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A. SPECIFIC AIMSTaste solution acceptance is a complex behavior that can be easily measured. We propose to refine

existing methods so they can be used to screen large numbers of mice for aberrations in taste solution acceptance. To do this will require three specific aims:

Specific Aim 1. Fine-tuning the long-term, two-bottle choice test. The most commonly used test to examine the acceptability of taste solutions in mice is the long-term, two-bottle choice test. We propose to fine-tune the already well-established methods for this test. Specifically, we will compare the response of groups of C57BL/6J (B6) and 129/SvJ (129) mice tested during systematic manipulations of drinking bottle spout position, the number of drinking bottles, the test duration, the maintenance diet, and the subjects' age. We will also determine which taste solutions produce carry-over effects (that is, influence solution intake in subsequent tests), and explore procedures to eliminate them. These experiments will allow us to optimize test conditions so as to maximize the likelihood of finding mice with aberrant taste solution acceptance.

Specific Aim 2. Optimizing test conditions to conduct brief-exposure tests using a lickometer. A complimentary method of assessing taste phenotypes is to conduct brief-exposure tests using a lickometer. In this aim, we propose to automate the equipment required for such tests. We will also establish appropriate solution concentrations and test conditions to optimize the likelihood of discovering mice with aberrant taste phenotypes.

Specific Aim 3. Establishing reference data for subsequent identification of mice with aberrant taste phenotypes. The previous aims will establish the best methods for taste phenotyping large numbers of animals. In this aim, we propose to use these methods to test large numbers of B6 and 129 mice, 24 other "reference" strains, 7 strains with known taste deficits, and 8 groups of mice with surgical or dietary manipulations. This will establish reference data for subsequent mass screening of mice and demonstrate the feasibility of detecting genetic differences in taste phenotypes.

A section of the proposal is devoted to administrative issues, including the procedures we will use to disseminate test methods and results. This includes developing a detailed training manual, publishing a database of results, and exploring other ways of providing detailed methods and reference data to interested parties. B. BACKGROUND AND SIGNIFICANCE

The RFA for this proposal provides ample justification for phenotyping large numbers of mice, and we will not repeat the arguments here. Instead, we will discuss why it is important to conduct research on taste solution acceptanceA1 in the mouse, and summarize potential approaches to do this. Why study taste solution acceptance?

There are at least two reasons to study taste solution acceptance. First, studying what an animal drinks tells us about mechanisms of taste perception. Loss of taste reduces intake of palatable solutions and increases intake of unpalatable ones. Understanding the mechanisms of taste perception has implications for health and wealth (e.g., formulation of foods and drinks). Second, taste solution acceptance is a complex behavior that is influenced by physiological state. Disturbances in physiology are frequently expressed as changes in ingestive behavior. This can be quite specific. For example, disturbances of sodium balance increase intake of NaCl solutions(e.g.,30,39,89,91), disturbances that produce hypocalcemia lead to increased intake of calcium solutions(e.g.,110,113), protein deficiency leads to increased protein intake(e.g.,40), and metabolic disturbances such as diabetes alter intake of sweet compounds(e.g.,109). Thus, abnormalities in taste solution acceptance provide a non-invasive indication of dysfunction of many physiological mechanisms involved in homeostasis. Taste and genetics in the mouse

Primarily because of historical antecedents and its larger size, the rat has been the favored rodent for taste perception studies. However, there have been exceptions from "classical" genetics, most notably the development and characterization of mouse strains with various sensitivities to bitter compounds18,19,42,126.

1A?To simplify description, we use the term "taste solution" in the general sense, to refer to any solution or suspension that is ingested, even though these fluids may have trigeminal, olfactory and/or nutritive effects. We use "solution acceptance" to refer to solution intake or preference. We discuss the advantages of various measures of solution acceptance on p29.

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Recent interest promulgated by the revolution in genetic methodologies has redirected several investigators with experience in chemosensory research to join in the hunt for genes involved in taste perception. One breakthrough has been the production of mice with a knockout of the -G protein subunit gustducin gene, which have diminished sensitivity to sweetness and bitterness45,61,62,64,65,106,127. Another has been the localization of a pair of quantitative trait loci on chromosome 4, one of which is the Sac locus60, which together account for more than 50% of the genetic variance in the intake of sweet solutions by C57/BL6 and 129/J mice and their hybrids3. The behavioral data here are complimented by electrophysiological recordings showing that differences in transduction (or a peripheral sensory process) can account for the disparate preference for sweetness shown by the two parent strains3,4,69. Very recently, two G protein-coupled receptors in the apical sensing-end of taste receptors have been characterized (TR1 and TR2)26, and one appears to be localized in the same portion of chromosome 4 as the Sac locus26. It is therefore possible that genetic methods have already exposed a receptor of major importance for the perception of sweetness.

These successes have provided a new impetus to the study of taste perception, and it seems likely that several genes underlying taste receptor structure and function will be identified in the next few years. Nevertheless, many puzzles remain. For example, the TR1 and TR2 receptors are not co-expressed with the -G protein subunit of gustducin. Thus, gastducin-coupled taste receptors remain to be discovered. Moreover, besides peripheral taste reception, there are complex mechanisms responsible for taste coding and integration of sensory input into behavioral output. Many genes must be involved in these higher levels of taste function and ingestive behavior, but they are unknown.

The influence of genes exerting a large contribution to taste perception will be easy to phenotype and thus the genes should be relatively easy to identify. However, as attention turns to genes with smaller or less significant effects on taste perception, it will become progressively harder to discern their contribution. Whereas it has been possible to isolate QTLs with major effects on the acceptance of sweetness and alcohol with groups of several hundred F2 hybrid mice(e.g.,3,16,75,93), it is likely that this strategy will require several thousand animals for genes with lesser effects or epistatic contributions. Similarly, NIH is planning a multicenter initiative to screen many thousands of mice with mutations. Before such an investment in time and money begins, it is prudent to establish methodologies for testing taste perception and acceptance that meet several criteria, listed below. 1. Each test must be rapid or at least require little time of the investigators. This is because of the large

number of subjects involved. A corollary is that the tests involve simple procedures that relatively unskilled laboratory personnel can perform routinely.

2. Each test must be sensitive. Obviously, insensitive tests may fail to detect animals with subtle taste deficits. The more sensitive the test, the more likely it will discriminate animals with unusual phenotypes. Generally, sensitivity can be increased by repeated or prolonged testing, but this is not a viable option for rapid screening of mutagenized mice (see Criterion 1, above). More important for studies of taste perception is the choice of compounds and concentrations of taste solutions to test. Variation in response is seriously curtailed if the taste solutions are highly palatable because all animals respond maximally, leading to ceiling effects. Similarly, highly unpalatable solutions are ingested in such small amounts that floor effects restrict variation.

3. Each test must be highly reliable. If a test is unreliable it is useless for genetic studies. False positive results precipitate an unfruitful investment involving genotyping and/or breeding mice with normal phenotypes. False negative results hide rare mutations. Thus, errors in testing must be kept to an absolute minimum. Like sensitivity, reliability often comes at the price of repeated or prolonged testing but this is not an option for rapid screening.

4. Each test must be independent of other tests. Because it is most efficient for the same animal to be tested more than once, it is essential that tests with one taste compound do not influence the response to other compounds. More generally, the lack of invasive treatments or permanent effects of each test on behavior is particularly important for studies of mutagenized mice because they may be used to investigate a number of phenotypes in addition to taste perception.

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5. Each test must be transferable to other laboratories. A test must be sufficiently robust that it produces the same results with minor variations in conditions, such as different cage sizes, diet, lighting or temperature. If not, it is critical that these conditions are specified and controlled.

Methods currently available to assess taste perception in rodentsHow do the methods that are currently available to assess taste perception in rodents stack up against

these criteria? Below is a description of current methods, together with their advantages and disadvantages from the perspective of screening huge numbers of animals. All the methods have problems. Most of them have so many disadvantages they cannot be adapted to screen large numbers of mice, and we do not intend to pursue them further here. However, we believe the first two have advantages that outweigh the disadvantages, and they are thus worthy of pursuit.

Long-term, two-bottle choice test. The two-bottle choice test (a.k.a. two-bottle preference test or two-tube choice test) has been the standard, workhorse method of assessing taste solution acceptance in mammals since the work of Richter in the 1930's(e.g.,6,22,88,90,99,114,130). In its simplest form, the animal is presented with two drinking tubes, one containing water and the other a taste solution. It is common to conduct sequential 48-h tests involving a range of ascending concentrations of the taste solution being examined. Because rodents can have pronounced side preferences the position of the bottles is switched every 24 h. Variations on the basic theme involve tests using shorter or longer durations, tests with both choices being a taste solution, and tests with three or more choices (often called "cafeteria" experiments). Some of the advantages of this method are that the procedure is very simple and low-tech, and many mice can be tested simultaneously (we have tested >250 at once). There is also a large body of existing evidence, including a growing literature with mice as subjects, to draw from. The measure of preference (intake of solution/total intake) is, within limits, performance- and body size-independent (see also Table 1). There are three main disadvantages of this method. First, although daily measurements can be made very quickly, testing a series of compounds takes substantial time (weeks-to-months) because each test requires a minimum of 48 h. Second, there is no attempt to confine the taste solution to the oral cavity, so intakes and preferences reflect postingestive events as well as chemosensory ones. Third, most likely because of postingestive factors, long-term two-bottle tests are not always independent. There are strong carry-over effects to contend with, although these are usually ignored (see Section C.2 and Experiment 1f, below).

A few studies, particularly in the field of alcohol research, have used long-term tests in which the animal consumes all its fluid from a single bottle. There is no water available. However, this has all the disadvantages of the long-term, two-bottle test with none of the advantages. The only justification for conducting this form of test is to force the animals to drink a non-preferred solution. In this case, the animal's dislike for the solution is pitted against its thirst. Because thirst is determined by both the amount and osmotic load of the ingested solution, interpretation is generally impossible. Although the long-term one-bottle test can be a useful treatment to induce alcohol dependence or hypertension (with NaCl as the drinking solution), we do not consider it a viable measure of taste solution acceptance.

Brief-exposure test using a lickometer. The major problem with the long-term preference test is that it does not provide a "pure" measure of taste; the oral and postingestive effects of the taste solution are confounded. One method that has been used successfully to characterize oral effects without postingestive ones is to conduct a short test. This requires the assumption that postingestive effects are not manifest immediately. Early investigators used 15- or 30-min tests but it is clear that this allows plenty of time for the expression of some postingestive events (e.g., osmotic inhibition of intake). Based on studies primarily in rats, a general consensus developed that tests must be 2-3 min or less in order to minimize postingestive factors (e.g.,29,101,124,125). As the tests became shorter, several problems emerged. One was that it was difficult to make animals drink during short tests without first depriving them of water. Thirsty animals tend to "guzzle" the first solution they come across rather than select among choices. Almost universally, the solution to this problem has been to conduct one-bottle tests (see67 for an exception). Short-term tests are not long enough for thirst to develop so access to water (the 2nd choice) is unnecessary. The second problem is that volume intakes during short-term tests are very small, particularly in small species like the mouse. It becomes impractical to measure volumes of solution ingested under ~1 ml because spillage can contribute more than this. The solution has been to adopt various devices that record individual licks, using a lickometer.

