Circadian activity patterns of inbred and outbred mice

strains.Mariel Ferragamo

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.

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.

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.

Prior ResearchOther 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).

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?

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.

Experiment: Running Wheel

• Activity of the animals was recorded using running wheel data.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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

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.

Data: meansBalb/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

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.

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.

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?

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.

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.