Multi-Center Trial of a Standardized Battery of Tests of Mouse Behavior A project funded by the NIH Office of Behavioral and Social Science Research via

Multi-Center Trial of a Standardized Battery of Tests of Mouse Behavior

A project funded by the NIH Office of Behavioral and Social Science Research via

NIAAA and NIDA.

Investigators:

John Crabbe, Doug Wahlsten, Bruce Dudek

Web site: http://www.albany.edu/psy/obssr

StaffStaff

Albany Edmonton Portland

Jennifer FogartyKristina HenricksChristopher DowningJaya Singh

Melike SchalomonSharon Doerksen

Pamela MettenStephen BoehmTamara PhillipsJanet DorowCharlotte WengerSue Burkhart-KaschJason SibertCarrie McKinnonCedar Nolte

Goals of the Study

• Evaluate mouse phenotypes at three sites using simultaneously performed identical procedures.

• Establish the feasibility of implementing precise test protocols in multiple sites.

• We sought to maximize standardization of protocols across site, often to a level of minor nuance, and at times perhaps not the “best” methodology possible for a task.

• Examine stability of strain differences across three laboratories, shipping status, and gender.

• Evaluate a broad-based behavioral test battery.

• Generate a roadmap for future studies designed to focus on narrower domains of phenotypes.

GROUPING FACTORS

Genotype (Stock)• A/J

• C57BL/6J

• BALB/cByJ

• DBA/2J

• 129/SvEvTac

• 5HT1B++

• 5HT1B--

• B6D2F2

Site• Albany

• Edmonton

• Portland

Sex• Female

• Male

Shipping Status• Bred In House

• Shipped

• Thus the design is an 8 x 3 x 2 x 2 factorial.

• 96 cells.

• Targeted n=4/cell (384 planned), based on power analysis.

• 379 tested, 378 completely.

• No empty cells

Design Specifications

Replications

• Testing protocol required replications.

• Two replications yielded n=64 per site per replication. This produced a target of n=2 per cell across all cells in each replication.

• One site ran a third replication to fill some cells (n=6).

Chronology

• Breeding stock received 12/2/97

• Matings set 1/13/98

• First litters born ~ 2/1/98

• Shipped mice for testing received 3/18/98 to 4/2/98

• First replication begun 4/20/98

• Second replication begun 4/27/98

• Third replication begun 5/4/98 (Edmonton Only)

• Age at testing initiation: ~ 65-75 days

Features of Husbandry Equated Across LabsFour time frames may differ in the requirements to some extent.

1. After arrival from supplier but before mating: housed with same sex

2. Mated pairs before and after delivery of litter

3. Weaned offspring prior to the start of behavioral testing

4. During Behavioral Testing

After arrival of Breeding Stock

Food Purina 5001 (4.5% fat)

Bedding Bed-O-Cob 1/4"

Nesting material None

Cage top Stainless steel bars (passive filter top in Portland)

Animal marking nil

Animal weighing nil

Mice/cage 4 or 5 maximum

Lights on/off 0600/1800

Cage changing weekly

Features of Husbandry Equated Across Labs, cont’d

Mated pairs (items the same as after arrival are not repeated)

Food Purina 5020 (9% fat)

Nesting material 1 Nestlet

Marking none

Weighing at mating

Mice/cage 1 female, 1 male

Male present with litter? Yes; postpartum pregnancy allowed

Inspection of new litter Count live and dead pups; remove dead, put live in nest

Culling of newborns Remove only the obvious runts for ethanasia

Cage changing Weekly

Cage changing with litter Do not disturb cage until litter is 4 days old or younger; then clean cage with fresh nestlet

Features of Husbandry Equated Across Labs, cont’d

Weaned offspring

Age at weaning Aim for 21 days; may be ± 1 day on weekend


Food Purina 5001

Mice/cage Same sex, 4 or 5 maximum

Marking nil

Body Weight Record at weaning to closest .5g

During behavioral testing

Mice/cage 2 (housed 2 weeks prior to testing; random culling)


Marking Sharpie pen on tail

Weighing prior to day 5 activity test

Handling Hand with surgical glove

Cage changing After Day 5 testing

Targeted Phenotypic Domains

• Reproductive performance

• Basic growth characteristics

• Behavioral test battery

• Basic neuroanatomical characteristics

Variables Matched Across Site

• Chronology for breeding stock receipt, matings, receipt of shipped mice, and test battery beginning dates.

