Mouse models of
neurodegenerative disorders
The importance of vision in
mouse behavior
Richard E. Brown, Aimee Wong, Timothy O’Leary & Rhian Gunn
Psychology Department and Neuroscience Institute
Dalhousie University
Halifax, Nova Scotia, Canada B3H 4J1
What about the mice?
• Mouse strains: inbred, mutant, transgenic
• High throughput analysis
• Ethological/psychological approach
Examples of projects in my laboratory (1999-2005)
1. MHC-congenic mice differ in anxiety and locomotor behaviour but not in learning
and memory. [Behavior Genetics, 1999, 29, 263 – 271] [Behavioral Brain Research, 2003, 144, 187-197]
2. p75-NGF receptor knockout mice show age-related decline in learning and memory.
[Neurobiology of Aging, 2000, 21, 125 – 134]
3. CD-1 mice. Effects of Ritalin on behavioural development.
[Developmental Psychobiology, 2001, 39, 216 – 228] [Developmental Psychobiology, 2002,41, 123-132]
[Pharmacology, Biochemistry & Behavior, 2004, 77, 491-500]
4. CD-1 mice. Effects of Ritalin in the developing striatum.
[Neuroreport, 2004, 15, 1045-1048] [Dev. Brain Research, 2002, 135, 71-77]
5. C57BL/6J mice. Sex differences in effects of diazepam on avoidance learning.
[Pharmacology, Biochemistry and Behavior, 2002, 72, 13 – 21]
6. C57BL/6J vs DBA/2J: differences in anxiety, locomotor behaviour and learning and
memory. [Genes, Brain and Behavior, 2002, 1, 96-110]
7. C57BL/6J vs. DBA/2J: Differences in maternal behaviour.
[Physiology and Behavior, 1999, 67, 599-605] [Physiology and Behavior, 2002, 75, 551-555]
Examples of projects in my laboratory (cont.)
8. C57BL/6J vs DBA/2J: Differences in hypothalamic structure and in parental
behaviour. [Brain Research, 2002, 952, 170 - 175]
9. BALB/c vs. C57BL/6J VS. 129Sv: Differences in olfactory sensitivity and
forebrain neuroblast migration. [Neuroscience, 2003, 118, 263-270]
10. Fmr1-tm1Cgr Fragile X mouse: A phenotypic and molecular characterization.
[Genes, Brain & Behavior, 2004, 3, 337-359]
11. JAX Phenome Project: Visual abilities of 14 strains of mice.
[Genes, Brain & Behavior, 2005, in press]
12. JAX Phenome Project: Strain differences in the MWM, Barnes Maze, Rotarod,
and pain threshold. [in progress]
13. C57BL/6J vs. DBA/2J: Differences in aging, vision, learning and memory.
[in progress]
14. Coloboma mice: Are they a good model for ADHD? [in progress]
15. Effects of mup-75 saporin on brain and behavior. [in progress]
Our Lab
Timothy O’Leary
Richard Brown
Nikki Hoffman
Rhian Gunn
Duyen Ngyuen
Aimee Wong
Deborah Ikede
(not shown)
Jackie Benedict
Andrew Johnston
Jeff McCrossin
Our Mice: JAX Phenome Project
Differences in anxiety, locomotor behaviour and spatial learning in 14
strains of mice.
C57BL/6J
DBA/2J
Molf/Ei
Other: C3H/HeSnJ-Cm
August 2005
John A. King, ed. Biology of Peromyscus
(Rodentia). Special Publication No. 2, The
American Society of Mammalogists, 1968.
