The authors would like to acknowledge salary awards, equipment grants, and operating funds from the Fonds de la recherche en sant&eacute; Qu&eacute;bec (FRSQ), Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Concordia University Research Chairs Program (CRUC), as well as the thoughtful feedback on this manuscript from Dr. Jane Stewart.

1. Animal Housing
<ol>
	<li>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.</li>
	<li>Bedding changes: Animal handling as well as changes in cages or bedding can all have non-photic effects on circadian rhythms21-23, 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
</ol>
2. Data Collection (See Figure 1 - Vitalview Hardware Configuration)
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>Computer hardware: Our running-wheel recordings are made with Vitalview, a hardware and software platform developed by Mini Mitter (<a href="http://www.minimitter.com/vitalview_software.cfm" target="_blank">http://www.minimitter.com/vitalview_software.cfm</a>). However, there are other recording platforms such as ClockLab, developed by Actimetrics (<a href="http://www.actimetrics.com/ClockLab/" target="_blank">http://www.actimetrics.com/ClockLab/</a>). 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.</li>
</ol>
3. Data Recordings
<ol>
	<li>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).</li>
	<li>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.</li>
</ol>

<ol>
	<li>Pittendrigh, C. S., Daan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Analysis+of+Circadian+Pacemakers+in+Nocturnal+Rodents.+V.+Pacemaker+Structure:+A+Clock+for+All+Seasons.">A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. V. Pacemaker Structure: A Clock for All Seasons.</a> J. Comp. Physiol. 106, 333-355 (1976).</li><li>Pittendrigh, C. S., Daan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Analysis+of+Circadian+Pacemakers+in+Nocturnal+Rodents.+IV.+Entrainment:+Pacemaker+as+Clock.">A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. IV. Entrainment: Pacemaker as Clock.</a> J. Comp. Physiol. 106, 291-331 (1976).</li><li>Pittendrigh, C. S., Daan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Analysis+of+Circadian+Pacemakers+in+Nocturnal+Rodents.+III.+Heavy+Water+and+Constant+Light:+Homeostasis+of+Frequency?.">A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. III. Heavy Water and Constant Light: Homeostasis of Frequency?.</a> J. Comp. Physiol. 106, 267-290 (1976).</li><li>Pittendrigh, C. S., Daan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Analysis+of+Circadian+Pacemakers+in+Nocturnal+Rodents.+II.+The+Variability+of+Phase+Response+Curves.">A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. II. The Variability of Phase Response Curves.</a> J. Comp. Physiol. 106, 253-266 (1976).</li><li>Pittendrigh, C. S., Daan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Analysis+of+Circadian+Pacemakers+in+Nocturnal+Rodents.+I.+The+Stability+and+Lability+of+Spontaneous+Frequency.">A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. I. The Stability and Lability of Spontaneous Frequency.</a> J. Comp. Physiol. 106, 223-252 (1976).</li><li>Ralph, M. R., Foster, R. G., Davis, F. C., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+suprachiasmatic+nucleus+determines+circadian+period.">Transplanted suprachiasmatic nucleus determines circadian period.</a> Science. 247, 975-978 (1990).</li><li>Moore, R. Y., Eichler, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+a+circadian+adrenal+corticosterone+rhythm+following+suprachiasmatic+lesions+in+the+rat.">Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat.</a> Brain Res. 42, 201-206 (1972).</li><li>Stephan, F. K., Zucker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+drinking+behavior+and+locomotor+activity+of+rats+are+eliminated+by+hypothalamic+lesions.">Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions.</a> Proc. Natl. Acad. Sci. U.S.A. 69, 1583-1586 (1972).</li><li>Abe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+isolated+brain+regions.">Circadian rhythms in isolated brain regions.</a> J. Neurosci. 22, 350-356 (2002).</li><li>Yamazaki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+central+and+peripheral+circadian+oscillators+in+transgenic+rats.">Resetting central and peripheral circadian oscillators in transgenic rats.</a> Science. 288, 682-685 (2000).</li><li>Lamont, E. W., Robinson, B., Stewart, J., Amir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+and+basolateral+nuclei+of+the+amygdala+exhibit+opposite+diurnal+rhythms+of+expression+of+the+clock+protein+Period2.">The central and basolateral nuclei of the amygdala exhibit opposite diurnal rhythms of expression of the clock protein Period2.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 4180-4184 (2005).</li><li>Amir, S., Lamont, E. W., Robinson, B., Stewart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circadian+rhythm+in+the+expression+of+PERIOD2+protein+reveals+a+novel+SCN-controlled+oscillator+in+the+oval+nucleus+of+the+bed+nucleus+of+the+stria+terminalis.">A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis.</a> J. Neurosci. 24, 781-790 (2004).</li><li>Yoo, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 5339-5346 (2004).</li><li>Guilding, C., Piggins, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenging+the+omnipotence+of+the+suprachiasmatic+timekeeper:+are+circadian+oscillators+present+throughout+the+mammalian+brain?.">Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?.</a> Eur. J. Neurosci. 25, 3195-3216 (2007).</li><li>Boulos, Z., Terman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+availability+and+daily+biological+rhythms.">Food availability and daily biological rhythms.</a> Neurosci. Biobehav. Rev. 4, 119-131 (1980).</li><li>Boulos, Z., Rosenwasser, A. M., Terman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+schedules+and+the+circadian+organization+of+behavior+in+the+rat.">Feeding schedules and the circadian organization of behavior in the rat.</a> Behav. Brain Res. 1, 39-65 (1980).</li><li>Verwey, M., Amir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food-entrainable+circadian+oscillators+in+the+brain.">Food-entrainable circadian oscillators in the brain.</a> Eur. J. Neurosci. 30, 1650-1657 (2009).</li><li>Davidson, A. J., Poole, A. S., Yamazaki, S., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+food-entrainable+circadian+oscillator+in+the+digestive+system?.">Is the food-entrainable circadian oscillator in the digestive system?.</a> Genes Brain Behav. 2, 32-39 (2003).</li><li>Hara, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+feeding+entrains+liver+clock+without+participation+of+the+suprachiasmatic+nucleus.">Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus.</a> Genes Cells. 6, 269-278 (2001).</li><li>Damiola, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+feeding+uncouples+circadian+oscillators+in+peripheral+tissues+from+the+central+pacemaker+in+the+suprachiasmatic+nucleus.">Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.</a> Genes Dev. 14, 2950-2961 (2000).</li><li>Mrosovsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+response+curves+for+social+entrainment.">Phase response curves for social entrainment.</a> J. Comp. Physiol. A. 162, 35-46 (1988).</li><li>Cain, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reward+and+aversive+stimuli+produce+similar+nonphotic+phase+shifts.">Reward and aversive stimuli produce similar nonphotic phase shifts.</a> Behav. Neurosci. 118, 131-137 (2004).</li><li>Antle, M. C., Mistlberger, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clock+resetting+by+sleep+deprivation+without+exercise+in+the+Syrian+hamster.">Circadian clock resetting by sleep deprivation without exercise in the Syrian hamster.</a> J. Neurosci. 20, 9326-9332 (2000).</li><li>Banjanin, S., Mrosovsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferences+of+mice,+Mus+musculus,+for+different+types+of+running+wheel.">Preferences of mice, Mus musculus, for different types of running wheel.</a> Lab Anim. 34, 313-318 (2000).</li><li>Verwey, M., Lam, G. Y., Amir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+of+PERIOD1+expression+in+the+dorsomedial+hypothalamic+nucleus+in+the+absence+of+entrained+food-anticipatory+activity+rhythms+in+rats.">Circadian rhythms of PERIOD1 expression in the dorsomedial hypothalamic nucleus in the absence of entrained food-anticipatory activity rhythms in rats.</a> Eur. J. Neurosci. 29, 2217-2222 (2009).</li><li>Gooley, J. J., Schomer, A., Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dorsomedial+hypothalamic+nucleus+is+critical+for+the+expression+of+food-entrainable+circadian+rhythms.">The dorsomedial hypothalamic nucleus is critical for the expression of food-entrainable circadian rhythms.</a> Nat. Neurosci. 9, 398-407 (2006).</li></ol>

