Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

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Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Transcript of Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Page 1: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Page 2: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Circadian Rhythms of Food Intake (John Lighton, PhD)

• What is your Aim?

• Essential Tech, Tools and Approach

• It’s All About Data Resolution (Resting EE, Activity, and Food Intake)

• Environmental Influences

• Some Additional Tech and Closing Thoughts

Page 3: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in

the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools

and laboratory services.

Page 4: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

John Lighton, PhD

President & Chief Scientist, Sable Systems International

Copyright 2015 InsideScientific & Sable Systems International. All Rights Reserved.

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Question: What is Your Aim?

Ultimately, that’s up to you & your research questions! But -

• Proximately, you probably need to obtain data…

• Now: Describing proximate ends to achieve your ultimate goals

• Also some interesting findings… – On intake (food, water), and output (metabolic heat and activity)

– These data should be synchronized – These data should have high temporal resolution – The data resolution should be high – Analytical flexibility should be maximal – The more data modalities, the better…

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THE CAGE…

• The ideal is…

• Home Cage

• Ambient RH

• Autoclavable

• BPA-free

• No seal required

• Metabolic

measurement

optional

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MASS MONITORING

• Req’d for Gravimetric

Intake Monitoring

• Force Transducer =

Easy, Cheap

• Limited Range

• Poor Resolution

• OK for Student Labs

• The Alternative…

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MASS MONITORING

• Real Load Cell (the

standard for accurate

mass measurement)

• Lab Balance

Resolution, Range

• Radical Innovation

(compact, intelligent,

retains individual

calibration)

Addresses Resolution

Challenges and issues

of “dead volume”

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FOOD INTAKE

• Universal Load Cell

• Real-time measurement

• Recorded to disc at 1Hz

• 2 mg resolution,

1 kg range

• Crumb tray – no spillage

• Grille spacing reduces

caching = accurate intake

• Multiple grilles available

• Fine granularity gives

insight into behavioral

patterns

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Raw Feeding Data

• Stores hopper mass vs. time

• Recorded to disc at 1Hz

One Feeding Episode

• Analyze behavior

• Determine individual

feeding episode intake

rates

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Processed

Feeding Data

• Outputs cumulative

intake vs. time

• Shows rate of intake,

structure

• Can output binned data

• Integrates with other

cage data

• Fully traceable

FOOD INTAKE

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Processed

Feeding Data

• Outputs cumulative

intake vs. time

• Shows rate of intake,

structure

• Can output binned data

• Integrates with other

cage data

• Fully traceable

Food Selection

• Determine food preference

• Control access to

either/both foods

• Integrates with other

cage data

FOOD INTAKE

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FOOD INTAKE ANALYSIS StartDate StartTime EndDate EndTime InterUp_minUp_min Up_g Up_g_min Stud_t Prob RFID

