Mouse gene knockout illuminates how light resets clock

A key role in synchronizing daily rhythms to the day/night cycle has been traced to a light-sensitive protein in the eye, by knocking out the gene that codes for it. Mice lacking a gene for the photopigment melanopsin show a dramatic deficiency in their ability to regulate their circadian rhythms by light. The discovery, by National Institute of Mental Health (NIMH) grantees, helps unravel the heretofore elusive mechanisms by which day/night cycles regulate such rhythms in mammals. NIMH grantees Ignacio Provencio, Ph.D., Uniformed Services University of the Health Sciences (USUHS), and Steve Kay, Ph.D., The Scripps Research Institute, and colleagues report on their findings in the December 13 Science.*

In a similar knockout mouse study reported in the same issue of Science, another research team, led by NIMH grantee Norman Ruby, Ph.D., Stanford University, also found melanopsin to be a “significant contributor” to circadian function.**

Each day, a clock in the brain’s hypothalamus that governs daily rhythms – sleeping/waking, body temperature, eating, arousal. — is reset by light detected in the eyes. Yet, how this works has been a mystery. Light can still reset the clock even if the rods and cones, the photoreceptors in the retina for vision, are removed, but not if the eyes are removed. Hence, scientists have hypothesized that the eyes must contain a system of photoreceptors for resetting the clock that is separate from the system for sight.

Retinal ganglion cells, which contain melanopsin, have emerged as a prime candidate only within the past year. While most of these cells are wired to parts of the brain involved in vision, about one or two percent of those in a rodent’s retina project to other areas, including the clock, located in an area of the hypothalamus called the suprachiasmatic nucleus.

“Unlike the rods and cones, this light-detection system is thought to respond to the level of illumination rather than to images,” explained Provencio. “It may have an important impact on general well being, since among other functions, light levels and time of day can modulate mood, activity levels, and even performance.”

Using mouse embryonic stem cells, Provencio, Kay and colleagues altered the gene to create a strain of mice that lacked a functioning gene for melanopsin. The mice appeared healthy and showed normal activity rhythms as they ran on wheels in constant darkness. This suggested that melanopsin is not involved in the normal functioning of the clock itself.

Then, the researchers exposed the melanopsin knockout mice to 15 minutes of blue light at a time in their cycle when normal mice show strong phase delays — alterations in the time of onset of activity in response to light. The mice lacking melanopsin showed significantly less phase delay than normal control mice, likely because of reduced sensitivity in signals from the retina to the clock. To confirm this deficit in light input, the knockout mice were exposed to constant white light, which normally would trigger phase adjustments resulting in a longer internal clock day than in constant darkness. The melanopsin-deprived mice showed a shorter lengthening of their internal clock day than the control mice.

“Light input to the clock was significantly reduced in the melanopsin-deficient animals,” said Provencio. “The sensitivity of their circadian system to light was reduced by 50 to 80 percent.”

Although the study shows that melanopsin significantly influences the resetting of the clock at three different light intensities, exactly how this protein translates light into a neural signal isn’t yet known.

The researchers propose that melanopsin is required for normal setting of the brain’s clock by light, but that other mechanisms for light input also play a role, since the animals still show some phase shifting. As in plants and flies, “independent photoreceptors with overlapping roles may function to adapt the organism to the natural changes in light quality and irradiance,” they suggest.

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Also participating in the study were: Drs. Satchidananda Panda, Trey Sato, John Hogenesch, Genomics Institute of the Novartis Research Foundation; Drs. Ana Maria Castrucci, Mark Rollag, USUHS; Dr. Willem DeGrip, University of Nijmegen.

Other funders of the Provencino/Kay study included the American Cancer Society, the European Union and the Novartis Science Foundation. The Ruby study was also supported by grants from the National Heart, Lung, and Blood Institute (NHLBI), the National Institute on Drug Abuse (NIDA), and Deltagen, Inc.

* Satchidananda Panda, Trey K. Sato, Ana Maria Castrucci, Mark D. Rollag, Willem J. DeGrip, John B. Hogenesch, Ignacio Provencio, and Steve A. Kay, Melanopsin (Opn4) Requirement for Normal Light-Induced Circadian Phase Shifting, Science Dec 13 2002: 2213-2216.

** Norman F. Ruby, Thomas J. Brennan, Xinmin Xie, Vinh Cao, Paul Franken, H. Craig Heller, and Bruce F. O’Hara, Role of Melanopsin in Circadian Responses to Light, Science Dec 13 2002: 2211-2213.

NIMH, NHLBI and NIDA are part of the National Institutes of Health (NIH), the Federal Government’s primary agency for biomedical and behavioral research. NIH is a component of the U.S. Department of Health and Human Services.