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There are several types of lickometer, differing in the method they use to detect licks. The earliest models involved embedding a phonograph needle into a drinking spout so that when a rat drank from the spout, the vibrations were picked up by the needle, then amplified, and the output of the amplifier fed into a chart recorder. By far the most common method now in use involves passing an undetectable current (<1 µA) through the drinking spout. When the animal drinks it becomes part of a circuit from the spout to the cage floor, which is grounded, and this change in conductivity is amplified, shaped, and recorded by a computer. Another method involves incorporating a miniature strain gauge in the drinking spout so that the pressure of each lick can be recorded. There are also methods in which the movement of the tongue is detected when it crosses a light beam mounted in front of the drinking spout. Weijnen has published several excellent reviews that outline the advantages and disadvantages of each type of lickometer122-125.

A typical experiment involves recording lick rates during the first 2 min after a period of water deprivation. The number or rate of licks is used as a measure of taste acceptance. The advantages of this method are that it provides a "true" measure of taste solution acceptance. The apparatus required is simple. It can be obtained commercially from several vendors or easily fabricated in-house. Because most lickometers are portable, it is also possible to test animals in their home cages. Although the vast majority of work has used the rat as the subject, mice have also been successfully tested (see Section B.4)32,46,53. Another advantage is that with short-term tests carry-over effects are eliminated101. There are also disadvantages of the brief test approach. First, because the tests are short, it is difficult to maintain sensitivity. In particular, a careful balance must be made between the severity of water deprivation and taste solution palatability so that the subject drinks some solution but does not drink at maximal rates (to avoid floor or ceiling effects). This requires careful pilot work. Second, although many animals (typically 8-24) can be tested simultaneously and each test is very short (<5 min), it is impractical to test the animals more than once a day. It may therefore take several weeks of testing to screen several compounds in each animal (see also Table 1).

Gustometer. A variant of the brief exposure test involves measuring intakes using a gustometer20,63,76,85,97,104,105,120. This apparatus consists of a sound proof chamber containing an animal cage with a retractable drinking spout. Typically, the thirsty rat is presented with a taste solution for 10 sec, and lick rates are monitored. In the most sophisticated equipment, the spout is automatically retracted, purged of taste solution, rinsed with water, refilled with another taste solution, and reintroduced to the animal. Simpler designs involve rotating a carousel containing 12-24 prefilled bottles, which allows access to the next bottle. Because exposure to each solution is very short, it is possible to test many solutions (10-20) before satiety occurs. A variation of this method, in which rats receive electric shocks if they continue to drink (or stop drinking, depending on the protocol) various taste solutions, or press one of two bars, has been used to examine gustatory thresholds and taste generalization(e.g.,105). The gustometer has several advantages, most notably the capability to test several taste solutions in a single short (<10 min) trial. However, from the perspective of screening thousands of mice, it has many disadvantages. To our knowledge, the system has not been used to test mice. Indeed, even for rats, there are only limited data, and there have been no studies comparing the system's sensitivity to more conventional methods. We suspect sensitivity is low considering the short test duration and highly motivated state of the subjects. The apparatus is complex, requiring an air pressure source, multiple solenoid valves, and servomotors, as well as intricate "plumbing" of taste solution reservoirs to the dispenser. This is expensive and the complexity leads to increased likelihood of breakdowns. Although the actual test is short, extensive pre-training is required in order to make the subjects drink when required. Typically, multiple solutions are presented to the animal in a random sequence, but solutions ingested toward the end of the test session are disparately affected by previous intakes and the progression of satiety. Normally, this can be controlled for by using a counterbalanced order of solution presentation when groups of subjects are tested, but this is not an option for identification of individual outlying subjects.

Taste reactivity and oral reflexes. In this method, the facial expressions of animals are videotaped. A taste solution is introduced into the mouth through an implanted oral cannula. Stereotyped ingestive behaviors (e.g., rhythmic mouth movements, tongue protrusions, head shakes, forelimb flailing) are scored from the videotape by investigators blind to the test solution the animal received. The primary advantage of this method is that it can be used in severely compromised animals, including young pups. However, the invasive procedures, extensive time required for testing, and limited sensitivity preclude this as useful for screening large numbers of animals. To our knowledge, it has been reported in mice only once51.

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Sham feeding. It is possible to minimize postingestive events using the sham feeding preparation, and thus isolate orosensory signals. Typically, the animal is given an esophageal or gastric cannula which, when unplugged, allows ingested liquids to drain out, and thus eliminates postgastric signals. Although this method has been used extensively in the rat and dog, it has not been attempted in the mouse. Among other problems, the difficult surgery and extensive time required to maintain cannulated animals precludes this as a useful approach for screening large numbers of subjects.

Conditioned taste aversion methods. If an animal becomes sick after consuming a distinctive flavor it will later avoid the flavor. The avoidance generalizes to similar flavors and this property has been used to determine whether various taste solutions have common orosensory properties(e.g., 55,71,92,129. Ninomiya has used the method with some success to characterize strain differences in response to different sweeteners70 and even identify a QTL for sweetness69. Typically, animals are adapted to a severe (>23 h) water-deprivation schedule over several days and then given to drink a "target" taste solution for 10 min. Immediately afterwards, they are injected with the poison, LiCl. After a day or two to recover from illness, the animals receive daily tests with taste solutions of interest. It is often necessary to re-affirm the aversion by repeating the initial target taste-poison trial every few days. Generalization of the aversion is determined by comparing the intake of solutions ingested after poisoning to the intake of the same solutions by animals not conditioned to avoid the target solution. This method has the advantage that it provides a novel measure: the subjects' perception of the similarity of two tastes. However, it is not suitable for mass screening because only one target taste solution can be examined without carry-over effects, extensive daily testing is required, differential sensitivity to the toxicity of LiCl can affect the results, and the subject cannot easily be used as its own control. Moreover, even with the luxury of large, homogenous groups of animals to test, there are thorny issues involving the interpretation of changes in fluid intake as due to generalization of the target taste aversion, accentuated neophobia, or altered motivation to drink.

Gustatory electrophysiology. Considerable progress has been made toward understanding the mechanisms of gustation by making electrophysiological recordings from gustatory afferent fibers while a taste stimulus is applied to the tongue(e.g.,13,17,24,25,36,74,95,128). This procedure requires the subject to be anesthetized, a gustatory nerve (generally the chorda tympani nerve) to be exposed by surgery, and the nerve laid on recording electrodes. The subject's mouth is held open and taste solutions are allowed to flow through the oral cavity for a few seconds. Water "wash-out" periods are interspersed between taste solution presentations. The integrated response of the nerve or change in frequency of individual nerve fibers is amplified and recorded. The major advantage of this method is that the event being measured is peripheral and so transduction events can be isolated from central processing. Another advantage is that each animal can be tested with many taste stimuli (20-40) in a single session, without concerns about satiety or other motivational confounds. However, there are many disadvantages: The procedure is terminal and the dissection procedure requires consummate skill. Electrophysiological recording is also not very amenable to finding individual differences because there is wide variation in response due to the placement of the electrode relative to the nerve. Finally, the terminal nature of the procedure precludes efficient breeding strategies. These disadvantages make gustatory electrophysiology clearly inappropriate for mass screening of mice.C. PRELIMINARY RESULTS

Our laboratory is one of very few actively pursuing the genetics of taste solution acceptance. Some of this work can be found in the eight papers in the Appendix. Manuscripts (MSs) 1-5 describe research on taste solution acceptance in mice. MS6 and 7 describe research in rats, which demonstrates the range of taste solutions that can be tested and the utility of the "lickometer" apparatus to be used in Specific Aim 2 and 3. The final paper (MS8) is a review on the issue of specific appetite. This is included because it contains a comprehensive discussion of the pitfalls of taste solution research.

Most of the results presented in this section come from completed, unpublished experiments and have direct pertinence for the methods and goals of this project. 1. Studies using long-term, two-bottle choice tests to examine taste solution acceptance

We have conducted many experiments comparing the intake of various taste solutions by various strains of mice. The largest have involved phenotyping 670 F2 B6x129 mice, 450 F2 NZBxCBA mice, over 600 backcross mice congenic for the Sac locus, and ~670 mice from different inbred strains. It would be a

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formidable and somewhat immoderate task to test every taste solution with every strain of mouse. Instead, our general approach has been either to survey a large number of taste compounds in just two strains or a smaller group of taste solutions in several strains. We have concentrated on the C57BL/6ByJ (B6) and 129/J (129) strains because early work showed these strains differed in NaCl acceptance12. This was a propitious choice given that the C57BL/6J strain appears to be the most common choice for mutagenesis studies and the 129/SvJ strain has been used as a background strain for knock-out mice generation.

An unpublished example of multiple phenotyping in the B6 and 129 strains is our work on sweeteners. Using standard 48-hr, two-bottle choice tests, we have obtained concentration-intake functions from B6 and 129 mice given 18 different sweeteners (Fig. 1). We found that the mice showed three patterns of response. For some sweeteners, B6 mice had lower preference thresholds and higher intakes than did 129 mice but both strains exhibited a strong preference for high concentrations (e.g., sucrose, maltose, saccharin, acesulfame, and SC45847). For other sweeteners, the B6 mice had strong preferences but the 129 mice did not strongly prefer any concentration (e.g., D-phenylalanine, D-tryptophan, L-proline and glycine). The third group of sweeteners includes compounds that taste sweet to humans but neither strain showed a preference for them (e.g., aspartame, thaumatin, glycyrrhizic acid, neohesperidin hydrochalcone, and cyclamate). These results, together with data from gustatory electrophysiology and human psychophysics, argue that sweetness is not a unitary phenomenon but instead there are several types or configurations of (the) sweet receptor (see Section D, below).

With respect to the approach using multiple strains and few phenotypes, the most ambitious example is our recently funded grant (DC-03854) to assess strain differences in the response of 28 strains of mice to various salts (NaCl, KCl, CaCl2, NH4Cl), umami-like compounds (MSG, inosine 5'-monophosphate), acids (hydrochloric, citric and L-glutamic), bases (NaOH, Ca(OH)2), and irritants (capsaicin and menthol). At the time of writing (April 1999), we have completed assessment of all 28 strains with NaCl and 13 strains with all the other salts and acids8. There are several purposes to such studies, including identification of common strain patterns (and thus perhaps underlying genes), and common behavioral responses. However, this work is important for this proposal because it demonstrates our experience in finding appropriate test conditions and taste solution concentrations for conducting two-bottle choice tests. We will build on this expertise to help design the experiments proposed here.

It is noteworthy that conducting 48-h two-bottle choice tests can be done on a large scale. We have successfully tested cohorts of more than 250 F2 B6 x 129 hybrid mice with a series of 12 taste solutions. The tests can also produce meaningful results with animals with motor deficits. In collaboration with Dr. Ralph Puchalski at this institute, we have recently found that mice with a knock-out of the Isk gene, which encodes for a potassium channel found in kidney and tongue, had attenuated NaCl solution acceptance relative to their wild-type controls. We could tell this from preference scores even though some of the animals had gross motor problems, including intense circling behavior similar to that seen with middle ear disease78.