Mouse Ethogram
List with descriptions of the postures and movement patterns for adult peromyscus
5. Placing – the foodstuff is dropped
I. The isolated animal
6. Pushing with forepaws – the foodstuff is packed at the site
7. Covering – soil or material is placed over the assembled foodstuffs
A. Sleep and resting
1. Curled – head tucked under its body; this posture with the weight resting on the F. Digging
1. Forepaw movements
hindlegs and head
2. Kick back
2. Stretched – has not tucked its head under the body bu t rests
3. Turn and push
B. Locomotion
G. Nest-building
1. On a plane surface
1. Gathering
2. Climbing
2. Pushing and patting with forepaws
3. Swimming
3. Combing (lateral movements of forelimbs)
C. Care of the body surface and comfort movements
4. Molding – the nest cup is shaped by the turning movements of the animal
1. Washing
H. Exploring and foraging
2. Scratching
1. Elongate posture (stretch attend)
3. Sneezing or coughing
2. Upright
4. Sandbathing
3. Testing the air
5. Stretch
4. Sniffing the substrate
6. Yawn
5. Alarm postures
7. Shake
a. Rigid upright
8. Defecation – may be dropped as animal is moving
b. Freezing (on all fours)
9. Urination – stands still for a moment on all fours
II. Social behavior
10. Scent marking (perineal drag)
A. Initial contact
D. Inge stion
1. Naso-nasal
1. Manipulation (of the food with forepaws)
2. Naso-anal
2. Lapping
3. Mutual grooming
3. Gnawing
B. Contact-promoting and sexual behavior
4. Chewing
1. Circling
5. Swallowing
2. Follow and driving
E. Gathering foodstuffs and caching
3. Male patterns
1. Picking up
a. Mounting
a. With forepaws
b. Thrusting
b. With incisors
c. Intromission
2. Carrying in mouth
d. Ejaculation
3. Dragging with incisors
4. Female patterns
4. Digging
a. Raising the tail
b. Lordosis (elevation of hindquarters while assuming a frozen posture)
5. Post-copulatory washing
C. Approach, flight, attack, and other agonistic patterns
1. Turn toward
2. Approach
a. Slow approach (body contours relaxed)
b. Elongate approach
3. Threat (proper)
4. Rush
5. Chase
6. Flight
7. Move away (not fleeing; an orientated avoidance)
8. Bite
9. Locked fight
10. Modified fight
11. Side display
12. Uprights
13. Submission
14. Defeat
15. Kicking
16. Attack leap
17. Escape leap
18. Tooth-chattering
19. Pattering (with the forefoot)
D. Miscellaneous patterns seen in a social context
1. Sandbathing
2. Marking
3. Pilo-erection
4. Trembling of the body
E. Maternal patterns
1. Parturition crouch
2. Grasping of the neonate with the incisors
3. Manipulating the neonate with the forepaws
4. Patting and pushing the neonate with the forepaws
5. Grooming
6. Retrieving
7. Pulling under (with forepaws)
8. Nursing (brooding) posture
American Psychologist, 1961, 16, 681-684
THE MISBEHAVIOR OF ORGANISMS
KELLER BRELAND AND MARIAN BRELAND
Animal Behavior Enterprises, Hot Springs, Arkansas
In our attempt to extend a behavioristically oriented approach to the engineering
control of animal behavior by operant conditioning techniques, we have fought a
running battle with the seditious notion of instinct. It might be of some interest to
the psychologist to know how the battle is going and to learn something about the
nature of the adversary he is likely to meet if and when he tackles new species in
new learning situations.
Thirty-eight species, totaling over 6,000 individual animals, have been conditioned,
and we have dared to tackle such unlikely subjects as reindeer, cockatoos, raccoons,
porpoises, and whales.
Emboldened by this consistent reinforcement, we have ventured further and further
from the security of the Skinner box. However, in this cavalier extrapolation, we
have run afoul of a persistent pattern of discomforting failures. These failures,
although disconcertingly frequent and seemingly diverse, fall into a very interesting
pattern. They all represent breakdowns of conditioned operant behavior.
The raccoon and the bank
A second instance involves a raccoon. The response
concerned the manipulation of money by the raccoon
The contingency for reinforcement was picking up the
coins and depositing them in a 5-inch metal box.
Raccoons condition readily, have good appetites, and
this one was quite tame and an eager subject. We anticipated no trouble. We started out by reinforcing him for
picking up a single coin. Then the metal container was
introduced, with the requirement that he drop the coin
into the container. Here we ran into the first bit of
difficulty: he seemed to have a great deal of trouble
letting go of the coin. He would rub it up against the
inside of the container, pull it back out, and clutch it firmly for several seconds. However, he
would finally turn it loose and receive his food reinforcement. Then the final contingency: we put
him on a ratio of 2, requiring that he pick up both coins and put them in the container.
Now the raccoon really had problems (and so did we). Not only could he not let go of the
coins, but he spent seconds, even minutes, rubbing them together (in a most miserly fashion), and
dipping them into the container. He carried on this behavior to such an extent that the practical
application we had in mind—a display featuring a raccoon putting money in a piggy bank—
simply was not feasible. The rubbing behavior became worse and worse as time went on, in spite
of nonreinforcement.
Breland & Breland 1961, pg 681-682
How exactly have our mice been
genetically manipulated
and what is the effect of this
manipulation on their brain and
behaviour?
Coloboma Mice: Are they
a good model of ADHD?
Chromosome
2
Coloboma (Cm) mice
•
Deletion of 2 cM segment of
chromosome 2
•
Hemizygous for these genes (50%
reduction in proteins):
–
–
–
–
–
–
JAG-1 (a signal ligand protein)
Plcb4 (phospholipase C ß4)
Cm (an eye protein)
SNAP-25 (a protein involved in
neurotransmitter release)
and 37 others
Cm and C3H mice have retinal
degeneration and are blind.
