Behavioral test paradigms - KAISTbs.kaist.ac.kr/~brain/Lecture/2010Spring/case... · 0 = Eyes wide...

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Behavioral test paradigms

Transcript of Behavioral test paradigms - KAISTbs.kaist.ac.kr/~brain/Lecture/2010Spring/case... · 0 = Eyes wide...

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Behavioral test paradigms

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The ethics of animal use

• Utilitarianism: the view that an action is right if and only if it produces a better balance of benefits and harms than available alternative actions

• Consider the “U.S. Government

Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training”

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U.S Government Principles (summary)

• Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society.

• The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. [Alternative] methods…should be considered.

• Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative.

• Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia.

• Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painless killed at the end of the procedure or, if appropriate, during the procedure.

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U.S Government Principles (continued)

• The living conditions of animals should be appropriate for their species and contribute to their health and comfort….veterinary care shall be provided as indicated.

• Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals.

• …exceptions…should be made by…an appropriate review group such as an IACUC

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The three “R’s”:

a consensus in the research community

•Reduce

•Refine

•Replace

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Paigen K, Eppig JT. A mouse phenome project.

Mamm Genome. 2000 Sep;11(9):715-7.

Moldin SO et al. Trans-NIH neuroscience

initiatives on mouse phenotyping and

mutagenesis. Mamm Genome. 2001

Aug;12(8):575-81.

Grubb SC et al. A collaborative database of

inbred mouse strain characteristics.

Bioinformatics. 2004 Nov 1;20(16):2857-9.

www.jax.org/phenome

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IACUC

• Must have at least a laboratory animal veterinarian, a

scientist, and a community member not affiliated with the

institution and who does not use animals in research

• Reviews all facilities and programs twice a year;

identifies and ensures correction of any deficiencies

• Reviews “Protocols”, the researcher’s or teacher’s

proposal for animal use

• Monitors animal use; can suspend any animal activity

that does not meet standards

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Factors control behaviors

• Epigenetic factors• Environmental factors• Experimenter

The three “E’s”:

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Crabbe JC and Phillips TJ (Nat Neurosci 6:440-2, 2003) Mother nature meets mother nurture

Epigenetic sources of behavioral differences in mice(Francis DD et al. Nat Neurosci 6:445-6, 2003)

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Intrauterine position in mice according to number of flanking males (0M to 2M).

In mice, 2M fetuses are exposed to a higher level of

androgens than 1M or 0M. Females adjacent to males

become partially masculinized in their behavior, and 2M

fetuses, both male and female, show increased body

weight, greater aggression and territoriality, and

enhanced male traits including reproductive behavior

(Ryan & Vandenbergh 2002). In rat, overall litter

male/female composition may be more important than

precise intrauterine position (reviewed by Ryan &

Vandenbergh 2002).

Conversely, proximity to female fetuses influences male

physiological development: prostate glands of males

flanked by two females were enlarged compared to

controls (Timms et al. 1999); estradiol is implicated

(vom Saal et al. 1997).

Lathe, R. (2004) The individuality of mice. Genes, Brain & Behavior 3 (6), 317-327.

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Environmental factors

• Housing environment• Enriched environment• Experimental environment

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The Mouse…Why?

Life span : 2 yearsPrenatal development: 21 daysAdolescence: 2-3 weeksSexually fertile: 4-6 weeksFull adult: 10 weeks

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The Mouse genome

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Man-Mouse Synteny(Greek, along band)

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Mouse IQ test

Developmental tests Learning and memory Ethological

• Righting reflex• Grasp reflex• Rooting and oriental • Acoustic startle• Ultrasonic vocalization• Forelimb grip strength• Pain tests

• Step-down avoidance• Olfactory discrimination• Various mazes• Olfactory discrimination• Visual discrimination

• Nesting building• Parental care of pups• Mating• Resident-intruder• Visible burrow• Elevated plus maze

AND MANY OTHERS…

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The most important factor for mouse IQ test

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This order was chosen in the attempt to go from the least stressful to the most stressful test. Animals were received at least 2 weeks

prior to the beginning of testing and given at least these 2 weeks for acclimation. They were group housed, 3–5/cage, and were at least8 weeks of age at the start of the experimental series in order to minimize variability due to developmental factors.

In addition, two partners (EMBL and Harwell) tested the influence of cage enrichment on behaviour in this test battery.

Results are currently being analysed and will be compared and evaluated in the next workpackage meeting at the end of April/04. The“working” SOPs will be audited accordingly.

