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

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Melanopsin and the Mammalian ipRGC

Among the nonvisual opsins, melanopsin has been the most extensively studied photopigment. It is expressed in a small subset of RGCs that project to sites in the brain not involved in the formation of images. Originally cloned from the photosensitive dermal melanophores of Xenopus laevis, homologs were subsequently identified in chicken and other nonmammalian vertebrates. A second melanopsin gene was recognized in the chicken genome and is also present in other vertebrate classes. However, based on chromosome synteny, it is clear that one of these two genes has been lost in mammals.

In the nonmammalian vertebrates, melanopsin is expressed in the retina and extraretinal tissues, including the

iris, brain, pineal, and skin. Many of these sites had been shown previously to be inherently light sensitive, although the photopigments mediating the sensitivity were unidentified. Unlike the broad anatomical distribution observed in nonmammalian vertebrates, in mammals, melanopsin expression is restricted to less than 2% of the RGCs. Notably, extraretinal expression has not been observed in any mammal.

Pituitary adenylyl cyclase-activating peptide and glutamate are coexpressed within the melanopsin-containing cells. Melanopsin is what confers photosensitivity upon these intrinsically photosensitive retinal ganglion cells (ipRGCs). Loss of melanopsin renders these cells nonresponsive to light.

Murine ipRGCs elaborate two or three primary dendrites which bifurcate within 50 mm of the perikaryon. The

dendrites ramify within the S1 (OFF layer) or S5 (ON layer) sublaminae of the inner plexiform layer. A small fraction of ipRGCs may have dendrites arborizing in both S1 and S5. Dendrites are studded with varicosities giving a rosary bead appearance. Melanopsin protein is found throughout the plasma membrane of the cell body, the segment of the axon contained within the eye, and the dendritic arbor. Arbors are sparse, regularly tiled across the retina with substantial overlap, and rather large, having mean field diameters of around 450 mm in mice and 600 mm in rats. The receptive field of ipRGCs corresponds to the dimension of the dendritic field, indicating that the entire arbor is capable of initiating phototransduction. Membrane density of melanopsin is about 10 000-fold lower than that of rods and cones. This is largely attributable to the lack of a specialized light harvesting organelle in ipRGCs comparable to the photopigment-dense outer segments of rods and cones. This relative paucity of photopigment in ipRGCs results in a very low photon-capture efficiency, thereby rendering this system effective only in very bright irradiances. Nevertheless, ipRGCs, similar to rods, can signal the absorption of single photons. The spectral sensitivity of dark-adapted melanopsin peaks in the blue wavelengths and coincides with the action spectra of many of the nonvisual responses previously described, including circadian photoentrainment and the pupillary light reflex of visually blind mice that lack rods and cones.

A comparison of the melanopsin peptide sequence against known opsins indicates that it more closely resembles the rhabdomeric opsins of invertebrates rather than the ciliary opsins of vertebrates. Not surprisingly, melanopsin employs a phototransduction cascade more similar to that of insect rhabdomeres rather than vertebrate outer segments. Illuminated amphibian melanophores darken and exhibit a light-stimulated increase in inositol trisphosphate and protein kinase C (PKC)-dependent phosphorylation. Presumably, these effects are mediated by a melanopsininitiated cascade because overexpression of melanopsin

hypersensitizes melanophores to light. Inhibition of phospholipase C (PLC), PKC, or chelation of intracellular calcium blocks melanophore darkening suggesting that this light-initiated response must activate a phosphoinositide signaling cascade. Heterologous expression of melanopsin in HEK293 cells renders them light sensitive and leads to a light-dependent increase in intracellular calcium and depolarization of membrane potential. Depolarization can be blocked by pharmacologic inhibitors of the Ga(q/11) subunit of guanine nucleotide-binding protein or PLC. These lines of evidence again suggest that light-activated melanopsin triggers a phosphoinositide signaling cascade similar to that of rhabdomeric photoreceptors. These same transduction components are found within ipRGCs and are critical for eliciting photoresponses. Furthermore, responses to light can be elicited in excised, inside-out patches of ipRGC membrane indicating that the critical signaling molecules are closely associated or are within the plasma membrane. A hallmark of rhabdomeric opsins is the formation of a stable red-shifted metastate in response to illumination. Whether melanopsin forms such a state remains equivocal, although some dark-adapted responses to blue light appear to be primed by preexposure to red light, which presumably photoconverts a red-sensitive metastate back to the blue-sensitive dark-adapted state. The general similarity to rhabdomeric signaling indicates that ipRGCs and invertebrate photoreceptors may have shared a common evolutionary ancestor.

ipRGCs project to central sites not associated with vision but implicated in regulating different forms of nonvisual photophysiology. For example, the SCN of the hypothalamus, the site of the primary circadian pacemaker, receives a robust input from ipRGCs. By contrast, the classical, nonphotosensitive RGCs provide minimal innervation to the SCN. Photoreceptor-mediated effects on the circadian axis occur exclusively through ipRGCs, suggesting that classical retinal rod and cone circuits impinge upon ipRGCs at the inner plexiform layer. Other principle central targets of ipRGCs include the IGL, the olivary pretectal nucleus, and the lateral habenula. The subparaventricular zone, the ventrolateral preoptic nucleus, and the ventral lateral geniculate nucleus are secondary, less intensely innervated targets.