From: http://www.informatics.jax.org/searches/
linkmap.cgi?chromosome=2&midpoint=78.0&
cmrange=1.0&dsegments=1&syntenics=0
July 4, 2005
Once you have a genetically
modified mouse, how do you
determine its behavioural
deficits?
And what can go wrong along the
way?
Thompson and Kim (1996)
Proc. Natl. Acad. Sci. USA, 93, 13 439.
Genetic lesions?
• What neural pathways are altered in genetically modified mice?
• What memory systems do these alterations effect?
• How do you analyze the effects of genetic lesions?
• Can you use double/triple dissociation tests?
The JAX Test Battery and Beyond
•
•
•
•
•
•
•
•
•
Morris Water Maze
Visual Water Box
Barnes Maze
Open Field
Light-Dark Transition
Test
Rotarod
Olfactory Discrimination
Task
Elevated Plus Maze
Elevated Zero Maze
•
•
•
•
•
•
Set-shifting
Olfactometer
Tail-Flick test
Hot Plate test
Pre-pulse Inhibition
Cued and Contextual Fear
Conditioning
• Tail Suspension Test
• 5-Choice Serial Reaction
Task
• Developmental Test
Battery
Learning and Memory Tests
Morris Water Maze
North
110 cm
West
Visible platform
position
(reversal)
water
Hidden platform
for reversal
3 days Acquisition (4 trials/day)
3 days Reversal (4 trials/day)
1 day Probe (1 trial)
1 day Visible Platform (4 trials)
60 seconds maximum/trial
South
Hidden platform
position for
acquisition
East
Strain Differences in MWM Results
60
Latency to
find
platform
DBA/2J
f)
60
40
30
60
50
40
30
20
20
10
10
MOLF/Ei
j)
50
Latency (sec)
Latency (sec)
50
BALB/cByJ
Latency (sec)
b)
40
30
20
10
0
0
Acq 1 Acq 2 Acq 3
Rev 1 Rev 2 Rev 3
0
Acq 1 Acq 2 Acq 3 Rev 1 Rev 2 Rev 3
Day
g)
Acq 1 Acq 2 Acq 3 Rev 1 Rev 2 Rev 3
Day
k)
Day
c)
1400
Distance (cm)
Distance (cm)
Swim
speed
1200
1000
800
600
1400
1200
1200
1000
1000
Distance (cm)
1400
800
600
400
800
600
400
400
200
200
200
0
0
0
Acq 1 Acq 2 Acq 3 Rev 1 Rev 2 Rev 3
)d Day
Acq 1 Acq 2 Acq 3 Rev 1 Rev 2 Rev 3
Day
)l
)h Day
001
001
001
08
08
08
04
02
06
04
02
02
0
0
0
dauq tcerroc ni emit %
dauq tcerroc ni emit %
dauq tcerroc ni emit %
tnardauq tcerroc ni emit %
04
06
tnardauq tcerroc ni emit %
06
tnardauq tcerroc ni emit %
Probe
quadrant
duration
Acq 1 Acq 2 Acq 3 Rev 1 Rev 2 Rev 3
Can Your Mice See?
• Many authors have suggested that performance in
behavioural tasks may depend on visual ability
Carman et al., 2003, Neurobiol Learn Mem, 78, 332-46.
Robinson et al., 2004, Behav Brain Res, 119, 77-84.
Thifault et al., 2002, Brain Res Bull, 58, 213-18.
Voikar et al., 2001, Physiol Behav, 72, 271-81.
• These warnings have gone unheeded, as many researchers
have used visually impaired mice in visuo-spatial tasks.
• We therefore examined the effects of differences in visual
ability on performance in commonly used behavioural
tests.
• Mice with impaired visual ability may perform poorly on
these cognitive tests, not because of cognitive impairment
but because of poor vision.
