Circadian Rhythms in Different Mice Strains

1. Circadian activity
patterns of inbred and
outbred mice strains.
Mariel Ferragamo

2. Circadian Rhythms
• A circadian rhythm is any biological process that
displays a consistent pattern within about 24 hours.
• Animals have an internal biological clock that maintains
their behavior in a circadian rhythm.
• Most animals do not have a perfect 24 hour internal
clock and if they are given no time cues will show a drift
of their day length.
This condition is known as free-running.
• When animals are given time cues then they can use
these to lock their internal rhythms to the external world.
This condition is known as entrainment.

3. Circadian Rhythms
• When we change our time of daily activity by
travelling across times zones we need to entrain our
internal clock to the new time. The greater the shift
in time, the harder it is for the body to catch up and
we may experience jetlag.
• Most mammals use light cues to entrain their
circadian rhythms.
• Mice are nocturnal; they are active at night in the
dark. They are good experimental subjects to test
circadian rhythms and entrainment to light cues.

4. Strains of Mice
• Laboratory mice are bred to have different
characteristics and behaviors.
• Mice strains are different types of mice, just as
labradors and terriers are to dogs.
• Inbred mice have very little genetic variation
whereas outbred mice are more different from
each other. This experiment uses two strains: the
inbred Balb/c, and the outbred CD1.
• Inbred strains can be very useful to help determine
the genetics of behaviors.

5. Prior Research
Other scientists have shown that the internal clock is
genetically determined and can differ between
strains of mice (Schwartz and Zimmerman, 1990).
Strains of mice can show differences in their free-
running day length and activity patterns (Schwartz
and Zimmerman, 1990).
One study using daily shift changes showed that CD1
mice try to use the onset of darkness as the cue for
entrainment (MCGowans Coogan , 2013).

6. My Questions
• Comparing my control group outbred mice to my inbred
group I can ask how much of the circadian behavior is
influenced by genetics and how much by environment.
• Do the different strains of mice show differences in their
internal day length and their light entrainment patterns?
• Are the inbred mice more similar in their activity than the
outbred mice?
• Is it easier to adjust to a shift of the clocks forward or
backward?

7. Experiment
• We used nine animals of each strain of mice (Balb/c and
CD1) and they were kept in a room with only artificial
lighting.
• Each animal had its own cage with a running wheel and
constant access to food and water.
• The mice were run in two different groups as we only
had 10 running wheels.
• The light schedules were set to 12 hours on and 12 hours
off, and were changed over the course of several
months.
• The mice experienced forward and backward schedule
changes of 2 hours, as well as a period of 2-3 weeks of
darkness.

8. Experiment:
Running Wheel
• Activity of the animals was recorded using running
wheel data.

9. Experiment
• We collected baseline data for the first 10-11 days, to
allow the mice to get used to their wheels.
• For each time shift the animals were given 5-7 days to
adjust before the next change. After 2 or 3 schedule
changes the lights were turned off and the mice were
kept in darkness.
• Red lights were used only for animal care during this
time. After the extended darkness period the lights
resumed with 12 hours on and 12 off.
• Running wheel activity of the animals was recorded by a
computer and the program “Clocklab” (Actimetrics)
was used to plot the data and calculate the times of
offset and onset of the running.

10. Data
• This graph is called an
actogram and is used to
display circadian activity.
• The y-axis represents the
date, the x-axis represents
the time (in a 24 hour day).
• The black lines show when
the animal was running.
• The red blocks show when
the lights were on.

11. Data
• The mouse is free-running when the lights are off
and the activity drift to an earlier start time each
day, shows that it has a shorter internal day than
24 hours.
• When the lights provide cues then the running is
maintained close to a 24 hour cycle.

12. Data:
Example Actograms
Balb/c CD1
This Balb/c mouse timed its running
very precisely to the offset of the
light and rarely ran in the light.
This CD1 mouse showed
more variability in the
timing of running
compared to the lights.

13. Data:
More Example Actograms
Balb/c CD1
0 4 8 12 16 20 0
50
45
40
35
30
25
20
15
10
5
1
Time in hours (midnight to midnight)
Day
This Balb/c mouse also timed its
running very precisely to the offset
of the light and rarely ran in the
light.
This CD1 mouse also
showed more variability in
the timing of running
compared to the lights.

14. Data
• The Clocklab program determined the time that
each mouse started its main running bout each day.
We subtracted this onset of running time from the
time that the lights turned off. This difference shows
how precisely the mouse times its running with the
lights.
• We calculated the mean time difference for each
strain to see if they differ in entrainment precision. We
calculated the standard error of the time difference
to see how much the precision varies among mice in
each strain.

