Download Basic Mouse Neurobehavioral Exam SHIRPA Goals Effect of

2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Resources
Basic Mouse Neurobehavioral
Exam
(or how to give a mouse a physical)
Julie Watson MA Vet MB DACLAM
• Samuel Irwin - First Phenotyping Screen
– Psychopharmacologia (Berl.) 13 222-257 (1968)
• Jacqueline Crawley
– Crawley,J and Paylor,R. Hormones & Behavior 197-211 (1997)
– “What’s Wrong with my Mouse?” Wiley-Liss 2000.
• SHIRPA stage I
– Mamm Genome. 1997 Oct;8(10):711Rogers DC, Fisher EM, Brown SD, Peters J, Hunter AJ, Martin JE
• Websites:
– Eumorphia
http://www.eumorphia.org/EMPReSS/servlet/EMPReSS.Frameset
– MRC Mutagenesis Program
http://www.mgu.har.mrc.ac.uk/facilities/mutagenesis/mutabase/
SHIRPA
Goals
• SmithKline Beecham Pharmaceuticals
• Harwell, MRC Mouse Genome Centre and
1. Detect abnormalities likely to affect
future behavioral tests
Mammalian Genetics Unit
• Imperial College School of Medicine at St
•
Mary's
Royal London Hospital, St Bartholomew's
and the Royal London School of Medicine
Phenotype
– Blindness
– Physical defects
– Deafness
•
• Assessment
Effect of Background Strain
Age-related hearing
loss & vestibular
defects (<3m)
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•
•
•
•
Many 129 strains
A/J
C57BR, C57L
DBA
I, LP, NOD, ALR, ALS
• C57BL/6J @12-18m
Blindness
(rd1 gene)
•
•
•
•
•
•
•
•
FVB
C3H
BUB
CBA
SJL
SWR
NON
P, PL
Goals (2)
2. Suggest further testing
– Presence of abnormal behaviors
– Absence of normal behaviors
– Motor or neurological deficits
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Behavioral Phenotyping Level 1 Screen
Accession #_________
Date ________Investigator ____________________Genotype ___________________________________
Pitfalls
• Variations due to:
– Husbandry methods (see John Crabbe, Science 1999)
– Individual mouse variables Age/sex/background
strain/social dominance
– Environment: Noise/temperature/humidity/location etc.
– Testing conditions: Location, time of day
• Detect only sizeable deficits
– Background strain not inbred. F1’s, N2’s
– Are controls comparable ?
– Insufficient numbers
• Statistical analysis ? - ordinal data
Background strain(s)_____________Inbred / N#___ Tg /TM KO/KI/Cond
Key: 0 = zero; 1 = slow or reduced; 2 = normal; 3 = hyper
Animal #
WT
Hemi
-/Animal #
WT
DOB/Age
Sex
Sex
Weight (g)
Condition Score
Empty Cage 2 mins:
Gait abnormal
Y
Posture abnormal Y
Freezing
Y
Wild running
Y
Stereotypies
Y
Escape
Y
Fur color
N
N
N
N
N
N
M
F
DOB/Age
Weight (g)
Condition Score
Exploring
0..1..2..3 Empty Cage 2 mins:
0= <1 side; 1 =< 1 circuit;
Gait abnormal
Y N
2= multiple circuits; 3= frantic
Posture abnormal Y N
Freezing
Y N
Digging
0..1..2..3
Wild running
Y N
Grooming
0..1..2..3
Stereotypies
Y N
Rearing
0..1..2..3
Escape
Y N
DORSAL
Hemi
Exploring
0..1..2..3
0= <1 side; 1 =< 1 circuit;
2= multiple circuits; 3= frantic
Digging
Grooming
Rearing
0..1..2..3
0..1..2..3
0..1..2..3
VENTRAL
DRAW
Bald patches/abnormalities
Bald patches?
Y.. N Piloerection?
Y..N
DRAW
Bald patches/abnormalities
Bald patches?
Y.. N Piloerection?
Physical abnormality Y..N
Y..N
Physical abnormality Y..N
Whisker damage
-/M F
Fur color
DORSAL
VENTRAL
Y..N
Whisker damage Y..N
Body tone
0..1..2..3 Whisker response
Body tone
0..1..2..3 Whisker response
NA 0..1..2..3
NA 0..1..2..3
Petting escape 0..1..2..3 Ear twitch
0..1..2..3 Petting escape 0..1..2..3 Ear twitch
0..1..2..3
Passivity
0..1..2..3
Palpebral reflex 0..1..2..3
Trunk curl
0..1..2..3 Forelimb place
Righting
0..1..2..3
Visual placing 0..1..2..3
0 ..1...2...3
RL withdraw 0..1..2..3
Biting
Reach c touch 0..1..2..3 Clicker
NA
Grip: >60
• Genotyping reliability ?
Gene Name_____________
Passivity
0..1..2..3 Palpebral reflex 0..1..2..3
Trunk curl
0..1..2..3 Forelimb place 0 ..1..2..3
Righting
0..1..2..3 RL withdraw 0..1..2..3
0..1..2..3 Visual placing 0..1..2..3
Biting
0..1..2..3
0..1..2..3 Reach c touch 0..1..2..3
Clicker
0..1..2..3
<60 time
_______
Grip: >60
<60 time
_______
Notes: _______________________________________________________________________________
____________________________________________________________________________________
Biosafety Cabinet
Initial Information
Simple Equipment
Observation in Cage
– Gait, posture, general appearance
– Are normal behaviors present?