Visual Water Box
(Prusky et al., 2000)
Visual Discrimination
Pattern Discrimination
Visual Acuity
Water (S-) Hidden Platform
(S+)
Pretraining (1 day, 12 trials)
Visual Discrimination (8 days, 8 trials/day)
Pattern Discrimination (8 days, 8 trials/day)
Visual Acuity (8 days, 8 trials/day)
Vision Results
20
20
40
20
DBA/2J
0
0
1
2
3
4
5
6
7
1
8
2
3
4
5
6
7
0
8
40
C3H/HeJ
FVB/NJ
Molf/Ei
SJL/J
1
2
3
4
5
6
7
CAST/Ei
SM/J
SPRET/Ei
20
0
8
80
80
80
60
60
60
40
40
129S1/SvImJ
C57BL/6J
DBA/2J
20
2
3
4
5
6
7
C3H/HeJ
FVB/NJ
Molf/Ei
20
2
3
4
5
6
7
3
2
1
8
4
5
7
6
% Correct
60
40
7
8
CAST/Ei
SM/J
SPRET/Ei
1
2
3
4
5
6
7
8
40
20
0.64
0.62
0.55
0.53
0.57
C3H/HeJ
FVB/NJ
Molf/Ei
SJL/J
20
0.43
0.55
6
100
60
0
0.53
0
0.43
0.64
0.62
0.55
0.53
0.43
0.32
DBA/2J
0
8
80
0.32
20
129S1/SvImJ
C57BL/6J
0.32
20
A/J
AKR/J
BALB/cByJ
5
40
80
0.17
40
% Correct
40
0.64
60
0.62
60
0.57
80
4
20
100
80
0
40
0
1
3
60
SJL/J
0
8
2
80
Visual Acuity: percent correct over 8 spatial frequencies
(c/deg)
100
0.17
% Correct
100
1
20
0.17
0
A/J
AKR/J
BALB/cByJ
1
100
% Correct
100
% Correct
100
% Correct
100
0.57
% Correct
Pattern Discrimination: percent correct over 8 days
0
CAST/Ei
SM/J
SPRET/Ei
0.64
129S1/SvImJ
C57BL/6J
A/J
AKR/J
BALB/cByJ
60
0.62
40
40
60
0.57
60
0.55
60
80
0.53
80
0.43
80
0.32
80
100
0.17
100
% Correct
100
% Correct
100
% Correct
% Correct
Visual Discrimination: percent correct over 8 days
How much of the variability in
performance between strains can
be accounted for by differences
in visual ability?
• Correlation
• Regression analysis
Morris Water Maze: Correlation of visual
ability with latency to find platform
Latency to find the platform is influenced by visual ability.
60
129S1
A/J
50
AKR/J
BALB/cByJ
40
30
C3H/HeJ
C57BL/6J
20
CAST/Ei
10
DBA/2J
FVB/NJ
SM/J
SPRET/Ei
Reversal latency (sec)
60
MWM reversal latency (sec)
MOLF/Ei
SJL/J
0
50
40
30
20
10
0
40
50
60
70
80
VD day 8 (% correct)
90
100
r = -.775, p < .01, R2 = .601
Morris Water Maze: Correlation of
visual ability with swim distance
Swim distance is influenced by visual ability.
129S1
A/J
1600
AKR/J
BALB/cByJ
1200
1400
1000
800
C3H/HeJ
C57BL/6J
600
400
CAST/Ei
200
DBA/2J
FVB/NJ
SM/J
SPRET/Ei
Reversal Distance (cm)
1400
MWM reversal distance (cm)
MOLF/Ei
SJL/J
0
1300
1200
1100
1000
900
800
700
600
500
400
40
50
60
70
80
VD day 8 (% correct)
90
100
r = -.718, p < .01 , R2 = .516
Morris Water Maze: Correlation of visual ability
with time spent in correct quadrant
Time spent in the correct quadrant the Morris Water Maze is
not influenced by visual ability
70
129S1
A/J
60
50
AKR/J
BALB/cByJ
40
C3H/HeJ
C57BL/6J
30
CAST/Ei
10
20
DBA/2J
FVB/NJ
SM/J
SPRET/Ei
% time in correct quadrant
100
% time in the correct quadrant
MOLF/Ei
SJL/J
0
80
60
40
20
0
40
50
60
70
80
VD day 8 (% correct)
r = .347, ns
90
100
What about sex differences?
35
C57
30
25
20
15
10
Acq 1
30
25
20
15
10
5
Rev 3
Rev 2
Rev 1
Acq 3
Acq 2
Acq 1
5
DBA
Day
Day
No sex differences: F(1,10) < 1.0
Sex by day interaction: F(5,50) = 3.11, p<0.05
60
Female
55
Male
50
45
129
35
30
25
20
55
Female
52.5
Latency to Platform (s)
Male
50
47.5
C3H
45
42.5
40
37.5
35
32.5
15
No sex differences: F(1,22) < 1.0
No sex by day interaction: F(5,110) < 1.0
Rev 3
Rev 2
Rev 1
Acq 3
Rev 3
Rev 2
Rev 1
Acq 3
Acq 2
Acq 1
Day
Acq 2
30
10
Acq 1
Latency to Platform (s)
No sex differences: F(1,22) < 1.0
No sex by day interaction: F(5,110) = 1.18, p = 0.32
40
Rev 3
40
Male
Rev 2
Male
Rev 1
45
Female
60
55
50
45
40
35
Acq 3
Female
Acq 2
50
Latency to Platform (s)
Latency to Platform (s)
Latency to platform in Morris Water Maze
Day
No sex differences: F(1,17) < 1.0
Sex by day interaction: F(5,85) = 2.51, p<0.05
100
What about age differences?