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2. Order and concentration effects in long-term taste testsSeveral methodological issues can limit interpretation of the long-term two-bottle

choice test. Particularly pernicious when testing several taste solutions in the same animal is the existence of carry-over effects. The ingestion of one solution can influence intake of another solution on the next test. Figure 2 (previous page) shows a severe example in 12 B6 mice that were tested with 6 NaCl concentrations; half the mice received them in ascending order and the other half received them in descending order. Intake and preferences for NaCl were dramatically affected by solution presentation order. We have seen similar effects with other strains, and some (but not all) other taste solutions. Such carry-over effects can be tolerated in studies with counterbalanced designs but this luxury is not available for mass screening of individuals. In Section D (Experiment 1f), we propose experiments to identify and control for carry-over effects.3. Assessment of taste responses in mutagenized mice

In collaboration with Dr. Maja Bucan (Dept. Psychiatry, Univ. Pennsylvania), who is a consultant for this project, we have conducted a pilot study to determine the practicality of screening large numbers of mutagenizedA2 mice. Based on our work with B6 mice described above, we selected concentrations of several taste solutions that produced a clearly expressed and homogeneous behavioral response. A cohort of 180 male and female C57BL/6 mice were tested. These mice were the progeny of male C57BL/6 mice that had been injected with N-ethyl-N-nitrosourea (i.p.) and subsequently mated with intact C57BL/6 females. They were transferred to our facility after they had been phenotyped for circadian activity in Dr. Bucan's laboratory. Ten solutions were tested, using 48-h or 96-h two-bottle choice tests, according to the general methods outlined below.

Analysis of frequency distributions (Fig. 3) showed that not all solutions tested were equally appropriate as taste stimuli for mutation screening. The variability of preferences for 10% ethanol, 0.1 mM citric acid, 250 mM NaCl and 0.03 mM quinine was such that reliable deviations could not be detected (preference scores of individuals that did not discriminate taste solution from water were within ± 3 interval). However, 4% sucrose, 2 mM saccharin, 30 mM d-phenylalanine, 50 mM citric acid, 0.3 mM quinine and 1 mg/l capsaicin did appear to be appropriate for screening.

Several mice avoided 0.3 mM quinine much less than did the rest of the animals, which could be due to mutations. The two mice deviating most were crossed with non-deviating mice; and their progeny were retested. Unfortunately, we did not see any evidence for genetic transmission of abnormal quinine avoidance in these progeny.

This pilot experiment taught us several things. In particular, the choice of appropriate taste solution concentrations is critical for producing sensitive tests. For example, it would be impossible to detect a mouse with abnormal ethanol preference using these methods. We do not know if this is a problem specific to the concentration of ethanol we used, a result of carry-over effects from previous tests, or some other methodological problem. Nevertheless, we were able to successfully test multiple taste phenotypes in a large cohort of mutagenized mice. 4. Lickometer studies of NaCl and alcohol acceptance

We have also conducted studies involving giving mice brief exposures to taste solutions with a lickometer. For example, in one experiment, B6 and 129 mice were water deprived during the first 6 h of the dark period. We then monitored lick rates during the first 15 min of fluid access each day. During the first week, the mice received water. During the second, they received 10% ethanol during the

2A?In most cases, including this one, it is the progeny of mutagenized mice, not the mutagenized mice themselves that are tested. However, for simplicity of description, we refer to the offspring with potential mutations as "mutagenized".

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FIG. 3. Frequency distributions of 180 mutagenized mice given 48- or 96-h two-bottle choice tests with 10 taste solutions.

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15-min test. During the third week, the mice were given constant access to 10% ethanol (data not shown), and then, during the fourth week, they were retested with 10% ethanol. There were no differences between B6 and 129 mice in water intake (Fig. 4). However, the B6 mice licked more for ethanol on every trial. Repeated testing, and even continuous exposure to ethanol for a week had no influence on the mice's lick rates, indicating there were no carry over effects evident in these brief-exposure tests. 5. Summary of Sections B and C

Taste solution acceptance is a complex behavior that gives insight into physiological state as well as taste perception. It involves many genes but, so far, only a few of them have been discovered. It can be measured easily and noninvasively. There are several methods of taste phenotyping but only two have been used to any extent in mice, and these two are the only ones with more advantages than disadvantages for mass phenotyping (see Table 1).

We have experience with both candidate methods of phenotyping. We have extensively phenotyped a large number of mouse strains for a few taste solutions, and two strains (B6 and 129) for many taste

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FIG. 4. Cumulative number of licks by B6 and 129 mice given 15-min tests drinking water (top row), 10% ethanol (middle row), and then 10% ethanol after 2 wk continuous access to ethanol. The aberrant lick rates on the first Wednesday test were due to a technical error.

Table 1. Comparison of the advantages and disadvantages of long-term, two-bottle choice tests and brief-exposure, lickometer tests

Feature Long-term, two-bottle choice test Brief-exposure, lickometer test

Equipment required Drinking tubes Drinking tube, lickometer amplifier, computer interface, computer

Dependent variables Taste solution and water intake, total fluid intake, taste solution preference

Lick rate and number

Test duration Typically 48 h per taste solution, with additional "wash-out" days

Typically 2 min per taste solution with 24 h between tests

Test capacity >300 mice/day. Limited only by time required to collect data.

8-24 simultaneous tests, limited by equipment but many replications can be

conducted each day Existing literature Strong Small, particularly for mousePerformance Fairly independent of mouse's physical

competencePhysical competence can affect licking rate

Scope Wide. Can detect aberrant intakes due to postingestive factors as well as chemosensory ones.

Narrow. Detects aberrant intakes due to chemosensory factors only.

Interpretation Complex because oral and postingestive factors are confounded

No postingestive factors to worry about

Independence Carry-over effects can confound intake on later tests

No carry-over effects

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solutions. Most relevant for this project, we have conducted a pilot study in which 180 mutagenized mice were screened with 10 taste solutions, using 48-h two-bottle tests. We have also conducted studies to examine the stability of licking responses in brief-exposure tests. Both two-bottle choice tests and brief-exposure tests can potentially be used for screening large numbers of mice. However, we believe the standard methods can be improved. The following section outlines potential improvements and how we will go about evaluating them.

D. RESEARCH DESIGN AND METHODSGeneral methods

All experiments will use mice purchased from Jackson Laboratories or bred in-house. Unless otherwise noted the mice will be individually housed under "standard conditions", which for our facility involves a 12:12 h light/dark cycle with lights off at 6 p.m., a temperature of 23 ± 1°C, and humidity of 45-85%. Each cage is made of plastic and measures 31 x 19.5 x 13 cm. The lid is made of a stainless steel grill with a wedge-shaped recession to hold pelleted food and a water bottle. Cages are held on two-sided racks with 12-16 mice per shelf and 5 or 6 shelves. The food is Teklad Rodent Diet 8604. Bedding is pine shavings.

Generally, the mice will be 7-8 weeks old when we begin testing them, and will have been adapted to individual housing and vivarium conditions for at least 10 days. Our work in the past has concentrated on the C57BL/6ByJ and 129/J strains for the reasons given above (Section C.1). Because slightly different substrains are used more prevalently by the mouse genetics community and are thus more likely to be targets for mutagenesis studies, all work with B6 and 129 mice in this project will use the C57BL/6J and 129/SvJ substrains (see98 for discussion of genetic variation among 129 substrains). Several studies have compared taste acceptance of various B6 and 129 substrains, and the differences were small57-60. It is extremely unlikely, and would be extremely informative, if there were differences in taste solution acceptance between mice of the same strain but different substrain.

Body weights will be collected from each mouse at the beginning and end of each test sequence. The mice will also be examined for disease or other problems each time their bedding is changed, every 3-4 days.Drinking tubes

Construction. The "bottles" or "tubes" used for two-bottle choice tests are in fact the barrels of graduated 25-ml polystyrene serological pipettes (Fisher, Springfield, NJ). A 6.4-cm long stainless steel sipper tube (Unifab, Kalamazoo, MI) is inserted into one end of the pipette and the other end is plugged with a rubber stopper. The sipper tubes have 3.175-mm diameter holes. The drinking tubes will be inserted into the mouse cage so that the tips extend 2.5 cm into the cage. Unless otherwise noted, the two tips will be ~2 cm apart.

Measurements and analyses. For long-term tests, intakes will be measured at the same time every day (in the middle of the light period) by reading values from the graduated scales on the side of the burettes. This allows an accuracy of ~0.2 ml. Extensive experience has shown that spillage and evaporation from these tubes is minimal (<0.05 ml/day) and so will be ignored.

In general, daily intakes of each solution and water will be averaged to provide 24-h daily intakes. In addition to these "raw" intakes, several others will be derived. These include intakes corrected for body weight (intake/body weight x 1000 g), total intakes (water intake + taste solution intake), and taste solution preference (taste solution intake/total intake). Some investigators include corrections for surface area or other factors related to mouse size(e.g.,86). Generally these measures covary closely unless body weights vary considerably or intakes are very extreme, allowing basement or ceiling effects. We have discussed the advantages and disadvantages of each of these measures at length (6,9 in Appendix). Suffice it say, here, we will conduct analyses using all the derived dependent variables in order to compare which produce the most easily interpreted results. Solutions to be tested

The selection of appropriate taste solutions to test is among the most critical methodological challenges. Fortunately, we have had considerable experience testing a wide range of taste solutions in mice and rats (see MSs 1-7 in Appendix), and we intend to draw upon this to select panels of solutions to be screened.

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Traditionally, taste perception has been divided into four primary tastes; sweet, salty, sour, and bitter. However, in the last few years, this quadripartite domination has been challenged, if not abandoned. It now appears that at least three of the primary taste qualities have subqualities (sweet10,34,54; salt31,43,48,52,94,100; bitter11,126) Moreover, there are some tastes that cannot easily be categorized as sweet, sour, salty, or bitter (see Table 2). The evidence for some nontraditional tastes, such as umami, is extremely solid50; for others it is less complete and controversial(e.g., 79,80,96). Some compounds have trigeminal and/or olfactory effects and so are not specifically taste stimuli. We are well aware of these issues but they are largely irrelevent for the purposes of finding mice with abnormal phenotypes. We believe the best strategy is to first identify animals with aberrant intake of any solution, and then worry about whether this is due to dysfunctional mechanisms involved in taste, the trigeminal sense, motivation, etc.

Table 2. Chemosensory qualities and candidate taste modalities and subqualities, with examples and concentrations of solutions to be tested

Quality Subqualities Examples Conc. to be tested Refs

Sweet sucrose-like sucroseglucosesaccharin

120 mM120 mM 2 mM

3,4,6,16,59,60,75 Unpublished results

amino acid-like d-phenylalanine 30 mMaspartame-like aspartame 1 mM

Salt amiloride-blockable sodium channels

NaCl 75 mM 300 mM

4-6,9,12, Unpublished results

nonspecific/mineral KClCaCl2

400 mM 100 mM

Sour none HClcitric acid

30 mM 50 mM

4 Unpublished results

Bitter quinine-like quinineHCl 0.3 mM 5,18,19,42,47,57,58,126SOA-like SOA 1 mM

Umami none MSG 300 mM 7 Unpublished results

Starch or maltodextrin

unclear Polycosemaltodextrincornstarch

60 g/l 60 g/l 60 g/l

Unpublished results

Fat unknown corn oil 10 g/l Unpublished results

Texture and irritation

touch/texturetemperaturepain

capsaicinethanol

1 mg/l 10 g/l

4,6,16,93Unpublished results

Notes: The strength of the evidence for various modalities and subqualities varies substantially (see text). The "concentration to be tested" is determined from previous data primarily from our laboratory (given in References column). SOA = sucrose octaacetate, MSG = monosodium glutamate, IMP = inosine 5'-monophosphate.

