Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

Summary

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Abstract

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day^1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).

Although circadian rhythms in running-wheel activity are typically linked to the SCN clock^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Protocol

1. Animal Housing

1. Cage: In order to record the running-wheel activity of an individual rodent, each cage should house a single rodent and running-wheel. Because running wheels can be considered a form of enrichment, all rodents in any study should have similar access to a running wheel.
2. Bedding changes: Animal handling as well as changes in cages or bedding can all have non-photic effects on circadian rhythms^21-23, so, cages with mesh-flooring are ideal because they minimize contact with the animal. Notwithstanding the availability of such a tray-system, bedding changes should be avoided during critical phases of an experiment. Alternatives include using longer-lasting bedding, which would allow for more infrequent cage changes, or changing cages on a pseudo-random schedule.
3. Isolation Boxes: Cages should be kept in isolation boxes that are sound-attenuated, light-controlled, and well-ventilated. Depending on the size and configuration of the isolation boxes, the number of cages within each box will typically range from 1-8. When housing multiple cages in a single isolation box, one should be aware that various odors and sounds coming from other animals can have confounding effects on the circadian behavior of individual animals. To avoid these problems, one should attempt to house one cage per isolation box.
4. Ventilation: Adequate air flow is imperative to making the boxes a comfortable home environment for rodents. The fan in each box should be hooded, so as to prevent light from outside the box from reaching the inside. Also, fans will typically remove air from the box and blow it into the room. Small light-tight vents allow air to enter the isolation boxes from several points, and help to avoid uncomfortable breezes. In order to verify that there is adequate ventilation, the temperature inside the isolation box (when closed for several hours, with the lights on) should be virtually identical to the temperature in the room where it is housed.
5. Lighting: Environmental light intensity should be the same in all cages. Arrange a single light at a similar location above each cage, and always use the same brand/type of bulb. Use moderate intensity illumination (100-300 lux) at cage level. Avoid excessively high illumination levels, which are more likely to produce direct changes on behavior attributable to the light rather than the circadian system, per se (e.g. masking).
6. Darkness/Dim red lighting: If it is necessary to handle or treat animals in the dark (e.g. in constant darkness or nighttime), night vision goggles should be used. Alternatively, because the circadian system is relatively insensitive to red-wavelengths, dim red lighting can be used. The specific red light you use should be tested to ensure it does not alter running-wheel activity (e.g. masking) or adjust the circadian clock (e.g. produce a phase shift).

2. Data Collection (See Figure 1 - Vitalview Hardware Configuration)

1. Running wheels: The diameter and ergonomics of the running wheel will change the amount of use²⁴. Thus, use smaller and lighter wheels for mice, and larger heavier wheels for rats. When washing and re-installing wheels, ensure that the wheels are able to spin unobstructed, do not "wobble", and that the recording micro-switches are activated by each turn of the wheel.
2. Micro-switches: Each revolution of the running wheel should activate a magnetic or mechanical micro-switch. Information from the micro-switch is transmitted via a single channel and recorded by a computer which can bin the data over time (e.g. every 2, 5, 6, or 10 min).
3. Computer hardware: Our running-wheel recordings are made with Vitalview, a hardware and software platform developed by Mini Mitter (http://www.minimitter.com/vitalview_software.cfm). However, there are other recording platforms such as ClockLab, developed by Actimetrics (http://www.actimetrics.com/ClockLab/). Both platforms bring together data from many single-channel sources (e.g. a single micro-switch activated by a single running wheel) into a single computer file. Data from individual channels can then be graphed and analyzed separately at a later date.

3. Data Recordings

1. Files: The above mentioned software platforms can be used to separate out single channels so that individual files are created for each running-wheel record. Such data are best visualized and graphed with specially designed programs such as Actiview (Minimitter, Bend, OR), Circadia, or Clocklab (Actimetrics, Wilmette, IL) which can all produce periodograms and actograms. However, single-channel files can also be opened and analyzed using general spreadsheet programs such as Excel (Microsoft, Redmond, WA).
2. Calculating Circadian Time (CT): CT 12 is, by definition, the onset of running-wheel activity in nocturnal rodents. In parallel with the 24 hr day, by convention, one circadian day is broken into 24 circadian hr. Accordingly, if the circadian free-running period is 24 hr 30 min as measured by the wall clock, CT 0 will occur approximately 12 hr 15 min after CT12.

Results

1. Computer programs: Specialized computer programs are typically used in the generation of actograms and the calculation of circadian period. These programs include, but are not limited to, Actiview (Minimitter, Bend, OR) and Circadia.
2. Actograms: Actograms provide a graphic illustration of the daily patterns of running-wheel activity. There are single-plotted (x-axis = 24 hr) and double-plotted (x-axis = 48 hr) actograms. Both methods plot sequential days from top to bottom, but double-plott...

Discussion

Monitoring daily activity rhythms using running wheels is the most commonly used and reliable method for assessing the output of the master circadian clock in nocturnal rodents. Wheel-running activity, however, is only one of many aspects of behavior and physiology that can be monitored continuously. Although the vast majority of running-wheel activity occurs during the night, over 30% of the total wakefulness occurs during the daytime^25,26. Other endpoints can be used to assess circadian rhythms, including ge...

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
Vitalview Card & Software	Mini Mitter	#855-0030-00	(Bend, OR, USA)
DP24 Dataport	Mini Mitter	#840-0024-00	(Bend, OR, USA)
QA4-Module	Mini Mitter	#130-0050-00	(Bend, OR, USA)
Magnetic Switch	Mini Mitter	#130-0015-00	(Bend, OR, USA)
C-50 Cable assembly	Mini Mitter	#060-0045-10	(Bend, OR, USA)
Rat running wheel assembly	Mini Mitter	#640-0700-00	(Bend, OR, USA)
Cage and tray support	Mini Mitter	#640-0400-00	(Bend, OR, USA)
Useable cut away cage	Mini Mitter	#664-2154-00	(Bend, OR, USA)
Grid floor for cage	Mini Mitter	#676-2154-00	(Bend, OR, USA)
Waste tray	Mini Mitter	#684-2154-00	(Bend, OR, USA)
Lamp housing	Microlites Scientific	#R-101	(Toronto, ON, Canada)
4W Fluorescent lamps	Microlites Scientific	#F4T5/CW	(Toronto, ON, Canada)
Isolation chambers	Custom built		28"H x 20"W x 28"D ½" Black Melamine.