• Exploring, thigmotaxis, digging, grooming,
rearing
– Are abnormal behaviors present?
• freezing, wild running, stereotypies, seizures,
pruritus
WEIGHT
PERTINENT INFORMATION
TESTER BLINDED TO GENOTYPE
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Digging
Rearing/Escape
Subtle Deficits
Obvious Deficits
Limited Rearing
Abnormal Behavior
Physical Exam
Ulcerative Dermatitis
• Pick up, record abnormal physical features
– Whisker loss, bald patches
• barbering, fighting, dermatitis
– Unkempt haircoat, piloerection
• sick mouse
– Eyes, legs, tail
• Genetic/congenital defects, fighting
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Normal Whiskers
Barbering
Ref. Kalueff et al. Behavioral Processes 2005
• Usual: barbering by socially dominant mouse
– Requires cooperation
• All mice in cage may barber if e.g. overcrowding
stress
• More social barbering = less physical aggression
• Whiskers are important
– Bitten off, not pulled out
– Used for object & texture discrimination
– Exploration, balance and orienting
Tests of
General Reactivity
Whisker loss
Four Tests
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•
•
•
Normal Response to Approach
Response to approach
Body tone
Petting escape
Passivity
Normal Body Tone
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
High Body Tone
Petting Escape
Unusual Escape Attempts
Passivity
Tests of
Postural Reactions and Reflexes
Trunk Curl
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•
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•
Trunk curl
Righting reflex
Forelimb proprioceptive positioning
Rearlimb withdrawal
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Righting Reflex
Proprioceptive Positioning
Withdrawal - Fast
Withdrawal - Slow
Tests of
Facial Nerve: Sensory & Motor
Ear Twitch
• Ear twitch
• Whisker response
• Palpebral reflex (V, VII)
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2006 Johns Hopkins Phenotyping Symposium
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Ear Twitch – Whole Head
Response
Whisker Response
Palpebral Response
Sight
• Placing – visual II
• Placing – tactile
Visual Placing
Tactile Placing
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Hearing Test
Clicker
Clicker
Other Tests
Test for
Provoked Aggression
• Provoked Aggression
• Grip Strength
Grip Strength Normal
Grip Strength Abnormal
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Impact of Whisker Deficits,
Pruritus on Behavioral Tests
Impact of Blindness on
Behavioral Tests
• Lack of Whiskers
– Anxiety tests e.g. visual cliff, elevated T maze –
• Learning and memory tests
• Water maze, contextual fear conditioning
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–
–
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animal cannot learn?
has limited spatial abilities?
Consider alternatives
2 lever or hole-poke test in operant chamber
• Skew response compared with mice with whiskers
• ? Remove whiskers
• Itchy skin (B6 dermatitis)
– open field, aggression
• hyperirritability,
• increased activity
• increased aggression
• Anxiety tests
• Visual cliff, elevated T maze, light- dark box
– Low anxiety?
Impact of Hearing Deficits on
Behavioral Tests
Examples of Further Testing
• Cued fear conditioning
– Measures ability to learn and remember auditory cue
• Alternative: contextual fear conditioning
• Ability to recognize and remember place
• Prepulse inhibition
– Startle reflex to loud sound inhibited by quieter
sound that immediately precedes it
– Measure of sensorimotor gating (filtering out
irrelevant material by brain)
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Open Field
Light Dark Box
Elevated Plus Maze
Accelerating Rotarod
Cued and Contextual Fear Conditioning
Morris Water Maze
• Alternative: use puff of air instead of tone
Open Field
Light Dark Box
• Test of anxiety
• Conflict between
exploring novelty and
avoiding light open
spaces
•General test of motor activity
•Infrared beams or video
•Distance traveled
•Pattern of movement
•Discrete movements e.g. rearing
• Strain variations
• Responsive to
anxiolytics
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Elevated Plus Maze
•Test of anxiety: prefers dark
enclosed arms
•Score entries into arms over 5
min trial
•Anxiolytics increase entries into
open arms - widely used in drug
discovery
Accelerating Rotarod
1. Single trial - motor
coordination and
balance
• 4-40 rpm over 5
mins
• Latency to fall
2. Successive trials –
motor learning
PICTURE CREDITS: EUROPEAN MOLECULAR BIOLOGY LABORATORY
Cued and Contextual
Conditioning
Morris Water Maze
•Learning and memory test
•Pair footshock with auditory cue –
see freezing behavior
•Day 2 return to same chamber –
Freezing = memory of place - i.e.
contextual fear
Opaque water bath
Containing hidden
platform
Visual cues
•Move to different chamber, test
freezing (control for context)
•Give auditory cue in new chamber
– freezing = memory of tone - cued
fear
Probe Trial
Did they Learn?
Computer
Tracking
via video
camera
•Most stressful test – do last
•Pretraining – place on hidden platform
•Training – 60 sec swims to find
platform, then time on platform
•Train until WT make criteria (e.g. <10
secs to find platform)
• Probe trial
Visual Cliff
•Test of sight –
plexiglass cover to
apparent cliff
•Place on border
•10 trials – see which way
steps off
•Relies on ability to see
cliff
•Original research
showed blind strain 50/50
•Vs. sighted strains 80%
on solid side
•Confounded by other
senses (whiskers)
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2006 Johns Hopkins Phenotyping Symposium
Watson Clinical Phenotyping
Acknowledgements
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Janice Clements
Christian Newcomer
Jacqueline Crawley
Michela Gallagher
Cory Brayton
Nadine Forbes
Questions?
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