Behaviour Studies

Pharmacological Research, 2009Pharmacological Research, 2009Methods in Behavioral PharmacologyMethods in Behavioral PharmacologyMethods in Behavioral PharmacologyMethods in Behavioral Pharmacology

Allan V Kalueff, PhDAllan V Kalueff, PhDJan 26 2009: TUMC PharmacologyJan 26 2009: TUMC PharmacologyJan 26, 2009: TUMC PharmacologyJan 26, 2009: TUMC Pharmacology

Learning ObjectivesLearning Objectives

U d d b i i i l f h i Understand basic principles of neurophenotyping research: Why? How? Tests vs. Screens

List the three tiers of behavioral phenotyping of t i itransgenic mice

Describe multiple assays for motor dysfunction in rodents

Describe rodent models of depression and anxiety Describe micro- and macro-behavioral approaches to

neurophenotyping research

Why do we need behavioral tests to understand human brain disorders?

Why use animal models?y

Ethical considerations

Time

Fi l Fiscal

Experimental control Experimental control

Behavioral tests are used to study:Behavioral tests are used to study:

Effects of environment (e.g., stressors) on behavior Adverse effects of genetic mutations Gene x Environment interactions Side effects of drugs Drug interactions Pharmacological effects of drugs in experimental

models of brain disordersmodels of brain disorders Drug x Gene x Environment interactions

Why use behavioral models?

Throughput

Cost Ph i l i l C l itCost Physiological Complexity

Modified from Kokel and Peterson, 2008

Why theWhy the mouse?

Mice and humans share over 90% of genes

Easy to breed, can be housed in large #s Precise gene targeting available At least 80 strains have abnormal depression or

anxiety-related phenotypes Similarities to human neural circuits Similarities to human neural circuits

Quantifying behavioral responsesQuantifying behavioral responses

Scoring customized for specific subjects/test

E i t h ld b Experimenters should be:1)blind to treatment2) high in inter/intra rater reliability2) high in inter/intra rater reliability3) consistent (time, season, place)

Pletnikov, 2006

Three tiers of behavioral phenotyping (C l 2000)(Crawley, 2000)

Tier 1. General Observations Do animals look healthy? Do animals look healthy? Do they gain weight normally? Are they grooming normally? y g g y Do they move around the cage properly? Do they show reflex responses, such as eye

blink, ear twitch, whisker twitch, and righting reflex?

Three tiers of behavioral phenotyping (C l 2000)(Crawley, 2000)

Tier 2. Motor functions and Sensory function Motor function (e.g. gait analysis or motor activity) Hearing (e g startle) Hearing (e.g. startle) Tactile sensation (e.g. von Frey hairs) Thermal sensation (e.g. paw withdrawal to heat) Vision. Visual cliff test, with glass floor. Smell. Novel odor test, i.e. vanilla painted onto a wall.

Tier 3. Specific tests in Basic Science Research on the pharmacology of Neurological and Psychiatric Diseases

Model vs. Screen

Modeli d i d d i inducing a depressed or anxious state (e.g., Open field test novelty)

Changes in physiology and behaviorg p y gy Emphasis on construct and etiological

validity

Screen Often used to test drugs or Test genetically altered model Emphasis on face and predictive

validityvalidity

Traditional tests of affective states (emotionality)

Anxiety Depressiony p

Open field test Forced swim testOpen field test Elevated plus maze

Forced swim test Tail suspension test

Animal models of depression

Porsolt test (Forced Swim test)

Based on learned helplessness

Quantifies number and duration of Quantifies number and duration of immobility episodes

Tail-suspension test

immobility indicates depression

Tail-suspension test

dry version of the forced swim test

El t d l dElevated plus and zero mazes

Rat exposureSocial interaction test

Novelty testsyOpen field test

Measures distance moved, vertical rears, and time spent in the center vs. the periphery

Light/dark box

Q tifi b f t i d tiQuantifies number of entries and time spent in the in the lighted area. Exploratory behaviors vs. anxious behaviorsbe a o s

Open field and exploratory strategiesIn addition to assessment of the amount of behavior (i.e., frequency and duration measures), analyses of quality of behavioral represent an important part of behavioral phenotyping:

spatial temporal spatio-temporal characteristics

What? When? Where?

