Design and Methods - Mutations in Taste
Logistics - Identification of mutations is conducted in two stages: primary screening at The Jackson Laboratory, and secondary screening at Monell. In the high-throughput primary screen, mice are tested with a wide range of taste stimuli and nutrients, and following identification of deviants and heredity confirmation, groups of mutant mice are shipped to Monell for further characterization of the mechanisms underlying the deviant behavior.
Rationale - To be applicable for primary screening, methods assessing taste-related behavior should: (i) provide essential information about underlying mechanisms; (ii) reliably characterize a single individual rather than a group; (iii) require little time and labor; and (iv) be compatible with other phenotyping assays that will be conducted before or after them. For that purpose, we have critically considered numerous approaches, including long-term single- or two-bottle tests, short-term intake or lick recordings, assessing taste reactivity based on behavioral responses to orally infused stimuli [245], using various types of gustometers [246-248], and sham feeding of animals with esophageal or gastric cannulae. Our experience strongly suggests that the two-bottle tests are the most suitable for primary screens for mouse taste mutants. The use of other, more laborious, methods will be justified at the level of secondary screening. We note that the tests we propose may be further improved for screening mutagenized mice, and studies towards this end are underway (details can be found at http://www.monell.org/MMTPP/).
Primary screens
Mutagenized mice are placed in individual cages equipped with two bottles (see details in http://nmf.jax.org/biblio.html#249>249). During this test, mice receive sequentially eight solutions representing different taste qualities and nutrients: 2 mM saccharin, 10 mM citric acid, 0.1 mM quinine, 300 or 450 mM NaCl, 75 mM CaCl2, 10 mM inosine monophosphate (IMP) and 1 mg/l capsaicin. These concentrations are chosen based on extensive previous studies using different genotypes, and are the most appropriate for C57BL/6 mice. Each solution is tested for 4 days, with 3 days of water only between the testing of different solutions. A complete screen cycle takes 7 weeks. Deviating animals are re-tested to confirm the phenotype. Body weights of individual mice are determined in the beginning and the end of the testing period. Testing the solutions of different taste qualities on the same animals assesses whether the effect of a mutation is specific to a certain stimulus or whether it is generalized (for example, due to changes in taste buds or their innervation). In any case, the mechanisms affected by mutations will be characterized more intensively through the use of secondary screens.
Secondary screens
Depending on the mode of inheritance, mice homozygous for the mutant allele, wild-type homozygotes, and/or heterozygotes are used in groups of 5-10 mice each (when possible, littermates are used), plus non-mutagenized C57BL/6J mice are used as an additional control. Conducting a battery of experiments with each group of deviating and control mice takes approximately 3 - 5 months.
In the secondary screens, we utilize strategies similar to those we have been using to characterize mechanisms underlying strain differences in two-bottle tests. The particular tests included in the secondary screen depend on the taste modality affected by a mutation. In all cases, we use two-bottle tests with series of concentrations of a compound by which the deviant was found, to provide additional information (e.g. on thresholds of preference or avoidance, concentrations that show the greatest genotype difference). The concentration series also allow us to distinguish between perceptional (if differences between mutant and wild-type animals occur already at low concentrations of solutions with negligible nutritive value) and post-ingestive (if the difference appears only at high concentrations) mechanisms of acceptance. The role of taste perception is assessed using short-term lickometer tests. If we suspect that deviants are insensitive to a stimulus, we examine the hypothesis by using conditioned taste aversion (CTA) tests [250, 251]. Animals may be indifferent to a solution in the two-bottle tests even if they perceive its taste, but experience the taste as hedonically neutral (neither aversive, nor palatable). CTA results tell us whether animals can distinguish a test solution from a vehicle.
Deviations in acceptance of many compounds suggest a mechanism involving a generalized effect on the taste system. If this is the case, we count the number of fungiform papillae on the tongue [252] as a way to characterize structural and/or developmental abnormalities in the peripheral taste organ or nerve structure. Deviations in acceptance of specific compounds involve secondary two-bottle preference tests with ascending series of additional related compounds. The purpose of these tests is to distinguish among several factors that can affect taste acceptance, e.g. perception of one or another taste component of this compound, its nutritive value, specificity of changes within sub-qualities of the same taste quality, or changes in specific appetites. If we find a deviation in saccharin acceptance, we test additionally other artificial sweeteners and sucrose. For citric acid deviants, we test HCl; for quinine we test caffeine, cycloheximide, propylthiouracil and phenylthiocarbamide; for CaCl2: KCl and calcium lactate; for Inosine 5’-monophosphate: monosodium glutamate and glutamic acid; for capsaicin: menthol and nicotine; and for NaCl: KCl and sodium bicarbonate. The mice deviating in NaCl acceptance could also be submitted to sodium depletion [250] or mineralocorticoid hormone injections [253], treatments that usually increase NaCl consumption.