Functions of ipRGCs

The role of ipRGCs in mediating nonvisual responses to light has been largely elucidated through the use of genetic mouse models. Mice null for melanopsin exhibit rather modest phenotypes. The magnitude of light-induced phase shifts of circadian locomotor activity rhythms is attenuated in melanopsin knock-out mice to about 40% of that observed in wild-type sibling controls (Figure 3(a)). The lengthening of circadian period observed in wild-type

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Figure 3 Circadian responses to light in retinal mutants.

(a) Irradiance-dependent circadian phase shifting in wild-type and melanopsin-null mice. Mice lacking melanopsin were deficient in light-induced circadian phase shifting at all irradiances tested. (b) Melanopsin-null mice also lacking rods and cones fail to show synchronization (entrainment) of circadian locomotor activity to the light:dark cycle; white is the time that lights are on. Actograms are double plotted (i.e., horizontal lines correspond to 48 h) and darkness is indicated by a gray background. By contrast, the wild-type mouse is well entrained.

mice that are maintained in constant light is also diminished in melanopsin knockout mice. However, entrainment to standard light:dark cycles appears to be unaffected in mice lacking melanopsin. Other nonvisual responses such as the pupillary light response, the acute inhibition of activity by light (masking), and the suppression of the melatonin biosynthetic pathway are unaffected in melanopsin knockout mice. Interestingly, many of these same responses show no deficits in murine genetic models lacking rods and cones. Combined, these results suggest some level of redundancy between the rod/cone system and ipRGCs. To test this possibility, melanopsin-null mice have been crossed with mice either lacking functional rods and cones or lacking rods and cones all together. These animals exhibit no responses to light (Figure 3(b)). Essentially, they behave as though they have been bilaterally enucleated.

Many of the retinorecipient structures such as the SCN that mediate nonvisual responses to light receive negligible, if any, input from rod and cone pathways.

However, melanopsin-knockout mice with light-insensitive ipRGCs continue to exhibit some degree of light-driven nonvisual responses. This raises the possibility that in addition to their inherent photoreceptive capacity, ipRGCs may also serve to convey light signals initially detected by rods and/or cones to brain sites such as the SCN. Indeed, specific ablation of ipRGCs mimics the previously mentioned results obtained in animals lacking functional rods, cones, and ipRGCs. These results indicate that ipRGCs, while intrinsically light sensitive, also transmit information generated by the classical photoreceptors.

Apart from its role in entraining circadian rhythms, the eye is a circadian oscillator itself. This was first demonstrated in the isolated eyes of gastropods, which show a circadian rhythm in the firing frequency of compound action potentials. Shedding of disks from the distal tips of rods is also circadian and persists in isolated amphibian and mammalian eyes whose optic nerves have been severed. Teleost eyes exhibit circadian rhythms in retinomotor movements of photoreceptors and the migration of melanosomes within cells of the retinal pigment epithelium. Syntheses of melatonin and dopamine are perhaps the best-studied circadian outputs of the eye. These amines are synthesized in antiphase; melatonin levels peak in the night and dopamine levels peak during the day. This phase relationship is maintained in constant darkness indicating that these rhythms are indeed circadian. However, in the absence of melatonin, dopamine is only acutely synthesized in response to light and is no longer produced in a circadian fashion.

Determining which ocular cell types harbor the circadian clock has proved to be difficult. Isolated amphibian eyecups continue to produce melatonin rhythmically, even when the vast majority of inner retinal neurons are lesioned. These findings suggest that the photoreceptors contain a competent clock and the biosynthetic machinery to make melatonin. Additionally, action spectral analyses in the amphibian retina implicate the principal green-sensitive rods as critical to the acute light-mediated regulation of ocular melatonin synthesis. Whether these cells are the actual site of the amphibian retinal clock remains to be determined.

The mammalian eye also contains a circadian clock. Cultured hamster retinas maintained in constant darkness produce melatonin rhythmically with a period near 24 h. Retinas from tau hamsters whose period of locomotor rhythms are significantly shorter than those of wild-types also exhibit rhythms in retinal melatonin with periods that parallel the shortened activity cycles. Importantly, these rhythms can be reset by pulses of light demonstrating that similar to the amphibian eye, the mammalian eye is an autonomous circadian system with a circadian pacemaker that drives a measurable output, which can be reset if it comes out of phase with the light:dark cycle.

In summary, the mammalian eye has a dual function. Similar to the ear which supplies auditory and vestibular

input to the brain, the eye provides visual input for the formation and interpretation of images and nonvisual input for the regulation of a myriad of light-regulated physiology such as the entrainment of circadian rhythms. The primary central circadian pacemaker within the SCN is entrained by light, the most reliable of external, daily time cues. The photoreceptors mediating photoentrainment include the classical photoreceptors (rods and/or cones), known primarily for their role in vision, and unique RGCs, which are inherently light sensitive because they express the photopigment melanopsin. The relative insensitivity of ipRGCs suggests that the classical photoreceptors mediate photoentrainment at low light levels, while ipRGCs are responsible for entraining the SCN at higher irradiances. However, even at low light levels, photoreceptive signals transmitted through rod or cone pathways reach sites of the brain involved in nonvisual photoresponses via the ipRGCs.