40
95
35
90
85
B6.mpc1d
DBA/2J
Latency (sec)
D2.mpc1b
75
%
30
C57BL/6J
80
B6.mpc1d
C57BL/6J
25
D2.mpc1b
DBA/2J
70
20
65
60
15
55
10
50
age 1
6 mos.
age 2
12 mos.
age 1
age 3
18 mos.
6 mos.
Visual Discrimination in the
Vision Task
age 2
12 mos.
age 3
18 mos.
Latency to find platform in
the Morris Water Maze
800
60
700
50
Distance (cm)
500
B6.mpc1d
C57BL/6J
400
D2.mpc1b
DBA/2J
300
200
% of time in correct quadrant
600
40
B6.mpc1d
C57BL/6J
30
D2.mpc1b
DBA/2J
20
10
100
0
age 1
6 mos.
age 2
12 mos.
age 3
18 mos.
Swim distance in the MWM
0
age 1
6 mos.
age 2
12 mos.
age 3
18 mos.
% time in correct quadrant in the MWM
• D2 mice perform better than other strains on the
vision test and Morris Water Maze at 4 months of
age.
• D2 mice develop glaucoma after 9 months of age.
• As D2 mice age, their performance in the vision
test and MWM declines, but performance of B6
mice improves.
• Visual ability may account for:
– 45% of the variability in latency to reach the
platform in the MWM at 12 months of age
– 60% of the variability at 18 months of age
Learning and Memory:
Barnes Maze (Pompl version)
Escape box
Acquisition
68 cm
Reversal
1 day habituation
4 days acquisition (4 trials)
4 days reversal (4 trials)
1 day probe (1 trial)
5 minutes maximum/trial
Pompl PN et al., Adaptation of the circular
platform spatial memory task for mice: use
in detecting cognitive impairment in APP(SW)
transgenic mouse model for Alzheimer’s
disease. J Neurosci Methods, 1999; 87, 87-95.
Barnes
Maze
Results:
sighted
vs.
blind
strains
Errors
40
40
35
40
35
AKR/J
C57BL/6J
30
30
25
20
15
15
10
10
15
10
5
0
0
Acq 1
Acq 1
Acq 2
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
300
Acq 2
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
Acq 1
Acq 2
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
Rev 4
300
300
C3H/HeJ
FVB/NJ
A/J
129S1/SvImJ
250
C57BL/6J
250
0
Rev 4
Rev 4
Latency to Escape Box
AKR/J
Molf/Ei
250
SJL/J
Balb/cByJ
DBA/2J
Balb/cJ
200
200
150
Latency (sec)
Latency (sec)
200
150
100
50
50
Acq 1
Acq 2
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
Acq 1
Rev 4
Acq 2
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
0
Rev 4
Acq 1
Time in Correct Quadrant (Probe Day)
% Time in Correct Quadrant
35
30
25
20
15
10
40
40
35
35
% Time in Correct Quadrant
40
50
0
0
30
25
20
15
10
5
C57BL/6J
DBA/2J
Acq 3
Acq 4
Rev 1
Rev 2
Rev 3
30
25
20
15
5
0
129S1
Acq 2
10
5
0
150
100
100
% Time in Correct Quadrant
20
5
5
SJL/J
30
Errors
Errors
Errors
Molf/Ei
Balb/cJ
25
20
FVB/NJ
35
Balb/cByJ
DBA/2J
25
Latency (sec)
C3H/HeJ
A/J
129S1/SvImJ
0
A/J
AKR/J
Balb/cByJ
Balb/cJ
C3H/HEJ
FVB/NJ
Molf/Ei
SJL/J
Rev 4
Barnes Maze: Correlation of visual
ability with errors & latency
Number of errors, latency during reversal and % time in correct quadrant
are not dependent on visual ability.
250
200
175
Probe Day:
150
125
100
50
75
50
25
0
40
50
60
70
80
90
100
% Correct Visual Discimination Day 8
r= .232, ns
16
Mean Reversal Errors
14
12
10
% Time in Correct Quadrant
Mean Reversal Latency
225
40
30
20
10
0
8
40
6
4
50
60
70
80
90
% Correct Visual Discimination Day 8
2
0
40
50
60
70
80
90
% Correct Visual Discimination Day 8
r= -.409, ns
100
r=.154, ns
100
Barnes Maze - what strategies did mice use?
