Sprengel 07/10Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
The Mouse as Animal Model
1. Genetic manipulation is possible Transgenic mice Gene targeted mice
2. Three month generation time3. Behavioural test are available4. Physiology is well established
Wednesday, July 14, 2010
Sprengel 07/10
candidate gene phenotypic analysis
Reverse Genetic Approach
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
1) Pharmacological study
Drug treated versus naive mice naive
Comparative Phenotyping
Wednesday, July 14, 2010
Sprengel 07/10
1) Pharmacological study
Drug treated versus naive mice naive
2) Genetically modified mouse line
Genetic manipulated versus WT mice
Comparative Phenotyping
Wednesday, July 14, 2010
Sprengel 07/10
First question for setting up theexperimental design
Genetic Background
(Mouse strain)
C57Bl6
Wednesday, July 14, 2010
Sprengel 07/10
First question for setting up theexperimental design
Genetic Background
(Mouse strain)
C57Bl6
C57Bl6/J C57Bl6/N
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
C57BL/6J, C57BL/6N, and C57BL/6C mice in a behavior test battery.
700–2,200 mice in total.
Significant differences among the substrains were found in the results of various behavioral tests, including the open field, rotarod, elevated plus maze, prepulse inhibition, Porsolt forced swim, and spatial working memory version of the eight-arm radial maze.
Small genetic differences may have a great influence on behavioral phenotypes. Thus, the genetic background of different substrains should be carefully chosen, equated, and considered in the interpretation of mutant behavioral phenotypes
Wednesday, July 14, 2010
Sprengel 07/10
Gene targeted mice
ES cell line SV129
X
C57Bl6/J
Wednesday, July 14, 2010
Sprengel 07/10
Gene targeted mice
ES cell line SV129
X
C57Bl6/J
F1 hybridSV129- C57Bl6/J
Wednesday, July 14, 2010
Sprengel 07/10
Gene targeted mice
ES cell line SV129
X
C57Bl6/J
C57Bl6/J
20backcrosses(3 years)F1 hybrid
SV129- C57Bl6/J
Wednesday, July 14, 2010
Sprengel 07/10
F1 hybrid C57Bl6/J20
backcrosses(3 years)SV129- C57Bl6/J
Solid phenotypes Phenotypes on C57Bl6/X background
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
Transgenic mice
multiple foundershave to be analysed
Wednesday, July 14, 2010
Sprengel 07/10
Transgenic mice
multiple foundershave to be analysed
Gene-targeted mice
one founder issufficient for theanalysis
Wednesday, July 14, 2010
Sprengel 07/10
Transgenic lines
Multiple founders have to be analyzed
Offsprings from two independent founders, with two different integration sites have to be analysed in behaviour!
Four groups of mice (10 -12 mice per group)
Wednesday, July 14, 2010
Sprengel 07/10
Conditional transgenic miceGroup 1: Gene “ON”Group 2: Gene “Off”
Site-effect of the drug needs attention.
Transgenic lines
Wednesday, July 14, 2010
Sprengel 07/10
Possible confounding influences of the laboratory environment were studied in several inbred strains and one null mutant
by simultaneous testing in three laboratories on a battery of sixbehaviors.
Apparatus, test protocols, and many environmental variables wererigorously equated.
Despite standardization, there were systematic differences in behavior across labs
Wednesday, July 14, 2010
Sprengel 07/10
Reliability of behavioral phenotyping is not challenged seriously by experimenterand laboratory environment as long as appropriate standardizations are met and suitable controls are involved.
Lewejohann et al., Genes, Brain and Behavior (2006) 5: 64–72.
Wednesday, July 14, 2010
Sprengel 07/10
Starting The Experiment
Set up multiple breedings
21 days + two month
het x wthet
wt
Transgenic
Wednesday, July 14, 2010
Sprengel 07/10
Starting The Experiment
Set up multiple breedings
21 days + two month
het x wthet
wt
Transgenic
het x het
KO
het
wt
Gene targeted
Wednesday, July 14, 2010
Sprengel 07/10
The Experiment
Wednesday, July 14, 2010
Sprengel 07/10
The Experiment
Move the mice to the facility where the behavioural experiments should be performed
single housing with toys (enriched environment)without toys
start handling each mouse 10 to 20 min per day
diet down to 85 % body weight
PRE - HANDLING
Wednesday, July 14, 2010
Sprengel 07/10
Mouse handling prior to behavioural testing
Mice were taken out of their cage by the tail and placed on the experimenter's arm wearing a lab coat.
This was done until the mice were used to the experimenter and to the fact that they were taken out of the cage.
