confounding variables in the behavioural phenotyping … · behaviour genetics and the central...

Confounding variables in the

behavioural phenotyping of genetically

modified mice

Richard E. Brown

Psychology Department, Dalhousie University

Halifax, Nova Scotia

B3H 4J1 Canada

[email protected]

AbstractInbred, mutant and transgenic mice are used as animal models for a multitude of human disorders such as Fragile X syndrome, Alzheimer’s disease, ADHD, Parkinson’s disease, Friedreich’s Ataxia and many others. There has not, however, been a systematic comparative approach to the study of transgenic mice. Our lab has, therefore, undertaken to compare the behavioural phenotypes of a number of strains of inbred mice and mouse models of neural disorders, including Fragile X disease and Alzheimer’s disease. Our aim is to determine how genetic differences between transgenic mice affect commonly used measures of anxiety, locomotion, learning and memory and how the behaviours of different mouse models of Alzheimer’s disease change with age. This presentation examines the problem of determining the behavioural deficits in mouse models, outlines the types of errors that can be made and discusses the ways to correct these errors in order to conduct reliable and valid studies of behaviour. Some issues that will be raised are the problems of testing aging mice, the presence of background strain differences, the interaction between sensory, motor and cognitive deficits in mouse behaviour, the effects of apparatus design and procedural differences on behaviour, and the problem of experimenter error. The take home message is that the analysis of behaviour has much in common with analytical chemistry: you need to have a lot of controls and you cannot skip any steps in the protocol.

Our Lab

Dr. Richard Brown

Research Assistants:

Rhian Gunn

Undergrads:

Ahmed Hussin

Caitlin Blaney

Hector Mantolino

Anthony Diab

Grad Students:

Tim O’Leary

Leanne Fraser

Kurt Stover

Kyle Roddick

What is a Neurodegenerative Disease?

1. A type of neurological disorder marked by the loss of specific nerve cells.

2. Incurable disease caused by gradual loss of the neurons, often leading to death.

3. A disorder caused by the deterioration of certain nerve cells (neurons). Changes in the these cells cause them to function abnormally, eventually bringing about their death.

4. Hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction.

Examples of Neurodegenerative Diseases

• Alzheimer’s Disease

• Parkinson’s Disease

• Creutzfeldt-Jakob

Disease

• Multiple Sclerosis

• Lewy Body Disease

• Amyloid Lateral

Sclerosis

• Prion Disease

• Schizophrenia

• Glaucoma

Inbred, mutant, knockout &

transgenic mice - definitionsInbred mice: Mice derived from a single ancestral pair and

mated brother to sister for 20 generations or more are called

inbred strains of mice.

Mutant mice: A mouse with a gene mutation that confers a

phenotypically identifiable difference from the “wild-type”

genotype.

Knockout mice: Mice that have a target gene silenced or no

longer expressed.

Transgenic mice: Mice that have had new genes introduced

into their germ line.

Behaviour Genetics and The Central Dogma

Genes Brain Behaviour

Altered

Genes

Altered

Brain

Altered

Behaviour

BUT

What about environmental influences?

Brain

Genes Behaviour

Environment

Genes x Environment Brain Behaviour

?

??

How should testing proceed?

• Screen prospective mice on a battery of tests

over age

• Longitudinal and cross-sectional studies

• Only when the behavioural deficits have been

quantified and replicated search for neural

correlates

• Search for drugs or other treatments to

reverse the neural and behavioural deficits

A theoretical age-related disease curve in mice

The expected effects of treatment on age-related

diseases

Glaucoma as a

neurodegenerative disease

• causes irreversible vision loss

• characterized by progressive retinal ganglion cell death, neural atrophy and axon degeneration

• caused by elevated eye pressure (elevated intraocular pressure) and mechanisms independent of IOP

• cell death via trans-synaptic degeneration

[Gupta, N. & Yucel, Y.H. (2007). Curr Opin Opthalmol, 18:110-4.]

The DBA/2J mouse model of glaucoma

• Age-related intra-ocular pressure

• Age-related retinal ganglion cell

degeneration

• Age-related loss of visual function

• Due to mutations of GpnmbR150X and

Tyrp1b(Anderson et al., 2001, Nat Genet, 30, 81-5)

Visual Water Box(Prusky et al., 2000)

Pattern Discrimination

Visual 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)

Wong & Brown, 2006

Genes Brain Behav, 5: 389-403.

Wong & Brown,

2007, Neurobiol

Aging, 28:

1577-93

Age-related visual loss in DBA/2J mice but not in C57BL/6J mice

How does visual loss affect

cognitive function?