The presence of an ocular photoreceptive system that is anatomically distinct from the rods and cones raises the possibility that individuals suffering from blindness due to photoreceptor loss may retain a full healthy complement of ipRGCs. Some blind individuals do indeed lack cognitive vision but maintain an ability to regulate pineal melatonin and remain synchronized to the prevailing day:night cycle. The high incidence of ocular infection in the blind drives many clinicians to replace the eyes with prosthetics. Care should be taken to consider the anatomical source of blindness. Those suffering from photoreceptor-derived or cortical blindness may develop circadian-based maladies upon removal of the eyes, thereby exacerbating an already diminished quality of life.

See also: The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Regulation of Ion Channels in Photoreceptors; Evolution of Opsins; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The

Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Rods; Rod and Cone Photoreceptor Cells: Inner and Outer Segments.

Further Reading

Berson, D. M. (2007). Phototransduction in ganglion-cell photoreceptors. Pflugers Archiv 454: 849–855.

Berson, D. M., Dunn, F. A., and Takao, M. (2002). Phototransduction by retinal ganglion cells that set the circadian clock. Science

295: 1070–1073.

Cahill, G. M. and Besharse, J. C. (1993). Circadian clock functions localized in xenopus retinal photoreceptors. Neuron 10: 573–577.

Campbell, S. S. and Murphy, P. J. (1998). Extraocular circadian phototransduction in humans. Science 279: 396–399.

Czeisler, C. A., Shanahan, T. L., Klerman, E. B., et al. (1995). Suppression of melatonin secretion in some blind patients by exposure to bright light. New England Journal of Medicine

332: 6–11.

Do, M. T., Kang, S. H., Xue, T., et al. (2009). Photon capture and signalling by melanopsin retinal ganglion cells. Nature 457: 281–287.

Graham, D. M., Wong, K. Y., Shapiro, P., et al. (2008). Melanopsin ganglion cells use a membrane-associated rhabdomeric phototransduction cascade. Journal of Neurophysiology 99: 2522–2532.

Guler, A. D., Ecker, J. L., Lall, G. S., et al. (2008). Melanopsin cells are the principal conduits for rod–cone input to non-image-forming vision. Nature 453: 102–105.

Hattar, S., Liao, H. W., Takao, M., Berson, D. M., and Yau, K. W. (2002). Melanopsin-containing retinal ganglion cells: Architecture, projections, and intrinsic photosensitivity. Science 295: 1065–1070.

Keeler, C. E. (1927). Iris movements in blind mice. American Journal of Physiology 81: 107–112.

Kumbalasiri, T. and Provencio, I. (2005). Melanopsin and other novel mammalian opsins. Experimental Eye Research 81: 368–375.

Provencio, I. (2007). Melanopsin cells. In: Hoy, R. R., Shepherd, G. M., Basbaum, A. I., Kaneko, A., and Westheimer, G. (eds.) The Senses: A Comprehensive Reference. Oxford: Elsevier.

Tosini, G. and Menaker, M. (1996). Circadian rhythms in cultured mammalian retina. Science 272: 419–421.

Wright, K. P. Jr. and Czeisler, C. A. (2002). Absence of circadian phase resetting in response to bright light behind the knees. Science 297: 571.

Glossary

Circadian oscillator – A system that generates self-sustained oscillations or rhythms of about 24 h. Current models include self-sustained transcription and translation feedback loops that operate at the cellular level to provide outputs that are circadian in nature.

Cyclic GMP-gated cation channels

(CNGCs) – The nonselective cation channels that are activated through direct binding of cyclic nucleotides onto the channel proteins. In general, cyclic-nucleotide gated channels are heterotetrameric complexes consisting of two or three different subunits (a, ß, and g), with important channel properties determined by the subunit composition.

ENSLI amacrine cells – A class of retinal amacrine cells that release the peptide, somatostatin. These cells get their name because they are immunoreactive for enkephalin, neurotensin, and somatostatin (ENSI, enkephalin-, neurotensin-, and somatostatin-like immunoreactive).

L-type voltage-gated calcium channels (L-VGCCs) – The membrane channels that mediate a voltage-dependent and depolarization-induced calcium influx. They regulate diverse biological processes such as contraction, secretion, neurotransmission, differentiation, and gene expression in many different cell types. The L-VGCCs are composed of a pore-forming a1-subunit and the auxiliary b-, a2d-, and g-subunits. They can be blocked by divalent cations (e.g., cobalt) and organic L-VGCC antagonists, including dihydropyridines, phenylalkylamines, and benzothiazepines.

Retinoschisis – An X-linked retinal dystrophy that features disorganization of retinal cell layers, disruption of the synaptic structures and neurotransmission between

photoreceptors and bipolar cells, and progressive degeneration of rod and cone photoreceptor cells. Retinoschisis results from mutations in retinoschisin.