The criteria we have used for selecting taste solutions are as follows: 1. The solution must represent a specific taste quality or nutrient, with at least circumstantial evidence for its transduction. 2. The solution concentration must evoke a clear response. This can be either preference or avoidance, but not indifference because indifference cannot be distinguished from a failure to perceive. 3. There must be little non-genetic (within-strain) variation in response to the taste solution. Having stated the criteria, we admit that because of limited information about the underlying taste transduction mechanisms we are forced to make compromises. One is that the specificity of some representative taste solutions for their corresponding taste qualities is not always clear. Thus, for example, KCl is both a non-sodium mineral and a bitter compound. (This lack of

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Table 2a. Technical notes about preparing taste solutions: Some of the compounds listed in Table 2 are insoluble in water but for simplicity, we refer to them as solutions. Malto-dextrin, cornstarch, and corn oil will be held in suspension by homogeniz-ation with 0.1% xanthan gum. Capsaicin will be first dissolved in 90% ethanol and then diluted to the appropriate concentration with water, giving a final ethanol concentration below taste threshold (0.1%). For these compounds, two-bottle tests will involve a choice between the taste "solution" and its vehicle (either 0.1% xanthan gum or 0.1% ethanol). For brief-exposure studies, a sub-threshold concentration of NaCl (e.g., 0.1 µM) will be added to the insoluble suspensions to provide the electrical conductivity required for the contact lickometer to work.

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specificity is similar to the nonselective actions of many neurotransmitter agonists and antagonists). In such cases, it may require more than one taste solution to represent a taste quality. We also include alcohol as an irritant, although it has complex orosensory properties (e.g., sweetness, bitterness, irritation, odor)51, primarily because of the practical importance of understanding alcohol consumption.

We are keenly aware that when testing mutagenized mice, it will be necessary to balance between providing a comprehensive scan on the one hand and keeping the number of tests within manageable limits on the other. Clearly, we could increase the chances of finding taste aberrations by testing each animal with other compounds (not listed) and multiple concentrations of each solution. However, screening even the test panel we have offered will be a major (although manageable) undertaking. We believe it is better at this stage to provide data on a wide variety of taste solutions rather than restricting analysis to just a few. We discuss later how we will prioritize which solutions to test if time constraints dictate a more limited approach (see Making recommendations about how and what to test, below).

An approach we have considered to reduce the number of test solutions is to mix two or more taste solutions together. If an aberrant phenotype is found with a cocktail then its components could later be presented individually to find the source of the aberration. This would be a very efficient strategy, but we doubt it will work. Mixtures do not necessarily taste like the sum of their components (taste synergy is common) so it would be unclear what was being tested. A loss of sensitivity to one taste solution might be masked by increased sensitivity to another. Moreover, the sources of variance will increase (due to each taste solution and their interactions), making it less likely we will detect modest aberrations. If an animal cannot detect the taste of a mixture, it will also fail to detect a single component. But if an animal cannot detect a single component, it still might be able to detect a mixture. Thus, testing single components will detect more deviations. We think this idea warrants pilot work but we do not propose to test it formally here.

Short test sequence. For most of the experiments listed in Specific Aim 1 there is no reason to believe that the results are likely to depend on specific taste solutions so it will be neither necessary nor efficient to test a large array. Consequently, we will use a condensed, or short test sequence for these experiments (see Table 3)

These solutions were chosen to encompass the four classic taste qualities (sweet, salty, sour, and bitter). The concentrations were chosen based on our previous work and existing literature involving tests of hundreds of B6 and 129 mice and their hybrids4,6,12,42. This work indicates the two strains find the chosen concentrations of saccharin and NaCl moderately palatable and those of citric acid and quinine moderately unpalatable. The B6 and 129 mice respond differentially to all four solutions (B6<129 for sweet; B6>129 for salty, sour and bitter; see Appendix). Our extensive experience with these solutions indicates that if carry-over effects exist, they are minimal and not a problem (75 mM NaCl can produce very mild carry-over effects under some conditions (see Section B, above), and so will be tested last in each series).Specific Aim 1. Fine-tuning the long-term, two-bottle choice test

The long-term two-bottle choice test is the most accepted method of examining taste solution acceptance in animals and, unlike other methods of taste phenotyping, there is a strong literature using mice as subjects. The method is simple to perform and the equipment required (drinking tubes) minimal. As discussed above (Section B and Table 1), the two-bottle choice test has many advantages for mass phenotype screening and few disadvantages. However, the requirement to screen large numbers of mice raises a number of methodological issues and questions that have not been addressed. The goal of this specific aim is to select the most efficient strategies for conducting choice tests.

General method . The experiments will use groups of male mice aged 2 months at the start of testing (unless otherwise specified). Experiments 1a - 1e will involve groups of 16 B6 and 129 mice. These strains differ in preference for the taste solutions to be tested4,6,9. For most experiments, each mouse will be tested several times with the "short test sequence" identified in Solutions to be tested (Table 3), above. Unless otherwise mentioned, each solution will be presented for 48 h, with the position of the water and taste solution drinking tubes switched after 24 h. The taste solutions will always be given in the same order, but

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Table 3. Solutions to be used in the short test sequence

water 2 mM saccharin 50 mM citric acid 0.3 mM quinine hydrochloride 75 mM NaCl

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the order of each sequence of tests will be counterbalanced according to a modified Latin square design. This will ensure that approximately equal numbers of mice from each strain are exposed to each test condition at the same time.

General statistical analyses. In each of the experiments in this section we propose to compare the same animals tested under various test conditions. Although it will be interesting to see if the manipulations influence mean intakes of solutions, for the purposes of this project it will be more important to assess whether there are differences in the heterogeneity of the response (i.e., differences in variance, assessed by a Test for Homogeneity of Related Variances21). In general, tests that result in low within-strain variability will be considered better than those that have high within-strain variability. However, because it is feasible to have low variance but low predictive power if the test also results in strain means that are close together, the best measure of the "success" of each treatment will be its ability to discriminate between the two strains of mice. This can be assessed by comparing the F statistics for similar between-strain comparisons from each pair of conditions.

Simply comparing F statistics will not provide a measure of whether one F value is significantly greater than another. To do this, we will use the following analysis: Data for each test will be converted to z scores based on the mean and variance of the B6 mice. This will provide z scores for the 129 mice based on their separation from the B6 mice. The 129 mice z scores obtained from each condition can then be compared by t-test, and the resulting t value assessed for significance using standard criteria (i.e., p<0.05). Similar statistical methods are used to discriminate signal from noise in taste psychophysics (e.g,, d' or m41,72).Experiment 1a - Influence of drinking bottle spout position on test sensitivity

In the typical two-bottle test, each mouse is allowed to choose between a taste solution and water. In our experiments, which are typical of those conducted by other investigators, two drinking tubes rest in the space that is normally occupied by a water bottle, such that the spouts penetrate the mouse cage about 2 cm apart. Food is available from a hopper adjacent to the (mouse's) left-hand drinking tube. The extent to which the position of the tubes influences solution preference has not been systematically examined. However, there are occasional observations that suggest this is not a trivial issue. For example, rats have been observed to use "oral mixing" by rapidly alternating between drinking from one tube and then another1. This behavior occurred when the drinking spouts were close together (2.5 cm) but not far apart (11.5 cm). It is also well known that mice can have strong side preferences. When two similar solutions or two tubes of water are presented it is rare for intakes from each tube to be identical, and not uncommon for mice to drink >85% from one tube. There are several studies showing that paw preference differs between strains of mice(e.g.,14,15) and it seems reasonable to assume that this may generalize to drinking tube position preference. It is also possible that some mice simply prefer to drink from the tube closest to the source of food or edge of the cage.

Two methods have been used to control for side preferences. One is to counterbalance across groups, so that half the animals in each group receive the taste solution on the left and the other half receive it on the right(e.g.,22). This is not feasible when looking for individual differences, as is the case here. The other method is to switch the position of the tubes half way through the test, so that each tube is presented on each side for an equal time. This dictates that the test duration must be 48 h (or a multiple of 48 h) because the diurnal cycle makes it impossible to switch bottles at earlier times and still match exposure. A 48-h test is no problem for most experiments, but with many solutions and thousands of subjects, reducing the time of each test from 48-h to 24 h could save substantial amounts of time and money. The purpose of this study is to determine whether the distance apart of the drinking spouts affects solution intake and/or preference.

Method . The cleanest design would be to compare intakes from spouts placed at several distances apart. However, this is impractical because a standard mouse cage is 19.5 cm wide, and half this width is occupied by the food hopper. Thus, without removing the food, the range available is only 2 - 8 cm (allowing space for the drinking tubes). Moreover, because the position of the food hopper may affect side preferences, this factor should also be taken into account. Thus, we plan to test groups of 16 male B6 and 129 mice under 5 conditions, in counterbalanced order:

(1) "standard" conditions, with the drinking spouts placed 2-cm apart(2) "standard" conditions, with the drinking spouts placed 8-cm apart (3) food on the floor, with the drinking spouts placed 2-cm apart

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(4) food on the floor, with the drinking spouts placed 8-cm apart(5) food on the floor, with the drinking spouts placed 18-cm apartIn each condition, each mouse will receive the "short series" of taste tests identified in Solutions to be

tested, above. In the last three conditions, a few grams of food will be placed in a pile in the middle of the back wall of the cage, equidistant and as far away as possible from the drinking spouts. Because the mice are likely to spread the food around the cage this is not a perfect control for food position, but we will look for any marked patterns in where each mouse chooses to place its food.

Analyses of results and interpretation. The results will allow us to compare the extent to which taste solution intake depends on (1) the distance between the drinking spouts, and (2) the proximity of a spout to food. The effect of interspout distance will be assessed using planned comparisons between conditions 1 vs 2, and 3 vs 4 vs 5. The effect of food proximity will be assessed using planned comparisons between conditions 1 vs 3, and 2 vs 4. A number of subsidiary calculations will allow us to assess whether the expression of side preferences is influenced by spout position. For example, comparison of the variance between the first test in which a choice of water vs. water is presented will give a measure of side preference independent of the effect of taste solutions.

From a practical viewpoint, the most satisfying result would be if the various conditions had no effect on solution intake or preference. This would allow us to continue with less concern about these factors, and it would not be necessary to formalize as "critical" this aspect of test methodology in the protocol sheets we will develop. If there are substantial differences in intake, we would then choose the condition that produced the largest differences between the two strains of mice, and use this in all subsequent work.Experiment 1b - Comparison of the sensitivity of two- versus three-bottle tests

For the reasons discussed above, the two-bottle test has been the mainstay of taste solution testing for over 60 years. It was clear from the beginning that side preferences influence choice, particularly when the subject is indifferent to the taste solution or finds it only barely discriminable from water. The remedy has been to control for side preference by switching the drinking tube position half-way through the test. This extraneous variable adds to the variation in response of individual subjects. To obviate this problem, it would be much better to eliminate than control for side preferences. The only practical way we can think of to do this is to present the taste solution flanked on either side by water (or vice versa). In this experiment we plan to investigate whether this three-bottle method has significant advantages over the two-bottle one.

