麻豆传媒

IT SOUNDS like such a daft thing to do. First, take 15 men and women, slide
thermometers up their rectums, and make them spit every hour. Next, wake them up
in the middle of the night and fit frumpy, long, light-tight skirts around their
waists. Finally, flood the backs of their knees with super-bright
light鈥攁nd briskly pack them off to bed again.

If this sounds ridiculous, what Scott Campbell and Pat Murphy found when they
did this experiment was outrageous. With such a regime, they discovered that
they could reset their subjects鈥� biological clocks鈥攃locks that tick away
in all of us, governing when we sleep, when we wake and a host of other
physiological events.

But it wasn鈥檛 the mention of the spit or the rectal probe that made Campbell
of Cornell University Medical College in White Plains, New York, slightly
nervous about broaching the topic while relaxing at a colleague鈥檚 home last
year. In his line of work, such words are freely tossed about at the dinner
table, as taking temperatures and measuring salivary hormones are standard ways
to track people鈥檚 rhythms. Indeed, bizarre experiments are nothing new in a
field in which scientists have done everything from sending boxes of bees around
the world to give them jet lag to putting people into total isolation in caves
for months.

No, it was the knees that made Campbell nervous. Scientists have known for
decades that light can shift our biological clocks鈥攍ight coming in through
the eyes, that is. Finding that a dollop of light behind the knees could
do the trick was tantamount to saying we have eyes, of a kind, in our skin.

鈥淲e were astonished. We were bowled over,鈥� says Al Lewy of Portland鈥檚 Oregon
Health Sciences University, who was entertaining fellow rhythm researchers at
his home when Campbell first described his bizarre findings, which were
subsequently published in Science (vol 279, p 396). 鈥淚f true, this is
such a very, very important finding.鈥�

It鈥檚 medically important because a host of people鈥攂lind insomniacs,
groggy shiftworkers, bleary-eyed travellers and those with seasonal
depression鈥攃ould be treated with light on the backs of their knees while
they slept. And scientifically important because it would show that human beings
are different from all other mammals so far studied. 鈥淭he knee experiment is
completely outside of what anybody expected based on experiments that have been
done over and over again with lab animals鈥攁nd that is really odd,鈥� says
Mike Menaker, rhythm researcher at the University of Virginia in
Charlottesville.

It is no surprise, then, that while some voices are enthusiastic鈥斺�滻t鈥檚
the most exciting thing to hit our field in nearly twenty years,鈥� comments
rhythm researcher Charmane Eastman of Rush-Presbyterian-St Luke鈥檚 Medical Center
in Chicago鈥攐thers are less so. 鈥淚 am highly sceptical,鈥� says Joe Takahashi
of Northwestern University in Evanston, Illinois. 鈥淭his needs to be confirmed in
other labs.鈥�

Right or wrong, the knee experiment is simply one strand of research in a
field that is coming into its own with a vengeance. Studying biological clocks
was once considered downright quirky. Nowadays, you need only flip through a
random Science, Nature or Neuron and some dazzling
new clock finding will jump out at you. Elegant experiments are revealing how
clock genes work together to make us tick
(This Week, 18 July 1998, p 6).
Timekeepers continue to show up in all kinds of body parts where they weren鈥檛
supposed to exist. And rhythm researchers are getting a handle on the question
they鈥檝e grappled with for decades鈥攚hat are the organs and molecules that
keep our body clocks marching smartly in step with the 24-hour world in which we
live?

It鈥檚 no wonder that biological rhythms exist. After all, conditions on Earth
change rhythmically as the Sun rises and sets, and so life evolved rhythms as
well, choosing favoured times for sleeping, waking, foraging and so on. These
rhythms, in turn, rely on bona fide biological clocks鈥攅xperiments with
hosts of species, Homo sapiens among them, show that rhythms
keep beating even when creatures are isolated, in caves or windowless labs, kept
oblivious to the real time of day.

Left to their own devices, though, body clocks lack the precision of fine
Swiss timepieces. Away from the Sun, a creature鈥檚 鈥渢rue鈥� rhythm shows its face,
and it鈥檚 rarely exactly 24 hours. If animals and plants couldn鈥檛 reset their
clocks, they would soon run into trouble as their timing drifted slowly out of
phase with the Sun, causing them to emerge from burrows or unfurl their leaves
at odd, inadvisable times.

Natural selection鈥檚 solution? Reset clocks each day, using sunlight. Just how
that takes place has been one of the long-standing mysteries in the science of
daily鈥攐r circadian鈥攔hythms. Somehow, organisms must detect light,
send signals to the body鈥檚 circadian pacemaker, and then push the clock hands
forward or back a bit as appropriate.