Circadian Oscillators Regulate

the Functions of the Visual System

Circadian oscillators are biological clocks that exist in almost all living organisms on the earth from bacteria to humans, with persistent rhythmic periods close to 24 h (circa dian) even in the absence of external timing cues. The circadian oscillators coordinate rhythmic changes in biochemistry, physiology, and behavior of living organisms, so that organisms can be synchronized with the 24-h oscillations of the external environment. The molecular nature of circadian oscillators varies from species to species. A generalized model for the generation of circadian rhythm involves the transcription, translation, and feedback of clock genes and their own transcriptional products. It is composed of two interlocking transcription–translation feedback loops as well as post-translational modulations. Collectively, these components comprise the core oscillator or the circadian oscillator. Visual systems have to detect images despite large daily changes in ambient illumination between day and night, and intrinsic circadian oscillators in the retina provide such a mechanism for visual systems to initiate more sustained adaptive changes throughout the course of the day. The retina is a heterogeneous tissue with multiple cell types organized in several cell layers. Early studies of circadian regulation in Xenopus and chicken retinas indicated that retinal circadian clocks are mainly located in the photoreceptors. Later research revealed that there are multiple oscillators present in various retinal cells in a species-dependent manner. The overall circadian regulation of the retina relies on the synaptic circuitry and feedback modulation among different retinal oscillators, so that the retina is able to anticipate and adapt to sustained daily illumination changes as well as acute light/ dark adaptation.

Circadian Regulation of Photoreceptors

The circadian oscillators in photoreceptors are endogenous and able to function independently in the absence of other retinal inputs. These photoreceptor oscillators lead to morphological, physiological, biochemical, and molecular changes that ultimately regulate photoreceptor function and physiology in a circadian fashion. In vertebrate rod photoreceptors, outer segment shedding and renewal is a continuous process, but its rate is under circadian control. In some teleosts and anurans, the

inner segments of rod and cone photoreceptors undergo contraction and elongation (i.e., retinomotor movement) in response to changes in ambient illumination as well as in the circadian cycles. While cones remain in a contracted state during the day, rods contract at night. Photoreceptors form specialized ribbon synapses with secondary neurons such as horizontal or bipolar cells. The numbers and ultrastructures of synaptic ribbons in both photoreceptors and bipolar cells undertake changes in relation to the time of day and light intensities. In avians, amphibians, reptiles, and other lower vertebrate species, melatonin is synthesized and secreted from photoreceptors at night, while its synthesis is inhibited by light. The transcription of arylalkylamine N-acetyltransferase (AANAT), the melatonin synthesis enzyme, is under circadian control. While the synthesis and release of dopamine from retinal amacrine cells shows light-driven and circadian fluctuations, the circadian nature of dopamine is dependent on the melatonin rhythm. In addition to the circadian oscillator genes, several photoreceptor-specific genes, ion channels, and enzyme activities are also under circadian control that ultimately contribute to the circadian regulation of photoreceptor physiology and function.

Ion Channels in Photoreceptors

Ion channels are macromolecular pores that allow charged ions to move across cell membranes and contribute to the excitability of neurons and muscles. Electrical signals are generated and modulated as different types of channels open and close in response to neurotransmitters, hormones, membrane potential changes, mechanical forces, and other agents. There are two major classes of ion channels: voltage-gated and ligand-gated ion channels. Voltage-gated ion channels open or close their pores in response to membrane potential changes. Ligand-gated ion channels gate ion movements and generate electrical signals in response to specific chemicals such as neurotransmitters or cyclic nucleotides. Vertebrate photoreceptors are highly polarized, so the distribution of ion channels on the plasma membrane is spatially compartmentalized. The outer segment membrane contains cyclic guanosine monophosphate (cGMP)-gated ion channels that are closed by light, whereas the inner segment and the synaptic terminal are furnished with at least six different ion channels. These six ion channels include:

1.hyperpolarization-activated and cyclic-nucleotide- modulated cation channels;

2.L-type voltage-gated calcium channels (L-VGCCs);

3.calcium-activated potassium (KCa) channels;

4.noninactivating voltage-gated potassium channels;

5.calcium-activated chloride channels; and

6.cGMP-gated ion channels.

Thus far, cGMP-gated ion channels and L-VGCCs have been studied the most rigorously, especially with regard to their gating properties and physiological functions in photoreceptors. Both ion channels are under circadian control. In the following sections, the circadian regulation of cGMP-gated ion channels and L-VGCCs are reviewed in detail.

Circadian Regulation of cGMP-gated

Cation Channels

Cyclic GMP-gated cation channels (CNGCs) are nonselective cation channels that belong to the family of cyclic-nucleotide-gated channels. In general, cyclic- nucleotide-gated channels are heterotetrameric complexes consisting of two or three different subunits (a, b, and g), with important channel properties determined by the subunit composition. Activation of these channels is through direct binding of cyclic nucleotides onto the channel proteins. The CNGCs of rods and cones have structurally similar but distinct a- and b-subunits. Rods contain CNGCa1-subunits that are more sensitive to calciuminduced inhibitions, while cones contain CNGCa3subunits that are not as sensitive to calcium. In the vertebrate retina, phototransduction is mediated by a G-protein-coupled cascade that results in changes in the gating of CNGCs in the outer segments of rods and cones. Light initiates a fall in intracellular cGMP as the end point of a G-protein-mediated phototransduction cascade, resulting in closure of CNGCs, reduced cation influx, and membrane hyperpolarization. In the dark, the intracellular cGMP concentration is relatively high. This causes tonic activation of these channels and a steady transmembrane influx of sodium (Naþ) and calcium (Ca2þ) ions. Therefore, CNGCs carry the photoreceptor dark current and serve essential roles in the light-dependent changes in photoreceptor membrane potential and subsequent neural processing.