Method. Groups of 16 male B6 and 129 mice will be tested. Each mouse will be tested three times with the "short sequence" of solutions identified in Solutions to be tested, above. During one series, the solutions will be presented using the standard two-bottle test procedure. During another, the mice will be presented with three drinking tubes spaced evenly apart (the distance between spouts will be determined from the results of the previous experiment). The middle tube will contain the taste solution and both the two outer tubes will contain water. The third series will be similar to the second, except both outer tubes will contain taste solution and the middle tube will contain water. Each of the five tests in each series will last 48-h. Tube positions will not be altered in the 3-tube conditions.

Analyses of results and interpretation. When the mice are tested with three tubes, intakes from the two tubes containing the same fluid will be combined to obtain a single value for intake of water or taste solution. These values will then be used for statistical analyses in a similar manner to the previous experiment.

The main question to be answered by this experiment is whether three-bottle tests produce better discrimination between the two groups of mice than do two-bottle tests. Is the reduction in variance produced by eliminating side preferences worth the added complexity of the three-bottle test?

The main potential advantages of the three-bottle test are that it could (a) reduce variability, and (b) cut testing time in half, from 48 h to 24 h per test. A more subtle but equally important potential advantage is that it will increase the range of preference variability. With a two-bottle test, the range of preferences varies from 50% (indifference or undetected) to 100% (intake of only the test solution) for solutions that are liked, or 50% to 0% (total avoidance) for solutions that are disliked. Thus, the effective range for any solution concentration is 50%. With a water-solution-water three-bottle test, the expected level of a solution that the mouse cannot detect or finds indifferent is 33%, so the effective range is 67% (i.e., 33% - 100%) for a palatable solution and 33% for an unpalatable one. The situation is reversed for the solution-water-solution

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three-bottle test. Thus, by judiciously choosing the type of three-bottle test it may be possible to substantially increase the range, and thus sensitivity of the test, which would increase the likelihood of spotting mutant mice with only moderately altered taste preferences.

A question that arises naturally from this line of inquiry is "Why stop at three bottles?" Is it feasible to provide each mouse with a choice between many bottles of various taste solutions and allow it to choose? With enough bottles available simultaneously, it would be possible to test all the solutions listed in Table 1 at once! Versions of this "cafeteria choice" method have been used extensively with rats (e.g.,87,89,118). However, there are both practical and theoretical reasons it will not work for individual animals, the greatest being the enormous within-subject variability. Nevertheless, if time is available, we may conduct studies using the cafeteria choice method using some of the mice that complete Specific Aim 2 (see below).Experiment 1c - Influence of test duration on test sensitivity

In general, the longer the test, the more stable are average intakes. Typically, two-bottle tests are conducted for 48 h, but occasionally shorter or longer intervals are used. There is no rigorous basis to determine whether longer tests produce significantly more reliable results. To rectify this, in this study, we will compare the results of tests differing in duration.

Method. Groups of 16 male and female B6 and 129 mice will be tested. Each mouse will be tested several times with the "short sequence" of solutions identified in "Solutions to be tested", above. The type of test (two- or three-bottle) will depend on the results of the previous experiment. The duration of the tests will be 1 (only if a three-bottle test is used), 2, 4, or 6 days.

Analyses of results and interpretation. The main question to be answered by this experiment is whether longer tests produce significantly greater stability of intakes and thus greater discrimination between 129 and B6 mice than do shorter tests. It would be most propitious if test duration had little or no effect on the stability of intakes because it would then be safe to recommend using 2 day tests, or even 1-day tests if the 3-bottle method is successful. If test duration significantly affects the results, the advantages of increased test sensitivity will have to be carefully weighed against the disadvantage that less taste solutions can be assessed in the same time.

Several interesting subsidiary analyses will be conducted. One will be to determine whether intakes during the 1st, 2nd, and 3rd 2-day period of 6-day tests are similar, both to intakes during the other two periods and to intakes when the tests are only 2 or 4 days long. Animals frequently show neophobia to unpalatable taste solutions. For many compounds, such as sucrose and NaCl, they have higher intakes on the first day of a test than subsequent days (perhaps because of the postingestive modulation of intake). Judicious statistical comparisons will allow us to analyze the impact of these factors on the variability of intake during the first 2-day period.

Both male and female mice will be tested in this experiment to determine whether the periodicity of the females' ~4 day estrus cycle affects solution intake. If this is a major contributor to variability it would be reflected by lower variability in 4-day tests than either shorter or longer tests. Although there is evidence for estrus-related periodicity of taste solution intake in rats, the effects tend to be fairly small(e.g.,49,119). If they prove to be more substantial in mice, we would be forced to recommend mutagenesis phenotyping tests utilize females during a specified phase of estrus, which would add the burden of monitoring the estrus cycle and substantially increase the duration of each experiment, or, more likely, confine testing to male mice only. Experiment 1d - Influence of maintenance diet on taste solution test sensitivity

Typically, the contribution of diet to taste solution preference is ignored, and to our knowledge, there are no studies of diet-taste interactions performed with mice as subjects. However, there are many examples from the rat literature. Some are very obvious. Feeding an unpalatable diet increases intake of sucrose and other palatable drinks (see111). Feeding a high-salt diet increases water intakes (in order to counteract the osmotic effects of the dietary salt), which can decrease preference scores if taste solution intakes are not also increased. There are also more subtle nutrient-dependent effects. Rats fed high-fat diets show stronger preferences for a variety of fats and fat-like compounds than do controls fed low-fat diets81-83. The gustatory electrophysiological response to sodium is modulated by dietary sodium content24,77. These diet-taste interactions are not always straightforward. For example, we found that modest manipulations of diet calcium content influenced intake of 24 of 35 taste solutions tested, including

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representatives of all the taste qualities listed in Table 122,111. We emphasize that the dietary manipulations can be subtle and still have large effects on intake. Rats fed diet containing 129 mmol Ca2+/kg (the level in AIN-76A diet) drank twice as much 300 mM NaCl as did rats treated identically except fed a diet containing 250 mmol Ca2+/kg (the level in Purina chow). The experiment below is designed to investigate whether taste solution intake is influenced by differences in diet that are likely to occur between different institutions.

Method. Groups of 16 male B6 and 129 mice will be tested according to a between-subject design. It is more efficient to use a between-subject design for this experiment because it requires at least two weeks for the mice to adapt to a new diet. One group from each strain will be fed (a) Teklad Rodent Diet 8604, (b) Wayne lab Blox, (c) Purina rodent chow no. 5001, (d) Purina rodent chow no. 5012, (e) Lab Diets Rodent Diet no. 5001 (also called Richmond Standard Diet), (f) AIN-76A diet, (g) AIN-93 diet. The first five diets listed are cereal-based diets ("chows") in common use in institutions with large mouse colonies. Purina chow no. 5012 is a high-energy diet designed for breeding colonies whereas the other four diets are maintenance diets. The last two diets listed are semisynthetic diets recommended for rodents by the American Institute of Nutrition2,84.

The mice will be adapted to the appropriate diet for at least 3 weeks then given the "short sequence" of solutions identified in Solutions to be tested, above. The precise test methods will be chosen based on the results of the previous experiments but will be identical for each diet condition.

Analyses of results and interpretation. Analyses will be conducted in the same manner as for the previous experiments. It will be interesting to determine which, if any, diet produces the largest differences between B6 and 129 mice. Note that this experiment is designed to give a practical and not a theoretical outcome. The diets differ along many dimensions (e.g., texture, palatability, energy, nutrient and micronutrient contents) and it will not be possible to determine why some diets produce clearer differences among the groups than others. It would take an extensive series of follow up studies involving systematic variation of diet composition to isolate the variables involved. But for the purposes of the mutagenesis project this is not necessary.

One comparison of particular interest will be to determine whether the semisynthetic diets produce clearer results than do the chows. The ingredients in chow are controlled to some extent, but there can be large fluctuations within each brand, depending on the sources of protein available to the manufacturer during production. On the other hand, the formulations of AIN-76A and AIN-93 are rigorously fixed, and there should be little or no difference in these diets depending on the source of ingredients. It is noteworthy that the leading journal in the nutrition field (J. Nutr.) has been known to reject articles if the animals are fed chow because its ingredients are unknown. Nutritionists consider chow with the same horror that a physiologist would consider bathing tissue in diluted sea water rather than Ringer's solution, or a geneticist would use random-bred rather than inbred strains. If the results of our tests are auspicious we would recommend the use of defined semisynthetic diets for all experiments in the mutagenesis program.

It is possible that diet composition could be manipulated in a manner that increases choice test sensitivity. For example, adding NaCl to the diet would increase fluid intake, and may thus increase signal-to-noise (i.e., intake-to-spillage and evaporation). Although we do not specifically propose to follow this approach here, we intend to conduct pilot experiments to assess if it is a worthwhile approach. Experiment 1e - Characterization of changes in taste solution acceptance with age

There has been very little work on the effect of age on taste solution intake, even in rats35,38,56,66,73, and what work there is has concentrated on the transition from pup to weanling. Because the mutagenesis program is likely to require that taste phenotyping be coordinated with other behavioral tests, it will not always be possible to specify the age at which mice will be available for testing. In this experiment, we will determine to what extent age influences taste solution intake.

Method. Groups of 16 male and female B6 and 129 mice will be tested according to a mixed design. One group of each sex and strain will be tested with the short sequence of taste solutions, starting at the following ages: 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 75, 100 weeks. The ages are chosen to span the life cycle, with greater concentration on younger mice, when physiological changes are occurring most rapidly, and which will probably be most relevant for mutagenesis phenotyping. Both sexes will be tested because, unlike the previous manipulations, there is reason to believe sex hormones will have different effects at different times (e.g., before and after puberty). We anticipate testing all the mice simultaneously. This will

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require careful coordination as we will have to maintain younger mice in our own colony for several months before they are old enough to test. We also intend to retest the youngest groups repeatedly as they age, so that we can obtain both cross-sectional and longitudinal data on the effect of age on taste preferences.

Analyses of results and interpretation. The results will be analyzed in a similar manner to the previous experiments, except sex will be included as an additional factor. This experiment will provide much-needed information about changes in solution acceptance across the life cycle. The results will be useful in setting appropriate age ranges for phenotyping mutagenized mice. It will also provide valuable normative data for comparing studies using animals of different ages. The use of both cross-sectional and longitudinal designs in the same experiment will provide a cross-check on the assumption that there are no effects of previous experience with the taste solutions tested here on subsequent taste solution intake.