You鈥檇 think that the first step鈥攄etecting the light鈥攚ould be the
no-brainer part. After all, we have eyes stuffed with light-sensing cells called
rods and cones, which are stuffed, in turn, with light-sensing pigments called
rhodopsins. If this system鈥檚 good enough to see with, shouldn鈥檛 it work
perfectly well for resetting body clocks, too?

For some reason, nature opted to make things more complicated. Horseshoe
crabs have sensors in their tails that are designed to shift rhythms. Eyeless
fruit flies can still reset their clocks. Amphibians, birds and reptiles have
light sensors deep in their brains and, strange as it might sound, sunlight can
penetrate through the skull and tickle those sensors, too. For rhythms, the eye
is by no means the only game in town.

Except, that is, in mammals. Time after time, scientists like Menaker have
shown that they can shift the circadian clocks in mice and hamsters by flooding
the animals with light during the night. But without eyes, the animals鈥� clocks
don鈥檛 shift. This absolute reliance on eyes is a puzzle, says Menaker. Perhaps
it鈥檚 a vestige of a time in our evolutionary past when mammals were all
nocturnal. Venturing out only at twilight and dawn, our small, burrow-dwelling
ancestors couldn鈥檛 depend on enough light getting in through the skull. So the
deep brain sensors were dismissed from the job. The eyes got a hefty
promotion.

The weird thing, though, is that we use a totally different part of the eye
to sense the light that resets our rhythms鈥攏ot the 鈥渟eeing鈥� part at all.
This perplexing paradox was unearthed in 1991 by rhythm researcher Russell
Foster, who was working with Menaker. They found that a certain mutant strain of
mouse鈥攐ne in which the light-sensing part of the eye, the retina, decays
after birth鈥攃ould shift its clock perfectly well in response to light.
鈥淲e were completely gobsmacked by those results,鈥� says Foster, now a lecturer at
Imperial College, London. Colleagues were too. Some simply refused to believe
it.

Could there be some mistake? The mutant mouse, Foster noted, had lost all of
its rods and most, but not all, of its cones. To rule out the possibility that
those lone remaining cones were doing the light-resetting, Foster recently
tested mice that lacked all rods and cones. Again, when he flashed light at
them, their clocks shifted perfectly normally.

This was odd. After all, there鈥檚 not much left in the eye when you obliterate
all the rods and cones: just sundry nerve cells, which are not thought to sense
light themselves. They don鈥檛 even contain rhodopsins.

Or so scientists thought. Then, this July, Foster鈥檚 group reported in
Nature (vol 394, p 27) that they鈥檇 found a strange, new gene for an
opsin鈥攖he protein that combines with a pigment to form
rhodopsin鈥攁fter trawling around in salmon DNA. The researchers dubbed the
protein 鈥渧ery ancient opsin鈥� because it is only distantly related to the rod and
cone opsins. What really excites Foster, though, is where the very ancient opsin
is made: in the parts of the eye that remain perfectly intact in those rodless,
coneless mice. And though the protein, so far, has only been fished out of
salmon and a school of its fine-finned relatives, Foster has preliminary
evidence that it鈥檚 there in his blind mice as well.

Although this very ancient opsin might be the key to resetting body clocks in
mammals, it isn鈥檛 the only contender. This January, another unusual
opsin鈥斺�漨elanopsin鈥濃�攎ade the news, this one present in the skin, eyes
and brains of frogs. Meanwhile, a decidedly non-opsin-like molecule has edged
onto the stage, and is jostling with the rhodopsins for clock-resetting
supremacy. This one鈥檚 called a 鈥渃ryptochrome鈥�.

The cryptochrome story stems from studies in a spindly weed called
Arabidopsis thaliana (or thale cress), the well-studied darling of plant
molecular geneticists. A certain mutant, scientists found, can鈥檛 sense blue
light properly鈥攁nd it can鈥檛 keep proper track of time. The plant doesn鈥檛
know when to flower. What could be wrong with it?

The mutant, it turns out, lacks a protein eerily similar in structure to
molecules that Aziz Sancar, a biochemist at the University of North Carolina at
Chapel Hill, has been studying diligently for decades鈥攚ithout a smidgen of
interest from rhythm researchers. The scientists may be excused for their lack
of interest, however, because as far as anyone knew, Sancar鈥檚 proteins were a
workaday set of enzymes鈥攃alled photolyases鈥攖hat repair damaged DNA.
To biochemists like Sancar, photolyases are interesting because they do their
mending job exotically, using light. But nobody thought they had anything to do
with rhythms.