• Food, bedding, light cycle.

• Details of Behavioral Test Battery Protocols!

• Apparatus.

Behavioral and Other Phenotypes

• Locomotor Activity. 15 min test in Accuscan Digiscan system.

• Elevated Plus maze. 5 min test.• Rota-rod coordination test.• Water Maze escape task.• Cocaine-stimulated locomotor activity.• Alcohol preference.• Neuroanatomical indices.

Test Battery Sequence

• Day 1: Baseline Locomotor Activity in Accuscan Digiscan automated activity monitors. (15 min test)

• Day 2: Elevated plus maze test. (5 min test)

• Day 3: Rota-rod test. (10 trials)

• Day 4: Water Escape test. (8 trials)

• Day 5: Cocaine-treatment activity test. (15 min test; 20 mg/kg, ip)

• Day 6-7: Null

• Day 8: Begin alcohol drinking test. (6 days)

Protocol Implementation

• Morning and afternoon sets of n=32 mice (16 cages) were run to complete the n=64/replication.

• Semi-blind testing was employed by using cage numbers rather than strain identity as identifiers (caveat regarding coat color).

• Testing order (which mouse genotype,sex, etc) was based on a constrained randomized sequence (although cage-pair mates were always tested in adjacent positions). This sequence varied across site.

• Same daily test routine for each squad of 2 or 4 mice was used on the first 5 tests.

• Documents describing protocols are available on the web site.

Current Status of Data Analysis• Database for combined sites constructed, and web site

created for data sharing.

• Database includes data from baseline activity test, rota-rod test, water escape test, cocaine activity test, elevated plus maze test, alcohol preference, and adult body weights.

• Statistical analyses of all tasks has been done and figures are available on the web site.

• An initial report of findings is published: Crabbe, Wahlsten and Dudek (1999) Science, 284:xx-xx.

51015202530

AlbanyEdmonton Portland

Mouse Stock

Bod

y W

eig

ht

(g)

51015202530

Body Weights for Mice Bred In House

Females

Males

Locomotor Activity in Eight Stocks at Three Sites

STOCK

A/J

C57

BL/

6J

BA

LB/c

ByJ

DB

A/2

J12

9/S

vEvT

ac

5HT1

B+/

+

5HT1

B-/-

B6D

2F2

15 M

IN D

IST

AN

CE

TR

AV

ELE

D (

CM

)

1000

2000

3000

4000

5000

6000Albany Edmonton Portland

* **

* *

Stock-Dependent Shipping Effects

STOCK

A/J

C57

BL/

6J

BA

LB/c

ByJ

DB

A/2

J

129/

SvE

vTac

5HT1

B+/

+

5HT1

B-/-

B6D

2F2

Mea

n H

oriz

onta

l Dis

tanc

e T

rave

lled

1000

2000

3000

4000

5000

6000 Bred Shipped *

*

STOCK-DEPENDENT SEX DIFFERENCES

STOCK

ME

AN

HO

RIZ

ON

TA

L D

IST

AN

CE

TR

AV

ELLE

D

1000

2000

3000

4000

5000

6000Female Male

*

*

DIS

TA

NC

E T

RA

VE

LL

ED

(C

M)

2000400060008000

1000012000

STOCK

2000400060008000

1000012000

COCAINE-STIMULATED ACTIVITY IN 15 MIN TESTSEIGHT GENOTYPES

2000400060008000

1000012000

15 min Baseline15 min Cocaine (20 mg/kg, ip)

