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Noninvasive, Automated Measurement of Sleep, Wake
and Breathing in Rodents
Sleep is critical to health and yet is rarely measured in animal models. This webinar highlights a noninvasive method for
studying sleep in rodents. Topics covered include 3Rs, user experience, automation, and data analysis.
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Noninvasive, Automated Measurement of Sleep, Wake
and Breathing in Rodents
Dr. Bruce O’HaraProfessor of Biology,
University of KentuckyCo-founder, Signal Solutions LLC
• The importance of sleep to health and disease
• Physiological outputs used to track sleep
• Benefits of noninvasive sleep studies
• Setting up a PiezoSleep Study
• Data Collection and Analysis
• Use studies: How noninvasive methods enhance research
Today’s Agenda
Why Sleep?
1. Strongly conserved in all mammals and perhaps all animals
2. Important for general health, performance and mood.
3. Still unclear WHY
Effects of Sleep Deprivationhttps://en.wikipedia.org/wiki/Sleep_deprivation
Major Sleep Discoveries Identified using Research Animals (primarily mice)
1. Biochemical, genetic and anatomical pathways involved in sleep
2. Relationships between sleep and disease
3. Behavior and sleep
4. Possible fundamental function of sleep
A core function for SLEEP?
Newly described “Glymphatic System” Brain clears cellular garbage through convective flow exchange between CSF and ISF which increases 5x during sleep!!!
Sleep Drives Metabolite Clearance from the Adult BrainLulu Xie1,*, Hongyi Kang1,*, Qiwu Xu1, Michael J. Chen1, Yonghong Liao1, MeenakshisundaramThiyagarajan1, John O’Donnell1, Daniel J. Christensen1, Charles Nicholson2, Jeffrey J. Iliff1, Takahiro Takano1, Rashid Deane1, and Maiken Nedergaard1,†
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880190/
https://www.youtube.com/watch?v=96aZtk4hVJM
Physiological Outputs Used to Monitor Sleep in Mice
EEG/EMG: measures the electrical activity of cortical neurons and muscles, respectively, to determine - wake, REM and NREM sleep.
Video and IR beam breaking: rely on gross body movements to distinguish inactive from active.
Piezoelectric sensors: detect gross and fine body movements, including breathing signatures, that characterize sleep and wake.
Physiological Outputs Used to Monitor Sleep in Mice
TOP: Piezoelectric Output. BOTTOM: Impedance Pnuemograph.Courtesy of Dennis Grahn and H. Craig Heller, Stanford University, who established that the sensitive piezo
signal could be useful as a measure of breathing and thus as a measure for monitoring sleep and wake as well.
Piezoelectric Output vs. Impedance Pneumograph
Methods for Recording Sleep in Mice
Preparing mouse for EEG sleep recording
Preparing mouse for PiezoSleep sleep recording
Traditional EEG-based Mouse Sleep Studies
PROS
+ Differentiates NREM, REM sleep states + Historically accepted method – Gold Standard+ Highly Accurate (when everything is working right!)_______________________________________________
CONS
- Invasive surgery- Mouse activity limited- Labor intensive > low throughput - Time intensive human scored EEG analysis- Technical expertise required for surgery and EEG scoring- Expensive
PiezoSleep Sleep Studies in Mice
PROS
+ Non-invasive > no surgery, no headmount
+ Simple to deploy
+ High through-put
+ Long term/lifetime studies
+ Automated, consistent sleep/wake scoring
+ Analysis Software > straight from data collection to analysis
+ Labor saving and cost effective
_________________________________________________
CONS
- No REM/NREM discrimination (beta)
- Unfamiliar technology
For many studies, general sleep-wake is sufficient and
quite valuable
Sleep-wake Monitoring Based on Piezoelectric Sensor Technology
Piezoelectric film on the cage floor transforms mechanical pressure into electrical signals
Classifier Extracts Sensor Signals Derived From Mouse Movement
Classifier analyzes features of the signal and generates a decision statistic, a measure of how well the signal meets our criteria for sleep.
Wake Signal = Varied Movement
2.5-3 Hz Breathing Rhythm of Sleep
PiezoSleep SystemData Collection and Analysis
Setting up a PiezoSleepStudy
To learn more about thePiezoSleep Mouse Behavioral
Tracking System,visit www.sigsoln.com
Thank you to our event sponsor SignalSolutions
Step 1: Collecting Activity Data with PiezoSleep
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
PiezoSleep Set up
Scalable Data Acquisition Hardware
Monitoring capabilities for 1 -80 mice
OPTIONS FOR:
Data Marking
Digital I/O
Analog I/O
Capabilities for integration with complementary systems (simultaneous video, EEG, etc) and for sleep feedback control (ex: sleep fragmentation system).
Calamari: 8 channels
Squid/Squid Plus: 16,32
Giant Squid: up to 80 channels
Step 2: Data Analysis with SleepStats
SleepStats Data Analysis Software
• Input is sleep/wake features collected in PiezoSleep
• SleepStats’ secondary algorithm refines the sleep/wake classification, and converts data to useful information such as….