15. Data:
Entrainment Precision
• This figure shows the average difference between
the time the lights went off and the onset of running
for each mouse strain (+ one standard error).
The Balb/c (red bar)
showed more
precise timing and
less variability than
the CD1 (blue bar).

16. Data
• The average difference in time between the light
turning off and the running was positive. This means
that the mice usually started running a bit before
the lights went out. The animals were anticipating
the time the lights would go off and their internal
clock matched the light schedule.
• Moving the lights forward or backwards 2 hours
makes the animals adjust to a new schedule.

17. Data:
Forward and Backward Shifts
• To compare the forward and backward light shifts
we calculated the mean time difference on the
day before the shift, the day of the shift and the
day after the shift for each mouse strain.
• On the day before the change the mice display the
time they are used to.
• On the day of the change the mice suddenly get a
new schedule and respond to it.
• The day after shows us how quickly they adapt to
the new time.

18. Data:
Forward and Backward Shifts
• For both shifts the Balb/c mouse shows more precise
entrainment and less variability. The backward shift
has better light entrainment on the day of the shift
and also on the next day than the forward shift.
Forward shift Backward shift

19. Data:
Forward and Backward Shifts
• With a backward shift, the mouse is not active and
suddenly the lights come on 2 hours early. The
Balb/c mice jump on the wheels right away. The
next day they are anticipating the new time and
are showing close entrainment.
• With a forward shift, the mouse starts running early
anticipating the lights going off but the lights stay
on for an extra 2 hours. The Balb/c mice quickly
recover the next day.

20. Data: means
Balb/c
(mean in hours)
CD1
(mean in hours)
Mann-
Whitney rank
test p<0.05
Free-running day length 22.87 23.61 * Strains differ
Day length with lights 23.96 23.87 same
Mean entrainment
difference
0.33 2.71 * Strains differ
Mean backward
entrainment
-0.03 1.18 * Strains differ
Mean next day (from
backward)
0.23 1.83 * Strains differ
Mean forward
entrainment
0.95 4.56 * Strains differ
Mean next day (from
forward)
0.37 3.89 * Strains differ

21. Conclusions
• When the mice were free-running the Balb/c had a fairly short
internal day length (mean 22.87 hours) which agrees well with the
results of other scientists for this strain (e.g. Schwartz and
Zimmerman found a mean of 22.94 hours)
• The CD1 free-running (mean 23.61 hours) is a little bit shorter than
24 hours which also is similar to the measurements of others for this
strain (McGowan and Coogan found a mean of 23.7 hours)
• Under the light conditions of 12 hours on 12 hours off both strains
of mice show a day length which is very close to 24 hours.
• Both mice strains adjusted to new light schedules so their
behavior was influenced by the light.

22. New Findings
• Previous research indicated that the time the lights went
off can be used as a cue for entrainment for mice. My
study showed that the Balb/c mice used this cue with
greater precision than the CD1 mice. As expected the
inbred Balb/c mice also showed less variability in their
responses.
• Both strains of mice adjusted more quickly to a
backwards shift. When the clock is shifted backwards it
more closely matches their short internal clock and this
makes it easier to adjust.
• The Balb/c mice entrained better to both new light
schedules than the CD1 mice.

23. Applications
• It is important to choose the right mouse strain for
particular research studies. The Balb/c mouse would be
a good model to study light entrainment.
• Mice strains that respond differently to schedule
changes would allow a comparison in a study on jetlag.
The different effects on health and cognition could be
measured using physiological measures such as blood
pressure or blood sugar, as well as behavioral tests of
memory.
• Do mice that entrain easily suffer fewer bad effects of
jetlag? Or would it be easier for the outbred mice who
pay less attention to the time cues?

24. References
• McGowan, N.M. & Coogan, A.N. (2013) Circadian
and behavioral responses to shift work-like
schedules of light/dark in the mouse. Journal of
Molecular Psychiatry, 1(7), 1-11.
• Schwartz, W.J. & Zimmerman, P. (1990) Circadian
Timekeeping in Balb/c and C57BL/6 inbred mouse
strains. The Journal of Neuroscience, 10(11), 3685-
3694.

25. Acknowledgments
• Thank you to the following for making this possible:
o Gustavus Adolphus College for allowing the experiment to
be conducted in the animal facilities – IACUC approved.
o Dr. Janine Wotton, for mentoring me and assisting with this
project.