As biologists get to grips with the effects of light on the human circadian
clock, the hunt for clues as to what makes it tick at a cellular and molecular
level is gathering momentum. Present knowledge is fragmentary, but the circadian
clocks of other animals, particularly sea slugs and the fruit fly, are providing
some answers.
A turning point came three years ago when biologists traced the human
circadian clock to two small regions of the brain’s hypothalamus, known
as the suprachiasmatic nuclei (SCN). The giveaway clue was the discovery
inside the SCN of receptors for melatonin, a hormone long linked to circadian
rhythms. Each no bigger than a grain of sand, the SCN lie at the base of
the brain, near to the optic tract, a nerve that runs directly to the retina.
People with tumours near their SCN lose their circadian rhythms, as
do animals whose SCN have been surgically removed. Moreover, the transplantation
of SCN tissue from rat fetuses to adult rats with damaged SCN restores their
circadian rhythms – all of which is compelling evidence that the SCN are
the site of the mammalian ‘master clock’.
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Whether mammals possess additional biological clocks is a subject of
much debate. Conclusive evidence is lacking, says Peter Morgan of the Rowett
Research Institute in Aberdeen, but the phenomenon of ‘split’ rhythms –
in which some but not all of the body’s circadian rhythms are disrupted
– points to the existence of secondary clocks.
Morgan and his colleagues are studying the seasonal rhythms of sheep.
They believe mammals with tightly controlled seasonal reproductive cycles,
such as sheep and ferrets, possess a second clock, in the brain, with a
cycle lasting a year rather than a day. As the length of the day shortens
in the autumn, the clock instructs the animals to become reproductively
active.
The location of this ‘circannual’ clock remains stubbornly elusive.
But as with the circadian clock, melatonin may hold the key. Released by
the pineal gland in darkness, melatonin acts as a chemical timekeeper, setting
both daily and annual hormonal rhythms. By unravelling the chain of biochemical
events triggered by its secretion, Morgan and his colleagues hope to find
the circannual clock.
Recently, they identified what could be a key kink in the chain: a region
of the sheep’s pituitary gland, known as the pars tuberalis, which possesses
melatonin receptors and responds biochemically to the hormone. The researchers
believe the pars tuberalis functions as a relay device, responding to
melatonin by dispatching another hormone. The assumption is that this hormone
(whose identity is still uncertain) then acts on the sheep’s circannual
clock, regulating the activities of its cells in some way.
The researchers’ ultimate goal is to find a way of manipulating the
reproductive cycles of sheep. The natural cycle is awkward for farmers because
it results in a seasonal glut of meat. Many would prefer production to be
even throughout the year. So far tests at the Rowett Institute show that
melatonin capsules can be used to shift the breeding season forward by several
months. The snag is that to produce this effect, the melatonin capsules
must be given daily for prolonged periods – an impractical task for farmers.
All biological rhythms, from short heartbeats to long hormonal cycles,
originate in the rhythms of cells. A pineal gland removed from a chicken,
for example, continues to display a circadian rhythm of melatonin release.
It also does so when cut into pieces. Likewise, neurons removed from the
SCN of a mammal continue to secrete peptides in a rhythmic fashion when
cultured in the laboratory.
Yet exactly how such cellular rhythms become synchronised to the daily
light-dark cycle is shrouded in speculation. At the cellular and molecular
level, says Morgan, biologists have only a ‘very rudimentary understanding’
of the biological clocks of mammals. The problem is exacerbated by the fact
that the SCN have not one but many physiological roles, and it is not known
which subset of their thousands of cells functions as the circadian clock.
Researchers do know that melatonin suppresses electrical activity within
the SCN. But the identities of the affected cells are still a mystery. Similarly,
evidence is emerging (mainly from experiments on rats) that light stimulates
the firing of neurons within the SCN, as well as triggering the activation
of certain genes. The optic nerve may act as the clock’s periscope, enabling
it to sense light; but, again, firm evidence is lacking.
For other creatures, however, the picture is much clearer. The circadian
clocks of gastropods (sea slugs) such as Aplysia have been pinpointed
to groups of ‘pacemaker’ neurons clustered inside the creatures’ eyes.
The comparative simplicity of the gastropod nervous system means researchers
can detect circadian rhythms of electrical activity in these neurons – something
which is still impossible in studies of the mammalian circadian clock.
The pacemaker neurons of eyes removed from sea slugs continue to diplay
a circadian rhythm of electrical activity which can be manipulated with
pulses of artificial light. What drives such rhythms? A key finding from
research on Aplysia is that the rhythms are sustained by the synthesis
of proteins inside the neurons, at certain points in the daily cycle. Chemical
compounds known to block protein synthesis shift the phase of the rhythms
and lengthen their circadian cycle.
Earlier this year, Arnold Esken and his colleagues at the University
of Houston, Texas, went a step further by showing that Aplysia’s rhythms
also depend on its pacemaker neurons being able to copy, or transcribe,
genes into messenger RNA – the first step in protein synthesis.
None of this is a surprise to Jon Jacklet of the State University of
New York in Albany, who discovered the sea slug’s circadian clock more than
20 years ago and has been studying it ever since. Complex cellular events
such as the activation of genes and protein synthesis are only to be expected,
says Jacklet, given the slowness of circadian rhythms compared with most
simple biochemical processes. Jacklet believes the cellular events that
keep the sea slug’s clock ticking over are likely to be common to the circadian
clocks of other animals, including humans.
The race is now on to find out exactly how particular genes and proteins
regulate the electrical activities of Aplysia’s pacemaker neurons. A major
influence on the firing rate of any neuron is the concentration of calcium
ions in its cytoplasm. So many biologists are trying to forge links between
genes activated during the circadian cycle and the cellular events that
control the release or uptake of calcium ions.
The creature whose circadian rhythms are best understood at the genetic
level is the fruit fly, Drosophila. During the 1980s, researchers led by
Michael Young of the Rockefeller University in New York discovered what
appears to be a ‘master’ gene for the fly’s daily rhythms. Mutations in
the gene, which is known as per, can lead to unusually short (19-hour) or
long (28-hour) circadian rhythms. However, the function of the protein encoded
by the gene is still a mystery. The speculatation is that it regulates circadian
rhythms either by influencing the way neurons communicate, or by switching
on other genes. Confusion surrounding the whereabouts of the fly’s circadian
clock is a major obstacle to progress.