Learning and Memory Tests

u

110

S h

water

Hiddenplatformfor reversal

cm

North

West

o t

East

Visible platformposition(reversal)

Hiddenplatformposition foracquisition

Morris Water Maze

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

Age-related failure of visuo-spatial learning and memory in

DBA/2J but not C57BL/6J mice

Wong & Brown,

2007, Neurobiol

Aging, 28:

1577-93

Age-related vision loss can be

delayed with Timoptic-XE

Visual detection task Pattern discrimination task Visual acuity task

40

50

60

70

80

90

100

% C

orr

ect

1 2 3 4 5 6 7 8

Day

0.00%

0.25%

0.50%

30

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50

60

70

80

90

100

% C

orr

ect

1 2 3 4 5 6 7 8

Day

0.00%

0.25%

0.50%

12 month old mice receiving 0% Timoptic-XE performed significantly worse (P<.05) than

mice receiving 0.5% or 0.25% Timoptic-XE in the visual detection, pattern discrimination and

visual acuity task.

(Wong, 2010 PhD Thesis)

0

10

20

30

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60

70

80

90

100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

% C

orr

ect

Spatial frequency (c/deg)

Age-related decline in Morris Water

Maze performance can be

prevented with Timoptic-XE

0

10

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ten

cy (

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c)

Acq

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v 3

0.00%

0.25%

0.50%

Day

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900

Dis

tan

ce

(c

m)

Acq

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v 1

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v 2

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v 3

0.00%

0.25%

0.50%

Day

0

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40

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80

100

% T

ime

Correct Opposi te Left Right

Cell

0.00%

0.25%

0.50%

12 month old mice receiving 0% Timoptic-XE had a significantly longer latency and swim

distance to find the platform (P<.05) than mice receiving 0.5% or 0.25% Timoptic-XE. There

were no significant differences between drug groups in the % time spent in the correct

quadrant in the probe trial.

(Wong, 2010 PhD Thesis)

Therefore the DBA/2J mouse

model of glaucoma shows1. Age-related decline of retinal ganglion cells.

2. Decline of visual function.

3. Treatment with Timoptic-XE delays decline of visual function

4. No cognitive deficits when vision is normal.

The aging DBA/2J mouse has visual, not cognitive deficits.

These effects can be dissociated using behavioural analysis.

What about mouse models of

Alzheimer’s Disease?

There are many different transgenic mouse models of Alzheimer’s

Disease. JAX (Bar Harbor, Maine) list over 60 different AD

models. Each model is created by different combinations of

genetic manipulations.

The hypothesis is that:

Alzheimer’s gene ->

Alzheimer’s-like neural degeneration ->

Age-related cognitive dysfunction

A theoretical age-related disease curve in mice

The expected effects of treatment on age-related

diseases

Questions about mouse models of

Alzheimer’s Disease1. Do mouse models of Alzheimer’s Disease show an

age-related decline in cognitive function which differs significantly from control strains?

2. Can you distinguish Alzheimer’s mice from mouse models of other neurodegenerative disorders?

3. Are deficits in test performance due to sensory or cognitive deficits?

4. Do treatments for Alzheimer’s Disease delay this age-related decline in cognitive function?

What have we learned after testing mice for 10 years?

1. What mice to test? Background Strains & Sensory

System BiasStrain 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

Alzheimer’s mice in th e Brown Lab.

Strain JAX # Express Background

Single Transgenic

*

B6.Cg-Tg(PDGFB-APP)5Lms/J

“I5” or “AP PInd”

004662 Human APP wit h the Indiana

mutation (V717 F)

C57BL/6J x DBA/2J

F1 cross as controls

Single Transgenic

*

B6.Cg-Tg(PDGFB-APPSwInd)20Lms/J

now

B6.Cg-Tg(PDGFB-APPSwInd)20Lms/2J

“J20” or “APPSwInd”

004661

006293

Human APP wit h both

Indiana (V717F ) and Swedish

mutation (K670N/M671L)

C57BL/6J x DBA/2J


Also #4662 as control

Double Transgenic

*

B6C3-Tg(APP695)3DboTg(PSEN1)

5Dbo/J

“3D5D” or “APP+PS1”

003378 Chimeric human/mouse APP

with Swedish mutation

(K679N/M671L = APP695)

and mutant human presenilin

1 with A246 E mutation

C57BL/6 x C3H/He

B6C3F1/J as controls

Double transgenic

**

B6C3-Tg(APPSwe,PSEN1dE9)85Dbo/J

“D85” or “APP+PS1dE9”