Given the dearth of existing data on this topic, it is difficult to know what results to predict. Reproductive steroids influence taste preferences in rats (e.g., 68), so it is likely that the physiological changes associated with puberty (at about 6 weeks in mice) will influence intakes, and that effects of sex will be seen in all but the youngest groups tested. Physiological effects related to growth, which is more rapid in young than old animals, may also influence solution acceptance. Data from humans suggest that taste acuity deteriorates slightly in the aged27,121 so we may see some corresponding changes in solution acceptance in mice. Finally, older mice have had more time for uncontrolled environmental factors to influence their behavior so we expect to see greater variability in the responses of older than younger mice. It will be important to establish whether these are sufficiently large as to preclude screening old animals.Experiment 1f Assessment and elimination of carry-over effects induced by drinking taste solutions

For maximum validity and flexibility it is crucial that taste solution acceptance tests are independent of each other. That is, a mouse's experience with one taste solution must not influence, or carry-over to, its acceptance of solutions consumed later. Our previous work suggests this is not a problem for the compounds used in the short test sequence (except perhaps NaCl), but there are several examples where such carry-over effects occur after other compounds are ingested. There has been very little work to determine the causes of carry-over effects. There is no a priori way to know which taste solutions produce carry-over effects, so this needs to be determined empirically.

There are two questions. First, which taste solutions produce carry over effects? Second, if a compound produces carry-over effects then can they be eliminated? In this experiment, we address the first question by screening candidate taste solutions for their potential to produce carry-over effects. We also propose follow up experiments to neutralize carry over effects of affected taste solutions by interpolating "wash-out" days between taste tests.

Method. The approach will be to determine whether intake of a high concentration of a taste solution (the "target" concentration, listed in Table 1) influences subsequent intake of a low concentration. Each of the taste solutions listed in Table 1 will be tested in a separate experiment involving two groups of 12 male B6 mice given three 48-h two-bottle tests. For both groups the first and third tests will be a choice between water and half the concentration of the taste solution listed in Table 1. The second test will be a choice between water and the target concentration for one group or two tubes of water for the other. Thus, for example, one group of mice will receive successive tests with (1) water vs. 15 mM d-phenylalanine, (2) water vs. 30 mM d-phenylalanine, and (3) water vs. 15 mM d-phenylalanine. The corresponding control group will receive (1) water vs. 15 mM d-phenylalanine, (2) water vs. water, and (3) water vs. 15 mM d-phenylalanine,

Analyses of results and interpretation. Carry-over effects will be implied from a difference in solution acceptance on the final test between the groups previously given the target taste solution relative to those that received only water. The use of a pre-test (the first 48-h test) allows the effect of the target solution to be assessed within-subjects, and thus adds power to the design. It also will indicate if the less concentrated solutions produce carry over effects if there are differences in solution intake between the first and third tests of the control group (that is, the group that receives water between the 1st and 3rd tests).

Our working hypothesis is that there are two causes of carry-over effects: The first is due to a "taste" solution perturbing normal homeostatic mechanisms. For example, when a mouse ingests sucrose solution for several days it gains weight. Weight-loss related anorexia will influence intake of taste solutions ingested during the following few days. The second mechanism involves the association of taste cues with a solution's postingestive consequences. If a taste solution has aversive physiological consequences (e.g.,

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causes malaise) the mouse will learn to avoid that taste solution and solutions with similar orosensory properties. Similarly, if a taste solution has beneficial postingestive consequences, the mouse will learn to prefer it and similar solutions (i.e., Conditioned taste aversions and preferences; see our review112). A distinction between oral and postingestive actions is consistent with data showing that oral-oral associations can occur only when two solutions are presented in close temporal contiguity (i.e., <1 min apart), whereas oral-postingestive associations can occur with much longer delays (i.e., >1 h)33,37,44,116.

Based on our previous work and this hypothesis, we anticipate that carry-over effects will be produced by solutions with pronounced physiological effects, such as sucrose (calories) and 300 mM NaCl (osmotic effects). Drinking substances with few or no postingestive actions, such as d-phenylalanine and sucrose octaacetate will not carry-over to subsequent tests.

A thorny methodological issue is the appropriate choice of test solutions for this experiment. We chose to use test and target solutions that were identical in all aspects except concentration because solutions with similar tastes should allow maximum generalization. This seems to be the most conservative strategy. The alternative strategy, of testing each taste solution after pre-exposure to each of the other 20 solutions would be a horrendous experiment to conduct (i.e., over 400 combinations to investigate). If the proposed tests do not reveal carry-over effects between taste solution concentrations it is highly unlikely that they will exist between taste solutions.

Follow-up experiment. Once a solution that produces carry over effects is identified, we will attempt to determine whether it can be dissipated using a "washout" period. With selected taste solutions, we will repeat the initial experiment except there will be a period of 0, 24, 48, or 96 h between the 2nd and 3rd tests, during which time the mice will receive two tubes of water to drink. The goal will be to determine what is the shortest "washout" period that allows extinction of the carry-over effects. Elimination and control of carry-over effects. With the information from our pilot work and this experiment we will be able to establish series of tests that produce independent results. If a taste solution is found to produce carry-over effects, we will first determine whether there are other solutions that can provide as much information. For example, it may be possible to use nonnutritive saccharin rather than sucrose as a test of sweetness perception, or sucrose octaacetate rather than quinine hydrochloride as a test of bitterness perception. For some compounds, such as NaCl, there is no satisfactory substitute but compelling reasons to include them in the test series (see Section B). In these cases, the results of the follow-up experiment will allow us to include the appropriate number of wash-out days in the test series, and/or to test the compound at the end of the series.

We believe the proposed experiments will characterize most, if not all, carry-over effects. However, as discussed above, it remains possible that intake of one taste compound will interfere with subsequent intake of another taste compound. Some carry-over effects may also be strain- or even sex-specific. The existence of such effects would be evident from the results of Experiment 3, which involves testing all the compounds in several mice strains of both sexes. In the unlikely event that they occur, we may need to repeat parts of this experiment using the affected strain and sex in order to characterize and then isolate them.Specific Aim 2. Developing methods for brief-exposure tests using a lickometer

The two-bottle choice test that forms the foundation of Specific Aim 1 has many advantages but three serious disadvantages. First, it requires a relatively long time to collect data on a number of taste solutions. Second, carry-over effects can influence the results. Third, the test results reflect both orosensory and postingestive factors, making interpretation difficult. In Specific Aim 2 we propose to develop a method that circumvents these problems: the brief-exposure or "lickometer" test (see Table 1).Description of equipment currently in use

We have considerable experience using lickometers to measure fluid intake in rats23,117 and have recently adapted our equipment to test mice115. The basic theory and history of the lickometer is outlined above (Section B) and given in detail in several reviews122-124. Nearly all of this work involves testing drinking behavior in rats. There have been a few studies using mice32,46 but these have been little more than demonstrations that the method is possible. The biggest problem in testing mice rather than rats is ensuring that the animal forms a circuit while drinking. Unlike rats, which are generally housed in steel cages, mice

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are generally housed in plastic cages with shavings on the floor. In order to use a contact lickometer it is necessary for the mouse's feet (or other body part) to be grounded while they drink. One method to do this has been to provide a "grab bar" which the mouse holds while it drinks32. In our laboratory, we have found it more satisfactory to let the mouse stand on a metal plate in order to drink (see Fig. 5). Specifically, a 25 x 8 cm piece of 1/16" steel sheet is bent into a distorted Z shape so that it provides a metal floor suspended below the drinking spout. Four holes drilled through the steel sheet attach it to the steel cage lid with nuts and bolts. One bolt also holds the ground wire. Except for the tip, the metal spout of the drinking tube is sheathed in Tygon tubing so that it does not contact the metal lid or plate. An insulated cable held in place by the sheathing runs to the lickometer circuitry. The cage modifications can be made for under a dollar and require less than 5 min to prepare. Once made, the modified cage lids can be washed using automatic cage or tunnel washers without problems and the steel sheet can be removed without damage to the cage lid. A similar installation can be used for cages with filter tops with little difficulty.

Once a good circuit between spout, mouse, and floor has been made, the rest is easy. There are several lickometer amplifiers available commercially (e.g., TSE systems, model 2.07; Columbus Instruments drinkometer; Stoelting, model 57450), all of which use currents far too low to be detectable by mice (<1 µA). We use ones made by Med Associates (ENV-250C). The output of the amplifier is then fed into a computer. Again, there are several commercially available interfaces that can do this and at least one system that combines the lickometer amplifier and computer input into one box. We use A-bus components made by Alpha Products (Fairfield, CT). The output of the lickometer amplifier activates a latched relay (model LI-157) connected to a PC Bus adapter (MB-120). These components have the advantage of being relatively cheap (about $400 for 8 complete lickometer circuits, including a 24 v power supply and cables), and the same equipment can be interfaced to any computer with an RS-232 interface.

In our case, we use an old 80286 computer running DOS but the same data collection program will run under any operating system. The software to record lick patterns was written by the PI and comprises of approximately 20 lines of QBASIC code. Briefly, the input port is scanned once every 0.1 sec (maximum lick rates are about 6 Hz in the mouse) and the status of each latched switch is checked. When a switch closure is detected this implies the mouse is drinking, and the time of this event is saved on disk. To prevent a single contact being recorded as multiple licks, a subroutine ensures that each lick is finished (the switch state returns to zero) before another lick can be registered.

After a test is complete, the data are immediately copied to a floppy diskette and, from there, to another computer. Fine analysis can be done using Excel or other spreadsheet programs. We have found that analyses using the total number of licks in a 2 min test are adequate for nearly all situations. Other groups testing rats have used lick rates in the first few seconds (first bout lick rates), interbout intervals, and intrabout lick rates (e.g., 29,103) but only with longer tests. The decline in licking rate has been taken as a correlate of the accumulation of postingestive factors28,103. We intend to collect and analyze this information but, to simplify description, refer to the number of licks as the only dependent variable. Current method

The present equipment is set up to test 12 mice at a time, and we typically conduct 4 or 5 replications (48 or 60 mice) per day by staggering replications about 20 min apart. It is easy to increase the number of lickometers, and indeed, in some studies we have run 32 lickometers simultaneously during long-term (24 h) tests. However, for the 2-min tests that will be used here, there are some practical problems to overcome. Each test is conducted as follows. All the mice are adapted for several days to a mild water deprivation schedule (typically, water is removed for the first 6 h of the dark period). Tests are conducted at the end of the deprivation period. As quickly and quietly as possible, a drinking tube is placed onto the cage of each

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Metal plate (8 cm wide)

MousePlastic cage

Metal drink ing spout

Metal cage top

(insulated from cage top)

Attachment bolts

Taste solution

Wire to lickometer c ircuitryWire to ground

FIG. 5. Diagram of cage modified for detection of individual licks by a mouse (lickometer). Note that the mouse must stand on a metal plate to reach the drinking spout.

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mouse to be tested. The computer software detects when each mouse begins to lick (this is defined as at least 5 licks in less than 2 sec; a criterion is used to avoid considering accidental contacts while the tubes are being positioned or inadvertent touching of the drinking tube by the mouse). At 2 min after the first lick, the computer displays that time is up (the word "End" appears next to the circuit number on the screen), and the investigator removes the drinking tube. Because the mice start drinking at different times (unless they are well trained and strongly deprived), it can take as long as 30 min to conduct a test. During this time, the investigator must remain vigilant in order to remove tubes as each mouse completes its 2-min test. If the tubes were left on the cages, mice that begin drinking first would receive more exposure to the taste solution than mice that are reticent to start drinking.A problem with the current method and its solution

This method is fine for experiments involving only a few subjects but would become impractical when scaled up to test dozens of mice at once. The biggest problem is it will become tricky to remove lots of drinking tubes at the appropriate times. It is also likely that the activity of the investigator disturbs some mice, adding to variability in their drinking bout lick rates.

