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Validation of a high-throughput mouse behavioral testing protocol and application to the BXD genetic reference population for gene mapping of multiple pain traits
William R. Lariviere, PhD1, Jason Spence, BSc2, Roumyana Kirova, PhD2, and Elissa J. Chesler, PhD2. (1) Anesthesiology, University of Pittsburgh School of Medicine, A1305 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, (2) Mammalian Genetics & Genomics, Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6445
Genetic factors contribute to individual differences in pain sensitivity in humans and nociception in animals. Molecular and behavioral characterization of mouse genetic reference populations including recombinant inbred (RI) mice can be used to determine the responsible genes, their mechanistic effects, and the specific tissues involved. High-throughput behavioral testing protocols are required for efficient assessment of mouse nociception in large populations and mutant screens. These often controversial protocols involve repeated testing, which via habituation to stressors or anticipation of aversive stimuli may interact with organismic variables including sex to mimic or confound the effects of genetic variation. Prior to gene mapping and systems genetic analysis of several nociception traits in a panel of 80 RI mouse strains, we have designed a phenotyping protocol at ORNL in which a single technician can assay 50 mice per week on 5 nociception measures. We have validated a protocol using a replicated latin-square design for testing the same mouse on the Von Frey test of mechanical sensitivity; the tail clip test of mechanical nociception; tail withdrawal from hot water; the hot plate test; and Hargreaves' thermal plantar test. Results show virtually no influence of carry-over effects or repeated testing on these assays. Our results demonstrate the validity of our protocol, and provide a method of validation of related high-throughput testing protocols. We have also developed a simple desktop database system to facilitate implementation of the latin-square approach as part of the testing protocol. The approach is currently being applied to testing a panel of 80 RI strains on the above mentioned traits for gene mapping and complex genomic analysis. These data will be presented, including heritability estimates, novel gene mapping results, their integration with neurobehavioral, immunological, and physiological phenotypes for the same mice, and transcript expression levels in multiple brain regions and other tissues.