Circadian organization of the mammalian retina: from gene regulation to physiology and diseases

Abstract

The retinal circadian system represents a unique structure. It contains a complete circadian system and thus the retina represents an ideal model to study fundamental questions of how neural circadian systems are organized and what signaling pathways are used to maintain synchrony of the different structures in the system. In addition, several studies have shown that multiple sites within the retina are capable of generating circadian oscillations. The strength of circadian clock gene expression and the emphasis of rhythmic expression are divergent across vertebrate retinas, with photoreceptors as the primary locus of rhythm generation in amphibians, while in mammals clock activity is most robust in the inner nuclear layer. Melatonin and dopamine serve as signaling molecules to entrain circadian rhythms in the retina and also in other ocular structures. Recent studies have also suggested GABA as an important component of the system that regulates retinal circadian rhythms. These transmitter-driven influences on clock molecules apparently reinforce the autonomous transcription-translation cycling of clock genes. The molecular organization of the retinal clock is similar to what has been reported for the SCN although inter-neural communication among retinal neurons that form the circadian network is apparently weaker than those present in the SCN, and it is more sensitive to genetic disruption than the central brain clock. The melatonin-dopamine system is the signaling pathway that allows the retinal circadian clock to reconfigure retinal circuits to enhance light-adapted cone-mediated visual function during the day and dark-adapted rod-mediated visual signaling at night. Additionally, the retinal circadian clock also controls circadian rhythms in disk shedding and phagocytosis, and possibly intraocular pressure. Emerging experimental data also indicate that circadian clock is also implicated in the pathogenesis of eye disease and compelling experimental data indicate that dysfunction of the retinal circadian system negatively impacts the retina and possibly the cornea and the lens.

Keywords: Circadian; Cornea; Dopamine; Electroretinogram; Melatonin; Photoreceptors; Retina; Retinal pigment epithelium.

Figure 1. The circadian clock system in the mammalian retina controls several functions

Many studies have shown that rhythms in the eye are under direct control of the retinal circadian clock system. Recent studies have also indicated that the many different cell types within the eye contain circadian clocks that interact to modulate many ocular functions. Shown here are several known circadian processes in the retina and the eye, with their approximate location identified by retinal layer. RPE = retinal pigment epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer.

Figure 2. The molecular mechanism generating the circadian oscillation is composed of two feedback loops

In the positive feedback loop the transcription factors Bmal1 and Clock activate the transcription of Period (Per) and Cryptochrome (Cry) genes, as well as that of clock-controlled genes (CCGs). In the cytoplasm, PERIOD and CRYPTOCHROME proteins heterodimerize, enter the nucleus and interact with BMAL1/CLOCK, inhibiting the transactivation of their own promoters (negative feedback). BMAL1 and CLOCK also stimulate the transcription of Rev-Erba and Rora (Adapted from Tosini et al., BioEssays, 2008).

Figure 3. Bioluminescence Rhythms from mPer2Luc Mouse Retinal Explants

(A) Long-term culture of an intact mouse retinal explant showing persistent circadian rhythms in PER2::LUC expression. Arrows indicate times of media changes. (B) Representative DIC image of vertical retinal sections. Retinal explants were cultured, and vertical retinal slices were cut with a tissue slicer at 150 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments. (C) Flat-mount view showing tyrosine hydroxylase immunoreactivity in cultured retinal explants. The immunoreactive cells with relatively large somata and two to three thick primary processes that arise from the cell body are dopaminergic amacrine cells, whereas the immunoreactive cells with relatively small cell body and very few processes are type 2 catecholamine amacrine cells. (adapted from Ruan et al., , Plos Biol. 2008).

Figure 4. Differential Clock Gene dependence of Retinal and SCN Neural Circadian Clocks

Simplified core clock gene diagrams as in Figure 1. Genes and action lines shown in red depict genes which when knocked out individually severely attenuate clock function in retina or SCN, while knockout of those shown in green has minimal effect. Note that the retinal clock is more vulnerable to single gen knockouts than the SCN.

Figure 5. Neurochemical Outputs of the Retinal Circadian Clock

Melatonin secreted from photoreceptors at night (black) modulates both the scotopic (dark-adapted) and photopic (light-adapted) ERG b-wave. This is denoted as arrows to rod and cone bipolar cells, which are the primary site of production of the scotopic and photopic b-waves, respectively, although the sites of action for these effects may include other cells. Melatonin also antagonizes dopamine release from dopaminergic amacrine cells. Clock-driven dopamine release during the day phase of the retinal circadian rhythm (red) acts to uncouple the electrical synapses between rods and cones, to increase the amplitude of the photoptic ERG b-wave, and to antagonize melatonin release. Light-driven dopamine release (yellow) has the additional effects of uncoupling AII amacrine cells and horizontal cells, as well as increasing the amplitude of the ERG b-wave and decreasing the amplitude intrinsically photoreceptive ganglion cell responses.

Figure 6. Regulation of retinal melatonin levels by light and the circadian clock

During the night cAMP levels in the photoreceptors are elevated thus activating PKA and Aanat gene transcription. Phosphorylated AANAT (pAANAT) associates with 14-3-3 proteins, which activate and stabilize the enzyme resulting in increased conversion of serotonin to N-acetylserotonin, and ultimately to melatonin. Light at night exposure decreases cAMP levels resulting in dephosphorylation of AANAT and its subsequent degradation by proteasomal degradation. The circadian clock controls melatonin levels by directly regulating Aanat transcription and by gating the cAMP signaling cascade (adapted from Tosini et al., Exp. Eye. Res. 2013).

Figure 7. Dopamine modulates the circadian rhythm in visual processing

Light-adapted ERG b-wave amplitudes at CT 6 (filled circles and triangles) or CT 18 (open circles and triangles) plotted as a function of light adaptation time in Ctrl or rTHKO mice during DD1 (A) or DD2 (B). (A) There are significant day/night difference during DD1 for both genotypes (B) On the second day in DD, no significant day/night differences are observed in rTHKO mice, for Ctrl mice; however, day/night differences persist in Ctrl mice. (C) Contrast sensitivity is significantly reduced in rTHKO mice at three of the six tested spatial frequencies. (D) Acuity threshold is significantly lower in rTHKO mice compared with control mice. All data are represented as means ± SEM. (Adapted from Jackson et al., J. Neurosci. 2012).

Figure 8

A model for mid-day contrast sensitivity regulation. Dopamine, through a D4 receptor pathway, regulates the rhythmic expression of Npas2 in the GCL. NPAS2 in the retinal ganglion cells regulates the rhythmic expression of the Adcy1 gene in the GCL, which ultimately modulates day-time contrast sensitivity. CLOCK, in part through an Adcy1 independent mechanism, also regulates the circadian rhythm of contrast sensitivity. (Adapted from Hwang et al., J. Neurosci. 2013)

Figure 9. Representative examples of PER2::LUC bioluminescence rhythm in RPE

(A) A clear circadian rhythm IN PER2::LUC bioluminescence can be recorded in the RPE. The circadian rhythm in the bioluminescence damp-out after 6–7 days. (B) A medium exchange will reinitiate the circadian rhythm in these cultures thus allowing the recording for several weeks. The arrows indicated the day when the old medium in the culture was replaced with new one (Adapted from Baba et al., Molecular Vision, 2010).