To eliminate this problem, we propose the following solution. Each drinking tube will be mounted on a motor-driven retractable module (Med Associates ENV-252M; $350 each). This unit holds the drinking tube outside the mouse cage, but can be controlled by a computer-activated 24 v switch closure to insert and retract the tube into the cage. We will modify our software to automatically insert all drinking tubes at the beginning of the test and retract them as each mouse finishes drinking at the end of its 2-min bout.

The proposed equipment modification should allow us to test a virtually unlimited number of mice simultaneously (limited only by the cost of equipment). However, for the optimization and validation tests proposed here it will be sufficient to conduct tests on 24 mice at once, with two or more staggered replications each day. We thus now turn our attention to optimizing the test conditions. Experiment 2a - Optimizing test conditions for lickometer measurements

The most important factor in conducting short-term tests is to ensure that the mice drink appropriate amounts of taste solution. If a mouse is too thirsty or its taste solution is too palatable it will drink at maximal rates throughout the test. If, on the other hand, the mouse is not thirsty enough or the taste solution is too unpalatable it will drink little or nothing. Conditions that induce very low or very high intakes must be avoided in order to maximize test sensitivity. We address this problem in the following experiment.

Method. The basic method will be to test various concentrations of each of the taste solutions listed in Table 1 in order to find which support moderate rates of ingestion. Groups of 16 male B6 and 129 mice will be adapted for 3-5 days to a water deprivation schedule, with no water available during the first 6 h of the dark period. Under these conditions, we find that mice drink water promptly when it is returned but maintain normal weight gain (see Section C.4). The mice will then be tested with ascending concentrations of each of the taste compounds listed in Table 1. The specific concentrations to be tested will be chosen based on our work with long-term choice tests, but it is anticipated that 4-5 concentrations of each compound will be tested, spanning the range from avidly- to barely-ingested. Each series will begin with a test with water (or vehicle for the insoluble compounds). Because it is highly unlikely that carry-over effects are produced by short-term tests (see below), we will use the same animals repeatedly to test several solutions. For convenience, and to save time, we expect to test four batches of mice simultaneously. It may not be possible to find appropriate concentrations of the more palatable test solutions (e.g., sucrose, saccharin) because of ceiling effects on intake. If this occurs, separate tests will be conducted for these compounds, using non-deprived mice.

Analyses of results and interpretation. The primary dependent variable for all analyses will be the number of licks made in each 2-min test (see Description of equipment currently in use, above). We will generate concentration-response curves for each of the compounds listed. Concentrations to be used for subsequent studies will be chosen based on (a) the maximal separation between B6 and 129 mice, using the same methods as analysis as for Specific Aim 1, or (b) if there is no difference in response between the two strains, the concentration producing 50% of the maximal response.

We do not anticipate observing carry-over effects with these brief-exposure tests. We have not observed them in pilot work with mice, and a study designed to detect them in rats found no evidence for them101. Moreover, as carry-over effects are most likely the result of the association of orosensory cues with their

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postingestive consequences (see above) and the brief-exposure tests have few, if any, postingestive consequences, their existence seems implausible. Nevertheless, we will be on the look out for any such effects. In particular, the results of tests with water to drink (between each concentration series) will allow us to determine whether changes in response have occurred. If it appears that carry-over effects or other changes related to repeated testing influence the results, this will be clarified by testing additional groups of naive mice.

Additional studies and interpretative issues. There are many factors that might influence the results of lickometer tests. These can be divided into physical factors (e.g., the position of the drinking tube, size of the spout aperture, test duration) and animal factors (e.g., mouse age, sex, deprivation state). It would certainly be possible to characterize and then optimize the contribution of each of these factors, but we think this is unnecessary. For the reasons outlined in Section B, the contribution of factors other than taste solution palatability and thirst deprivation state is unlikely to be very great.

We also do not plan to run test-retest reliability studies, in which the response of the same mice is measured two or more times. This is a commonly undertaken during the development of a test but it seems inappropriate here. Test-retest reliability depends on the correlation between scores on repeated tests. A high correlation coefficient can be obtained when each subject has a similar score on each test and there are large differences between subjects. In our case, it is important that we do not have large differences between subjects (of the same strain) because if we did, we would not be able to identify mice with outlying responses. For our purposes, the best measure of test reliability is the variance in response of identically-treated mice of the same strain.

One issue is that interpretation of the lickometer tests involves the assumption that the mice are competent to drink and that water deprivation makes them equally thirsty. This will not be a problem for the proposed experiments because we already know that B6 and 129 mice drink equivalent amounts of water after water deprivation (see Preliminary Results). However, the same assumption cannot be made for mutants. We will be able to recognize these animals by aberrant lick rates during tests with just water to drink.

One possibility that may be worthwhile pursuing is whether the mice can be tested more than once a day. It might, for example, be possible to test an unpalatable taste solution after water deprivation during the dark period, and a palatable solution without water deprivation during the light period. The automated taste solution delivery equipment will make this feasible without requiring round-the-clock availability of an investigator. Specific Aim 3. Establishing reference data for subsequent identification of mice with aberrant taste phenotypes

The previous experiments will help outline the most sensitive test methods for taste phenotyping, as well as identify the range of ages at which mice can be tested. With our procedures optimized, it will now be time to develop them by providing reference data for several strains of mice. This will allow selection of strains for mutagenesis and outcrossing for mapping studies. It also demonstrates the feasibility of detecting genetic differences, and provides a scale for assessing the strength of a mutation's effect. Our approach involves three components. First, we will test large numbers of B6 and 129 mice. This will provide reference data about the variability of response in these two strains. Second, we will screen smaller numbers of 24 additional strains of mice. This will compliment and extend our ongoing small grant, which involves conducting two-bottle choice tests of NaCl, other mineral salts, and monosodium glutamate in 28 strains (NIH DC-03854). Third, we will test 12 groups of mice with known taste deficits. Experiment 3 - Collecting normative data from reference strains and strains with unusual taste phenotypes

Choice of mice to be tested. The main two reference strains to be tested will be B6 and 129 mice. We have chosen to focus on these strains because (a) they are currently being used for mutagenesis studies, (b) they are likely to be the first strains in which the genome will be completely sequenced, (c) they are among the most common strains in use for nongenetic as well as genetic studies, and (d) we already have considerable experience with them. We propose to test groups of 50 male and 50 female mice of these strains and groups of 16 male and 16 female mice of the other strains and groups (see below). The relatively large group sizes were chosen because (a) it is important to obtain accurate measures of variance,

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including frequency distributions, and this requires more subjects than is usual, and (b) it is extremely critical to avoid collection of a non-typical sample distribution because these data will be used for many subsequent comparisons. The greater number of subjects ensures that the sampling distribution will be closer to the population distribution.

We will test the following 24 strains of mice: A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57L/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/H1J, LP/J, NOD/LtJ, NZB/B1NJ, P/J, PL/J, RBF/DnJ, RF/J, RIIIS/J, SJL/J, SM/J, SWR/J, SEA/GnJ, CAST/Ei, SPRET/Ei, We are currently testing these strains as part of our small grant, DC-03854 (the grant includes 28 strains; these 24 plus B6, 129 (see above), and CBA/J and SWR/J, which have known taste deficits (see below). Although several of the strains have been tested with one or two taste compounds by other investigators, and they will all be tested with salts and acids as part of DC-03854, there have been no concerted attempts to examine other taste modalities. Other reasons to choose these strains are: (1) Their diverse ancestral origins, which increases the chances of detecting variation in chemosensory responses, and increases the number of polymorphic markers that could be used for genetic mapping in future studies. (2) Two inbred strains representing a different subspecies of M. musculus (M. musculus Castaneus) and a different mouse species (M. spretus) are also included. They are extremely valuable for genetic mapping because of their large number of polymorphic markers. (3) These strains include progenitors of 12 sets of recombinant inbred strains (AKXD, AKXL, AXB, BXA, BXD, BXH, BXJ, CXB, CXJ, NXSM, SWXJ, SWXC), which could be used in future studies for genetic mapping. (4) They include strains in which polymorphisms of microsatellite markers have been extensively characterized, which would increase the effectiveness of genetic mapping in future studies.

The following mice with known abnormalities of taste solution acceptance (relative to the B6 strain) will be tested. (i) Several strains of mice and congenics with known abnormalities in bitter taste, including SWR/J, SW.B6-Soab and C3.SW-Soaa mice. The SWR/J mice are an inbred strain homozygous for the soa-a [taster] allele. The SW.B6-Soab are a congenic line currently available at approximately generation F10N21. The C3.SW-Soaa mice have been maintained by lineal backcrossing to C3 inbred mice. They carry the soa-a [taster] allele from the SWR/J inbred line and are currently available at generations 28-29. We have recently received breeding stock of these mice from Dr. Glayde Whitney (Dept. Psychology, Florida State University). (ii) Mice with abnormalities of sweet taste. Of course, we will have 129 mice in this category because we have already demonstrated differences in sweet taste between B6 and 129 mice. In addition, we will test 129.B6-Sac congenic mice, which are currently in generation N5, and homozygous partially congenic mice, which will be available in July 1999. We also will test alpha-gustducin subunit knock-out mice (both homozygotes and heterozygotes), obtained from Dr. Robert Margolskee (Mount Sinai School of Medicine, NY). These mice have deficits in both bitter and sweet perception127. (iii) Mice with abnormalities of salt taste. Once again, our work suggests that B6 and 129 mice differ substantially in response to NaCl. In addition, we will test NZB mice because we have found these animals are extremely avid consumers of NaCl, and CBA/J mice because these animals strongly avoid it9. If additional strains of mice with taste deficits come to light they will also will be tested.

Finally, we intend to test B6 animals given manipulations that are known to influence taste solution acceptance. This includes (i) selective denervation of the tongue by transection of the chorda tympani nerves, glossopharyngeal nerves, or both chorda tympani and glossopharyngeal nerves. Studies conducted primarily in rats show that chorda tympani transection influences the response to NaCl, whereas the glossopharyngeal nerves convey information about sweetness, and both pairs of nerves convey information about bitterness102,107,108. We have experience performing these nerve transections in mice (see Fig. 5). The completeness of surgery will be verified by staining the tongue and counting taste buds at postmortem. Other procedures to be used include (ii) adrenalectomy, which interferes with sodium metabolism and induces a specific sodium appetite (see MS8 in Appendix), (iii) streptozotocin-induced diabetes, which interferes with blood glucose regulation and alters sweetness acceptance (in rats)109 (iii)

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Control Glossopharyngea Chorda tympani0

1

2

3

4

5

Flui

d in

take

, ml

75 mM (0.5%) NaCl

Water

FIG. 3. Example of our work with gustatory nerve transections in B6 mice. The chorda tympani nerves were accessed through the tympanic membrane (n = 5). The glossopharyngeal nerves were accessed by tunneling under the digastric muscle (n = 3). Control mice received sham surgery (n = 4). At 13 days after the surgery, the mice were presented with 75 mM for two days, given together with water. Note that bilateral transection of the chorda tympani nerve eliminated the normal aversion of this strain to NaCl solution.

