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What Sets the Biological Clock?

Most people run on an internal 24-hour cycle, synchronized to the light and dark cycles of the outside world. Information about external luminescence is conveyed to the suprachiasmatic nucleus (SCN) of the hypothalamus, which incorporates it into what is known as the circadian rhythm, or biological clock. In cold-blooded vertebrates, deep-brain photoreceptors allow for photoentrainment, the process by which the eyes facilitate setting of the circadian clock. Mammals do not have these receptors;

By | June 10, 2002

Most people run on an internal 24-hour cycle, synchronized to the light and dark cycles of the outside world. Information about external luminescence is conveyed to the suprachiasmatic nucleus (SCN) of the hypothalamus, which incorporates it into what is known as the circadian rhythm, or biological clock.

In cold-blooded vertebrates, deep-brain photoreceptors allow for photoentrainment, the process by which the eyes facilitate setting of the circadian clock. Mammals do not have these receptors; instead, mammalian eyes collect light and send the information back to the SCN through the optic nerve, a pathway called the retinohypothalamic tract (RHT). This is known, in part, because mice with removed eyes cannot reset their clocks.

Oddly enough, about half of all blind people can photoentrain, as can mice that lack functional rods and cones, implying that another receptor capable of processing light exists in the eye. Tracer studies, in which a marker traverses the neuron from one end to the other, indicated more than 20 years ago that a small subset of cells found in the innermost part of the retina innervates the SCN. These retinal ganglion cells (RGCs), which were not known to contain any photopigments of their own, were thought merely to relay information from the rods and cones. But a spate of work, culminating in papers from at least five laboratories earlier this year,1-5 has shown that RGCs are intrinsically photosensitive, and has all but confirmed the identification of the responsible photopigment as well.

Assembling the Clues

As a graduate student of Russell Foster at the University of Virginia in the early 1990s, Ignacio Provencio used mice with degenerate retinas to investigate "nonvisual photoreception." Much of that work involved assessing the light-response characteristics, the so-called action spectrum, as well as tracer and biochemical studies. The research suggested that these mutant mice used an opsin—the photopigment family found in rods and cones—to process luminance. Absent another candidate, Foster's group then speculated in 1997 that cones, having leaked through the mutation, were capturing enough luminescence information necessary to keep the biological clock in sync. Another possibility implicated a heretofore unknown photoreceptor that was doing the work.

Mark Rollag's lab at the Uniformed Services University in Bethesda, Md., was studying the dispersion of dermal pigments in response to light. Provencio joined the group as a postdoc, and in 1998 the researchers announced they had cloned an opsin from the granules in frog skin cells.6 In situ hybridization showed that melanopsin message was expressed both in the frog brain and iris, organs previously identified as photosensitive. The researchers also found melanopsin mRNA in the retina, but not in the photoreceptor layers housing the rods and cones. For Provencio, "This rang a bell."

Courtesy of Jens Hannibal

Melanopsin Captured: A rat retinal ganglion cell containing Melanopsin (green) located in the membrane of both soma and dendrites and PACAP (red), a neurotransmitter of the retinohypothalamic tract.

Another clue became apparent as the researchers found melanopsin homologues expressed in the ganglion cell and amacrine layers of the retina. In 2000 they reported, "The anatomical distribution of melanopsin-positive retinal cells is similar to the pattern of cells known to project from the retina to the suprachiasmatic nuclei of the hypothalamus, a primary circadian pacemaker." 7 The pattern was similar to what Provencio had seen in his tracer studies.

Provencio, now a principle investigator at Uniformed Services, and Rollag and collaborators went on to make antiserum against melanopsin, and used it to further define protein distribution. In January 2002, they reported visualizing "an expansive photoreceptive 'net' in the mouse inner retina."1 The following week, Johns Hopkins' King-Wai Yau and colleagues confirmed that finding in the rat.2 Such an arrangement would ideally position these RGCs to capture and sum ambient light, rather than to distinguish light from point sources as a visual system must do. "So we were looking at the possibility that maybe some RGCs were directly light-sensitive and could send that information to the SCN," Provencio said.

Piecing the Puzzle Together

By this point, several other labs had heard the bell's toll. David Berson's lab at Brown University had done some "very elegant studies," Provencio notes, in which electrical impulses were recorded from RGCs that had been retrograde labeled by injecting a fluorescent tracer into the SCN.