ALBANY

EDMONTON

PORTLAND

Water Escape Performance at Trial 4Expressed as Trial 1 latency - Trial 4 latency

STOCK

Trial 4

La

tency

- T

ria

l 1 L

ate

ncy

-10

-5

0

5

10

15

20 Albany Edmonton Portland

Water Escape to Visible Platform

Trial Block

1-2 3-4 5-6 7-8

Me

an E

scape L

ate

ncy

(se

c)

5

10

15

20

25

30

A

C

D2

B6

129 (all 3)F2

Proficient Level

2

4

6

8

AlbanyEdmonton Portland

Mouse Stock

Eth

anol

Con

sum

ptio

n (g

/kg)

2

4

6

8

Average Four Day EtOH Consumption (g/kg)

Females

Males

Ethanol Preference Scores in Eight Stocks at Three Sites

STOCK

Eth

anol

Pre

fere

nce

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 Albany Edmonton Portland

Total Arm Entriesthe Elevated Plus Maze

in Eight Stocks at Three Sites

STOCK

Me

an T

ota

l Arm

En

trie

s

5

10

15

20


*

** * * * * *

Open Arm Time in the Elevated Plus Maze (five min test)


STOCK

A/J

C57

BL/

6J

BA

LB/c

BY

J

DB

A/2

J

129/

SvE

vTac

5HT1

B+/

+

5HT1

B-/-

B6D

2F2

Mea

n O

pen

Arm

Tim

e (s

ec)

25

50

75

100

125


*

* **

*

*

Center Square Time in the Elevated Plus Maze (five min test)


STOCK

A/J

C57

BL/

6J

BA

LB/c

BY

J

DB

A/2

J

129/

SvE

vTac

5HT1

B+/

+

5HT1

B-/-

B6D

2F2M

ean

Cen

ter

Squ

are

Tim

e (s

ec)

25

50

75

100

125

150

175

200 Albany Edmonton Portland *

*

*

*

End Arm Foray Index (Closed - Open) the Elevated Plus Maze


STOCK

A/J

C57

BL/

6J

BA

LB/c

BY

J

DB

A/2

J

129/

SvE

vTac

5HT1

B+/

+

5HT1

B-/-

B6D

2F2

Mea

n C

lose

d -

Ope

n F

ora

ys

0

2

4

6

8

10Albany Edmonton Portland *

* **

General Conclusions From Present State of Data Analysis

• For the baseline activity, relatively good stability of the genetic differences is seen across site with virtually no meaningful influence of shipping status.

• Cocaine stimulated activity does vary considerably across site, but in an interactive manner with genotype.

• Strong patterns of genetic influence in the water escape task are only partly consistent across site and shipping status.

• Strain differences in alcohol consumption are relatively stable across site although a main effect of site is present.

• Elevated Plus Maze indices show considerably less cross site stability.

• The cross-site stability of strain differences is thus likely to be highly task-dependent.

Highlights of Lessons from the Multi-Center Standardization Trial

1. High quality data can be obtained simultaneously in several labs.

2. Logistics are much more challenging than for the typical study done in one lab.

3. Principal glitches can be avoided in future studies.

4. Anomalies or accidents afflicting one lab do not undermine the entire study.

5. a. A large advantage is conferred by the use of automated data collection.

b. Testing 2 mice at one time is very difficult without automated apparatus.

c. Video tape recording for later scoring from tapes is a markedly inferior method.

Highlights of Lessons from the Multi-Center Standardization Trial (cont’d)

6. We need to know more about the reliability of each test in order to plan future studies.

7. We need to standardize the stimulus surroundings for each test, not just the apparatus.

8. We should decide in advance what statistical analysis program will be used.

9. In the future, we should strive for optimal rather than convenience solutions.

Arrangement of apparatus in one lab