004462 Chimeric human/mouse APP

with Swedish mutation

(K670N/M671L) and mutant

human presenilin 1 (dE9)

C57BL/6J x C3H

B6C3F1/J as controls

Double transgenic

**

B6SJL-Tg(APPSwFlLon,

PSEN1(M146L;L286V) 6799Vas/J

“5xFAD”

006554 Human APP wit h Swedish

(K670N,M671L), Florida

(I716V) and London (V717I)

mutations, PS 1 with M146L

and L286V mutations

C57BL/6J x SJL/J


Triple transgenic

**

B6;129-Psen1tm1MpmTg(APPSwe,

tauP301L)1Lfa/J

“3xTg -AD”

004807 Human APP with Swedish

mutation (APP69 5), human

Tau mutatio n (P301L),

chimeric human/mouse

presenilin -1 (M146V)

C57BL/6 x 129S1


Alzheimer’s mice in the Brown lab

* = used to have

** = currently have

2. How should mice be housed?

Effects of social isolation on neural-

behavioural development

(“isolation syndrome”)

• Retarded growth

• Elevated CORT levels

• Hyperactivity in a novel environment

• Increased anxiety

• Reduced ability to shift attention

– Impaired reversal learning

• Impaired spatial learning

• Neophobia for novel environments

(Hellemans, K.G.C. et al., 2004. Dev Brain Res 150, 103-115.)

Effects of environmental enrichment on the brain(Mattson et al., 2001, Mech Ageing Dev, 122, 757-78; Lewis,

2004, Ment Retard Dev Disabil Res Rev, 10, 91-5; Mohammed

et al., 2002, Prog Brain Res, 138, 109-33; Spires & Hannan,

2005, FEBS J, 272, 2347-61.)

Increased weigh t of cerebral cortex

Increased ChE activi ty

Increased complexity of dendriti c arbors

Increased number of synapses in hippocampus and cerebellum

Prevents age-related loss of synapses in the hippoc ampus

Facilit ates neurogenesis

Promotes recovery from neural injury

Improves recovery from shock

Reduced Aß levels and amyloid deposits

Increased BDNF and NGF in hippo campus

Increased gene expression

Kolb et al., 1998. Neurosci Biobehav Rev, 23, 143-59.

Separation of group-housed mice leads to “depressive-like”

behaviour.

3. What tests to use?

The test battery approachLEARNING & MEMORY

• Morris Water Maze

• Barnes Maze

• Rotarod

• Cued and Contextual Fear Conditioning

• Olfactometer

• Radial Arm Maze

• Conditioned Taste Aversion

• Novel Object Recognition

EMOTIONALITY

• Elevated Plus-Maze

• Open Field

• Light/Dark Transition Box

• Tail Suspension Test

• Forced Swim Test

DEVELOPMENTAL TEST BATTERY

SPECIES-TYPICAL BEHAVIOURS

• Social Transmission of Food Preference

• Social recognition/preference

• Nest building

• Hoarding

SENSORY SYTEMS

• Visual Water Box

• Conditioned Odour Preference

• Prepulse Inhibition/Auditory Startle

• Tail Flick Test

• Hot Plate Test

MOTOR COORDINATION

• Rotarod

• Balance Beam

• Paw Slip Test

ATTENTION

• 5-choice Serial Reaction Time Task

I5 (APPInd) J20

(APPSwInd)

3D5D (APP +

PS1)

D85 (APP +

PS1dE9)

5xFAD

EPM X X

Light/dark box X X X

Open Field X X X X X

FST & TST X X

Rotarod X X X X

Vision Task X X

Odor Task X X X X

PPI X X

MWM X X X X X

Fear

Conditioning

X X X X

STFP X X X

Nesting X X X

Tests completed, October 2010

4. Methodological issues

Longitudinal vs. Cross-sectional studies

We decided to do both

Learning & memory vs. performance

% correct

Latency

Learning strategy

Because performance can be affected by sensory &

motor as well as cognitive deficits, we used both latency

(performance) and accuracy (memory) scores.

What ages to test?

How many tests per mouse

test battery?

3, 6, 9, 12, 15 months of age

4, 8, 12 months of age

4, 8, 12, 16, 20, 24 months of age

6, 12, 18, 24 months of age

3? 6? 10? 12?

We usually opt for 8-12 tests in an increasing order of severity.