In chick cone photoreceptors, the apparent affinity of CNGCs for their activating ligand is under circadian regulation. There is roughly a twofold change in the apparent affinity of CNGCs for cGMP throughout the course of a day, with the affinity substantially higher at night than during the day even in constant darkness. Such changes in channel affinities can be expected to occur especially at the lower range of cGMP concentrations ( 7 mM in the dark) that are within the photoreceptor physiological range. Other biophysical features of the gating of CNGCs, such as unitary conductance, numbers of cGMP-binding sites on the channels, density of channels in

the plasma membrane, and the maximum current amplitudes do not vary as a function of the time of day.

The ion channel gating properties of photoreceptor CNGCs can be modulated by multiple processes, including direct phosphorylation or dephosphorylation of the channel subunits and the binding of Ca2þ/calmodulin or related molecules. Dephosphorylation of CNGCs on serine/threonine residues or tyrosine residues causes an increase in the apparent affinity of CNGCs for their activating ligand. Binding of Ca2þ/calmodulin or phosphorylation of CNGCs causes these channels to shift to a lower affinity state for cGMP. The clock regulation of cone CNGCs entails a post-translational modification of the channel molecules. More specifically, it is the circadian rhythmicity of tyrosine phosphorylation that underlies the circadian modulation of cone CNGC affinity to its ligand. Inhibition of tyrosine kinases during the day increases the apparent affinity of CNGCs for cGMP, whereas inhibition of tyrosine phosphatases at night produces the opposite effect. While the protein expression and phosphorylation of the channel pore-forming CNGCa3-subunits remain constant throughout the course of the day, tyrosine phosphorylation of an auxiliary subunit (probably the b-subunit, 85 kDa) displays a circadian rhythm. During the daytime, tyrosine phosphorylation on this 85-kDa protein is twice as high as at night. Therefore, circadian rhythmicity of tyrosine phosphorylation on the 85-kDa auxiliary subunit of cone CNGCs provides one of the final steps in the circadian regulation of CNGCs.

The CNGCs in photoreceptors are essential components of visual phototransduction cascades. As such, it is possible that they also play a role in the light entrainment of the circadian oscillators in photoreceptors. As the gating of CNGCs is under circadian control in cone photoreceptors, these channels represent a potential example of an entity that is both an input to and an output from the circadian oscillator. Roenneberg and Merrow have presented models of circadian oscillator systems in which pathways that lead to entrainment of the core oscillators (i.e., the circadian inputs) can themselves be regulated by the oscillators (i.e., they are also components of circadian outputs). One feature of these models is that they contain additional feedback loops that can markedly enhance the stability of the overall oscillator system. Therefore, the circadian regulation of CNGCs in retinal photoreceptors represents an adaptation to enhance the stability of the circadian oscillators in photoreceptors.

Signaling Pathways Leading to the Circadian Regulation of CNGCs

Even though the connection from the molecular oscillator to the regulation of CNGC affinity rhythm is still not completely understood, the small guanosine triphosphate (GTP)ase Ras and mitogen-activated protein kinase

(MAPK) signaling pathway and calcium-calmodulin kinase II (CaMKII) are involved as circadian outputs to regulate CNGC rhythms. The activities of Ras and MAPK are themselves under circadian control and oscillate concurrently with the CNGC affinity rhythm. The activity of CaMKII also displays a circadian rhythm, but it runs antiphase to the MAPK rhythm, and CaMKII is a downstream target of MAPK. Perturbation of the activities of Ras, MAPK, or CaMKII causes phase-dependent changes in the gating properties of CNGCs. Furthermore, this Ras–MAPK–CaMKII pathway serves as part of a common circadian output pathway to regulate other molecules in photoreceptors (see Figure 1). However, neither MAPK nor CaMKII directly phosphorylates CNGCs in a circadian fashion. It is their downstream targets leading to tyrosine phosphorylation on the auxiliary subunit of CNGCs that ultimately govern the circadian regulation of CNGC gating properties.

The expression and activation of adenylate cyclase in the retina are under circadian control, and the cyclic adenosine monophosphate (cAMP) content in retinal photoreceptors is maximal at night. In avian retinas, this cAMP rhythm not only drives the rhythm of melatonin synthesis and secretion, but it also serves as part of the circadian output and leads to MAPK activation and modulation of the CNGC affinity rhythm. Besides serving as a circadian output to regulate ion channels, cAMP can stimulate retinomotor movements. In Xenopus retina, cAMP signaling resets circadian oscillators within photoreceptors. Hence, the second messenger, cAMP, may well serve as part of both input and output pathways in photoreceptors.