Today, it is becoming clear that they do鈥攐r at least their distant
cousins do. The plant protein, unlike a bona fide photolyase, can鈥檛 mend DNA.
But it can absorb light. Thus, though the molecule, called a cryptochrome,
clearly evolved from a photolyase, its modern job appears to be different: it鈥檚
a photoreceptor, one that鈥檚 probably used for clock-resetting. And not just in
plants.

Mammals have cryptochromes, too: last May, Sancar and his postdoc Yasuhide
Miyamoto reported that mouse eyes鈥攊ncluding the parts that are crucial for
clock-resetting鈥攁re stuffed with cryptochromes. So are the skin and those
brain regions known to be crucial for proper timekeeping. Fruit flies have them,
as well: this month, in a paper published in Cell (25 November issue) a
team led by geneticist Jeff Hall and his postdoc Ralf Stenewsky from Brandeis
University in Waltham, Massachusetts, reported that flies lacking cryptochrome
can鈥檛 reset their clocks properly. Perplexingly, though, Sancar and Takahashi
report in the 20 November issue of Science that a mouse lacking one of
its cryptochrome genes is more sensitive, not less, to resetting with light.

So which is the long-sought circadian photoreceptor: an opsin or a
cryptochrome? The answer may be fuzzy but diplomatic: organisms may have more
than one molecule for the job. And that鈥檚 not all they may have more than one
of.

Some creatures, it鈥檚 now known, have a collection of clocks in their
bodies鈥攁 surprising finding for rhythm scientists. They鈥檝e long thought
animals had one or just a few pacemaker organs, happily ticking away and sending
signals round the body to make all tissues act in properly rhythmic ways. In
mammals, for instance, a brain region called the suprachiasmatic nucleus
contains the main body clock, which is reset each day by the eyes.

The first shocker came last year when Hall and Steve Kay of the Scripps
Research Institute in La Jolla reported that fruit flies have clocks simply
everywhere. Not just in the brain, but in the wing, thorax, abdomen and in
squishier organs like the guts and testes. These clocks tick away very nicely,
with no help from the brain鈥攅ven when they鈥檙e severed from the head. They
reset themselves, too, when light shines on them鈥攁gain, independently of
the brain and the eyes.

Then, in June, a group headed by molecular biologist Ueli Schibler of the
University of Geneva reported that mammals, too, have far-flung clocks. They
found pacemakers in skin cells grown in a culture dish. Now others have shown
that tissues including the liver, muscle and testes (but for some reason not the
spleen) are all ticking away happily. The exception is becoming the rule. 鈥淭hese
days, if you don鈥檛 find rhythms in a particular tissue people will just say
`Well, you haven鈥檛 looked hard enough鈥�,鈥� says Steve Reppert, a neurobiologist at
Harvard Medical School.

What are these outposts good for? Nothing, perhaps. They might be useless,
evolutionary relics. On the other hand, they might be in charge of local
rhythms, controlling cell division, sperm production or digestion (with the main
brain clocks, such as the suprachiasmatic nucleus, remaining master-controllers
of some sort). After all, moth sperm is produced on a regular cycle. Amylase, a
starch-digesting enzyme, is made by our gut rhythmically. And fly rhythm
researchers, a somewhat vulgar set, have long entertained the possibility that
fruit flies defecate on schedule.

The fact that these far-flung clocks can reset themselves sounds, on the face
of it, like pretty good news for Campbell鈥檚 back-of-the-knee scenario. But there
are problems with this line of thinking. First, flies and mammals鈥攏ot to
mention people鈥攁re very different. Reppert has tried shining light on
cells from a mammal鈥檚 suprachiasmatic nucleus, but the clocks in that tissue
stay stubbornly right where they are. Schibler has tried shining light on his
cultured skin cells, and while he has not given up on the experiment, 鈥渙nly my
mother would believe the results I have so far鈥�, he says.

Another key difference is that clocks, in the case of flies, have only been
shown to reset locally, right at the place where the light was shone. No one has
ever demonstrated that aiming a tiny spotlight at a fly鈥檚 鈥渒nee鈥� can send
synchronising signals rippling through its body, tripping a global reset. (Which
is what must be happening in the Campbell case.) 鈥淲e鈥檙e not going to do this
experiment, it鈥檚 ridiculous,鈥� says Hall. 鈥淪till we鈥檙e desperately hoping that
someone else will, because it should be done.鈥�

But systemic clock-resetting could easily occur in mammals, argues Campbell,
if light alters the level of some chemical in our blood. That chemical
messenger鈥攕ay, nitric oxide, which is known to shift clocks鈥攃ould
travel through the bloodstream and nudge the brain鈥檚 master-pacemaker. And hey
presto! Whole-body clock resetting. In fact, light can affect blood chemicals,
notes Campbell: the very light-source he used in his knee experiment (a device
called a BiliBlanket) was designed to shine on the skin of jaundiced newborns,
to break down the bilirubin pigment that builds up in their blood.