4/5/2013 18:22:47 4/5/2013 18:24:24 0 1.63 0.017 0.011 10.78 0 45779

4/5/2013 18:24:40 4/5/2013 18:27:02 0.27 2.38 0.004 0.002 2.43 0.029 37355

4/5/2013 18:27:23 4/5/2013 18:28:08 0.35 0.77 0.013 0.017 20.21 0 45779

4/5/2013 18:28:46 4/5/2013 18:29:10 0.63 0.42 0.003 0.008 8.21 0 45779

4/5/2013 18:30:55 4/5/2013 18:32:02 1.75 1.13 0.009 0.008 26.44 0 37355

4/5/2013 18:37:02 4/5/2013 18:41:06 5 4.08 0.089 0.022 8.79 0 37355

4/5/2013 18:41:26 4/5/2013 18:46:00 0.33 4.58 0.068 0.015 77.59 0 45779

4/5/2013 18:48:30 4/5/2013 18:50:40 2.5 2.18 0.06 0.027 302.49 0 37355

4/5/2013 18:58:57 4/5/2013 18:59:08 8.28 0.2 0.006 0.032 16.39 0 37355

4/5/2013 19:27:38 4/5/2013 19:30:36 7.62 2.98 0.083 0.028 164.49 0 37355

4/5/2013 19:31:00 4/5/2013 19:32:44 0.4 1.75 0.072 0.041 150.41 0 45779

4/5/2013 19:35:09 4/5/2013 19:36:23 2.42 1.25 0.114 0.092 11 0 37355

4/5/2013 19:36:53 4/5/2013 19:39:01 0.5 2.15 0.016 0.008 7.64 0 37355

4/5/2013 19:39:11 4/5/2013 19:40:12 0.17 1.03 0.027 0.026 7.89 0 45779

4/5/2013 19:40:50 4/5/2013 19:41:17 0.63 0.47 0.003 0.006 5.99 0 37355

4/5/2013 19:41:32 4/5/2013 19:43:05 0.25 1.57 0.015 0.01 40.12 0 45779

4/5/2013 19:44:50 4/5/2013 19:45:21 1.75 0.53 0.005 0.01 13.2 0 45779

4/5/2013 19:53:24 4/5/2013 19:53:39 6.15 0.27 0.01 0.039 29.99 0 45779

4/5/2013 19:54:15 4/5/2013 19:58:09 0.6 3.92 0.146 0.037 441.09 0 37355

4/5/2013 20:03:56 4/5/2013 20:07:18 5.78 3.38 0.11 0.032 270.45 0 45779

4/5/2013 20:07:27 4/5/2013 20:07:50 0.15 0.4 0.061 0.152 5.23 0 45779

4/5/2013 20:27:15 4/5/2013 20:30:20 19.42 3.1 0.142 0.046 397.8 0 45779

4/5/2013 20:34:13 4/5/2013 20:36:19 3.88 2.12 0.075 0.035 275.58 0 45779

4/5/2013 20:37:13 4/5/2013 20:39:12 0.9 2 0.117 0.058 368.39 0 45779

• Available Over Any

Desired Interval

• Full List Of All Intake

Events

• Resolution 2 mg

• 10-20x Finer Resolution

Than Legacy Systems

• (Highlighted: Events

Visible Only to

Promethion)

• Statistical Verification of

Each Intake Event

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• Micro-Intake Events =

30%+ of All Ingestive

Behavior

• Proportion of Micro-

Intake Events is Higher

in the Photophase!

• Each = Initiation & Early

Termination of Ingestive

Behavior

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FOOD ACCESS CONTROL Access Control

Module

• Computer controlled

access to food

• Available for Mouse or

Rat food hoppers

• Intelligent Obstruction

Detection

• Access Control Door

• Connects to mass

sensor

• Light Source & Bedding

Temperature Sensor

• Customizable assays

(paired/yoked)

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Food Access Control

• Select from 8 preset assays

• Customize time and duration

• Individual cage setup

Select

Access

Control

Type

Add to your

custom

assay

Unlimited

number of

variations

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WATER INTAKE Water Intake

Monitor

• 2 µL resolution –

accuracy!

• Real-time measurement

• Key is to reduce leakage

• Increased granularity

gives insight into

behavioral patterns

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Raw Drinking Data

• Stores bottle mass vs. time

• Recorded to disc at 1Hz

Processed

Drinking Data

• Analyze

behavior

• Eliminate drift

• Determine

individual episode

intake rates

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BODY MASS Body Mass

Monitor

• 2 mg resolution

• Real-time measurement

• Recorded to disc at 1Hz

• Highly accurate

• Provides in-cage

enrichment

• Reduced technician

interaction

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Raw Body Mass Data

• Measure mass vs. time

• Ensure data is recorded to disc at sufficient

rate (1Hz) – typical log is every 15 min

• Is used about every 15 minutes

Track body

mass over

time

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Processed

Body Mass

Data

• Stores most recent

body mass for each

timestamp

• Synchronized with

other data

• No handling stress

Circadian Cycle

Clearly Visible

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VOLUNTARY EXERCISE

• Real-time measurement

• Magnetic Reed Switch

• Wheel Stop available

• Increased granularity

gives insight into

behavioral patterns

• Correlates with metabolic

data

Running

Wheel

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Raw Running Wheel Data

• Stores RPS vs. time

• Recorded to disc at 1Hz

Processed Running

Wheel Data

• Shows cumulative

distance run

• Can be binned and

synchronized with

metabolic data

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TOTAL ACTIVITY

• XY and Z IR arrays

• 1 cm beam spacing

• 0.25cm calculated centroid

• Real-time measurement

• Intelligent obstruction

detection

• Rearing captured with

Z-axis

• Highly accurate

• Not affected by static

obstructions

• Correlated with metabolic

data

• Fine & coarse motion are

separable

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Activity Analysis

• Total Activity

• Total Distance Traveled

• Rearing

• Activity associated with in-cage devices

• In cage position vs. time

• Customizable assays

• Position histograms

Activity Comparison

• WT vs. ob/ob

• Interaction with FH-MB

• Time spent time spent near food hopper

• Time spent at cage perimeter vs. inside

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Page 28: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