5. What about body weight?

24

26

28

30

32

34

36

38

40

42

44

46

Bo

dy

We

ight

(g

)

4 8 12 16

Age (months)

wt 2

APP+PS1dE9

wt 1

APP+PS1

APP+PS1 & APP+PS1dE9 mice

(longitudinal)

Genotype: NS

Age: p<.0001

Age x Genotype: p<.0001

Mice can range from 20 - 50 grams. Females weigh less

than males. Body weight affects behavior in some tests.

5xFAD mice

(cross-sectional)

Genotype: p = .076

Age: p<.0001

Age x Genotype: NS

0

5

10

15

20

25

30

35

Body

wei

ght (g

)

3m 6m 9m 12m

Age (months)

5xFAD

wildtype

6. What about visual deficits?

• 69 cm diameter

• 16 holes

• 15 cm high wall

• Intra-maze cues

• 69 cm diameter

• 16 holes•122 cm diameter

•16 holes

Pompl maze Small maze Large maze

0

2

4

6

8

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12

14

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18

Err

ors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 RT1 RT2

Day

Large

Small

Pompl

0

10

20

30

40

50

60

Tim

e (

sec)

ZC Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11 Z12 Z13 Z14 Z15

Zone

Large

Small

Pompl

Day

Err

ors

Zone

7. Which apparatus design to use? The Barnes Maze

8. What about the L:D cycle?

What is the light cycle in the housing room?We use a reversed 12:12h cycle, with the lights off from

9:30am to 9:30pm.

When in the light cycle should mice be tested?We test in the dark phase (usually from 10am - 6 pm).

9. What about experimenter errors?Error of

ApprehendingObserver 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.

Types of observer effects encountered

in ethological research.

From: Handbook of Ethological

Methods by Philip N. Lehner, 1979.

Garland: New York.

What cognitive (and other) deficits

have we found in Alzheimer’s mice?

- while controlling for all known confounds?

Multiple Memory Systems Approach

Thompson and Kim (1996)

Proc. Natl. Acad. Sci. USA, 93, 13 439.

Alzheimer’s Mouse Learning &

Memory ResultsSome examples

1. MWM

2. Barnes maze

3. Rotarod

4. Conditioned odor preference

5. Social transmission of food preference (STFP)

6. Cued & contextual fear conditioning

• Littermate controls

• Both males and females tested

• Age ranges from 3 - 24 months

MWM - APPswe/PS1dE9 mice

0

10

20

30

40

50

60

Lat

ency

to p

latfo

rm (s)

Acq

1

Acq

2

Acq

3

Re

v 1

Re

v 2

Re

v 3

wt

APPSwe/PS1dE9

At 12 months of age, APPSwe/PS1dE9 mice

took longer to reach the platform during

acquisition than wildtype mice (F(1,30) = 5.98,

p<.05). There was no effect of genotype

during reversal training.

% T

ime s

pent in

corr

ect quadra

nt (p

robe)

0

5

10

15

20

25

30

35

40

45

50

APPSwe/PS1dE9 wt

There was no effect of genotype on %

time spent in the correct quadrant or

number of annulus crossings

(platform crossings) during the probe

trial.

0

.5

1

1.5

2

2.5

3

Annulu

s C

rossin

gs (pro

be)

APPSwe/PS1dE9 wt

Problem

Virtually all tests of spatial memory

(hippocampal tests) are visual and vision

accounts for 53-61% of strain differences in

MWM learning and memory data (Brown & Wong, 2007.

Learn Mem, 14, 134-144).

Are there non-visual spatial memory tests?

We are examining olfactory spatial tasks.

MWM: APP+PS1 and APPswe/PS1dE9 AD

model mice

0

10

20

30

40

50

60

Lat

ency

to P

latfor

m (

s)

Acq

1

Acq

2

Acq

3

Rev

1

Rev

2

Rev

3

WT, Blind

WT, Norm al

Alz, Bl ind

Alz, Normal

However, some mice can be classified

as blind. When we separate mice based

on visual ability, we see a large effect of

visual ability at 16 months of age

(p<.0001) but no effect of genotype.

At 16 months of age, there is no

effect of genotype on latency to find

the hidden platform for both AD

strains, although APP+PS1 mice

take longer than APP+PS1dE9 mice

(p<.05).

0

10

20

30

40

50

60

Lat

enc

y to

pla

tform

(s

)

Acq

1

Acq

2

Acq

3

Re

v 1

Re

v 2

Re

v 3

Day

wt 1

APP+PS1

0

10

20

30

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50

60

Lat

enc

y to

pla

tform

(s

)

Ac

q 1

Ac

q 2

Ac

q 3

Rev

1

Rev

2

Rev

3

Day

wt 2

APP+PS1dE9

Vision task in APP+PS1 and

APPswe/PS1dE9 AD model mice

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30

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50

60

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80

90

100

% C

orr

ect

1 2 3 4 5 6 7 8

Day

wt 2

APP+PS1dE9

wt 1

APP+PS1

Mice were tested at 4 months of age.