CenterPeripheryCornersCorners

Anxiety

Increased thigmotaxis (peripheral vs. central activity) in BSERT-/- mice

Reduced exploration activity in SERT-/- mice

+/+ Reduced exploration activity in SERT-/- mice +/-

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Kalueff et al., 2007

Hallucinogens: Acid trip (LSD)

20 min 1 h 25 min 2 h 30 min 2 h 32 min 2 h 35 min 2 h 45 min I'm having a little trouble controlling this pencil. It seems to want to keep going

everything is changing color Everywhere Patient becomes startled by something on the floor

Patient is generally agitated, and becomes largely none-verbal. I am... everything is... changed... they're calling... your face... interwoven... who is... Patient mumbles

Patient sits on his bed. He reports that intoxication has worn

4 h 25 min 5 h 45 min 8 h 00 min

reports that intoxication has worn off except for the occasional distorting of faces

4 h 25 min 5 h 45 min 8 h 00 minRunning back and forth across the room

I can feel my knees again

Behavioral effects of LSD in rodents

Open field activity

Anxiety(5-HT1a)

Open field activity

+/+Saline +/+LSD

+/-Saline +/-LSD

Hyperactivity5-HT2a/c

+/-Saline +/-LSD

0 30 60 90 120 150Time, min

SHIRPA: gross phenotype assessmentSHIRPA: gross phenotype assessment

SmithKline Beecham Pharmaceuticals Harwell, MRC Mouse Genome CentreHarwell, MRC Mouse Genome Centre Imperial College School of Medicine Royal London Hospital, St. Batholomews Phenotype Assessment

Assessing motor phenotypesAssessing motor phenotypes

SHIRPA battery: a widely-accepted neurologicalSHIRPA battery: a widely accepted neurological battery involving a three-stage protocol

It is very basic, and includes measures of muscle function, cerebellar function, sensory function, neuropsychiatric function and autonomic functionneuropsychiatric function, and autonomic function

http://btc.bol.ucla.edu/shirpa.htm

Behavior recorded in Viewing Jar (I)

Body Position0 = Inactive0 = Inactive 1 = Active2 = Excessive Activity

Tremor0 Ab t0 = Absent1 = Present

Palpebral Closure0 = Eyes openy p1 = Eyes closed

Coat Appearance0 = Tidy and well groomed coat1 = Irregularities such as piloerection1 Irregularities such as piloerection

Whiskers0 = Present1 = Absent (include any further comments

Behavior recorded in Viewing Jar (II) Lacrimation

0 = Absent1 = Present

g ( )

1 = Present Defecation

0 = Present1 = Absent

Behaviour recorded in the Arena: Tansfer Arousal

0 = Extended freeze (over 5 seconds)0 Extended freeze (over 5 seconds)1 = Brief freeze followed by movement2 = Immediate movement

Gross Locomotor ActivityThe total number of squares the animal enters with all four feet in 30 sThe total number of squares the animal enters with all four feet in 30 s

Gait0 = Fluid movement and approximately 3-mm pelvic elevation1 = Lack of fluidity in movement (include comments eg. retropulsion,

th 3 l i l ti )more than 3 mm pelvic elevation)

Behavior recorded in Viewing Jar (III)

Tail Elevation0 = Dragging

g ( )

gg g1 = Horizontal extension2 = Elevated/straub tail

Startle Response0 = None0 None1 = Preyer reflex (backwards flick of the pinnae)2 = Reaction in addition to the Preyer reflex (e.g., Startled response)

Touch Escape0 = No response0 = No response1 = Response to touch2 = Flees prior to touch

Behaviour recorded above the Arena: Positional passivity

0 = Struggles when held by the tail1 = Struggles when held by the neck

2 = Struggles when laid supine2 Struggles when laid supine3 = No struggle

Behavior recorded in Viewing Jar (IV)

Skin Color0 = Blanched

Corneal Reflex0 = Present0 = Blanched

1 = Pink2 = Bright, deep red flush T k C l

0 = Present 1 = Absent

Contact Righting Reflex0 = Present1 Ab t Trunk Curl


Limb Grasping

1 = Absent Evidence of Biting

0 = None 1 = Biting in response to p g


Pinna Reflex0 = Present

g phandling

Vocalizations0 = None 1 = Vocal0 Present

1 = Absent 1 Vocal

Summary: SHIRPA batteryy y

A battery of tests that can be completed within a few A battery of tests that can be completed within a few minutes

Observation for normal and abnormal spontaneous b h i d t f ti it l lbehaviors, and measurements of activity levels, arousal, respiration, gait, muscle tone, reflexes, aggression, etc.

