Basic Mouse Neurobehavioral Exam

(or how to give a mouse a physical)

Julie Watson Vet MB DACLAM

Resources• Jacqueline Crawley

– Crawley,J and Paylor,R. Hormones & Behavior 197-211 (1997)– “What’s Wrong with my Mouse?” Wiley-Liss 2000.

• Samuel Irwin - First Phenotyping Screen– Psychopharmacologia (Berl.) 13 222-257 (1968)

• SHIRPA stage I – Mamm Genome. 1997 Oct;8(10):711-

Rogers 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/

• Clinical approach

Goals

1. Detect abnormalities likely to affect future behavioral tests

– Blindness– Physical defects– Deafness

Background Strain

Age-related hearing loss & vestibular

defects (<3m)

• 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

Pitfalls• Variations due to:

– Husbandry methods (see John Crabbe, Science 1999)– Age/sex/background strain– Noise/temperature/humidity/location etc.– Time of day

• Detect only sizeable deficits – Background strain not inbred. F1’s, N2’s– Are controls comparable ?– Insufficient group size

• Limited statistical analysis available - ordinal data

• Check genotyping reliability

Behavioral Phenotyping Level 1 Screen Accession #_________ Date________Investigator ____________________Genotype___________________________________ Background strain(s)_____________Inbred / N#___ Tg /TM KO/KI/Cond Gene Name_____________ Key: 0 = zero; 1 = slow or reduced; 2 = normal; 3 = hyper Animal # WT Hemi -/-

Animal # WT Hemi -/-

DOB/Age

Sex M F

DOB/Age

Sex M F

Weight (g) Condition Score 1..2..3..4

Fur color Weight (g) Condition Score 1..2..3..4

Fur color

Empty Cage 2 mins: Gait abnormal Y N Posture abnormal Y N Freezing Y N Wild running Y N Stereotypies Y N Escape Y N

_________________

Grooming 0… 1-2 3-6…>6 Digging 0 …1-2 3-6…>6 Escape 0… 1-2 3-6 >6

Exploring 0..1..2..3 0= <1 side; 1 =< 1 circuit; 2= multiple circuits; 3= frantic

Digging 0..1..2..3 Grooming 0..1..2..3 Rearing 0..1..2..3

Empty Cage 2 mins: Gait abnormal Y N Posture abnormal Y N Freezing Y N Wild running Y N Stereotypies Y N Escape Y N

Exploring 0..1..2..3 0= <1 side; 1 =< 1 circuit; 2= multiple circuits; 3= frantic

Digging 0..1..2..3 Grooming 0..1..2..3 Rearing 0..1..2..3

DRAW Bald patches/abnormalities

DRAW Bald patches/abnormalities

Bald patches? Y.. N Piloerection? Y..N Bald patches? Y.. N Piloerection? Y..N

Physical abnormality Y..N

Whisker damage Y..N Physical abnormality Y..N

Whisker damage Y..N

Body tone 0..1..2..3 Whisker response NA 0..1..2..3

Body tone 0..1..2..3 Whisker response 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 Passivity 0..1..2..3 Palpebral reflex 0..1..2..3

Trunk curl 0..1..2..3

Forelimb place 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 Righting 0..1..2..3 RL withdraw 0..1..2..3

Visual placing 0..1..2..3

Biting 0..1..2..3 Visual placing 0..1..2..3

Biting 0..1..2..3

Reach c touch 0..1..2..3 NA

Clicker 0..1..2..3 Reach c touch 0..1..2..3 Clicker 0..1..2..3

Grip: >60 <60 time _______

Grip: >60 <60 time _______

Notes: _______________________________________________________________________________

____________________________________________________________________________________

DORSAL DORSAL VENTRAL VENTRAL

BIOSAFETY CABINET

SIMPLE EQUIPMENT

WEIGHTPERTINENT INFORMATIONTESTER BLINDED TO GENOTYPE

Tests

• Behavior in Empty Cage:– Gait, posture, general appearance – Are normal behaviors present?

• Exploring, thigmotaxis, digging, grooming, rearing

– Are abnormal behaviors present?• freezing, wild running, stereotypies, seizures,

pruritus

REARING/ESCAPE

DIGGING

Knockout Mouse

LIMITED REARING

Tests

• Pick up, record abnormal physical features– Whisker loss– Bald patches or unkempt haircoat,

piloerection– Eyes– Legs and tail

NORMAL WHISKERS

WHISKER LOSS

General Reactivity

• Response to approach

• Body tone

• Petting escape

• Passivity

Normal Response to Approach

NORMAL BODY TONE

HIGH BODY TONE

PETTING ESCAPE

Unusual Escape Attempts

PASSIVITY

Postural Reactions and Reflexes

• Trunk curl

• Righting reflex

• Forelimb proprioceptive positioning

• Rearlimb withdrawal

TRUNK CURL

RIGHTING REFLEX

PROPRIOCEPTIVE POSITIONING

WITHDRAWAL - FAST

WITHDRAWAL - SLOW

Tests –V & VII NerveFacial sensory and motor

• Whisker response

• Ear twitch

• Palpebral reflex (V, VII)

EAR TWITCH

EAR TWITCH – WHOLE HEAD RESPONSE

WHISKER RESPONSE (?)

PALPEBRAL RESPONSE

Sight

• Placing – visual II

• Placing – tactile

VISUAL PLACING

Tactile Placing

Tests

• Clicker / hearing

• Pupil / response to light

• Biting / aggression

CLICKER

Provoked Aggression

Grip Strength Normal

Grip Strength Abnormal

Impact of Physical Deficits on Behavioral Tests

Impact of Blindness

• Learning and memory tests• Water maze, contextual fear conditioning

– animal cannot learn? – has limited spatial abilities?– Consider alternatives – 2 lever or hole-poke test in operant chamber

• Anxiety tests• Visual cliff, elevated T maze, light- dark box

– Low anxiety?

Whisker Deficits, Itchy Skin

• Lack of Whiskers– Anxiety tests e.g. visual cliff, elevated T maze –

• Skew response compared with mice with whiskers• ? Remove whiskers

• Itchy skin (B6 dermatitis) – open field, aggression

• hyperirritability, • increased activity • increased aggression

Hearing Deficits

• Cued fear conditioning– Use contextual fear conditioning

• Prepulse inhibition– Nb DBA still works

Examples of Further Testing

• Open Field

• Light Dark Box

• Elevated Plus Maze

• Accelerating Rotarod

• Cued and Contextual Fear Conditioning

• Morris Water Maze

Open Field

total activity, discrete movements, distance traveled rearing

Light Dark Box

• Conflict between novelty exploration and avoidance of light open spaces

• Strain variations

• Responsive to anxiolytics

Elevated Plus Maze

PICTURE CREDITS: EUROPEAN MOLECULAR BIOLOGY LABORATORY

•Exploration vs. aversion to open space - 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

Cued and Contextual Conditioning

•Learning and memory test

•Pair footshock with auditory cue – see freezing behavior

•Day 2 return to same chamber - Freezing is a measure of contextual fear

•Move to different chamber, test freezing (control for context)

•Give auditory cue in different chamber – freezing is measure of cued fear

Computer image

Morris Water Maze

Visual cues

Opaque water bathContaining hidden platform

Probe Trial

Acknowledgements

• Jacqueline Crawley

• Christian Newcomer

• Michela Gallagher

• Nadine Forbes

Questions?