Circadian Phase-Dependent Modulation of Cone CNGCs by Dopamine

While circadian rhythms can occur in retinal photoreceptors cultured in the absence of other functional cell types, multiple cell types contribute to the overall circadian control of the intact retina. In avian retina, melatonin inhibits the release of dopamine from a subpopulation of amacrine cells. Consequently, retinal melatonin and dopamine are in antiphasic circadian cycles. As a result, dopamine functions as a feedback signal from the inner retina that refines and modulates circadian control mechanisms within the photoreceptors. Dopamine evokes a phasedependent modulation of CNGC affinity rhythm in chick cone photoreceptors that is through the activation of the D2 family of dopamine receptors. Exposure to dopamine or D2 agonists causes a significant decrease in the apparent affinity of CNGCs at night but has no effect on CNGCs during the day. It is well established that D2 receptors are pertussis-toxin-sensitive G-protein- coupled receptors that mediate inhibition of adenylate cyclase and cause a decrease of cAMP formation in the

cAMP AANAT

Adenylate cyclase

Oscillator

Figure 1 This model illustrates the circadian rhythm in retina cone photoreceptors. Light and dark (day and night) signals from the environment enter the photoreceptor through cGMP-gated cation channels, since cGMP-gated cation channels are essential in phototransduction for light detection. The light/dark signals through the circadian input pathway entrain the photoreceptor core oscillator that cause the photoreceptor to be in sync with the environment. From the core oscillator through the circadian output, the activities of different molecules, including cGMP-gated ion channels,

L-type voltage-gated ion channels, and retinoschisin are under circadian regulation. This circadian output is composed of a series of signaling pathways. The circadian regulation of CNGCs in retinal photoreceptors represents an adaptation to enhance the stability of retinal circadian oscillators. It demonstrates a model that Roenneberg and Merrow have presented, in which the elements that lead to entrainment of the core oscillators (e.g., cGMP-gated cation channels) can themselves be regulated by the oscillators. One feature of this model is that it contains additional feedback loops that can

markedly enhance the stability of the overall oscillator system at the cellular level (photoreceptors only) and maybe at the retinal network level (the retinal oscillators). The circadian phasedependent regulation of cGMP-gated cation channels by dopamine and somatostatin is represented by brown and purple dotted arrows, respectively. The black dotted arrows indicate that there are multiple steps currently not known, while the solid arrows represent known signaling. Blue dotted arrows

represent that the secretion of melatonin and retinoschisin is under the control of L-type voltage-gated calcium channels. AANAT, arylalkylamine N-acetyltransferase; Ca2+, calcium ion; CaMKII, calcium-calmodulin kinase II; CNGC, cGMP-gated cation channel; DA, dopamine; D2, dopamine D2 receptor; G, G protein; L-VGCCa1, L-type voltage-gated calcium channel a1-subunit; SS, somatostatin; SSR, somatostatin receptor.

retina. However, the phase-dependent modulation of CNGCs in cone photoreceptors by dopamine does not involve pertussis-toxin-sensitive G protein or cAMP signaling, since exposure to cAMP or pertussis toxin does not reverse the phase-dependent modulation of dopamine on CNGCs. This circadian modulation of cone CNGCs by dopamine is partially through the MAPK–CaMKII signaling pathway.

Modulation of Cone CNGCs by Somatostatin

Somatostatin is released from a class of amacrine cells that are immunoreactive for enkephalin, neurotensin, and somatostatin, and thus are called ENSLI (enkephalin-, neurotensin-, and somatostatin-like immunoreactive) amacrine cells. There are two forms of somatostatin: somatostatin-14 (SS-14) with 14 amino acids and somatostatin-28 (SS-28) with 28 amino acids, and both forms are released from ENSLI cells. The release of somatostatin from the ENSLI cells is under circadian control, which is high at night and low during the day in mammalian retinas, and this rhythm is concurrent with the activities of these cells with a high sustained rate of activity in the dark and a low sustained rate of activity in the light. The five somatostatin receptors (referred to as Sst1–Sst5) are guanine nucleotide binding protein (G-protein)-coupled receptors. While all five subtypes of somatostatin receptors are expressed in mammalian retina, only Sst2–Sst5 are present in the chick retina. Both SS-14 and SS-28 modulate cone CNGC sensitivity to cGMP that depend on circadian phase and immediate history of illumination. Both SS-14 and SS-28 decrease CNGC sensitivity to cGMP at night, which are similar to the action of dopamine via the D2 receptor and resistant to pertussis toxin. In addition, SS-28, but not SS-14, evokes a transient increase in CNGC affinity to cGMP only during the early part of the day in cone photoreceptors that have been exposed to light for 1–2 h. This transient effect by SS-28 is mediated by pertussis-toxin- sensitive G-protein-coupled receptors and activation of phospholipase C and protein kinase C signaling cascades. Therefore, somatostatin modulation of retinal cones may serve to reinforce circadian processes intrinsic to the photoreceptors, and may also contribute to more rapid adaptive responses to changes in ambient illumination.