CALORIMETRY

Flow Generators

& Gas Analyzers

• Automated calibration

• Sequential or continuous

monitoring

• Can group for 4, 8,16, 24,

32 etc. cages

• Expandable and modular

• Water vapor dilution

correction

• Integrated O2, CO2, WVP

analyzers

• Pull-mode (negative

pressure)

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PULL MODE…

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MULTIPLEXED CALORIMETRY

• Metabolic measurement of multiple animals in sequence

• Economical – shares analyzers between animals

• We can reduce dwell time to < 15 sec, yielding a cycle time of 2 minutes/8, 16 or 24 animals

• This is < 50% of the time constant of the cages!

• Excellent for determining mean, resting, and active EE

• Even possible to correlate EE with activity

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CONTINUOUS CALORIMETRY

• Metabolic measurement of multiple animals simultaneously

• One analyzer chain per animal

• Time resolution for metabolic signals = 1 second

• This allows mathematical removal of washout effects

• Excellent for determining even the most fleeting and subtle metabolic signals

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DATA ANALYSIS Data

Synchronization

• Overlay metabolic data

with other continuously

monitored parameters

• All data are perfectly

synchronized and can be

exported to other

programs if required

(open formats)

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It’s All About Data Resolution

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BEHAVIORAL ANALYSIS

Raw Data Synchronization - Zoomed

•Body mass (black), food intake (red), wheel (gold), and water intake (blue)

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Automatic Behavior Extraction

EFODA Intake from food hopper A (significant intake found)

TFODA Interaction with food hopper A (no significant intake)

DWATR Intake from water dispenser (significant intake found)

TWATR Interaction with water dispenser (no significant intake)

WHEEL Interaction with wheel (>= 1 revolution)

IHOME Entered habitat (stable mass reading)

THOME Interaction with habitat (no stable mass reading)

LLNGE Long lounge (> 60 sec, no non-XY sensor interactions)

SLNGE Short lounge (5 - 60 sec, no non-XY sensor interactions)

EFODB Intake from food hopper B (significant intake found)

TFODB Interaction with food hopper B (no significant intake)

• Based On Sensor

Interactions

• Fully Automated

• No Video Analysis

Required

• All Animals

Simultaneously

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EACH BEHAVIOR HAS:

1. Date and time of start, end of behavior

2. Seconds duration

3. Mean X, Y position during behavior

4. Total distance in cm moved during behavior

5. Percent of time spent rearing during behavior

6. Quantification of behavior (behavior-dependent), e.g.: 1. Mass of food or water intake 2. Meters run on wheel 3. Body mass (in habitat)

7. Optional parameters (e.g. EE, RQ, Tb, HR, etc. etc.) as specified

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BEHAVIOR DATA LIST Start_Date Start_Time End_Time Durat_Sec Activity Amount Rear% X_cm Y_cm S_cm