If a subject group shows unusual behavior or function, further testing must be done in that domain

Motor problemsMotor problems

Disorders may have both peripheralDisorders may have both peripheral and central origins:

Cerebellum Cerebellum Brain stem Striatum Basal ganglia Motor cortex Spinal cordp Peripheral nervous system Musculoskeletal deficits

Gait assessment (I) Detects walking abnormalities

Easy to perform: place non-toxic paint on mouses feet

Sensitive to atypical patterns due to genetic alterations Sensitive to atypical patterns due to genetic alterations (see example below genetic mouse model of Huntington disease)

A) Wild type B) Mutant mice

Detloff, 2003

Gait assessment (II)

Ink is applied to the paw

( )Transgenic mice over-

expressing neurotrophin-3 The animal walks on paper The footprints are analyzed

for:

p g p

for: step length (SL) print length (PL) toe spread (TS) intermediate toe spread (ITS)

Taylor et al 2001

Problems: May be sensitive to procedure-evoked

Taylor et al, 2001

anxiety/stress

Swimming Assess ability to swim Abnormal patterns (vertical vs horizontal)Abnormal patterns (vertical vs. horizontal) Circling Divingg Sinking

www.umt.edu/urelations/rview/summer06/mice.htm

Normal horizontal swimming Abnormal vertical swimmingKalueff et al., 2006

Motor skills testsMotor skills tests

D i d d i (R i T ) Drug-induced turning (Rotation Test) Forelimb asymmetry (Cylinder Test) Beam walkingg Grip strength Grid walking Placing test Placing test Rotorod Landing Foot Spread Test

Skill d hi (f li b t t l) Skilled reaching (forelimb motor control)

Neurodetective International, 2008

Homecage activity chambers (I)Homecage activity chambers (I)

Normal behaviors to assess:

Di i Digging Grooming Thigmotaxis (staying close to

www med associates com

g ( y gthe walls)

Rearing Explorationwww.med-associates.com p

Homecage activity (II): general hypoactivity

Dramatic reduction of 24-h motor activity in SERT-/-motor activity in SERT-/-mice

Holmes et al., 2002

Homecage activity (III)

Abnormal behaviors to assess: Hyperactive running Stereotypes (jumping, circling, somersault)

S i Seizures Freezing/ inactivity episodes Overactive itchingOveractive itching Over-grooming and self-damage Overall impulsivity

Behavioral perseverations

Commonly seen behavioral perseverations:

Barbering Repetitive Jumping Bar-Mouthing Cage Top Twirling

www.aps.uoguelph.ca/~gmason/StereotypicAnimalBehaviour

Cage-Top Twirling Excessive Licking Excessive Grooming

www.nc3rs.org.uk

Specific animal tests for motor abilitySpecific animal tests for motor ability

Locomotory activity (gross assessment) Balance (e g Rotorod)Balance (e.g. Rotorod) Reflex testing Strength testing Fine motor analysis (FMA) Straight observation

Open field test (OFT)Open field test (OFT)

High/low activity levelg y Body posture Movement coordination

www med-associates com Rearing, exploring Additional movements (e.g. head twitches)

Thi i ( id f l )

www.med associates.com

Thigmotaxis (avoidance of open central areas)

Open-field testOpen field test

Open square or circular arenaOpen square or circular arena Typical parameters: 1. zones entered 2. time spent in periphery vs. center3. grooming time4 Rears4. Rears5. defecation Often use videotracking software (e.g.

Ethovision, HVS Image) providing distance traveled, speed, etc.

