Gustation *
Materials and Methods
(chemicals all from Sigma, Incorporation, USA)
Saccharin, sodium salt (hydrate)
Quinine Hydrochloride
Citric Acid, monohydrate
Inosine 5’-monophosphate (IMP, dihydrate)
Calcium Chloride (CaCl, dihydrate)
Sodium Chloride (NaCl)
8-methyl-N-vanillyl-6-nonenamide (Capsaicin)
Additional drink bottles/lines
Stock solutions to be made with 1 l purified water:
Saccharin, sodium salt (hydrate) 0.48 g
Quinine Hydrochloride 0.039 g
Citric Acid, monohydrate 2.10 g
Inosine 5’-monophosphate (dihydrate) 5.26 g
Calcium Chloride (dihydrate) 11.03 g
Sodium Chloride 17.53 g
Capsaicin is very volatile and may cause irritation upon contact with mucosal membranes. Therefore, work under hood and wear protective gear (gloves, mouth protection etc...).
Stock solution to be stored in refrigerator: 50mg capsaicin in 250μl EtOH (100%)
Experimental solution, room temperature: 5 μl of stock solution in 1l H2O
* (this behavioral screen is now performed at the Monell Chemical Senses Center under supervision of Dr. A. Bachmanov)
Primary Screen
Mice are placed in individual cages equipped with two drinking tubes. Following a training period of 2-3 days during which the animals can become accustomed to two drinking tubes and baseline water consumption will be recorded, the mice will receive 7 different substances to taste over the next seven weeks. The drinking fluid is available ad libitum, and the mice have a choice between water and the tested substance. Access to the tested substances is changed every day, i.e. it will be available alternately through the right or left drinking line. A testing period of 4 days is followed by a test-free and baseline-acquisition period of 2 days, before another substance will be tested. Body weight and consumption of water and tested substances are recorded over time and analyzed [249].
The sequence and concentrations of test substances were chosen based on extensive previous studies using different genotypes, and found to be the most appropriate for C57BL/6 mice: The mice are first exposed to 2 mM saccharin, followed by 0.1 mM quinine, 10mM citric acid, 10mm IMP, 75 mM CaCl, 300 mM NaCl, and 0.1% capsaicin in 10% ethanol; all solutions are presented at room temperature. Mice, which show preference for or avoidance of one or more of the tested substances will be further evaluated. However, at this time we have not used any secondary screens due to a lack of potential deviants.
Secondary screens
The choice of secondary screens depends on the results obtained in the primary screen, i.e. the substances tested are determined by the affected taste modality observed in the primary screens. Threshold of preference or avoidance and concentration at which a maximum genotype difference is observed will be determined; this allows distinction between perceptional (i.e. differences between mutant and wild-type mice that become apparent at low concentrations of solutions) and post-ingestive (i.e. differences between mutant and wild-type mice that become apparent at high concentrations of solutions) mechanisms underlying the observed phenotype.
In cases of suspected insensitivity to a stimulus (i.e. animals may be indifferent to a solution in the two-bottle tests even if they perceive its taste, because they perceive it as neither aversive, nor palatable) additional conditioned taste aversion (CTA) tests [250, 251] are performed.
Deviations in acceptance of many compounds suggest a mechanism involving a generalized effect on the taste system. Thus, we count the number of fungiform papillae on the tongue [252] as a way to characterize structural and/or developmental abnormalities in peripheral taste organ or nerve structure. Deviations in acceptance of specific compounds require secondary two-bottle preference tests using serial testing of 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 the compound, its nutritive value, specificity of changes within sub-qualities of the same taste quality or changes in specific appetites. Thus if we find a deviation in saccharin acceptance, we additionally test other artificial sweeteners and sucrose. For HCl deviants, we test citric acid; for quinine we test caffeine, cycloheximide, propylthiouracil (PROP), and phenylthiocarbamide (PTC); for CaCl2, KCl and calcium lactate; for MSG, inosine monophosphate 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 injections of mineralocorticoids [253], treatments that usually increase NaCl consumption. In mice with deviations in ethanol consumption, sensitivity to pharmacological effects of ethanol (locomotor activation or sedation) is studied (254, 255).