Circadian Regulation of L-VGCCs

L-VGCCs mediate a voltage-dependent and depolarizationinduced calcium influx and regulate diverse biological processes, such as contraction, secretion, neurotransmission, differentiation, and gene expression in many different cell types. The L-VGCCs are composed of a pore-forming a1-subunit and the auxiliary b-, a2d-, and g-subunits, and

they can be blocked by divalent cations (e.g., cobalt) and organic L-VGCC antagonists, including dihydropyridines, phenylalkylamines, and benzothiazepines. The a1-subunit serves as a voltage sensor to detect voltage changes across the plasma membrane, and it also controls the pore size to allow selective divalent cations to pass through. Mammalian a1-subunits are encoded by at least 10 distinct genes, and a1C, a1D, and a1F (also known as Cav1.2, Cav1.3, and Cav1.4, respectively) are expressed in the retina photoreceptors. Photoreceptors are nonspiking neurons, and they release glutamate continuously in the dark as a result of depolarization-evoked activation of L-VGCCs.

Circadian regulation of L-VGCCs has been observed in goldfish retinal bipolar cells, chick cone photoreceptors, and other nonretinal neurons. In both retinal cases, the average maximum current amplitudes of L-VGCCs are significantly larger at midnight than at midday. The activation voltages that elicit L-VGCC currents (i.e., current–voltage relationship) and the channel gating kinetics do not change throughout the course of a day. In chick retinas, the main factor contributing to the circadian regulation of L-VGCC current amplitudes is the expression of functional L-VGCCa1-subunits, and both messenger RNA (mRNA) levels and protein expression of VGCCa1D are rhythmic. The Ras–MAPK–CaMKII signaling pathway (Figure 1) serves as part of the circadian output to regulate L-VGCCs as well as CNGCs as described above. However, the varying maximum amplitudes of the L-VGCC currents is in stark contrast to the CNGC maximum currents, which remain constant throughout the day, and which instead exhibit changes in gating properties.

One major functional significance of the L-VGCC rhythm is the circadian control of melatonin release. In nonmammalian vertebrates, melatonin is synthesized and secreted from retinal photoreceptors and is under circadian control. Inhibition of L-VGCCs with dihydropyridines blocks the synthesis and release of melatonin. Another physiological aspect of the L-VGCC rhythm is the circadian control of retinoschisin secretion. Retinoschisin is a 224-amino-acid protein secreted mainly by retinal photoreceptors and bipolar cells. Retinoschisin is distributed throughout the retina but is mainly concentrated around outer and inner segments of photoreceptors and both retinal plexiform layers. Mutations in the retinoschisin gene (RS1) cause X-linked retinoschisis, a retinal dystrophy that features disorganization of retinal cell layers, disruption of the synaptic structures and neurotransmission between photoreceptors and bipolar cells, and progressive degeneration of rod and cone photoreceptor cells. Hence, retinoschisin is believed to play an important role in the development and maintenance of retinal cytoarchitecture. In chick retinas, the mRNA level and protein expression of retinoschisin are under circadian control, and the secretion of retinoschisin is higher at night than during the day. Inhibition of L-VGCCs with

dihydropyridines dampens the circadian rhythm of retinoschisin secretion where only nighttime secretion is affected. Therefore, the circadian control of L-VGCCs has a profound impact in regulation of photoreceptor physiology and synaptic transmission.

Circadian Regulation of Other

Photoreceptor Ion Channels: Potassium

Channels (K+ Channels)

Potassium (Kþ) channels are the most diverse ion channels with more than 100 genes of the pore-forming a-subunits identified to date. They can dampen membrane excitability and set the resting membrane potentials in neurons. According to their genetic homology and functional characteristics, there are four major families of Kþ channels: voltage-gated Kþ channels, Ca2þ-activated Kþ channels, inward rectifier Kþ channels, and leak Kþ channels. In photoreceptors, the dark inward current through CNGCs in the outer segment is balanced by a Kþ outward current in the inner segment, and this Kþ outward current is mainly carried out by the voltage-gated Kþ channels. The major subtypes of voltage-gated Kþ channels present in photoreceptors are delayed-rectifier Kþ channels (Kv1.2, 1.3, and 2.1) and A-type transient Kþ channels (Kv4.2). The Ca2þ-activated Kþ channels and outward-rectifying noninactivating Kþ channels (Kv10.2; eag2) are also found in photoreceptors.

Thus far, the reports on circadian regulation of Kþ channels have been limited to invertebrate studies. In Aplysia retinal pacemaker neurons (not photoreceptors), there is a robust circadian rhythm of potassium currents carried by voltage-gated Kþ channels (IKV), while A-type Kþ currents and Ca2þ-activated Kþ currents remain constant throughout the day. When IKV peaks during the late night (predawn), the compound action potential firing frequencies reaches its nadir. The circadian rhythm of IKV contributes to the circadian control of the frequency of compound action potentials in pacemaker neurons. The basal retinal neurons in the eye of the mollusk Bulla gouldiana express a circadian rhythm in optic nerve impulses. It is the circadian regulation of IKV, but not other outward Kþ channels, that drive the daily fluctuations in membrane conductance and membrane potential of these neurons.