There was no significant difference

between genotypes or between tg AD

mice and their respective wt.

0

20

40

60

80

100

120

% C

orr

ect

1 2 3 4 5 6 7 8

Day

wt 1, Blind

wt 1, Norm al

APP+PS1, Blind

APP+PS1, Norm al

However: All APP+PS1dE9 and wt 2

reached criterion. Of the APP+PS1

and wt 1 mice, 9 failed to reach

criterion and were classified as

“blind” (4 wt 1, 5 APP+PS1) and 8

mice reached criterion and were

classfied as “normal” (4 wt, 4

APP+PS1).

Within-strain (Mendellian) inheritance of retinal degeneration

genes confounds the results.

0

10

20

30

40

50

60

70

Lat

enc

y to

Pla

tfor

m (

s)

A1 A2 A3 R1 R2 R3

Day

tg

wt

Transgenic 5xFAD mice take significantly

longer to locate the hidden platform than

wildtype mice in the Morris water maze at

12 months of age (F(1,9) = 37.63,

p<.001).

0

10

20

30

40

50

60

Lat

ency

to P

latfor

m (

s)

A1 A2 A3 R1 R2 R3

Day

tg

wt

There is no deficit in learning the Morris

water maze at 9 months of age in

transgenic 5xFAD mice.

MWM - 5xFAD mice

Can they see? Not yet tested, have been genotyping for rd.

They have motor deficits on the Rotarod.

Barnes Maze APPswe/PS1dE9

mice

1

2

3

4

5

6

7

8

9

10

11

Nu

mbe

r o

f E

rro

rs

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

Day

Wild-type

APP/PS1

0

50

100

150

200

250

300

Lat

ency

to fi

nd e

scape

(s)

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

Day

Wild-type

APP/PS1

0

10

20

30

40

50

% T

ime

in C

orr

ect Zone

APP/PS1 Wild-type

APP/PS1 > wt (p<.01) APP/PS1 > wt (p<.01) APP/PS1 < wt (p<.05)

Transgenic APP/PS1 mice had a greater number of errors, a

longer latency to find the escape hole and spent less time in the

correct zone during the probe trial than their wildtype littermates at

16 months of age.

O’Leary & Brown, 2009. Behav Brain Res, 201, 120-127.

Barnes Maze 2 - APPswe/PS1dE9 mice

Wildtype mice used a spatial search strategy

significantly more than transgenic mice (F(1,17)

= 11.72, p<.005).

Wildtype mice spent more time in

the correct zone (ZC) during the

probe trial than transgenic mice

(F(1,15) = 5.43, p<.05).

Motor deficits in 5xFAD mice

Rotarod

0

25

50

75

100

125

150

175

200

Day

5 La

ten

cy t

o F

all

(s)

6-9 12-15

Age (Months )

tg

wtWildtype5xFAD

0

25

50

75

100

125

150

175

200

225

Da

y 5

La

ten

cy to

Fa

ll (s

)

6m 9m 12m 15m

Age (Months )

tg

wtWildtype5xFAD

Age: p<.01

Genotype: p<.01


Age: p<.0001

Genotype: p<.0001


0

20

40

60

80

100

120

140

160

180

200

220

Lat

enc

y to

Fa

ll (

s)

D1 D2 D3 D4 D5

Day

tg, longitud ina l

tg , cross-sectional

wt, longitudinal

wt, cros s-s ectional

0

20

40

60

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140

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Lat

ency

to F

all (

s)

D1 D2 D3 D4 D5

Day

tg

wt

Transgenic 5xFAD mice fell off

the Rotarod sooner than

wildtype mice at 12 months of

age (F(1,16) = 23.29, p<.001).

Transgenic 5xFAD mice that were tested

longitudinally performed equivalent to

wildtype controls at 12 months of age, while

those tested cross-sectionally performed

the worst (F(1,32) = 3.92, p = .056).

Cross-sectional vs longitudinal

testing on the Rotarod in 5xFAD Tg

AD mice

Mice tested in a cross-sectional study show deficits, but

those tested longitudinally do not.

