April 06, 2001
Chimeric Mice Reveal Clues to How Brain's Clock "Ticks"
By studying mice whose brains contain a composite of neurons that
produce normal and longer-than-normal circadian rhythms, researchers
are beginning to understand how neurons synchronize their oscillatory
behavior to control the body's 24-hour, internal clock.
The experiments represent the beginning of a new research direction,
say the scientists, that progresses beyond discovering the genes that
produce the internal clock machinery to exploring how brain cells
interact to produce coherent circadian rhythms.
The scientists also say that the technique of producing genetic
composite, or "chimeric," mice offers a promising way to study how
cells in different regions of the brain work together to produce
specific behaviors.
Most biological clocks operate on a 24-hour, circadian (Latin for
"about a day") cycle that governs functions like sleeping and waking,
rest and activity, fluid balance, body temperature, cardiac output,
oxygen consumption and endocrine gland secretion. In mammals, the main
circadian clock components reside in cells in the suprachiasmatic
nucleus (SCN) of the brain. Inside these cells, the molecular
components of the clock are "rewound" daily by the effects of light and
other stimuli.
In an article published in the April 6, 2001, issue of the journal
Cell, Howard Hughes Medical Institute investigator Joseph S.
Takahashi and Sharon Low-Zeddies, both at Northwestern University,
reported that they created more than 200 distinct chimeric mice whose
suprachiasmatic nuclei had differing ratios of normal and mutant
circadian neurons.
The mice were genetically engineered using a standard technique for
producing chimeric mice. The researchers combined eight-cell embryos
from wild-type mice with cells from embryos that contained a mutant
Clock gene, which produces a loss of circadian rhythms and a
period length of 27-29 hours in homozygous animals (mice with two
copies of the mutant gene).
These aggregate embryos usually spontaneously form a single embryo,
which can then be implanted in a surrogate mouse that would give birth
to a chimeric mouse. Since the wild-type mice were albino and the
mutant mice were pigmented, the scientists could determine which
animals were chimeric by their variegated coat colors and eye
pigmentation. Also, the Clock-mutant cells carried a genetic
marker for a characteristic dye, so that the scientists could
distinguish Clock cells from wild-type cells upon examining the
animals' brains.
The scientists measured the chimeric animals' circadian behavior
using standard analyses of the amount of time they spent on running
wheels in their cages. "One of the important facts established in
earlier research with rats and mice was that individual SCN neurons
could generate their own circadian oscillations in vitro," said
Takahashi. "Those experiments were important because they showed that
the circadian oscillator in mammals is cell-autonomous or
cell-intrinsic to SCN neurons."
A second important point learned from earlier studies, said
Takahashi, was that the Clock mutation reduced the amplitude and
lengthened the circadian rhythms of individual neurons in vitro.
Finally, he noted, researchers had found that rats and hamsters in
which the SCN had been lesioned lost circadian rhythm. Transplantation
of SCN tissue restored circadian rhythms in the animals.
All of these earlier experiments suggested that studying chimeric
Clock mice might offer new insights into how SCN cells work
together to generate the animals' circadian rhythms, said Takahashi.
Studying the chimeras would have a considerable advantage over studying
tissue-transplanted animals because the structure of the SCN would
remain intact, he said.
Low-Zeddies and Takahashi measured and analyzed the circadian
activity of chimeric mice whose SCNs ranged from mostly
Clock-mutant cells to mostly wild-type. Their studies showed
that behaviorally about one-third of the chimeric mice appeared to be
normal wild-type animals, one-third homozygous mutant and one-third
intermediate.
"This suggested that in order to dominate the animals' behavior, the
SCN had to have a majority of one cell type," said Takahashi. "That
might seem obvious, but it turned out that wasn't predictable because
lesion experiments showed that if you have just a few cells left in the
SCN, those are sufficient to generate rhythms," he said. "But we
clearly found that the SCN needs a majority of one cell type to
dominate behavior."
According to Takahashi, however, one of the most interesting
findings was that some of the intermediate chimeric mice behaved like
genetically mutant animals that were heterozygous-that is, each of
their cells contained one Clock-mutant gene and one wild-type
gene. Both the intermediate chimeric animals and heterozygous mutants
showed intermediate 25-hour circadian rhythms.
"This result argues strongly that cell-cell interactions and
integration of these periods must be occurring in these mice said
Takahashi. "And because the periods in such chimeras are coherent and
stable, the only way to get that is for all the cells to be
synchronized together."
Comparative analyses of the chimeric animals by Low-Zeddies and
Takahashi revealed that the period of circadian oscillation and the
amplitude of an animal's activity did not always co-vary. In contrast,
in Clock-mutant animals, the lengthening in circadian period is
always accompanied by a lowering of the amplitude of a mutant animal's
activity.
"We don't believe that anyone has found that period and amplitude
can vary independently," said Takahashi. "Such findings are so complex
and fine-grained, it would not have been possible without such a very
large number and range of animals."
Additional studies will be needed to understand the details of how
SCN neurons coordinate circadian rhythm, said Takahashi. However, he
said, this new strategy represents an important future direction for
understanding the physiological organization of circadian rhythms.
"Over the last four years, the field has been immersed in gene
discovery and description of the molecular mechanism of the circadian
clock in mammals and flies and other organisms," he said. "Of course,
the genes are important, but to understand the behavior of the animal,
we have to understand how cells interact in the brain to produce
coherent circadian behavior." Finally, Takahashi emphasized, the
chimera experiments demonstrate a new role for studies of such
animals.
"Chimera analysis has traditionally been applied to developmental
questions in mouse biology," he said. "But this study shows that it can
also be applied to study how brain structure governs behavior, which
has traditionally been thought of as too complex a mechanism to study
in this way."
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