonin receptors have been identified not only in several retinal neurons of the inner retina, but also in photoreceptors cells and RPE cells. The identification of melatonin receptors in photoreceptor cells was unanticipated, given that the photoreceptors are the site of retinal melatonin synthesis. The presence of melatonin receptors in photoreceptor cells suggests the possibility of what can be characterized as an intracrine (autocrine) signaling mechanism in response to melatonin, in which a cell synthesizes and releases a signaling molecule, and also has receptors to which the molecule binds and triggers an intracellular response. Another potential role of melatonin receptors in photoreceptor cells is that they may be involved in a negative-feedback mechanism which would enable melatonin to regulate the expression of the receptors to which it binds.

Melatonin Receptors in Photoreceptor Cells

Mel1a, Mel1b, and Mel1c melatonin receptor subtype protein and mRNA have been identified in the photoreceptors of nonmammalian vertebrates, and MT1 receptors in photoreceptors of the mammalian retina. The receptor immunoreactivity has been identified primarily in the photoreceptor membranes of the inner segments, although some cytoplasmic immunoreactivity has been reported for some subtypes, and may represent newly synthesized receptors that have not yet been transported to the plasma membrane, or receptors that have been internalized after activation.

Mel1b and Mel1c melatonin receptor RNA and/or protein are expressed in Xenopus photoreceptors, and the MT1 (Mel1a) receptor is localized to photoreceptors of the human retina. In the chicken retina, Mel1a immunoreactivity and melatonin receptor mRNA expression (Mel1a, Mel1b, and Mel1c) are localized to the photoreceptor layer. In the Xenopus retina, Mel1c immunoreactivity is observed in the plasma membrane of photoreceptor

inner segments, whereas Mel1b receptor immunoreactivity appears in a punctate pattern in the proximal portion of photoreceptor inner segments. The differential pattern of Mel1b and Mel1c may reflect differential regulation of expression or trafficking of melatonin receptors in photoreceptor cells.

Melatonin Receptors in RPE

The RPE is a monolayer of cuboidal cells located between the vascular choroid layer and the neural retina. It is very closely associated with the retinal photoreceptor cells. This close association reflects the vital function of the RPE to provide physical and metabolic support to the photoreceptors. Circadian signals may play a role in influencing the coordinated interactions between the RPE and its adjacent tissues. The RPE, photoreceptors, retinal neurons, and choroidal cells interact in a coordinated manner for optimal function. Melatonin may play a role in the timing of the circadian phagocytosis of shed photoreceptor outer segments. The distal tips of rod photoreceptor outer segments are shed on a circadian rhythm as part of a renewal process, with peak shedding occurring early in the light period. The shed outer segment tips are phagocytized by the RPE, and melatonin is thought to be involved in this process. Melatonin secreted from photoreceptors at night may activate melatonin receptors on the RPE to regulate some circadian activities of the RPE that are important for optimal photoreceptor activity.

Melatonin inhibits forskolin-stimulated cAMP synthesis in RPE cell cultures, and melatonin affects the RPE membrane potentials and resistances at the apical or basal membrane. The mRNA encoding all three melatonin receptor subtypes is expressed in Xenopus laevis RPE but the Mel1b receptor protein is localized only to the apical surface of the Xenopus RPE, and is not present on the basal surface. The Mel1c receptor protein has also been localized to the Xenopus RPE. The presence of melatonin receptors on the apical microvilli, which directly contact the photoreceptors, but not on the basal membrane of the RPE, suggests that the photoreceptors are more likely to be the source of melatonin that activate melatonin receptors on the RPE rather than melatonin that is produced by the pineal gland and secreted into the general circulation.

