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

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

StaffStaffAlbany Edmonton Portland

Jennifer FogartyKristina HenricksChristopher DowningJaya Singh

Melike SchalomonSharon Doerksen

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

Acknowledgements

• OBSSR, NIAAA, NIDA for funding.• R.H. Kant and Accuscan for generously providing

activity monitor and RotaRod apparatus.• Diligent postdocs, graduate students and institutional

staff at our three institutions who went to heroic efforts to ensure that the study could start on time in a coordinated fashion.

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 sex2. Mated pairs before and after delivery of litter3. Weaned offspring prior to the start of behavioral testing4. During Behavioral Testing

After arrival of Breeding StockFood Purina 5001 (4.5% fat)Bedding Bed-O-Cob 1/4"Nesting material NoneCage top Stainless steel bars (passive filter top in Portland)Animal marking nilAnimal weighing nilMice/cage 4 or 5 maximumLights on/off 0600/1800Cage 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 NestletMarking noneWeighing at matingMice/cage 1 female, 1 maleMale present with litter? Yes; postpartum pregnancy allowedInspection of new litter Count live and dead pups; remove dead, put live in nestCulling of newborns Remove only the obvious runts for ethanasiaCage changing WeeklyCage 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 offspringAge at weaning Aim for 21 days; may be ± 1 day on weekendNesting material NoneFood Purina 5001Mice/cage Same sex, 4 or 5 maximumMarking nilBody Weight Record at weaning to closest .5g

During behavioral testingMice/cage 2 (housed 2 weeks prior to testing; random culling)Nesting material NoneMarking Sharpie pen on tailWeighing prior to day 5 activity testHandling Hand with surgical gloveCage 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, 284:1670-1674 (June 4 issue).

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Mouse Stock

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Body Weights for Mice Bred In HouseFemales

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Locomotor Activity in Eight Stocks at Three Sites

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COCAINE-STIMULATED ACTIVITY IN 15 MIN TESTSEIGHT GENOTYPES

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

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

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Ethanol Preference Scores in Eight Stocks at Three Sites

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Total Arm Entriesthe Elevated Plus Maze

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


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


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


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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.

Summary and Implications• Some behavioral phenotypes can be expected to show stable, genetically

influenced, characteristics.• The reasons that some phenotypes may not show as much stability across

strains/genotypes are potentially many:– Method Variance and Measurment Reliability should play a role. Less

reliable indices should be less stable. Requirement for larger N’s.– Genotype x Environment Interaction may exist. Uncontrolled

environmental variation may exist and gene expression may vary with it. Both additive and non-additive sources of GxE may exist. As is often the case in behavior genetics, identification of the sources of environmental influence would be necessary before any real understanding of GxE can be expected.

– Site specific QTL??? It is theoretically possible that non-identical genes are influential at different sites (read environments). This kind of GxE would be accessible only in the context of gene mapping studies.

Summary and Implications, cont’d.• Are Behavioral Phenotypes Different?

– Behavioral Scientists are keenly aware of the need for careful control of protocols and standardization of methods, and influences of extraneous variables.

– Single tasks are not likely to provide strong characterization of complex psychological constructs. E.g., plus maze = anxiety???

– Some patterns of genetic influence on behavior can be highly stable. For example, body weight showed larger effect sizes for the strain by site interaction than locomotor activity and ethanol consumption. Similar findings exist for neuroanatomical measures.

Arrangement of apparatus in one lab