– Percent sleep (Daily or light vs dark)
– Bout length distribution (daily, light vs dark)
– Activity onset
– Peak activity
• Generates csv files of percent hourly sleep, average hourly sleep/wake bout length for further analysis in programs such as Excel.
Use Studies: How Non-invasive Methods Enhance Research
Biological Applications of the PiezoSleep System
Research
• Quantitative Trait Loci studies in BXD recombinant inbred lines and preCC mice
• Individual mutants and knockouts
• Testing effects of various drugs on sleep
• Assessing sleep quality in mouse disease models
• One of the most successful phenotypes at the Jackson Laboratory IMPC center.
• Sleep following Traumatic Brain Injury
Benefits
• Larger numbers of mice allow for accelerated discovery
• Easy data analysis software provides quick means to compare mice and design appropriate experiments
• Cost effective means to scale up and obtain results more quickly
• Noninvasive methods allow for studies that were not possible or problematic due to surgery required for EEG
Use Case 1: Identification of genes affecting sleep in the International Mouse Phenotyping Consortium
Piezoelectric film on the cage floor transforms mechanical pressure into electrical signals
18 research institutes and 5 national funders [click to learn more]
Phenotyping KOs at Jackson Lab IMPC center
• Unbiased selection of genes show highly significant effects on sleep
• High hit rate – currently 50/300 KO lines screened
• Broad overview; more granular analyses will help dissect complexity of sleep
− Purpose
− Function
− Pathways insights
• Breath rate algorithm may offer surrogate lung assessment; apneas
• Underscores importance of the IMPC/KOMP unbiased approach to annotating mammalian genome
Data can be found at the International Mouse Phenotyping Consortium (IMPC) website: www.mousephenotype.org
Noninvasive sleep monitoring in large scale screening of knockouts (KOMP2) produces high hit rate with implications for sleep and behavioral studies
Use Case 2: Identifying Sleep Modifying Substances
Treated vs untreated mice: Big picture view - overall percent sleep is similar
Hour by hour data exported from SleepStats provides more detail.
Mean hourly bout length per group
Despite similar overall percent sleep, treated mice have more long bout lengths
0
5
10
15
20
25
30
35
40
0 - 250 251 - 500 501 - 750 751 -1000
1001 -1250
1251 -1500
1501 -1750
1751 -2000
2001 -2225
2226 -2500
Freq
uen
cy
Bout Length in Seconds
Hourly Mean Bout Length Frequencies in Treated vs Untreated Mice
0 mg/kg dark
50 mg/kg dark
0 mg/kg light
50 mg/kg light
Especially during the light period!(normal rest period)
Time of dosing also appears to be relevant (not shown).
* used with permission
Use Case 3: Eyes Wide Shut – exploring sleep in other species
41
Mus musculus
African Spiny Mice (Acomys cahirinus) are one of several exciting new rodent models that have significant differences from standard lab mice that can better address important biological and biomedical issues
Watch the webinar to view this video
71
Simultaneous EEG, EMG and Piezo Monitoring of Spiny Mice
Courtesy of Chunang Wang, University of Kentucky
• REM/NREM discrimination
• Other behaviors – activity, feeding, drinking, seizures, cataplexy, tracking breathing variables(such as sleep apnea), general health monitoring
• Providing sleep feedback control for improved models and precision, such as sleep fragmentation studies, sleep specific hypoxia for better obstructive sleep apnea models.
• Other species – rat. Heart rate monitoring during sleep.
Future Applications
Noninvasive methods for monitoring sleep in research mice provide advantages over traditional methods:
Better for the mouse > no surgery, anesthesia or tethering needed
Simpler to employ > no technical expertise needed
Higher throughput > saves time, more data, higher statistical power
Quicker to results
Less expensive
Summary
SIGNAL SOLUTIONS, LLC
Kevin Donohue Mike Lhamon
Anuj Agarwal Rebecca Bernat
O’HARA LAB, UNIVERSITY OF KENTUCKY
Mansi Sethi Chunang Wang
Shreyas Joshi Ren Guierierro
Martin Stritz
SUNDERAM LAB, UNIVERSITY OF KENTUCKY
Dillon Huffman Asmaa Ajwad
Farid Yaghouby
UNIVERSITY OF KENTUCKY RODENT BEHAVIOR CORE
JACKSON LABORATORY
KENTUCKY SCIENCE AND TECHNOLOGY CORPORATION
FUNDING support for product development from DoD, NIH, Kentucky Cabinet for Economic Development
Acknowledgements
Thank You
Dr. Bruce O’Hara
Professor of Biology,University of KentuckyCo-founder, Signal Solutions [email protected]
For additional information on the products and applications presented during this webinar please visit www.sigsoln.com, and connect with Dr. Bruce O’hara by email.
