Behavioral test paradigms - KAISTbs.kaist.ac.kr/~brain/Lecture/2010Spring/case... · 0 = Eyes wide...
Transcript of Behavioral test paradigms - KAISTbs.kaist.ac.kr/~brain/Lecture/2010Spring/case... · 0 = Eyes wide...
Behavioral test paradigms
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”
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.
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
The three “R’s”:
a consensus in the research community
•Reduce
•Refine
•Replace
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
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
Factors control behaviors
• Epigenetic factors• Environmental factors• Experimenter
The three “E’s”:
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)
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.
Environmental factors
• Housing environment• Enriched environment• Experimental environment
The Mouse…Why?
Life span : 2 yearsPrenatal development: 21 daysAdolescence: 2-3 weeksSexually fertile: 4-6 weeksFull adult: 10 weeks
The Mouse genome
Man-Mouse Synteny(Greek, along band)
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…
The most important factor for mouse IQ test
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
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
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
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
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
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
A Six-lane treadmill for mice and rats helps determine the
various effects of exercise on genetically modified animals.
Exercise platform
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
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
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
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
Rapid Screening of electrocardiograms (ECG) and heart
rate variability can be performed on conscious mice and rats.
Cardiovascular system
EEG Analysis of Mouse Brain
Reference
electrode
Signal
electrodeAmplilifier
+
-
Digitizer
Data acquisition
& Analysis
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
Manual von Frey Automatic: electrotonic von frey
Pain tests: Mechanical
Pain tests: Thermal
2. Learning and memory
Explicit (Declarative) Implicit (Procedural)
Facts Events Skill and Habits Non-associativeAssociative learning
HabituationSensitizationClassical conditioning
E. Thorndike, First empirical and theoretical analyses of animal learning
Puzzle box, measuring escape latency
Learning curve
Classic maze
T-maze: Response learning
Y-maze: Novelty preference
Place learning vs. response learning
Complex maze: Cognitive map & Latent learning (Tolman, 1930)
Radial arm-maze: working memory
Morris water maze: Visual and Spatial learning
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)
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
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
Sample Trial
Choice Trial
Delay
Novel object discrimination test
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
Fear conditioning
Fear conditioning: Passive avoidance
3. Ethological tests
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
Elevated plus maze: Anxiety test
Zero-maze: Anxiety test
WILD MUTANT WILD MUTANT
Home cage vs. Enriched environment
Nesting behavior
Open field test
Locomotor, anxiety, curiosity, stereotypy, working memory…etc!
Social interaction: Barbering behavior
3 months housing in the same cage
Social interactions: dominance test
PLCb1 mutant are recessive
in social dominance test
10 cm 30 cm
3 cm
Dominance testSocial interactions in a cage
-/- +/+
-/- +/+
Testing device for Innate fear
10 cm
30 cm
Fear-eliciting
subject
3 c
m
Response of wild type mice to a ball of clothe
Innate avoidance of wild type mice to Cat’s hair
Lack of avoidance in PLCb1 KO mice to Cat’s hair
Innate avoidance of mice to rats
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
+/+;+/+ +/+;-/- -/-;-/-
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
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
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)
Others…