No conflicts of interest declared.

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 daytime25,26. Other endpoints can be used to assess circadian rhythms, including ge...

<table border="1">
	<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Vitalview Card &amp; Software</td>
 <td>Mini Mitter </td>
 <td>#855-0030-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>DP24 Dataport</td>
 <td>Mini Mitter </td>
 <td>#840-0024-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>QA4-Module </td>
 <td>Mini Mitter </td>
 <td>#130-0050-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Magnetic Switch</td>
 <td>Mini Mitter </td>
 <td>#130-0015-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>C-50 Cable assembly</td>
 <td>Mini Mitter </td>
 <td>#060-0045-10</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Rat running wheel assembly</td>
 <td>Mini Mitter </td>
 <td>#640-0700-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Cage and tray support</td>
 <td>Mini Mitter </td>
 <td>#640-0400-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Useable cut away cage</td>
 <td>Mini Mitter </td>
 <td>#664-2154-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Grid floor for cage</td>
 <td>Mini Mitter </td>
 <td>#676-2154-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Waste tray</td>
 <td>Mini Mitter </td>
 <td>#684-2154-00</td>
 <td>(Bend, OR, USA)</td>
 </tr>
 <tr>
 <td>Lamp housing</td>
 <td>Microlites Scientific </td>
 <td>#R-101</td>
 <td>(Toronto, ON, Canada)</td>
 </tr>
 <tr>
 <td>4W Fluorescent lamps</td>
 <td>Microlites Scientific </td>
 <td>#F4T5/CW</td>
 <td>(Toronto, ON, Canada)</td>
 </tr>
 <tr>
 <td>Isolation chambers</td>
 <td>Custom built</td>
 <td></td>
 <td>28"H x 20"W x 28"D &frac12;" Black Melamine.</td>
 </tr>
	 </tbody>
 </table>

recording and analysis of circadian rhythms in running-wheel activity in rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. 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 clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. 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.

<ol>
	<li>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.</li>
	<li>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...

Watch this Scientific Journal Video about Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents at JoVE.com

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

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.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

recording-analysis-circadian-rhythms-running-wheel-activity

Research

JoVE Journal

Neuroscience

21.1K Views.  McGill University. 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.