Melatonin Receptors in Inner Retinal Neurons

Using autoradiography with 125I-melatonin, it has been demonstrated that melatonin binding occurs in the inner plexiform layer (IPL) of many species. The IPL contains the synaptic terminals between bipolar cells, amacrine cells, horizontal cells, and ganglion cells. Since melatonin inhibits dopamine release from the retina, and highaffinity melatonin binding occurs in the IPL of the retina, the dopaminergic amacrine cell, which forms synaptic contacts in the IPL, has long been considered to be a

candidate for the site of action of melatonin in the inner retina. Another candidate cell for melatonin receptor expression is the GABAergic amacrine cell (GABA, gamma aminobutyric acid) of the INL since GABAA receptor antagonists block melatonin-induced suppression of dopamine release. This suggests that the effect of melatonin on dopamine release may not be mediated only by direct action on dopaminergic cells, but that indirect action on GABAergic amacrine cells may also contribute to the inhibition of dopamine release via melatonin.

The autoradiographic localization of melatonin-binding sites in cells of the inner retina has been confirmed by both in situ hybridization and immunocytochemistry. In Xenopus, Mel1b and Mel1c RNA expression is localized to the INL, GCL, and photoreceptor inner segments. In the chicken retina, the mRNA encoding the Mel1a, Mel1b, and Mel1c receptor subtypes is present in the INL, GCL, and photoreceptor inner segments. The INL contains the cell soma of bipolar, amacrine, horizontal, and Mu¨ller cells. In the human retina, Mel1b receptor mRNA is much more highly expressed than is Mel1a receptor RNA, suggesting that the Mel1b receptor has a more significant role in human retinal physiology.

Using antibodies against specific melatonin receptor subtypes for immunocytochemistry, all three melatonin receptor subtypes (Mel1a, Mel1b, and Mel1c) have been observed in the outer plexiform layer (OPL; the layer that contains the synaptic contacts between photoreceptors, bipolar cells, and horizontal cells) and the IPL. The MT1 (Mel1a) receptor has been localized to horizontal cells in several mammalian species, including human. All three melatonin receptor subtypes appear to be present in horizontal cells of fish and Xenopus retina. The MT1 (Mel1a) receptor has also been localized to AII amacrine and GABAergic amacrine cells of the mammalian retina.

Some Mel1a and Mel1c receptor immunoreactivity co-localizes with GABAergic and dopaminergic amacrine cells in the Xenopus retina. The presence of Mel1a and Mel1c receptors on dopaminergic and GABAergic amacrine cells is consistent with the observation that melatonin modulates the cyclic release of GABA and dopamine from retinal amacrine cells. In contrast, Mel1b receptor immunoreactivity does not appear to co-localize with markers for dopaminergic and GABAergic neurons in the Xenopus retina, suggesting that melatonin does not act directly on GABAergic and dopaminergic amacrine cells through the Mel1b receptor in this species.

In the Xenopus retina, the Mel1a, Mel1b, and Mel1c receptor proteins are differentially distributed throughout the retina. In the OPL, for example, presumptive horizontal cell processes are immunoreactive for Mel1a and Mel1b receptor subtypes, but the immunoreactive labels appear to be in different cell processes. Cell somas in the INL are immunoreactive either for Mel1b or Mel1a, or for Mel1a or Mel1c, but not for both. All three melatonin receptor

subtypes appear to be expressed in different populations of ganglion cells in the Xenopus retina, and the MT1 subtype is present in ganglion cells of the human and macaque retina.

Melatonin receptor mRNA and protein are rhythmically expressed in Xenopus and chicks, with peak levels of Mel1c expression occurring in the day. In chicks, the rhythms of Mel1a and Mel1b receptor protein generally appear to be the opposite to that of Mel1c, with lowest levels occurring in the early morning and higher levels in the evening. The patterns of cyclic rhythms appear to be distinctive for each receptor subtype in the retina. Circadian rhythms in melatonin receptor expression may perhaps be superimposed on the rhythm in retinal melatonin levels to provide an additional level of regulation of the responsiveness of retinal target to melatonin.

