The Retina and Other Light-sensitive Ocular Clocks

Abstract

Ocular clocks, first identified in the retina, are also found in the retinal pigment epithelium (RPE), cornea, and ciliary body. The retina is a complex tissue of many cell types and considerable effort has gone into determining which cell types exhibit clock properties. Current data suggest that photoreceptors as well as inner retinal neurons exhibit clock properties with photoreceptors dominating in nonmammalian vertebrates and inner retinal neurons dominating in mice. However, these differences may in part reflect the choice of circadian output, and it is likely that clock properties are widely dispersed among many retinal cell types. The phase of the retinal clock can be set directly by light. In nonmammalian vertebrates, direct light sensitivity is commonplace among body clocks, but in mice only the retina and cornea retain direct light-dependent phase regulation. This distinguishes the retina and possibly other ocular clocks from peripheral oscillators whose phase depends on the pace-making properties of the hypothalamic central brain clock, the suprachiasmatic nuclei (SCN). However, in mice, retinal circadian oscillations dampen quickly in isolation due to weak coupling of its individual cell-autonomous oscillators, and there is no evidence that retinal clocks are directly controlled through input from other oscillators. Retinal circadian regulation in both mammals and nonmammalian vertebrates uses melatonin and dopamine as dark- and light-adaptive neuromodulators, respectively, and light can regulate circadian phase indirectly through dopamine signaling. The melatonin/dopamine system appears to have evolved among nonmammalian vertebrates and retained with modification in mammals. Circadian clocks in the eye are critical for optimum visual function where they play a role fine tuning visual sensitivity, and their disruption can affect diseases such as glaucoma or retinal degeneration syndromes.

Keywords: amacrine cell; clock; cone; dopamine; entrainment; ipRGC; melatonin; molecular clock; retina; rod.

Figure 1

Circadian oscillations in vitro of eyecups from Xenopus leavis (A and B), individual photoreceptor layers from Xenopus leavis (C and D), isolated retinas from melatonin proficient C3H/He mice (E) and an isolated retina from a melatonin deficient C57BL/6 mouse (F). A and B are measurements of activity AANAT, a melatonin synthetic enzyme, at different times of day in a light-dark cycle (A) or constant darkness (B) in culture (from Besharse and Iuvone, 1983 with permission of the publisher). C shows light dependent phasing of melatonin release rhythms (measured every 2 hours) from two isolated photoreceptor layers maintained in flow through culture on opposite light dark cycles for two days followed by constant darkness. D is similar to C except that the cultures are in constant darkness and are maintained on opposite cycles of quinpirole, a dopamine D2/D4 agonist, instead of light for 2 days before continued culture in darkness (C and D are from Cahill and Besharse, 1993 with permission of the publisher). E. Average melatonin release from isolated retinas of melatonin proficient mice (from Tosini and Menaker, 1998 with permission of the publisher). F. PER2∷luc rhythm of a single isolated mouse retina over 29 days in culture with near continuous monitoring of luciferase activity. Note that the robust luciferase damps over time but its amplitude is partially restored when medium is changed (arrows at top) on the 12 and 24^th days (from Ruan, et al, 2008 with permission of the publisher).

Figure 2

In the retina the molecular clockwork has features like that in most cells of the body along with retina specific features. CLOCK/BMAL1 heterodimers drive circadian expression of clock component genes such as Per1, Per2, Cry1, and Cry2. They also drive many “clock controlled genes” such as Aanat and Usp2 that serve as retina specific circadian effectors. PER and CRY proteins provide negative feedback to inhibit CLOCK/BMAL1 regulated transcription. CLOCK/BMAL1 also drives expression of NR1D1, a nuclear receptor, which in association with RORA regulates BMAL1 expression in a pattern that is anti-phasic to Per and Cry genes as well as “clock regulated genes”. This regulatory loop reinforces the circadian oscillation initiated through PER and CRY negative feedback. NPAS2 can replace CLOCK within the molecular clockwork. This occurs in the retina within a subset of retinal ganglion cells that are important in the circadian regulation of contrast sensitivity.

Figure 3

In the Xenopus leavis retina there is a complex interplay of the light-dark cycle, the retinal circadian clock and dopamine and melatonin which are regarded as light- and dark adaptive effectors respectively (re-drawn with modifications and updated from Figure 1 in (Besharse & Green, 1996). Light directly regulates the phase of a retinal clock and also directly up-regulates dopamine release and down-regulates melatonin synthesis. Dopamine also modulates the phase of the clock via D2-like receptors, which in turn drives a rhythm of melatonin synthesis and release. There is also mutual antagonism in that melatonin inhibits dopamine release in circadian night and dopamine via D2-like receptors inhibits melatonin synthesis in the day. Of the D2-like receptor types in mice DRD4 plays a prominent role. However, dopamine phase shifts the PER2∷luc oscillator in mice via a DRD1 instead of a D2-like receptor.

Figure 4

There are similarities and significant differences in light and dopamine mediated phase regulation of the retinal clock in Xenopus leavis driving melatonin release and that in mice driving PER2∷luc expression. A. In Xenopus dopamine acting at D2-like receptors suppresses cAMP and increases PER2 expression in a pathway leading to both phase advances and phase delays. However, there is a dopamine and cAMP independent pathway through which light directly increases PER2 phase shifts the clock. B. In mice dopamine acting at D1 receptors (DRD1) phase shifts the clock. In addition, dopamine mediates the phasing effect of light as DRD1 antagonists block the phasing effect of light. There is evidence that retrograde signaling through melanopsin (OPN4) in ipRGCs modulates the effects of light on dopamine release from amacrine cells. However, OPN4 is not required for light induced phasing of the PER2∷luc rhythm and neuropsin (OPN5), also in a set retinal ganglion cells, is required. Current data imply that retrograde signaling from ipRGCs phase shift the clock through a pathway involving dopamine but it is unclear whether OPN5 is in a class of cells that is distinct from those expressing OPN4.

Figure 5

The eye is composed of multiple oscillators or clocks. The retina (A), RPE-choroid (B), cornea (C) and ciliary body (D) dissected from PER2∷luc mouse eyes on day one and cultured in darkness through 6 days in a Lumicycle™ device all exhibit sustained rhythms of luciferase activity with a period less than 24 hours and only moderate damping. The highly sensitive photomultiplier tubes permit detection of rhythmicity in each explant despite large differences in total photon counts. New data from the senior author's laboratory using methods reported previously (Baba et al., 2010, Ruan et al., 2008, Yoo et al., 2004). Although not surprising, this is the first report of such rhythmicity in the ciliary body.