Circadian Behavioral Analysis
Objective: Analyze circadian behavioral patterns to determine free-running period and confirm behavioral rhythmicity in mice using activity monitoring and periodogram analysis
Gather these items before starting the experiment. Check off items as you prepare.
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Protocol Steps
Animal acquisition and breeding
Acquire male and female C57BL/6 mice from Charles River Laboratories in equal numbers. Breed AQP4 KO mice in University of Rochester vivarium and backcross to C57BL/6 for 20+ generations.
Note: Equal numbers of males and females used to control for sex differences; minimum of five mice per group
View evidence from paper
“Male and female C57BL/6 mice (aged 3–5 months, weight between 25 g and 30 g) were acquired from Charles River Laboratories (Wilmington, MA) in equal numbers for each experimental group”
Housing setup
House mice either in a 12:12 light/dark cycle or under constant light with ad libitum access to food and water. For constant light experiments, house animals two per cage.
Note: Housing two per cage in constant light reduces risk of hypothermia; maintain humidity at 30-70%
View evidence from paper
“Mice were group-housed either in a 12:12 light/dark cycle or under constant light with ad libitum access to food and water”
Continuous activity monitoring
Monitor activity continuously using the Comprehensive Lab Animal Monitoring System (Columbus Instruments) with data recorded in 5-minute bins.
Note: Data collection must span minimum 10 days for accurate periodogram analysis
View evidence from paper
“Activity was monitored continuously in 5 min bins via the Comprehensive Lab Animal Monitoring System (Columbus Instruments)”
Circadian behavioral analysis
Analyze collected activity data using ActogramJ software to visualize circadian patterns and prepare data for periodogram analysis.
Note: Software processes continuous activity monitoring data
View evidence from paper
“Circadian behavioral analysis was completed with ActogramJ”
Free-running period determination
Determine free-running period of each cage using at least 10 days of activity data and a chi-square (χ²) periodogram analysis.
Note: Chi-square periodogram used to confirm behavioral rhythmicity and estimate experimental times
View evidence from paper
“Free-running period of each cage was determined using at least 10 days of activity and a χ² periodogram, which was used to confirm behavioral rhythmicity”
Activity onset determination
Estimate experimental times in conjunction with activity onset of the cage using periodogram results.
Note: Activity onset used to synchronize experimental timing with circadian phase
View evidence from paper
“χ² periodogram, which was used to confirm behavioral rhythmicity and estimate experimental times in conjunction with activity onset of the cage”