Small: Cued Training
Proportion of search strategy used
.7
.5
.3
.1
-.1
.9
.9
.7
.5
.3
.1
A2
R1
R2
A1
A2
Trial Block
.3
.1
A1
R2
.5
.3
.1
-.1
R1
R2
.7
.5
.3
.1
*Each block contained 8 trials
R2
Divided: No Cue Training
.7
.5
.3
.1
-.1
-.1
Trial Block
R1
.9
P roportion of search strategy used
.7
A2
Trial Block
Pompl: No Cue Training
P roportion of search strategy used
P roportion of search strategy used
R1
.9
A2
.5
Trial Block
Small: No Cue Training
A1
.7
-.1
-.1
A1
.9
Divided: Cued Training
Proportion of search strategy used
Random
Random
S
erial
Serial
S
patial
Spatial
.9
P roportion of search strategy used
Pompl: Cued Training
A1
A2
Trial Block
R1
R2
A1
A2
R1
Trial Block
R2
Correlation of visual ability with
frequency of center square entries
Anxiety: Open Field
Frequency of center square entries is influenced by visual ability.
9
8
7
129S1
A/J
6
5
AKR/J
BALB/cByJ
4
3
2
C3H/HeJ
C57BL/6J
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
1
0
Frequency of center square entries
CAST/Ei
MOLF/Ei
SJL/J
SM/J
SPRET/Ei
9
OFM centre square frequency
DBA/2J
FVB/NJ
7
5
2 days
1 trial/ day
5 minutes/trial
3
1
-1
40
50
60
70
80
VD day 8 (% correct)
90
r = -.696, p < .01 , R2 = .484
100
Correlation of visual ability with proportion
of time spent in light zone
Anxiety: Light-Dark Transition Test
Proportion of time spent in light zone is not influenced by visual ability.
.7
.6
129S1
A/J
AKR/J
BALB/cByJ
C3H/HeJ
C57BL/6J
.5
.4
.3
.2
.1
0
.7
CAST/Ei
MOLF/Ei
SJL/J
SM/J
SPRET/Ei
Proportion of time spent in light zone
.6
proportion time in light zone
DBA/2J
FVB/NJ
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
.5
.4
1 day
5 minutes
.3
.2
.1
0
40
50
60
70
80
VD day 8 (% correct)
r = .087, ns
90
100
Correlation of visual ability with
latency to fall
Locomotor Test: Rotarod
Motor learning is not affected by visual ability.
400
350
129S1
300
A/J
250
AKR/J
200
Balb/cByJ
150
C3H/HeJ
100
50
C57BL/6J
0
400
FVB/NJ
Molf/Ei
SJL/J
SM/J
Latency to fall on day 7 (sec)
DBA/2J
Latency to fall on day 7 (sec)
350
300
6 trials/day
7 days
48 rpm/min over 6 minutes
250
200
150
100
50
40
50
60
70
80
VD day 8 (% correct)
r = -.054, ns
90
100
Learning and Memory:
Olfactory discrimination task
Training
sugar
13 cm
odour pot
Prochip
30 cm
19 cm
Testing
Rose
compartment
Middle
compartment
Lemon
compartment
20 cm
4 days Training (4 trials/day)
10 minutes/trial
1 day Testing (3 minutes)
20 cm
opening
Rose
odour pot
opening
Prochip
69 cm
Lemon
odour pot
Olfactory Discrimination: Correlation of
visual ability with % digging in CS+
Mice with poor visual ability perform better at odour discrimination learning task.
100
80
60
40
20
0
% time digging in CS+
100
80
% digging CS+
129S1
A/J
AKR/J
Balb/cByJ
C3H/HeJ
C57BL/6J
DBA/2J
FVB/NJ
MOLF/Ei
SJL/J
SM/J
60
40
20
0
40
50
60
70
80
VD day 8 (% correct)
90
r = -.621, p < .05, R2 = .386
100
Contextual and Cued Fear
Conditioning
Visual ability influences context conditioning
in elderly (24 mo. old) mice.
No strain differences in cued conditioning -but all are deaf!
F(3,15) = 7.58, p < 0.01
F(3,15) < 1.0
What about hearing?
55
275
250
Barnes mean acq latency
MWM latency
50
45
40
35
30
25
225
200
175
150
125
100
75
50
20
25
20
30
40
50
60
70
80
ABR threshold Clicks
90
100 110
20
34
24
32
22
30
20
28
26
24
22
20
14
6
r = 0.261, R2 = 0.068
90
100 110
12
8
50
60
70
80
ABR threshold Clicks
100 110
14
16
40
90
16
10
30
50
60
70
80
ABR threshold Clicks
18
18
20
40
r = 0.389, R2 = 0.151
Barnes acq errors
MWM velocity
r = -0.421, R2 = 0.152
30
90
100 110
20
30
40
50
60
70
80
ABR threshold Clicks
r = -0.061, R2 = 0.004
What about hearing (2)?