Experimental procedures

Experiments without habituation ---- Experiments demanding habituation

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1. Developmental tests

Development of reflex responses (recording scores according to age)

-Righting reflex

-Acceleration righting

-Limb placing

-Postural flexion or extension

-Normal posture

-Forelimb grasp reflex (9% maximum until 9 days)

-Pivoting reflex

-Swimming (circling until 6-8 days)

-Straight-line waking

-Hyperkinesia (pop con stage (6-12)

-Vibrissa placing

-bar holding

-Cliff drop aversion

-Auditory startle

-Pain response

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SHIRPA (~phenotype assement)

http://www.mgu.har.mrc.ac.uk/facilities/mutagenesis/mutabase/shirpa_summary.html

Primary screening:

behavioral observation

Secondary screening

-Locomotor activity

-Food and Water Intake

-Balance and coordination

-Analgesia

-Histology

-Biochemistry

Tertiary screening

-Anxiety

-Learning and memory

-pre-pulse inhibition

-EMG

-EEG

-Nerve conduction

-MRI

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SHIRPA Primary1. Transfer arousal0 = Coma 1 = Prolonged freeze, then slight movement 2 = Extended freeze, then moderate movement 3 = Brief freeze (few seconds), then active movement 4 = Momentary freeze, then swift movement 5 = No freeze, immediate movement 6 = Extremely excited ("manic")2. Locomotor ActivityNumber of squares entered by all four feet in 30 seconds. 3. Palpebral Closure0 = Eyes wide open 1 = Eyes 1/2 closed 2 = Eyes closed4. Piloerection (모발 기립)0 = None 1 = Coat stood on end5. Startle Response90dB sound from clickbox 30cm above arena. 0 = None 1 = Preyer reflex (backwards flick of pinnae) 2 = Jump less than 1 cm 3 = Jump more than 1 cm6. Gait0 = Normal 1 = Fluid but abnormal 2 = Limited movement only 3 = Incapacity

7. Pelvic Elevation0 = Markedly flattened 1 = Barely touches 2 = Normal (3mm elevation) 3 = Elevated (more than 3mm elevation)8. Tail ElevationDuring forward motion 0 = Dragging 1 = Horizontally extended 2 = Elevated / Straub Tail10. Touch EscapeFinger stroke from above. 0 = No response 1 = Mild (escape response to firm stroke) 2 = Moderate (rapid response to light stroke) 3 = Vigorous (escape response to approach)11. Positional PassivityStruggle response to sequential handling. 0 = Struggles when held by tail 1 = Struggles when held by neck (finger grip, not scruffed) 2 = Struggles when laid supine (on back) 3 = Struggles when held by hind legs 4 = No struggle

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SHIRPA Primary

Behavior recorded in the Viewing JarThe animal is placed in the viewing jar for 5 minutes. This is located on top of a grid which is suspended above a piece of white paper. The following behaviours are recorded without disturbing the animal and the amount of urination or defaecation is monitored at the end of the observation period. Incidents of bizarre or stereotyped behaviour and convulsions are recorded separately.

1. Body Position0= Completely flat 1= Lying on side 2= Lying prone 3= Sitting or standing 4= Rearing on hind legs 5= Repeated vertical leaping

2. Spontaneous Activity0 = None, resting 1 = Casual scratch, groom, slow movement 2 = Vigorous scratch, groom, moderate movement 3 = Vigorous, rapid/dart movement 4 = Extremely vigorous, rapid/dart movement

3. Respiration Rate0 = Gasping, irregular 1 = Slow, shallow 2 = Normal 3 = Hyperventilation

4. Tremor0 = None 1= Mild 2 = Marke

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Description: In the grip strength test, the rodent is held by the nape of its

neck and the base of its tail. Its forelimbs are placed on the tension bar and

the rat is gently pulled back until it releases the bar. The distance the rodent

pulls the bar before it releases its paw-grip is measured in kilograms of

resistance by a strain gauge. Hindlimb strength can also be assessed.

Purpose: The purpose of the grip strength test is to evaluate the strength of

forelimb and hind limb muscles. This test is useful in detecting changes in

motor function and muscle strength associated with drugs or other

manipulations

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A Six-lane treadmill for mice and rats helps determine the

various effects of exercise on genetically modified animals.

Exercise platform

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Standard mouse boxes can be equipped to accommodate a

Running Wheel for mice. Cyclocomputers and a Data

Acquisition Program keep track of distance, frequency,

and time of exercise.

Running wheel for activity

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A Rota-rod is used for testing coordination and impairment

of locomotor agility of rats and mice.

The length of time the animal is able to maintain itself on

the rotating rod, and the speed of the rod at which the

animal falls are parameters describing an animal's agility.

Neuromuscular function

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Metabolic Cages determine VO2/VCO2 and respiratory

quotient in transgenic mice. This system is capable of

calculating overall metabolic fuel utilization (fat-protein-

carbohydrates).

Metabolic activity

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A Computerized Food and Drink Intake System collects

and delivers data to a computer for up to 72 hours without

disturbing the animals.

Food and water consumption

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Rapid Screening of electrocardiograms (ECG) and heart

rate variability can be performed on conscious mice and rats.

Cardiovascular system

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EEG Analysis of Mouse Brain

Reference

electrode

Signal

electrodeAmplilifier

+

-

Digitizer

Data acquisition

& Analysis

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A Bone Densitometer estimates bone

mineral content and density as well as fat

and lean mass in small animals and

excised bones.