Courtesy of Samer S. Hattar

Course Work: Axons work their way beyond theSCN nuclei to target other brain regions. The optic chasm has been cleared with benzyl benzoate benzyl alcohol solution

The researchers found that the cells were "robustly light-responsive, whereas other ganglion cells that we recorded in the same preparation were not," Berson says. The isolated retina had been photobleached, destroying the rods' and cones' abilities to transduce light, and so it was surprising that any light reactivity took place at all. This experiment was his first indication that those cells were actually acting like photoreceptors. "From there, it was a long series of increasingly Draconian measures to try to convince ourselves, and ultimately the world, that the light response we were seeing in these cells was in fact intrinsic to the cells themselves," Berson recounts. These measures included pharmacologically preventing the release of neurotransmitters; blocking the post-synaptic receptors; and microdissecting the labeled RGCs away from every other cell. What remained was "basically just a cell body stuck on an electrode." They always found the same result, that these RGCs depolarized in response to light. The dynamic range and threshold of the RGC's light response closely matched those expected for a cell responsible for photoentrainment: "The circadian entrainment mechanism integrates light energy over very long time scales, exhibits little adaptation, and responds poorly to brief stimuli," his paper reports. "Similar features were evident in the behavior of photosensitive ganglion cells."3

Show Me the Protein

Yau's group, working with Berson's, wanted to know where melanopsin was expressed, and where the axons of the melanopsin-containing neurons synapsed in the brain. They made polyclonal antibodies against rat melanopsin for use in immunohistochemical studies. In the few cells the antibodies recognized, melanopsin was found at the cell's surface and "everywhere in the cell—the cell body, all the processes, and including even the part of the axon on the retina, [but] disappears as it goes to the brain," Yau notes. This makes sense he explains, "because if indeed melanopsin is the photopigment that is intended to capture light, it should be located all over the cell to improve the chance that light will be absorbed."

To find out where the melanopsin-positive RGCs go when they leave the retina, Yau's group created transgenic mice in which an axonal targeting sequence, fused to a portion of the galactosidase gene, interrupted the melanopsin. The melanopsin promoter drives expression of the enzyme, so axons of melanopsin-expressing cells should stain blue with X-gal, which is exactly what was found. The labeled axons terminated in the SCN, with some fibers continuing along the optic tract to innervate the intergeniculate leaflet (IGL), ventral lateral geniculate (VLG), and the vicinity of the olivary pretectal nucleus (OPN). The IGL and VLG, along with the SCN, have previously been implicated in the circadian clock's entrainment, the paper notes, while the OPN helps to regulate the pupil's size in response to light. "Thus it appears that melanopsin-containing RGCs are generally involved in nonimage-forming visual functions."2

Great Minds Think Alike

Last spring, Harvard graduate student Josh Gooley was working in Clifford Saper's lab. He had read Provencio's earlier papers and wanted to test whether melanopsin is actually in the nerve cells involved in circadian rhythm. Saper recalls saying to Gooley, "Sure, that's easy. It would be a nice rotation project for you."

Gooley and his coworkers injected the SCN with a fluorescent marker, retrograde labeling a small, widely distributed subset of RGCs. They performed in situ hybridization with Provencio's melanopsin riboprobe and achieved equivalent results. When they did both together, looking for RGCs that terminate in the SCN and retinal cells that express melanopsin message, they found that most cells labeled by one method were also labeled by the other.4

Meanwhile, Jens Hannibal at the University of Copenhagen had already found that the RHTs make the neurotransmitter pituitary adenylyl cyclase-activating polypeptide (PACAP), and that PACAP-containing cells respond to light in situ. So when Provencio announced a new opsin located in a few cells of the retinal ganglion layer, Hannibal knew "these must be the same cells." His group began making riboprobes and antibodies, and (after some false starts) demonstrated that "the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells."5

Melanopsin is found on the surface of the RGCs that innervate areas of the brain involved in regulating circadian rhythm, pupillary response, melatonin release, and other light-dependent phenomena. These cells spread themselves thinly enough across the retina so that they don't interfere with visual perception, yet broadly enough that they could interpret the average illumination. They are photoresponsive with an action spectrum similar to that predicted for the circadian photoreceptor. Melanopsin is an opsin, like the photoreceptors in rods and cones (apparently not necessary for photoentrainment); it colocalizes with PACAP.

The evidence in favor of melanopsin being a photopigment is fairly compelling, although the researchers in the field have not bestowed an official "yes." For this to happen, they need to establish a few facts. Provencio puts it this way: "Nobody has yet shown that it can absorb light and function as a photopigment."

Josh P. Roberts ( a freelance writer in Minneapolis, MN.

1. I. Provencio et al., "Photoreceptive net in the mammalian retina," Nature, 415:493, Jan. 31, 2002.

2. S. Hattar et al., "Melanopsin-containing retinal ganglion cells: Architecture, projections, and intrinsic photosensitivity," Science, 295:1065-70, Feb. 8, 2002.

3. D. Berson et al., "Phototransduction by retinal ganglion cells that set the circadian clock," Science, 295:1070-3, Feb. 8, 2002.

4. J.J. Gooley et al., "Melanopsin in cells of origin of the retinohypothalamic tract," Nature Neuroscience, 4:1165, December 2001.

5. J. Hannibal et al., "The photopigment melanopsin is exclusively present in pituitary adenylate cyclase polypeptide-containing retinal ganglion cells of the retinohypothalamic tract," Journal of Neuroscience, 22:1-7, Jan. 1, 2002.

6. I. Provencio et al., "Melanopsin: An opsin in melanophores, brain, and eye," Proceedings of the National Academy of Sciences, 95:340-5, 1998.

7. I. Provencio et al., "A novel human opsin in the inner retina," Journal of Neuroscience, 20:600-5, 2000.


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