Conclusion

The circadian oscillators in the retina play important roles in the regulation of retinal physiology and function, including sensitivity to light, neurotransmitter release, gene expression, morphological changes at the synaptic terminals, metabolism (as pH changes), and rod– cone dominance. The photoreceptor components of

electroretinograms (ERGs), the electrophysiological recordings of retinal physiology, recorded from humans as well as animals, show daily rhythms. The circadian oscillators in photoreceptors are endogenous, and they can function independently without other retinal inputs. These circadian oscillators regulate retinomotor movement, outer segment disk shedding and membrane renewal, morphological changes at synaptic ribbons, gene expression, a delayed rectifier potassium current, the affinity of cGMP-gated ion channels, the L-type voltage-gated Ca2þ channel currents, and the activities of MAPK and CaMKII, among other photoreceptor activities. Photoreceptors are more sensitive to intense light damage during the subjective night than during the subjective day, even in animals that have been maintained in constant darkness for several days after circadian light–dark cycle entrainment. Several retinal degenerative diseases are associated with dampened or abnormal circadian rhythms in ERGs. In the Royal College of Surgeons rats, the first known animal model with inherited retinal degeneration, the diurnal changes in the c-wave of the ERGs are dampened prior to the beginning of retinal degeneration from postnatal day 17 to 24. Mutations in human cone–rod homeobox (CRX) are associated with retinal diseases, including cone–rod dystrophy-2, retinitis pigmentosa, and Leber congenital amaurosis, which all lead to blindness. In a mouse model of CRX mutation, the circadian rhythmicity of the ERG is abolished, and the mutant mice are not able to be entrained by light–dark cycles. The synthesis and release of dopamine from retinal amacrine cells is also under circadian control, and disruption of the circadian rhythmicity of dopamine synthesis causes a glaucoma-like disorder in quails. Fain and Lisman have proposed that circadian oscillators may provide a common pathway mediating several forms of retinal degeneration. Therefore, dysfunctions of circadian oscillators in the retina contribute to some forms of retinal degeneration, which in many instances may lead to blindness.

See also: Circadian Metabolism in the Chick Retina; The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Rhythms in the Fly’s Visual System; Fish Retinomotor Movements; Limulus Eyes and Their Circadian Regulation; Neurotransmitters and Receptors: Dopamine Receptors; The Photoreceptor Outer Segment as a Sensory Cilium.

Further Reading

Barnes, S. and Jacklet, J. W. (1997). Ionic currents of isolated retinal pacemaker neurons: Projected daily phase differences and selective enhancement by a phase-shifting neurotransmitter. Journal of Neurophysiology 77: 3075–3084.

Barnes, S. and Kelly, M. E. (2002). Calcium channels at the photoreceptor synapse. Advances in Experimental Medicine and Biology 514: 465–476.

Chae, K. S., Ko, G. Y., and Dryer, S. E. (2007). Tyrosine phosphorylation of cGMP-gated ion channels is under circadian control in chick retina photoreceptors. Investigative Ophthalmology and Visual Science 48: 901–906.

Chen, S. K., Ko, G. Y., and Dryer, S. E. (2007). Somatostatin peptides produce multiple effects on gating properties of native cone photoreceptor cGMP-gated channels that depend on circadian phase and previous illumination. Journal of Neuroscience

27: 12168–12175.

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

Kaupp, U. B. and Seifert, R. (2002). Cyclic nucleotide-gated ion channels. Physiological Reviews 82: 769–824.

Ko, G. Y., Ko, M. L., and Dryer, S. E. (2001). Circadian regulation of cGMP-gated cationic channels of chick retinal cones. Erk MAP Kinase and Ca2+/calmodulin-dependent protein kinase II. Neuron 29: 255–266.

Ko, G. Y., Ko, M. L., and Dryer, S. E. (2003). Circadian phase-dependent modulation of cGMP-gated channels of cone photoreceptors by dopamine and D2 agonist. Journal of Neuroscience 23: 3145–3153.

Ko, G. Y., Ko, M. L., and Dryer, S. E. (2004). Circadian regulation of cGMP-gated channels of vertebrate cone

photoreceptors: Role of cAMP and Ras. Journal of Neuroscience 24: 1296–1304.

Ko, M. L., Liu, Y., Dryer, S. E., and Ko, G. Y. (2007). The expression of L-type voltage-gated calcium channels in retinal photoreceptors is under circadian control. Journal of Neurochemistry 103: 784–792.

Ko, M. L., Liu, Y., Shi, L., Trump, D., and Ko, G. Y. (2008). Circadian regulation of retinoschisin in the chick retina. Investigative Ophthalmology and Visual Science 49: 1615–1621.

Michel, S., Manivannan, K., Zaritsky, J. J., and Block, G. D. (1999).

A delayed rectifier current is modulated by the circadian pacemaker in Bulla. Journal of Biological Rhythms 14: 141–150.

Molday, R. S. and Kaupp, U. B. (2000). Ion channels of vertebrate photoreceptors. In: Stravenga, D. G.,

Degrip, W. J., and Pugh, E. N., Jr. (eds.) Molecular Mechanisms in Visual Transduction, 1st edn., pp. 143–182. Amsterdam: Elsevier Science.

Pugh, E. N., Jr. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stravenga, D. G., Degrip, W. J., and Pugh, E. N., Jr. (eds.) Molecular Mechanisms in Visual Transduction, 1st edn., pp. 183–255. Amsterdam: Elsevier Science.

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