Bio-imaging tools

MicroPET

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Manual von Frey Automatic: electrotonic von frey

Pain tests: Mechanical

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Pain tests: Thermal

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2. Learning and memory

Explicit (Declarative) Implicit (Procedural)

Facts Events Skill and Habits Non-associativeAssociative learning

HabituationSensitizationClassical conditioning

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E. Thorndike, First empirical and theoretical analyses of animal learning

Puzzle box, measuring escape latency

Learning curve

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Classic maze

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T-maze: Response learning

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Y-maze: Novelty preference

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Place learning vs. response learning

Complex maze: Cognitive map & Latent learning (Tolman, 1930)

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Radial arm-maze: working memory

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Morris water maze: Visual and Spatial learning

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Esca

pe

Late

ncy

(sec)

Days2 4 6 8 10

0

20

40

60

2 4 6 8

Escape L

ate

ncy

(sec)

30

50

Blocks

10

Spatial Learning in PLCb1 -/- Mice

Visual cue Spatial cue

+/+ (n=10)-/- (n=10)

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Barnes maze used in studying spatial memory in the mouse. The mouse is put in the center of a

wooden platform. Because mice do not like open spaces they try to find some way to move out of it.

The only way they can escape from the exposed space is to find the one hole out of 40 that leads to

an escape hatch.

In our work with mice we have focused on memory for extrapersonal space as a model of explicit

memory, because spatial memory is well represented in rodents and has been particularly well

studied.

Barnes maze:

Place learning and working memory

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Tim

e to F

all

(sec)

1 2 3 4 5 6 7 8 9

50

100

150

200

250

300

350

400

Trials

Day 1 Day 2

+/+ (n=10)-/- (n=6)

Rotarod: Motor learning

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Sample Trial

Choice Trial

Delay

Novel object discrimination test

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Tim

e to F

all

(sec)

1 2 3 4 5 6 7 8 9

50

100

150

200

250

300

350

400

Trials

Day 1 Day 2

+/+ (n=10)-/- (n=6)

Motor Learning in PLCb1 -/- Mice

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Fear conditioning

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Fear conditioning: Passive avoidance

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3. Ethological tests

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Description: In the light/dark preference

test (test of emergence neophobia), the

activity of a rat or mouse in the light and

dark portions of a box are recorded. The

rodent’s avoidance of the lighted portion of

the apparatus suggests elevated anxiety,

while showing little or no preference for the

dark area of the enclosure suggests a lack

of anxiety.

Purpose: This test is useful in

determining the potential anti-anxiety or

anxiogenic effects of drugs or detecting

damage to the limbic system.

Light/Dark transition test

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Elevated plus maze: Anxiety test

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Zero-maze: Anxiety test

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WILD MUTANT WILD MUTANT

Home cage vs. Enriched environment

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Nesting behavior

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Open field test

Locomotor, anxiety, curiosity, stereotypy, working memory…etc!

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Social interaction: Barbering behavior

3 months housing in the same cage

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Social interactions: dominance test

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PLCb1 mutant are recessive

in social dominance test

10 cm 30 cm

3 cm

Dominance testSocial interactions in a cage

-/- +/+

-/- +/+

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Testing device for Innate fear

10 cm

30 cm

Fear-eliciting

subject

3 c

m

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Response of wild type mice to a ball of clothe

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Innate avoidance of wild type mice to Cat’s hair

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Lack of avoidance in PLCb1 KO mice to Cat’s hair

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Innate avoidance of mice to rats

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14

02468

1012

No. o

f e

ntr

y

gate 1 gate 2

02468

101214

gate 1 gate 2

048

1214161820

gate 1 gate 2

gate 1 gate 2

Subjects Inactive hair

Active hair

Rat

*

*

+/+ (n=10)-/- (n=10)

%N

o. o

f re

tre

at

0

20

40

60

80

100

0

5

10

15

20

25Pro

xim

ity

(cm

)

0

20

40

60

80

100

0

5

10

15

20

25

Pro

xim

ity

(cm

)

%N

o. o

f re

tre

at

Active hair

Rat

*

*

** N

o. o

f e

ntr

yN

o. o

f e

ntr

y

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+/+;+/+ +/+;-/- -/-;-/-

Lack of barbering behaviors in PLCb1 KO mice%

Am

ount of

whis

ker 100

50

0

Counting the number of whiskers (>1 cm )

before and after coupling

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Pre-pulse inhibition test

PS (Prepulse): 10 kHz, 85 dB, 20 msec

SS (Startle stimulus): 115 dB, 40 msec

ISI (interstimulus interval): 100 msec

% Prepulse inhibition =PS-SS coupling/ SS only x 100

Gravity sensor

Restrain box Speaker

Noise protection box

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Age: 8~11 weeks

+/+ (n=13; male=6 female=7)

+/- (n=4; male=3 female=1)

-/- (n=9; male=5 female=4)

-10

0

10

20

30

40

50

74+120 82+120

Trial Type

% o

f P

PI

Abnormal sensorimotor gating in PLCb1-/-

Pre-pulse inhibition (PPI)

Page 67: Behavioral test paradigms - KAISTbs.kaist.ac.kr/~brain/Lecture/2010Spring/case... · 0 = Eyes wide open 1 = Eyes 1/2 closed 2 = Eyes closed 4. Piloerection (모발기립) 0 = None

Others…