Rotarod Results in 2 strains of AD mice

tested longitudinally

0

25

50

75

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125

150

175

200

225

Lat

ency

to F

all (s)

1 2 3 4 5

Day

wt 2

APP+PS1dE9

wt 1

APP+PS1

4 months

0

25

50

75

100

125

150

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225

Lat

ency

to F

all (s)

1 2 3 4 5

Day

8 months

0

25

50

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100

125

150

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225

Lat

ency

to F

all (s)

1 2 3 4 5

Day

12 months

0

25

50

75

100

125

150

175

200

225

Lat

ency

to F

all (s)

1 2 3 4 5

Day

16 monthsNo differences between tg AD

mice and their respective wt in

motor learning.

Background strain differences:

APP+PS1 and wt 1 >

APP+PS1dE9 and wt 2 at all

ages, p<.01

Conditioned Odour Preference

Task

Prochip

sugar

odour pot

30 cm

19 cm

13 cm

69 cm

20 cm

20 cm

opening opening

Middle

compartment

Rose

compartment

Lemon

compartment

ProchipLemon

odour pot

Rose

odour pot

Training

Testing

4 days Training (4 trials/day)

10 minutes/trial

1 day Testing (3 minutes)

Olfactory Memory: Correlation of visual

ability with % digging in CS+

r = -.407, ns

VD day 8 (percent correct)

40

50

60

70

80

90

100

% d

igg

ing

CS

+

40 50 60 70 80 90 100

0

20

40

60

80

100

% digging in cs+

SM/J

SJL/J

Molf/Ei

FVB/NJ

DBA/2J

CAST/Ei

C57BL/6J

C3H/HEJ

Balb/cByJ

AKR/J

A/J

129S1

0

20

40

60

80

100

% digging in cs+

SM/J

SJL/J

Molf/Ei

FVB/NJ

DBA/2J

CAST/Ei

C57BL/6J

C3H/HEJ

Balb/cByJ

AKR/J

A/J

129S1

Brown & Wong, 2007

Learn Mem, 14:134-44.

All strains were able to learn odour discrimination and

remember that discrimination 24h later.

Conditioned Odour Preference

(Long-term olfactory memory)

0

20

40

60

80

100

% T

ime

Dig

ging

in

CS

+

4 8 12 16 20

Age

Wild type

APP+PS1

No significant difference

between genotypes at

any age.

Memory tests without

training at 8, 12, 16 and

20 months of age.

0

20

40

60

80

100

% T

ime

Dig

ging

in

CS

+

8 12 16

Age

APP+

Wildtype

Double Tg (APP+PS1) Single Tg (J20)

No significant difference

between genotypes at any

age.

Memory tests without

training at 12 and 16

months of age.

Conditioned Odour Preference 2

0

20

40

60

80

100

120

% T

ime

Dig

ging

in

CS

+

3m 6m 9m 12m

Age

5xFAD

wildtype

Memory for odour pairings

learned at 3 months of age.

No effect of genotype or age,

but a genotype x age

interaction (p<.05).

0

20

40

60

80

100

120

% T

ime

Dig

ging

in

CS

+

young old

Age

APPswe/PS1dE9

wildtype

There is no genotype difference

between the APPswe/PS1dE9

and its wildtype, nor is there an

effect of age (young = 6-8 months

of age, old = 21-25 months of

age) in % time spent digging in

the CS+. 24h memory.

Social Transmission of Food Preference

0

.2

.4

.6

.8

1

Pro

por

tion

of C

ued

Foo

d E

ate

n

3m 6m 9m

Age

5xFAD

wildtype

Genotype: NS

Age: NS

Genotype x Age: p<.01

Wildtype mice ate significantly

more cued food than 5xFAD mice

at 9 months of age (p<.05).

0

.1

.2

.3

.4

.5

.6

.7

.8

.9

1

Pro

port

ion o

f C

ued F

ood E

ate

n

APPSwe/PS1dE9 Wildtype

At 12 months of age, APPSwe/PS1dE9

mice ate less of the cued food than their

wildtype (p = .067).

Cued & Contextual Fear Conditioning

(Trace) in 5xFAD mice at 6 months of age

0

10

20

30

40

50

60

70

% T

ime

Fre

ezin

g C

on

text

wt 5xFAD

No effect of genotype in % time

freezing in context test.

0

10

20

30

40

50

60

70

% T

ime

Fre

ezin

g C

ue

pre-CS CS post-CS

5xFAD

wt

Transgenic 5xFAD mice froze more in

the cued test than wildtype (p<.05).

Mice froze more during the cue

presentation (CS) and post-cue

presentation (post-CS) than during the

pre-CS period (p<.001).No difference in context

conditioning.Transgenic mice better in

cued conditioning.