Measures both locomotor activity and anxiety y y

Spatial working and long-term memoryMovement duration(% of total)

8,0

10,0

SERT+/+SERT +/-

SERT -/-

Serotonin is involved in the regulation of memory and other cognitive functions

0,0

2,0

4,0

6,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

SSRIs have effects on memory in habituation tasks

W d OF d EPM h bit ti tTravelled distance (% of total)

6,0

8,0

10,0

We used OF and EPM habituation to assess spatial memory in SERT-/- mice

Normalized mouse activity (% of total)

0,0

2,0

4,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

y ( )

ConclusionsIncreased anxiety (previous slide)But: Normal spatial memory Vertical rears (% of total)

4,0

6,0

8,0

10,0

But: Normal spatial memory

0,0

2,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Rating scalesg

Scoring technique using a number to represent the d f b h i l itdegree of behavioral severity

0 1 20 1 2Normal behavior Intermediate motor

disturbancesConsistent abnormal motor coordination

Potential concerns: Statistics are non-parametric More quantitative than qualitative

Pletnikov, 2006

LocomotionLocomotion

Many potential confounds:

Habituation problems (too much/little) Habituation problems (too much/little) Testing time (consider circadian rhythms) Variability (e.g. interstrain)y ( g ) Problems with housing (e.g. multi-species odors,

sex pheromones) Effect of sound Effect of sound Floor/ceiling effects

Pletnikov, 2006

Beam walking test

Use food reward or dark escape" area as incentivep

beam width = difficulty

Endpoints recorded: Time to cross beam

F ll Falls Hind-leg slips

Problems:Problems: Often requires pre-training May involve motivational f tChang et al., 2005 factors

Rotorod (Rotarod)

A rotating bar, revolves at gconstant or increasing speeds

Latency to fall is primary endpoint

Typically, mouse performance as number of trials

Van Meer and Raber, 2005

RotorodM t b litiMotor abnormalities: Coordination Weakness Muscle tonicity Involuntary movements

Other domains: Sensory function

C iti bilit Cognitive ability Anxiety Non-motor seizures Chronic/systemic problems Problems: Cognitive phenotypes (e.g.

habituation) may affect motor performance) y pPletnikov, 2006

Measuring strengthMeasuring strength

Hanger test:

Time latency to fall fromTime latency to fall from an upside-down screen

phenome.jax.org/.../Lake3_Protocol

Rope climbing testRope climbing test

Ability to climb rope Latency to reach a 20 cm

markmark


Normal grip strengthNormal grip strength

Chimney test

Consists of a hollow tube large enough for a mouse g gor rat to fit inside comfortably

The animal is placed in the tube, and then the tube is positioned vertically, with the animals snout orientedpositioned vertically, with the animal s snout oriented downwards

The animal will attempt to keep itself from falling and will slowly walk backwards up to the top of thewill slowly walk backwards up to the top of the chimney

This measures the animals motor ability and di ticoordination

Hind-leg clasping reflexNormal reactionin a normal mouse

This mouse will appear normal in the cage but with you pick it up, it exhibits "clasping" rather th th l l tthan the normal plantar reaction

Clasping indicates neurological/motor i i t i i l

Ansorge et al 2006 impairments in animalsal. 2006

Davis, 2000Herzing, 2008 Davis, 2000

Foot-claspingp g

Example of a foot-clasping phenotype

Tanaka et al., 2004

Assessing other motor reflexesAssessing other motor reflexes

Ri hti flRighting reflex

Mice right themselves onto feet after put on their Mice right themselves onto feet after put on their backs, or dropped from some height (e.g. 20 cm) on a cushioned surface

Generally normal unless movement/vestibular disorders are present

Tail suspension test (abnormal spinning if vestibular Tail suspension test (abnormal spinning if vestibular problems)

Digging behaviorsDigging behaviors

A very common behavior in rodents Sensitive to stress, and anxiolytic/ anxiogenic

pharmaceutical compoundspharmaceutical compounds Marble-burying test often used to measure this

behavior

Marble burying/digging

To kick sand in someone's face is an archetypal agonistic interaction between humans

Rodents have been filmed kicking earth toward an approaching snake in their burrows They also buryapproaching snake in their burrows. They also bury noxious objects such as shock probes

M t b h i l i ti t ld th t bl Most behavioral scientists would assume that marbles are non-aversive to mice

Mice are probably not deliberately burying the marbles; they simply fall through the displaced bedding (MB test measures digging behavior)measures digging behavior)

Deacon, 2006

Digging Test Protocol

Digging is defined as coordinated movements of fore-Digging is defined as coordinated movements of foreor hind limbs that displace the substrate