5/26/2012 18:33:33 18:33:53 22 IHOME 21.896 0 8.8 22.3 0

5/26/2012 18:33:54 18:34:05 13 SLNGE 17 100 8.8 18.4 17

5/26/2012 18:34:06 18:35:46 107 WHEEL 56 0 9.1 3.8 0

5/26/2012 18:35:47 18:36:07 23 SLNGE 38 100 8.6 12.3 38

5/26/2012 18:36:08 18:37:51 110 WHEEL 79 0 9.1 3.8 0

5/26/2012 18:37:52 18:38:00 10 SLNGE 8 100 9.3 4.6 8

5/26/2012 18:38:01 18:38:07 7 DWATR 0.013 85.7 8.7 24.2 8

5/26/2012 18:38:08 18:38:25 19 SLNGE 29 100 8.3 22 29

5/26/2012 18:38:26 18:42:52 284 WHEEL 184 0 9.1 4.1 0

5/26/2012 18:42:52 18:43:03 12 SLNGE 4 100 9 4.3 4

5/26/2012 18:43:04 18:43:08 5 DWATR 0.052 80 8.3 16 17

5/26/2012 18:43:08 18:43:15 8 SLNGE 0 12.5 8.1 22.2 0

5/26/2012 18:43:16 18:43:21 6 THOME 0 83.3 8.7 21.8 0

5/26/2012 18:43:22 18:43:42 23 SLNGE 18 65.2 10.2 17.5 18

5/26/2012 18:43:43 18:43:45 3 WHEEL 44 0 9.6 4.8 0

5/26/2012 18:43:46 18:43:51 6 TFODA 0 100 9.5 16.1 18

5/26/2012 18:43:52 18:44:54 67 WHEEL 44 0 9.1 4.1 0

5/26/2012 18:44:55 18:45:18 26 SLNGE 20 69.2 9.9 6.5 20

5/26/2012 18:45:19 19:59:49 4756 IHOME 21.971 0 8.8 22 0

5/26/2012 19:59:50 19:59:57 9 SLNGE 1 100 8.8 21.9 1

5/26/2012 19:59:58 20:03:39 236 EFODA 0.058 42.4 9 20.2 2

Page 38: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

TIME BUDGETS

Behavior Minutes Percent

efoda 153.2 11.51

tfoda 3.6 0.27

dwatr 9.5 0.71

twatr 1.3 0.1

wheel 212.2 15.94

ihome 596.2 44.78

thome 5.6 0.42

llnge 281.4 21.13

slnge 68.3 5.13

Page 39: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

LOCOMOTION BUDGET (-WHEEL)

Behavior Meters Percent

efoda 13.5 14.37

tfoda 3.3 3.56

dwatr 6.4 6.79

twatr 0.8 0.82

ihome 0 0

thome 2.4 2.55

llnge 36.2 38.63

slnge 31.2 33.28

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LOCOMOTION BUDGET (+WHEEL)

Behavior Meters Percent

efoda 13.5 0.4

tfoda 3.3 0.1

dwatr 6.4 0.19

twatr 0.8 0.02

wheel 3241.2 97.19

ihome 0 0

thome 2.4 0.07

llnge 36.2 1.08

slnge 31.2 0.93

Page 41: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

TRANSITION PROBABILITY MATRIX

efoda tfoda dwatr twatr wheel ihome thome llnge slnge

efoda 0 0 10.64 2.13 2.13 6.38 0 12.77 65.96

tfoda 0 0 7.14 3.57 14.29 0 0 7.14 67.86

dwatr 5.13 2.56 0 0 0 2.56 0 2.56 87.18

twatr 11.11 22.22 0 0 11.11 0 0 22.22 33.33

wheel 0 3.06 0 1.02 0 0 0 5.1 90.82

ihome 0 0 0 1.39 0 0 0 34.72 63.89

thome 0 2.44 0 0 2.44 0 0 26.83 68.29

llnge 13.21 20.75 1.89 1.89 11.32 20.75 30.19 0 0

slnge 14.8 4 12.4 1.6 34 22.8 10.4 0 0

Page 42: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

EthoScan: Can Extract Behavior-Specific Data!

Activity Amount Rear% X_cm Y_cm S_cm temp light kcal_hr_2 RQ_2

LLNGE 28 14.8 8.4 7.6 28 21.10325 0.734515 0.34031 0.764798

EFODA 0.081 49.4 9.1 20.1 13 21.10951 0.739017 0.48767 0.800055

SLNGE 7 100 9.7 6.9 7 21.14026 0.738523 0.484761 0.790139

WHEEL 6 0 9.1 4 0 21.14551 2.797765 0.52275 0.817151

SLNGE 2 100 9 4.2 2 21.15659 3.756291 0.569091 0.818588

THOME 0 100 9 4.8 0 21.16365 3.747151 0.569289 0.80943

SLNGE 0 100 9 4.4 0 21.15832 3.740504 0.542788 0.822751

WHEEL 54 0 9.1 3.8 0 21.16305 3.725633 0.612379 0.826051

SLNGE 2 100 9.2 3.9 2 21.11449 3.715779 0.629385 0.832367

TFODA 0 100 12.3 17.3 5 21.11274 3.714397 0.642374 0.837089

SLNGE 29 77.8 7.6 10.7 29 21.10952 3.712675 0.642436 0.842363

DWATR 0.017 0 8.3 22 4 21.09149 3.712122 0.628371 0.843027

Page 43: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Resting EE…

Page 44: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Cause of this variation?

• Short Visits to Habitat

Correlate with High EE

• Long Visits = Separate

Data Population

• Why?

• To Answer – Need High

Temporal Resolution of

Metabolic Signals

IN ENRICHMENT HABITAT

Page 45: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

ACTH & Cortisol spike?

DETAIL OF EE IN HABITAT

• Cool-Down Period Lasts

~15 Min

• Variable Low Activity

Duration Correlates with

Low EE

• ANY Movement is

Detectable in Habitat

• EE Rises PRIOR to

Activity (Leaving

Habitat)

• How Can You Apply

This?