275
400
250
225
300
OFM line cross
RR day 7 latency
350
250
200
150
200
175
150
125
100
75
100
50
50
25
30
40
50
60
70
80
ABR threshold Clicks
90
100
20
110
.7
8
.65
7
.6
.55
.5
.45
.4
.35
.3
r = -0.262, R2 = 0.069
90
100 110
3
2
0
50
60
70
80
ABR threshold Clicks
100 110
4
.2
40
90
5
1
30
50
60
70
80
ABR threshold Clicks
6
.25
20
40
r = -0.509, R2 = 0.259
OFM centre sq freq
EPM prop on open
r = -0.348, R2 = 0.121
30
90
100 110
20
30
40
50
60
70
80
ABR threshold Clicks
r = -460, R2 = 0.212
What about body weight?
Rotarod
Mean Latency to Fall (s)
180
160
140
120
100
Female
Male
80
60
1
2
3
4
Day
5
6
7
F(1,318) = 5.005, p< 0.05
Female
Day 7 Mean Latency (s)
400
Male
F(1,320) = 61.214, p< 0.0001
350
300
250
200
150
100
Female: r = -0.403, p< 0.01, R2 = 0.162
Male: r = -0.240, p<0.01, R2 = 0.058
50
0
10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35
Day 7 Weight (g)
What about the environment?
•
•
•
•
physical housing
social housing
test procedure
experimenters
Improving housing conditions for laboratory mice: a
review of 'environmental enrichment'
Olsson I.A. & Dahlborn K. 2002. Laboratory Animals, 243-270
We have reviewed 40 studies carried out between 1987 and 2000, in
which preferences as well as the effect of housing modifications have
been studied.
Mice will work for access to nesting material and make use of this
material to make nests in which they rest.
They prefer a more complex cage to the standard cage and will also
work for access to cages with shelter and raised platforms.
On the basis of present knowledge, it is recommended that mice
should have access to nesting material. Strategies for future research
are outlined in the article.
The effects of methylphenidate are less important than social/physical
environment on developing C57BL/6J mice.
Open Field: Distance Traveled
MWM: Latency to Platform
Saline
55
50
MPH
45
40
35
30
25
MPH
2500
Distance (cm)
Latency to Platform (s)
3000
Saline
2000
1500
1000
20
15
500
0
Rev 3
Rev 2
Rev 1
Acq 3
Acq 2
Acq 1
10
Day 1
Day
F(1,78) < 1.0
F(1,78) < 1.0
55
Enrichment
3000
50
Enrichment
45
Isolation
35
30
25
Isolation
2500
Distance (cm)
40
2000
1500
1000
20
500
15
Day
F(1,78) = 4.44, p < 0.05
Rev 3
Rev 2
Rev 1
Acq 3
Acq 2
10
Acq 1
Latency to Platform (s)
Day 2
0
Day 1
Day 2
F(1,78) = 4.94, p < 0.03
J. Neurobiol. 54, 2003,
283-311.
Sources of “error” in mouse
behavior genetics experiments
1. Mice
•
•
Sensory system bias - vision, hearing deficits
Mouse misbehaviour [Breland & Breland 1961]
2. Environment
•
•
Physical environment (Enrichment)
Social environment
3. Experimenter error
4. Equipment error
Sensory System Bias
Strain
Type
JAX Number
Hearing
Vision
129S1/SvImJ
IN
JAX 002448
Normal
Normal
A/J
IN
JAX 000646
Deaf before 3 months
Albino
AKR/J
IN
JAX 000648
Normal
Albino*
BALB/cByJ
IN
JAX 001026
Deaf after 16 months
Albino
BALB/cJ
IN
JAX 000651
Normal
Albino
C3H/HeJ
IN
JAX 000659
Normal
Pde66rd1
C57BL/6J
IN
JAX 000664
Deaf after 16 months
Normal
CAST/EiJ
WD
JAX 000928
Normal
Unknown
DBA/2J
IN
JAX 000671
Deaf before 3 months
Glaucoma after 9
months
FVB/NJ
IN
JAX 001800
Normal
Pde66rd1
MOLF/EiJ
WD
JAX 000550
Normal
Pde66rd1
SJL/J
IN
JAX 000686
Normal
Pde66rd1
SM/J
IN
JAX 000687
Normal
Unknown
SPRET/EiJ
WD
JAX 001146
Normal
Unknown
Mouse Misbehavior
• Wild derived mice - often can not be tested on Rotarod,
Elevated Plus Maze, etc.
