Mouse Behavioral Phenotyping Laboratory

1. Consultation on appropriate and rigorous experimental design and optimal testing regimens for specific projects. Assistance with behavioral aims in grant proposals and IACUC protocols for behavioral studies.
2. Expert testing services, including the characterization of mutant mouse lines across multiple domains of function, and sleep phenotyping over days or weeks.
3. Training for students, post-doctoral fellows, and laboratory staff in appropriate methods for mouse behavioral testing and transformation of data from automated testing systems.
4. Services by special request, such as evaluation of neonatal and pre-weaning mouse pups, development and validation of new phenotyping approaches, implementation of drug regimens, blood or tissue collection, and workshops on a specific topic.

All behavioral testing procedures are conducted in strict compliance with the policies on animal welfare of the National Institutes of Health (stated in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council, 1996 edition), and are approved by the University of North Carolina Institutional Animal Care and Use Committee.

Motor function

• Open field test for activity and exploration (Fusion and VersaMax Systems, AccuScan Instruments)
• Grip strength measured by digital force meters (San Diego Instruments) or with a wire hang assay
• Accelerating rotarod test for motor coordination and balance
• Gait and stride analysis using the Catwalk system (Noldus Information Technology, Inc)
• Repetitive behavior, such as grooming and jumping, scored using Observer software (Noldus).

Sensory function

• Acoustic startle test for hearing and sensorimotor gating (SR-Lab system, San Diego Instruments).
• Buried food test for olfactory ability
• Visual cliff test
• Visible platform assay in a water maze
• Hot plate test for thermal sensitivity (IITC)

Tests for anxiety- and depression- related behavior

• Open field test, using time spent in the center region as an index of anxiety-like behavior
• Light/dark preference test in an open field (VersaMax Systems, AccuScan Instruments)
• Elevated plus maze or zero maze
• Marble-bury assay
• Forced swim, using measures of time spent immobile (Ethovision, Noldus)
• Sucrose preference, using a two-bottle choice test
• Behavior following restraint stress

Cognitive function

• Morris water maze and Barnes maze tests for spatial learning and cognitive flexibility during reversal learning.
• Y-maze test for working memory, measured by spontaneous alternation
• T-maze test for spatial learning, alternation, and reversal
• Delayed non-match-to- sample procedure for working memory
• Conditioned fear tests for contextual or cue-dependent learning (Near-Infrared Video Fear Conditioning (Med Associates))
• 5-choice serial reaction time task (5-CSRTT) for learning acquisition, visual attention, vigilance, and response inhibition (Med Associates)

Project initiation

• UNC investigators wishing to utilize the core should contact the director, Dr. Sheryl Moy, to discuss optimal study designs, feasibility for core use, and costs of testing.
• All projects utilizing the core must be approved by the UNC Institutional Animal Care and Use Committee (IACUC) before initiation. The director can provide drafts of amendments or sections to include behavioral testing in animal protocols.

Core charges

• Service costs are dependent on the specific test regimen and number of mice, with charges based on personnel effort for conducting studies, including testing time, set-up and clean-up, and data transformation and analysis. Please contact the director for project cost estimates.
• For training of students, post-doctoral fellows, and laboratory personnel, the core charges a $75.00 orientation and training fee. As a component of the UNC IDDRC, the core receives significant support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). Therefore, the core can provide subsidized rates and no-cost pilot studies and training services for IDDRC investigators.

Transfer of mice to the core facility

• The Mouse Behavior Laboratory is located in the Neuroscience Research Building (NRB), inside the Division of Comparative Medicine (DCM) animal vivarium. All transfers of mice to the core must first be approved by DCM.
• For each transfer group, the director will need an Excel spreadsheet with information about the mice, including:
  • Subject ID
  • Genotype or treatment
  • Sex
  • DOB
  • DCM cage card number
• Because single housing is stressful for mice, the core asks that each cage contain from 2-4 mice, separated by sex. For most projects, a maximum of 20 mice can be transferred to the core at one time.

The Behavior Laboratory provides consultation on study design to address issues of rigor, including use of littermate controls, consideration of sex, inclusion of appropriate subject numbers, balancing across cohort groups, and randomization of subjects to treatment conditions. Experiments in the laboratory are conducted by researchers blind to genotype, treatment, or other study variables. Statistical analyses include standard parametric approaches for data that are normally distributed, with appropriate post-hoc tests and consideration of multiple comparisons. In the case of non-normal data, nonparametric rank-based regression or other appropriate analyses are used. Reproducibility of data is confirmed, in part, by comparing behavior in wild type mice across projects and archived strain distributions and mutant line profiles. In addition, sets of C57BL/6J and other strains are periodically evaluated in the laboratory to ensure that assays are providing the expected indices for typical performance.

Guidelines from the UNC Office of Research Technologies

A. Eight steps to Rigorous and Reproducible Experiments at UNC:

• 1. If using a core facility, consult with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.
  2. Design your experiment with sufficient controls (rigor) and replicates (reproducibility).
  3. Assure that ALL of your reagents (antibodies, cell lines, mice) are fully validated.
  4. Have a clear and detailed protocol (SOP) and data analysis plan. Assure that the protocol is strictly followed or that any deviation is well documented.
  5. Assure that the staff or students performing the experiment are well trained and understand each step and the importance of performing them precisely.
  6. Use only well-maintained instrumentation, preferably maintained and operated in a core facility with expert staff (see #1 above).
  7. Document all steps, reagents, equipment and data analysis methods used in the experiment. Assure that the both the documentation and the data itself are properly stored in a safe data management repository.
  8. Acknowledge grants that support the research, including cores (by name) and core staff, in publications.

B. Guide to Rigor and Reproducibility for the Mouse Behavioral Phenotyping Laboratory

1. Consult with the core director in the planning stage: Sheryl S. Moy, Ph.D.
2. Subject numbers. Mouse behavior studies typically require 10 to 12 subjects per experimental group for adequate power in the statistical analyses. Thus, for a line of genetic mutants, an N of approximately 10 wild type and 10 mutant littermates is required. To evaluate effects of both sex and genotype, approximately 10 males and 10 females of each genotype will be required.
3. Ensure the use of appropriate controls for behavioral studies. In many cases, the optimal controls are wild-type littermates of mutant mice, derived from heterozygous breeding pairs. Biological variables that require consideration include age, sex, and genetic background. Other important factors are source of the mice (i.e. from a commercial laboratory or bred in-house), environmental conditions, and experimental history.
4. Housing conditions can affect mouse behavior. In general, mice for testing in the core should be group housed, with a maximum of 4 per cage. Individual housing should be avoided, since isolation can be stressful, especially for younger mice. Exceptions can be made for males separated for fighting or other reasons related to health, or when isolate housing is required for the experiment.
5. Acknowledgements. Please acknowledge use of core services and resources in publications and presentations. For example, “Assistance for this project was provided by the UNC Intellectual and Developmental Disabilities Research Center (NICHD; P50 HD103573; PI: Gabriel Dichter)”. Co-authorship of core faculty and staff is dependent on the significance of their contribution to the research. Please consult the website of the Association of Biomolecular Resource Facilities for policies on the appropriate inclusion of Core Laboratory Personnel in authorship of your publication: https://abrf.org/authorship-guidelines