Page 46: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

CONSISTENT > of EE before activity begins!

Page 47: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

It’s All About Data Resolution

Page 48: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Activity…

Page 49: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Variation caused by different running speeds

RUNNING WHEEL

• Most Energy-

Intensive

Behavior in Cage

• C57BL/6J Mice

Run ~6km in

Scotophase

• Variable

Metabolic

Signature

Page 50: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

MEASURING RUNNING ENERGETICS

Steve Wickler & Horse Rosemary Gillespie & Ant

Page 51: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

RUNNING MICE = DIFFICULT!

Frustrated mouse… Scared mouse…

CORTISOL!!

Page 52: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

We had a thought! Instead of a Treadmill…

Page 53: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Mouse 1 of 8 C57BL/6J male Body mass 24.89 g Ambient 21.04 °C

100% voluntary, stress-free locomotion

• muscle function • coordination • cardiovascular condition • free of stress bias

STRESSLESS LOCOMOTION ENERGETICS

Page 54: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

… AND SELF-CONSISTENT (8 MICE, 1 NIGHT)

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Data Also Consistent with Literature Values

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Food Intake…

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SHORT INTAKE DURATION = LOW INTAKE AMOUNT

Page 58: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

FOOD INTAKE – HIGH EE AT SHORT INTAKES

• Micro-Intakes More

Common in Photophase

• Often Occur After Brief

High EE (e.g. Wheel

Running) Episodes

• Feedforward?

Page 59: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

LEGACY INTAKE EVENT DETECTION…

• Adequate for Total Food

Intake Measurements

• Miss a Significant

Proportion (>30%) of

Ingestive Events

Page 60: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

HIGH RESOLUTION INTAKE EVENT DETECTION

• Full Complexity of

Ingestive Behavior is

Captured

• Made Possible by

Advanced Data

Acquisition Techniques

(1:500,000 Resolution)

• Statistically Verified

Page 61: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

ENVIRONMENTAL INFLUENCES

• Sound Level

• Light Level

• Occupancy

• Temperature

• RH%

• Barometric Pressure

Page 62: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

“darkness” (not)

Office light is on

Move around office

Lights off, close door,

then open it

ENVIRONMENTAL EXAMPLE

• This is from my office

• Moving around

• Closing the door

• Opening the door

• Exit sign down the

hall very dimly

illuminates the room

(practically darkness)

Page 63: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

OMG!

OMG!

OMG!

LOL

ENVIRONMENTAL EXAMPLE

• Environmental

factors influence

animal behavior and

metabolism

• Without recording

environmental

factors, such effects

add noise to the data

(= unexplained

variations)

Page 64: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

ENVIRONMENT EXPLAINED

Result: Cleaner

data with fewer

outliers

• Possible to explain

otherwise mysterious

responses by

experimental animals

Page 65: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

PROMETHION

Light Field Camera = Raw Light Data

Page 66: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Light Field Camera = Raw Light Data

Page 67: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Light Field Camera = Raw Light Data

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It’s All About Resolution

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Worried about Raw Data Information Overload?

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The Exact Information You Require

SCRIPT ACCESS…

Page 71: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Chapter 1. A Brief History of Metabolic Measurement

Chapter 2. Constant Volume and Constant Pressure Respirometry

Chapter 3. Coulometric Respirometry

Chapter 4. Constant Volume Techniques Using Gas Analysis

Chapter 5. Aquatic Oxygen Analysis

Chapter 6. Direct Calorimetry

Chapter 7. Measuring Field Metabolic Rates

Chapter 8. Flowthrough Respirometry: Overview

Chapter 9. Flowthrough Respirometry: The Equations

Chapter 10. Flowthrough Respirometry Using Incurrent Flow Measurement

Chapter 11. Flowthrough Respirometry Using Excurrent Flow Measurement

Chapter 12. Validating Flowthrough Respirometry

Chapter 13. Metabolic Data Analysis and Presentation

Chapter 14. The Varieties Of Gas Analyzers

Chapter 15. The Varieties Of Flow Meters

Chapter 16. The Varieties Of Activity Detectors

Chapter 17. The Varieties Of Scrubbers, Tubing And Tubing Connectors

Page 72: Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Thank You! For additional information on continuous or multiplexed metabolic measurements and behavioral systems please visit:

http://sablesys.com