Hearing & PPI deficits in 5xFAD mice

0

10

20

30

40

50

% P

PI

+4 +8 +12 +16 +20

Prepulse (Above background, 70dB)

APP/PS1, 12 months

APP/PS1, 8 m onths

APP/PS1, 4 m onths

Wild-type, 12 months

Wild-type, 8 m onths


APPSwe/PS1dE9 mice

Genotype: NS

Age: p<.05

5xFAD mice

Genotype: p<.05

Age: NS

0

200

400

600

800

1000

1200

1400

1600A

ver

age V

max

for

Initia

l Sta

rtle

(120d

B)

4 months 8 months 12 m onths

Age

APP/PS1

Wild-type

0

10

20

30

40

50

% P

PI

+4 +8 +12 +16 +20

Prepulse (Above background, 70dB)

APP/PS1, 12 months

APP/PS1, 8 m onths

APP/PS1, 4 m onths

Wild-type, 12 months



No genotype or age effects on

initial startle response.

-100

-80

-60

-40

-20

0

20

40

60

80

% P

PI

+4 +8 +12 +16 +20

Prepulse (above background, 70dB)

12-15, Wildtype

12-15, 5xFAD

6-9, Wildtype

6-9, 5xFAD

0

100

200

300

400

500

600

700

800

Aver

age

Vm

ax t

o

Init

ial S

tart

le (1

20d

B)

6-9 12-15

Age (months)

Wild type

5xFAD

No effect of age, but wildtype mice startle

significantly more than 5xFAD mice

(p<.0001).

Normal Abnormal

Cross-sectional studies

0

10

20

30

40

50

Co

nte

xt:

% T

ime

Fre

ezi

ng


APP/PS1

Wild-type

Cued and Contextual Fear Conditioning (delay) in APP+PS1dE9

mice and their wild-type controls at 4, 8 and 12 months of age

0

10

20

30

40

50

Co

nte

xt: %

Tim

e F

ree

zing

(7

d m

emo

ry)


APP/PS1

Wild-type

0

5

10

15

20

25

30

35

40

45

50

Cu

ed: %

Tim

e F

ree

zin

g

pre-CS CS

12 m onths , APP/PS1

12 m onths , Wild-type

8 months, APP/PS1

8 months, Wild-type

4 months, APP/PS1

4 months, Wild-type

0

10

20

30

40

50

Cu

ed:

% T

ime

Fre

ezin

g (

7d

me

mor

y)

pre-CS CS

12 m onths , APP/PS1

12 m onths , Wild-type

8 months, APP/PS1

8 months, Wild-type

4 months, APP/PS1

4 months, Wild-type

No effects of genotype were found in

% time freezing in the context.

Age: p<.02, 8> 4, 12

There was an effect of genotype as wt mice

froze more than the tg mice in the cued test

(p<.05).

Other possible confounds

Activity

Anxiety

Elevated plus-maze

Light/dark box

Open Field

Open field activity

0

5

10

15

20

25

30

35

40

45

50

Nu

mbe

r o

f R

ears

APP Wt 1 APP/PS1 Wt 2

0

2

4

6

8

10

12

Tim

e in

Cent

er (

s)


APP>Wt 1 (p<.05)

APP + Wt 1 > APP/PS1 +

Wt 2 (p<.001)

APP + Wt 1 >

APP/PS1 + Wt 2

(p<.001)

No difference between

genotypes

Background strain effects. The single transgenic APP (J20)

mice and their wildtype are more active than the double

transgenic APP/PS1 mice and their wildtype at 12 months of

age in a 10-minute trial in the open field.

0

50

100

150

200

250

300

350

Num

ber

of

Lin

e C

rosses


a a

bc c

a

bb

Elevated Plus-Maze

0

10

20

30

40

50

60

70

% T

ime S

pent in

Open A

rms

5xFAD Wildtype

6 month old 5xFAD mice spent more time in the

open arms than wildtype mice (p<.05). There was

no difference between genotypes on number of line

crosses. Wildtype mice reared more than 5xFAD

mice (p = .056).