Takahashi, Foster and others have their doubts about this blood idea: for
one, they don鈥檛 think a chemical like nitric oxide lasts long enough to carry
signals up to the brain. But their biggest problem with the knee effect is that
it contradicts the dogma that mammals need their eyes. Campbell reckons that
humans may be different because they aren鈥檛 nocturnal like the small mammals
studied to date. Further, rodents are furry and fur shields a mouse knee from
light. But Menaker doesn鈥檛 buy the fur defence. 鈥淔ur is trivial,鈥� he says. 鈥淚n a
black sheep it might not be trivial but in a white rat it鈥檚 trivial. And anyway,
we鈥檝e shaved them.鈥�

Campbell鈥檚 finding is also at odds with the fact that blind people who lack
eyes often can鈥檛 reset their clocks and suffer recurrent sleep problems as a
result. No one has investigated whether blind people in warmer climes, who are
more likely to expose their skin to light, are any less afflicted. 鈥淚 would love
to see somebody replicate Campbell鈥檚 findings before speculating on shorts for
the blind,鈥� says Peretz Lavie, sleep researcher at the Technion-Israel Institute
of Technology in Haifa, Israel.

At least half a dozen researchers are gearing up to do that right now,
including Charles Czeisler of Harvard Medical School, whose circadian research
facilities are as splashy as they come. 鈥淔rom everything we know it would seem
unlikely to be true鈥攂ut then again, if there were no surprises, everything
in science would just be dotting Is and crossing Ts,鈥� he says.

Of course, the marketplace works on a different timescale from that of the
laboratory. Companies have been clamouring to develop night-lights for the
knees, and Campbell is now collaborating with one to do so. The frenzy isn鈥檛
surprising, considering the range of people who would like to shift their
clocks. True, blasting eyes with bright light will reset clocks very nicely but
people generally don鈥檛 appreciate having to wake up in the middle of the night
to stare at fluorescent light bulbs. And night shiftworkers will do just about
anything they can鈥攅ven sporting their Raybans in the power-plant control
room鈥攖o keep that bothersome light away.

A knee-light would eliminate all those problems, and Kay鈥擭ew
Zealand-bound for a rhythm meeting鈥攚axes wistful at the prospect. 鈥淚f this
is repeatable, then here is something that comes out of these huge volumes of
circadian gobbledegook that can actually help people,鈥� he says. 鈥淚 sure wish I
had a small battery-bag of light-emitting jello to stick down my knickers so
that I could get on the plane, prop my feet up and know I鈥檇 be able to give a
decent talk on Monday.鈥�

BIOLOGICAL clocks must surely be super-important: why else would they be
found in practically every species scientists look at? Amazingly, this
assumption, though entirely reasonable, has not been tested until recently. Now
scientists working with bacteria and rodents have shown that critters with
abnormal circadian rhythms really do seem less 鈥渇it鈥�, evolutionarily speaking,
than their regular-as-clockwork counterparts.

Even the lowliest pond scum benefit from having clocks that run on time. In
one study, published in the Proceedings of the National Academy of
Sciences (vol 95, p 8660), Carl Johnson of Vanderbilt University in
Nashville and his collaborators compared the fate of different strains of
blue-green algae as they grew together in culture flasks. One mutant had a
鈥渟low鈥� biological clock in which the circadian cycle ran to 28 hours. Another鈥檚
ran fast, completing a daily cycle every 22 hours. When either was grown with
normal blue-green algae under a regular, 24-hour regime of 12 hours light, then
12 hours dark, the normal strain grew more rapidly and took over the flask.

If, instead, the lighting mimicked an unnaturally long day鈥�15 hours鈥�
light, then 15 hours鈥� dark鈥攖he 28-hour mutant won out. If the regime
imitated a shorter day (11 hours鈥� light, 11 hours鈥� dark), the 22-hour mutant
prevailed. So organisms whose rhythms most closely match the light-dark rhythms
of the world around them reproduce more rapidly鈥攁nd have an evolutionary
edge.

Lacking rhythms altogether can also be seriously dangerous to one鈥檚 health, a
discovery that Pat DeCoursey and colleagues at the University of South Carolina
in Columbia made quite by accident. The scientists were observing a population
of ground squirrels, half of which had been treated surgically to destroy the
brain鈥檚 circadian pacemaker. Normally, this species is active in the daytime,
but the surgically treated squirrels, having lost their sense of rhythm, took to
waking and feeding at night too. When a night-prowling predator unexpectedly
broke into the enclosure, it killed off twice as many rhythmless as normal
animals 鈥攑resumably because they were up and about instead of snoozing
safely in their burrows.