Wearing always the same lab coat is helpful for the habituation, because the mouse smell on the lab coat decreases the anxiety.
Before starting to train mice in appetitive motivated tasks (food reward) mice were habituated to condensed milk (diluted in water; 50/50 w/w) first in their home cage, then in an open field. Habituation to the condensed milk in the open field aimed to motivate the mouse to explore the environment for food reward.
Wednesday, July 14, 2010
Sprengel 07/10
Mouse handling prior to behavioural testing
Mice were taken out of their cage by the tail and placed on the experimenter's arm wearing a lab coat.
This was done until the mice were used to the experimenter and to the fact that they were taken out of the cage.
Wearing always the same lab coat is helpful for the habituation, because the mouse smell on the lab coat decreases the anxiety.
Before starting to train mice in appetitive motivated tasks (food reward) mice were habituated to condensed milk (diluted in water; 50/50 w/w) first in their home cage, then in an open field. Habituation to the condensed milk in the open field aimed to motivate the mouse to explore the environment for food reward.
Wednesday, July 14, 2010
Sprengel 07/10
Data from each mouse:
AppearanceNesting scoreTail suspension: neurodegenarationSwimming test Neophobia: latency to drinkOlfactory: latency to find buried foodAccelerating Rota-Rod: latency to fall offHorizontal bar score: latency to fall offOpen field: ActivityLight-dark-box evaluation: Anxiety
General animal handling prior to behavioural testing
Wednesday, July 14, 2010
Sprengel 07/10
Phenotyping
Wednesday, July 14, 2010
Sprengel 07/10
newborns (P0)
Wednesday, July 14, 2010
Sprengel 07/10
newborns (P0)
Wednesday, July 14, 2010
Sprengel 07/10
newborns (P0)
Wednesday, July 14, 2010
Sprengel 07/10
Pubs (P15-P21)
Wednesday, July 14, 2010
Sprengel 07/10
Adults (>P42)
Wednesday, July 14, 2010
Sprengel 07/10
Adults (>P42)
Wednesday, July 14, 2010
Sprengel 07/10
Adults (>P42)
Wednesday, July 14, 2010
Sprengel 07/10
Adults (>P42)
Wednesday, July 14, 2010
Adults (>P42)
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
Cognitive Behaviour
Morris Water Maze
spatial reference memory
Wednesday, July 14, 2010
Sprengel 07/10Water-Maze
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
0
10
20
30
40
50
60
70
0 1 2 3 4 5 6 7 8 9 10 11la
tency
(s)!
blocks of four trials!
GluR-A-/-!
wt!
water-maze
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
T-maze Y-maze evaluation: spatial learning
Puzzel box: cognitive performance
Fear conditioning: emotional learning
Olfactory learning:
Cognitive Behaviour
Wednesday, July 14, 2010
Spatial Working Memory
Sprengel 07/10
Wednesday, July 14, 2010
Spatial Reference Memory
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
T-maze Y-maze
Wednesday, July 14, 2010
Sprengel 07/10
Puzzle Box
Wednesday, July 14, 2010
Puzzle Box
Sprengel 07/10
Wednesday, July 14, 2010
Sprengel 07/10
NeurodegenerationTail suspension:
Wednesday, July 14, 2010
Sprengel 07/10
Emotional Learning
Passive avoidance
Wednesday, July 14, 2010
Sprengel 07/10
Emotional Learning
Passive avoidance
Fearconditioning
Wednesday, July 14, 2010
Sprengel 07/10
Fear conditioning
Wednesday, July 14, 2010
Sprengel 07/10
Aquisition
Context extinction
Wednesday, July 14, 2010
Sprengel 07/10
Olfactory Learning
Wednesday, July 14, 2010
Sprengel 07/10
Olfactory Learning
Wednesday, July 14, 2010
Sprengel 07/10
IntellicageNew Behaviour (Hans Peter Lipp)
www.newbehavior.com
Wednesday, July 14, 2010
Sprengel 07/10
Thank you !!!!
Wednesday, July 14, 2010
Sprengel 07/10
Building trust with your mouse is also absolutely fundamental for any training.
If the mouse feels uneasy about its surrounding and your presence, it will probably just try to escape by running along the wall for want of cover.
Such a frightened rodent won’t cross free and light area .
A stressed mouse can never concentrate
The mouse will be so nervous that it even refuses its favourite treats or other rewards outside the cage and, if ever, learn a lot more slowly.
http://mouse-agility.com
Thank you !!!!
Wednesday, July 14, 2010
Sprengel 07/10http://mouse-agility.comWednesday, July 14, 2010