• Molf
• Spret
• Cast
• Mutant mice
• Coloboma - walk backwards
• Peromyscus californicus - misbehave
QuickTime™ and a decompressor are needed to see this picture.
QuickTime™ and a YUV420 codec decompressor are needed to see this picture.
Experimenter Error
Error of
Apprehending
Types of observer effects encountered
in ethological research.
From: Handbook of Ethological
Methods by Philip N. Lehner, 1979.
Garland: New York.
Observer Error
Observer Bias
Observer
Effect
Error of Recording
Computational Error
Results
Error of apprehending - the position of the animal makes it difficult to observe the behavior.
Observer effect - the presence of the observer results in a change in the animal’s behavior.
Observer error - inexperience or poorly defined behavioral units.
Observer bias - the expectancies of the observer.
Error of recording - poor techniques and equipment, mental lapses in the observer and inexperience.
Computational error - errors in data transcription or inappropriate statistical tests.
Experimenter Error
• Not following the research protocol
• Intra-observer reliability
– Can the same observer obtain similar data again?
• Inter-observer reliability
– Can other observers replicate our measurements?
• Requires stability and accuracy in measurement.
• Time-sampling reliability
• Event-sampling reliability
Solutions? Lab protocols; trained observers
Videotape everything; reanalyze videotapes
Equipment Error
•
•
•
•
Solder joints broken (hardware)
Tracking program (software) errors
Baseline calculation for freezing behaviour
Computer glitches in programs running
equipment
• Computer glitches in data collection
(compounded by experimenter error)
Freeze threshold was 1.0
This shows an accurate record of
movements within the maze.
Freeze threshold was 1.0
Some tracking errors can be
detected easily by reviewing
the final movement
paths of mice.
Due to a light reflection, the
tracking system shows animal
movements that are outside of
the maze.
When zone regions are not
aligned properly in the elevatedplus maze, mice can move from
the open to the closed arms
without the tracking system
recording an entry into the
center zone.
The open field has lines drawn on
its floor in a 4 x 4 grid to measure
locomotion.
In the Limelight Software a grid is
arranged to match that of the open
field.
A line cross was recorded when
tracking errors occurred, where
the mouse was recorded as having
moved over the line indicating the
outer edge of the maze floor.
Freeze threshold was 1.2
Equipment Error
All pictures and thresholds were taken
with the plexiglass door of the sound
attenuation chamber down.
Freezethreshold
thresholdwas
was1.0
1.0
Freeze
Variations in the freeze threshold are
most likely due to differences in
illumination within the training
chamber.
The above thresholds are set so the
percent freezing is 100%, as the
inanimate object did not move during
testing.
How can you detect and correct
equipment error?
•
•
•
•
Don’t believe the manufacturers
Check all hardware and software
Trained users
Pilot studies
• Videotape and analyze by hand
What have we learned about
testing
mouse models of
neurodegenerative diseases?
Think like Sherlock Holmes
“This is indeed a mystery,” I remarked. “What do you imagine
that means?”
“I have no data yet. It is a capital mistake to theorize before one
has data. Insensibly one begins to twist facts to suit theories,
instead of theories to suit facts.”
(A Scandal in Bohemia)
“It seems most improbable.”
“We must fall back upon the old axiom that when all other contingencies
fail, whatever remains, however improbable, must be the truth.”
(The Adventure of the Bruce Partington Plans)
“You know my method. It is founded upon the observation of trifles.”
(The Boscom Valley Mystery)
“It is, of course, a trifle, but there is nothing so important as trifles.”
(The Man With the Twisted Lip)
Problems we have had with testing
mouse models of neurodegenerative
disease
• Are mice what they are supposed to be? (Fragile X, Coloboma)
• Is the Pompl version of the Barnes Maze a test of spatial learning?
• Are differences in learning and memory performance in the Morris
Water Maze and other tests confounded by vision?
• What other confounds are there in learning and memory tests?
–
–
–
–
–
Body weight
Hearing
Anxiety
Experimenter error
Equipment error
• Are results confounded by background strain, sex or age?
• When you buy a drug, does it do what the manufacturers claim it
does? (Mup75 saporin)
Conclusions
How do we improve the reliability and validity of
mouse models of neurodegenerative disorders?
1. Know thy mouse.
2. Understand gene - environment interactions.
3. Use a test battery approach.
4. Videotape experimenters to check accuracy.
5. Calculate equipment error.
The End
International Ethological Congress 2007
Dalhousie University
Halifax, Nova Scotia, Canada