Fill th 5 d ith d hi Fill the cage 5 cm deep with wood chips Several test cages can be run simultaneously Place a mouse in each cage and start the testPlace a mouse in each cage and start the test

timer. Test duration is 3 min. The latency to start digging, the number of digging

bouts and the total duration of digging arebouts and the total duration of digging are recorded

Deacon, 2006

Marble Burying Protocol

1 Fill the cage approximately 5 cm deep with wood1. Fill the cage approximately 5 cm deep with wood chip bedding

2. Place a regular pattern of glass marbles on the surface evenly spaced each about 4 cm apartsurface, evenly spaced, each about 4 cm apart

3. Place one animal in each cage and leave for 30 min

4 Count the number of marbles buried (to 2/3 their4. Count the number of marbles buried (to 2/3 their depth) with bedding

5. Alternatively, count the number of marbles buried fully partially (2/3 their depths) and non-buriedfully, partially (2/3 their depths), and non buried

D 2006Deacon, 2006Kalueff et al., 2006

Unusual escape attemptsUnusual escape attempts

When animal demonstrates abnormally active escape attempts

E.g. immediately after being placed on a surface theE.g. immediately after being placed on a surface the animal will jump/run away, rather than freezing

Could indicate hyperactivity, very high overall anxiety, hyperexcitability or other phenotypeshyperexcitability, or other phenotypes

If animal shows these abnormal behaviors, it needs further examination before being tested in other

diparadigms

Reflexes and postural reactionsReflexes and postural reactions

Common tests:

Trunk curl Trunk curl Rear-limb withdrawal Low/flat bodyy Tremor Hind-leg abduction

F li b iti i Forelimb positioning

Ethograms: behavioral microstructure (patterning): SERT-/- mice

5 min, observation cylinder test: ha, horizontal activity (number of ha episodes); va, vertical activity (protected rears); f, freezing episodes; g, grooming bouts; d, defecation; st, Straub tail. Line width reflects frequency of behaviors (circles) or their transitions (arrows)


transitions (arrows).

Macro-behavioral and Micro-behavioral Levels of Analysis

Stress Drugs

Genetic Mutations

Quality Quantity Both

Micro-behavioral Video TrackingModern tracking systems can analyze an individual animal in a full spectrum, recording the movement of specified body parts

It is also possible to assess regional distribution of physiological markers, such as regional body temperature

www.cleversysinc.com

Advantages of Micro-behavioral Analyses Certain abnormal behaviors can be detected, such as differences in swimming pattern in the Forcedswimming pattern in the Forced Swim Test

Video-tracking algorithmic computation reduces effects of manual scoring on reproducibility of data

Micro-behavioral analysis complements macro-behavioral endpoints, resulting in higher throughput modelsthroughput models

Juszczak et al., 2008

Animal Models of Psychiatric Disorders

Macro- Micro-

Anxiety

OCSD

UseOCSD

Depression

Schizophrenia

Frequently

SometimesSchizophrenia

Epilepsy

Serotonin Syndrome

Rarely

NoneSerotonin Syndrome

Tourettes Syndrome

Rett S ndromeRett Syndrome

Expanding neurophenotyping batteries

OCD screensPerseverations and stereotypies

Cognitive screensWithin- and between-trial habituation tasksBarnes maze, 3D-maze

Autism/sociability screens,

Spontaneous alternation tasksMismatch negativity

Schizophrenia-related tests

Aggression screens

p

Anhedonic depression

Chronic stressSocial defeat paradigm Early life stress

Behavioral effects of enrichment

Maternal phenotypesCross-fosteringNest-building phenotypes

Neurotoxicity syndromesSpontaneous (serotonin syndrome)Drug-potentiated

Drug abuse phenotypesEthanol-withdrawal anxietyEthanol-related behaviorsDrug preference

Oto-vestibular phenotypes

Bipolar depressionModels of mania g

Screens for hallucinogenic drugs

Ethics: The three RsEthics: The three R s

1) R d U i f i l i i t1) Reduce - Using fewer animals in experiments

2) Replace - replace the animal model in vivo with) p pin vitro or mathematical models, or with a species lower on the phylogenic scale.

3) Refine - eliminating or relieving pain suffered by the animals in experimentsanimals in experiments

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Behaviour Studies