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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
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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
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Why do we need behavioral tests to understand human brain
disorders?
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Why use animal models?y
Ethical considerations
Time
Fi l Fiscal
Experimental control Experimental control
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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
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Why use behavioral models?
Throughput
Cost Ph i l i l C l itCost Physiological Complexity
Modified from Kokel and Peterson, 2008
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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
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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
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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?
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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
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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
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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
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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
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El t d l dElevated plus and zero mazes
Rat exposureSocial interaction test
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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
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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
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Anxiety
Increased thigmotaxis (peripheral vs. central activity) in
BSERT-/- mice
Reduced exploration activity in SERT-/- mice
+/+ Reduced exploration activity in SERT-/- mice +/-
50
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120
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Kalueff et al., 2007
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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
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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
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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
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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
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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
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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)
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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
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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
0 = Absent1 = Present
Limb Grasping
1 = Absent Evidence of Biting
0 = None 1 = Biting in response to p g
0 = Absent1 = Present
Pinna Reflex0 = Present
g phandling
Vocalizations0 = None 1 = Vocal0 Present
1 = Absent 1 Vocal
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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
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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
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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
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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
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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
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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
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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
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Homecage activity (II): general hypoactivity
Dramatic reduction of 24-h motor activity in SERT-/-motor
activity in SERT-/-mice
Holmes et al., 2002
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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Measuring strengthMeasuring strength
Hanger test:
Time latency to fall fromTime latency to fall from an
upside-down screen
phenome.jax.org/.../Lake3_Protocol
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Rope climbing testRope climbing test
Ability to climb rope Latency to reach a 20 cm
markmark
Kalueff et al., 2007
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Normal grip strengthNormal grip strength
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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
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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
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Foot-claspingp g
Example of a foot-clasping phenotype
Tanaka et al., 2004
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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)
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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
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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
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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
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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
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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
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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
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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)
Kalueff et al., 2007
transitions (arrows).
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Macro-behavioral and Micro-behavioral Levels of Analysis
Stress Drugs
Genetic Mutations
Quality Quantity Both
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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
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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
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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
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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
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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