0

10

20

30

40

50

60

Num

ber of Lin

e C

rosses

5xFAD Wildtype

0

1

2

3

4

5

6

7

Num

ber of R

ears

5xFAD Wildtype

*

*

Light/dark box

0

2

4

6

8

10

12

14

16

Nu

mbe

r o

f trans

itio

ns

AP

PS

we

,PS

EN

1dE

9

wt1

AP

P69

5+P

SE

N1

wt2 0

1

2

3

4

5

6

7

8

9

Nu

mbe

r o

f he

adp

oke

s

AP

PS

we

,PS

EN

1dE

9

wt1

AP

P69

5+

PS

EN

1

wt2

0

20

40

60

80

100

% T

ime

in L

ight Zone

AP

PS

we,P

SE

N1d

E9

wt1

AP

P69

5+P

SE

N1

wt2

APP695+PSEN1 + wt2 >

APPSwe, PSEN1dE9 + wt

1

(p<.01)

APPSwe,PSEN1dE9 > wt1

(p<.05)


APP695+PSEN1 and wt2

APPSwe,PSEN1dE9 <

wt 1 (p<.05)


APP695+PSEN1 and wt

2.

The APPSwe,PSEN1dE9 tg strain and their wildtype are less active than the

APP695+PSEN1 and their wildtype at 12 months of age. While no differences were

found between APP695+PSEN1 and their wildtype, the APPSwe,PSEN1dE9 tg

mice were found to have a greater number of headpokes and spent less time in

the light zone than their wildtype, indicating greater anxiety.

a a

bb

a

b

b

b,c

b

a aa

What have we found about mouse models

of Alzheimer’s Disease?1. Do mouse models of Alzheimer’s Disease show an age-

related decline in cognitive function which differs significantly from control strains?

• So far, we have found few age-related declines in cognitive function in our mouse models. The APPswe/PS1dE9 model shows deficits in cued fear conditioning at 12 months and spatial learning in the Barnes maze at 16 months of age.

2. Can you distinguish Alzheimer’s mice from mouse models of other neurodegenerative disorders?

• Not yet tested.

3. Are deficits in test performance due to sensory, motor or cognitive deficits?

• We have found all: visual, auditory, motor & cognitive deficits

4. Do treatments for Alzheimer’s Disease delay this age-related decline in cognitive function?

• Not yet tested.

Alzheimer’s mice in th e Brown Lab.

Strain JAX # Evaluation of AD mice

Single Transgenic

*

B6.Cg-Tg(PDGFB-APP)5Lms/J

“I5” or “AP PInd”

004662 - impaire d performance in Morris water maze at 10

months of age

Single Transgenic

*

B6.Cg-Tg(PDGFB-APPSwInd)20Lms/J

now

B6.Cg-Tg(PDGFB-APPSwInd)20Lms/2J

“J20” or “APPSwInd”

004661

006293

- greater exploratory behaviour at 12 months of age

Double Transgenic

*

B6C3-Tg(APP695)3DboTg(PSEN1)

5Dbo/J

“3D5D” or “APP+PS1”

003378 - visual deficits du e to retinal degeneration gene

Double transgenic

**

B6C3-Tg(APPSwe,PSEN1dE9)85Dbo/J

“D85” or “APP+PS1dE9”

004462 - impaire d cued fear conditioning performance at 12

months of age

- impaire d in social transmission of food preference at

12 months o f age

- greater anxiety (measured in light/d ark box) at 12

months of age tha n wildtype

- poor Barnes maze performance at 16 months of age

Double transgenic

**

B6SJL-Tg(APPSwFlLon,

PSEN1(M146L;L286V) 6799Vas/J

“5xFAD”

006554 - impaire d hearing/P PI at 6 months of age

- less anxious (meas ured in elevated plus -maze) than

wildtype at 6 months of age

- impaire d in social transmission of food preference at

9 months o f age

- Rotarod defic its at 12 months of age

- impaire d memor y for odour discrimination learned 9

months previously at 1 2 months of age

Triple transgenic

**

B6;129-Psen1tm1MpmTg(APPSwe,

tauP301L)1Lfa/J

“3xTg -AD”

004807 Not yet tested

* = used to have

** = currently have

Is there a valid mouse model of AD?

Why have we found so few cognitive

deficits in AD model mice?

Possible reasons:• Background strain effects

• Confounds of sensory-motor deficits

• Longitudinal studies mask differences found in cross-sectional tests.

• Are we testing the most valid AD mouse models?

• Are we using valid and reliable tests?

• Do we have experimenter error?

• But - we did find deficits in blind mice that could be treated, thus we have faith in our procedures.

Solution: careful validation of mouse models of AD -dissociation of sensory-motor versus cognitive deficits; use of multiple memory tests.

A new gene x environment model for studying mouse models of

neurodevelopmental disorders

Environmental

Factors Neurochemistry (Extra- and intra-cellular)

Epigenetic mechanisms

Genetics (DNA)

mRNA

Protein Synthesis

Brain Development (Neural circuits)

Behaviour

The End

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