Effects of Melatonin on Retinal Function

Modulation of Neurotransmitter Release

Melatonin released from photoreceptors at night diffuses into the extracellular milieu and binds to melatonin receptors on dopaminergic amacrine cells. The activation of the melatonin receptors results in a decrease of dopamine release at nighttime. Thus, retinal dopamine levels are higher during the day and lowest during the night due to the circadian release of melatonin. Resultant lower dopamine levels at night cause a reduction in D2 dopamine receptor activity on photoreceptors, causing an increase in photoreceptor intracellular cAMP levels that in turn causes an increase in coupling of gap junctions between rod and cone photoreceptors so that rod input dominates the cone horizontal cells. The increased sensitivity of horizontal cells to light at nighttime is therefore mediated at least in part by activation of D2-like receptors by dopamine released from amacrine cells. A reduction in endogenous retinal dopamine levels causes hyperpolarization of horizontal cells and enhanced dark adaptation.

D1 dopamine receptors, which are positively coupled to cAMP synthesis, are located on horizontal cells. Melatonin may therefore postsynaptically regulate horizontal cell activity by inhibiting the stimulation of cAMP synthesis in response to D1 receptor activation. It may bind to receptors on GABAergic amacrine cells, stimulating them to inhibit dopamine release from nearby dopaminergic amacrine cells. In addition, since horizontal cells express melatonin receptors, and melatonin increases horizontal cell sensitivity to light, melatonin may act directly on horizontal cells to increase gap-junctional coupling of horizontal cells. Increased horizontal cell coupling would cause an increase in receptive field size, which would potentially increase the sensitivity of the retina to light during the dark period, since more second-order neurons would respond to a light stimulus.

Melatonin may therefore modulate dopaminergic transmission by a combination of directly reducing dopamine release from amacrine cells, and indirectly by stimulating GABAergic amacrine cells to inhibit dopamine release from dopaminergic amacrine cells, both of which would increase horizontal cell coupling. The resulting increased visual sensitivity at nighttime could be due to increased rod–cone coupling through dopamine binding to D2 receptors on photoreceptors, or to horizontal cell coupling stimulated by the binding of melatonin to melatonin receptors on horizontal cells. A summary diagram of the known locations of melatonin receptors and the possible interactions with the various target cells is presented in Figure 1.

Melatonin increases horizontal cell sensitivity to light in salamander retina, and also potentiates glutamateinduced currents from isolated cone-driven horizontal cells in carp retina by increasing the efficacy and affinity of the glutamate receptor. These observations suggest that melatonin acts directly on melatonin receptors of horizontal cells. Melatonin modulates cyclic guanosine monophosphate (cGMP)-dependent glutaminergic transmission from cones to cone-driven horizontal cells by activation of the Mel1a receptor, causes a depolarization of the H1 horizontal cell membrane potential, and reduces its light responses. These observations suggest that melatonin enhances the circadian sensitivity of rod photoreceptor signaling.

Melatonin has been shown in fish retina to potentiate responses of rod ON bipolar cells to simulated light flashes. This action of melatonin is mediated by the Mel1b receptor, and increases cGMP levels by inhibiting phosphodiesterase activity. Melatonin may bind directly to Mel1b receptors on rod ON bipolar cells to improve the signal/noise ratio for rod signals by enhancing signal transfer from rod photoreceptors to rod bipolar cells. The presence of melatonin receptors by immunocytochemistry has not yet been definitively established in bipolar cells.

Melatonin treatment of isolated rat retinal ganglion cells potentiates glycine-induced currents by increasing the efficacy and channel conductance of a glycine receptor. The inhibitory modulation of glycinergic inputs to ganglion cells may thus be strengthened by stimulation of melatonin receptor activation. This suggests that melatonin may regulate circadian changes in receptive field organization and light sensitivity by binding to melatonin receptors on ganglion cells.

Modulation of Photoreceptor Function

Several reports support the concept of a direct action of melatonin on retinal photoreceptor function. Melatonin induces membrane conductance changes in isolated frog rod photoreceptors, binds with low affinity to structures in the OPL in frog retina, enhances the rate of photoreceptor