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maintenance on a high-fat, low-carbohydrate diet, which facilitates fat metabolism and increases preferences for fat-like taste solutions81,82. The surgically compromised groups will have their own controls given "sham" surgery.

However, to quote from the RFA, we may "make necessary adjustments in scientific direction" if feedback from other scientists (including the Study Section panel) indicates that it would be valuable to test other strains as well, and have allocated funding in the budget to do so.

Methods. The experiment will be conducted in staggered replications of 96 mice. Each animal will be adapted to vivarium conditions for several days. It will be tested first with lickometer tests, and then with long-term choice tests. For the lickometer tests, each mouse will receive the complete set of taste solutions listed in Table 1 at concentrations determined in Specific Aim 2 (approximately 20 taste solution tests and tests with water every 4-5 days, requiring a total of 27 days to conduct). For the long-term choice tests, the mice will be given the same series of taste solutions at the concentrations listed in Table 1. The test conditions will be optimized based on the information obtained from the experiments conducted in Specific Aim 1. In particular, any potential carry-over effects will be eliminated by interpolation of wash-out days and careful arrangement of solution presentation order. It is anticipated that this portion of the experiment will require approximately 50 days.

Analyses of results and interpretation. Data analyses will be straightforward. For each test, means, variances, and frequency distributions for each group of mice will be calculated. For the larger groups (B6 and 129), measures of skewness and kurtosis will also be calculated. Differences between groups will be assessed using ANOVAs, with appropriate controls for the large number of comparisons to be made.

The descriptive statistics will allow us to set criteria for determining which mice have outlying phenotypes in subsequent studies. In our pilot work (see Section C), we used a criterion of diverging from the mean by more than three standard deviations to determine which animals to pursue as interesting mutant candidates. However, we do not consider it appropriate at this stage to set formal criteria. This is a decision that depends not only on statistical considerations but also on the cost and effort involved in subsequent confirmatory phenotyping, breeding, and genotyping, as well as the strain on available resources. If these follow-up steps are relatively inexpensive it will be possible to set a lax criterion with the understanding that a proportion of the mice singled out will not have mutations. The use of a more stringent criterion will minimize the misidentification of intact mice as mutants but will increase the probability that an interesting mutation will be missed.

The results from the groups of mice with known abnormalities of taste solution acceptance will illustrate the magnitude of the deficits we can expect to see in mice with taste-related mutations. They will also allow us to identify patterns of deficits; for example, do mice with increased acceptance of NaCl have reduced acceptance of sweet solutions (as appears to be the case in ratssee 111)? Such patterns might help to determine whether mice with borderline abnormal phenotypes on one measure are indeed mutants.

The experiment involves several tests in which the mice receive just water to drink. These tests are important because they establish baseline intakes, which is particularly critical for brief-xposure tests. Also, where side preferences for water differ from before to after a series of taste solutions, this can indicate the presence of carry-over effects. As discussed above, we will be on the look out for interactions between various taste solutions, as well as strain- and sex-specific carry-over effects. In the unlikely event that they occur, we may need to repeat parts of Experiment 1a using the affected strain and sex in order to characterize and then isolate them. An alternative would be to test separate groups of mice with the suspect taste solutions using a between-subjects design. If such effects cannot be eliminated or adequately controlled for, it may be necessary to exclude the taste solution from subsequent screens.

One possibility that we have not explicitly controlled for is that the procedures involved with testing mice in the lickometer will influence taste solution acceptance in subsequent long-term, two-bottle choice tests. For example, it is possible that the water deprivation schedule used during brief-exposure tests will produce long-lasting changes in physiology that could potentially affect taste solution acceptance. We think such interactions are very unlikely given the very mild deprivation schedule to be used (6 h/day). Nevertheless, if the results of long-term two-bottle choice tests differ from those we have already collected(e.g.,3-6,9) we will have to conduct additional studies to characterize this problem and control for it.

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The results will compliment and extend those found as part of our grant, DC-03854. This small project involves measuring taste solution concentration-response functions of 28 mouse strains using long-term taste acceptance tests. The work proposed here will confirm the general findings of DC-03854 but will not replicate the concentration-response functions. It will also (a) provide tests of many compounds not covered by the DC-03854 project (e.g., sweeteners, starches, fat, alcohol), (b) include more strains, and (c) involve brief-exposure tests. Comparison of the results found here with similar tests already conducted will give us an idea to what extent the "optimized" methods reduce within-strain variability.

It will be important to compare the results from brief-exposure lickometer tests to those from long-term choice tests. This will provide insight into the role of postingestive factors in determining solution acceptance in these strains. If an abnormal phenotype is identified by two-bottle choice tests but not brief-exposure tests, this would imply the underlying mutation involves postingestive mechanisms rather than orosensory ones. If, on the other hand, an abnormal phenotype is identified in brief- but not long-term tests, the implication would be that a mutation involving orosensory factors is present but can be masked by compensatory changes in postingestive mechanisms. Of course, some caution is required when making such comparisons because different concentrations of each taste solution may be tested in the brief- and long-term tests.

In addition to the theoretical implications to be gained from comparing brief- and long-term tests, there will also be some important practical ones. Comparison of the results will allow us to recommend whether comprehensive phenotyping requires the use of both types of test. If the results of both long-term and brief-exposure tests are congruent, it would be possible to eliminate one or other type of test. Which test would be eliminated depends on the relative sensitivity of the tests, access to test apparatus, the time available for testing, and similar factors, making a decision imponderable until the proposed studies have been conducted.

It should be noted that this study will provide an prodigious information about the taste perception of the "reference" and "abnormal" strains, which although not necessarily germane to this project will nevertheless be extremely valuable. The strain comparisons will allow hypotheses of single- or multi-gene inheritance can be tested, which will provide a background for subsequent chromosomal mapping and eventual positional cloning of these genes. This will be the first thorough analysis of the taste world of any of the strains to be tested.

Technical errors and their correction. All tests are subject to technical errors and taste phenotyping is no exception. Results are sometimes lost because of leaking stoppers and misread, blocked, or dropped drinking tubes. Although we have not kept statistics on this, we estimate that such errors invalidate as many as 1 out of every 75 tests. Technical errors occurring in the experiments proposed here are of little concern; all measures are based on group responses so data from the invalid test can simply be ignored. However, this becomes a much more serious issue when screening many individuals. Is the empty tube on the cage due to a leaking stopper or a mutation causing polydipsia? It will be necessary to reconduct tests with ambiguous results to either correct the error or confirm that an aberrant taste phenotype is present. Due care will be required interpreting data from retests, because of the increased potential for carry-over effects and other effects related to solution test order. We anticipate providing detailed instructions for dealing with technical errors as part of the protocol to be developed (see below).Time table for the proposed research

We intend to conduct the research in the order it is proposed. It will require the first 15 months of the project to conduct the experiments listed in Experiment 1. We will also fabricate and pilot test the automated lickometer system during this period. Specific Aim 2 will be conducted during the end of Year 1 and beginning of Year 2. Specific Aim 3, and any follow up studies that may arise, will be conducted during the 2nd and 3rd years.Making recommendations about how and what to test

This project will allow us to produce a series of recommendations about how and what to test but it will also require judgements based on experience in this area. Recommendations about how to test will be derived from the results of Specific Aims 1 and 2. The results of Specific Aim 3 will tell us whether long-term, brief-exposure, or both forms of screening are required. Recommendations about what to test will be derived from several sources. All three specific aims, and in particular Experiment 1f, will identify any

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solutions that produce carry-over effects. Some of these solutions can still be tested with careful controls (e.g., wash-out days, put at end of test sequence) but others may have to be eliminated. Experiment 3 will give us some relevant information about what to test because it will demonstrate how the response to each taste solution in mice with taste abnormalities differs from the response of reference strains. It will also show us how the acceptance of various taste solutions covaries (i.e., it will address questions such as "do mice that avoid SOA also always avoid quinine?"). It is likely that some covariance will occur (see, for example6), and that there will be no differences between any of the strains in response to some taste solutions. This information could potentially be used to eliminate redundant or uninformative solutions from the panel used for subsequent mutagenesis screening. However, we think this is premature. We may not have yet found appropriate strains to show a difference. The choice of taste solutions to be tested was made based on the criterion that each represent a different taste subquality, and thus presumably different taste mechanisms, each of which has the potential to be disrupted by mutations.

In practice, the size of the taste solution panel to be tested is likely to depend on a compromise between the desire to be thorough (and increase the chance of finding a mutation) and the time and resources available. Our recommendation would be to give a lower priority to taste solutions for which we have not found disparate phenotypes but we would advise testing even those taste solutions that do not differentiate among the groups we have tested if time and resources are available.

If the time available for a taste phenotype screen is very limited, the criterion for choosing which solutions to test will be based on the sensitivity of the test, the distribution of responses shown by reference strains, and the potential importance of understanding the behavior. An example of the latter criterion is that studying the acceptance of NaCl or alcohol has greater potential for understanding disease states (hypertension or alcoholism) than does studying Polycose intake, which appears to be important in the rat but not human96.Future research, and how we expect to fit into the mutagenesis program

Based on a very recent RFA (MH-99-007; Mouse mutagenesis and phenotyping: nervous system and behavior), it appears likely that several mutagenesis centers will be established, and these will either conduct their own phenotyping or provide mutagenized mice to other investigators. We hope to participate in either or both of these efforts. Although we do not have the expertise or capabilities to mutagenize our own mice, we foresee collaborating with institutions that do. Indeed, in addition to our work with Dr. Maja Bucan (Dept. Psychiatry, University of Pennsylvania; see attached letter), we have begun to collaborate with Dr. Kevin Seburn, who is the Physiogenomics Program Supervisor at the Jackson Laboratory. For the Jackson Laboratory project, we initially intend to provide taste phenotyping expertise and testing procedures that will be incorporated into a battery of tests of neurologic competence conducted in Bar Harbor. Under these conditions, we do not expect to be able to test the whole battery of taste solutions we have used here. However, later, if funding is available, we can envisage conducting our own, more thorough, screening, which would be dedicated to phenotyping taste, smell, and ingestive behaviors, and which would involve several colleagues here at the Monell Center. Of course, the tests developed with funding from this proposal will also be useful for screening phenotypes at other centers (e.g., obesity, digestive, renal, cardiovascular phenotypes) without our participation.

Another area where we hope to contribute to the mutagenesis program is in the characterization of mutant taste phenotypes once they have been discovered. Presumably, mutant mice and their affected offspring would be sent to us for testing by other groups. We could then determine whether their aberrant taste solution acceptance was due to dysfunction of chemosensory or postingestive mechanisms using brief exposure tests, the sham feeding method, and gustatory electrophysiology. If the deficit appears to be postingestive, we could characterize this further by conducting metabolic balance studies, analyzing blood metabolites and hormones, and measuring the animals' responses to directed metabolic challenges. Our laboratory has made extensive use of these methods for other purposes (see papers listed in CVs) and we foresee no problems adapting them to mutant mice.

Whatever our role (if any) in subsequent mutagenesis research, the experiments outlined in this project will provide a solid foundation for anybody about to undertake phenotyping taste solution acceptance in large numbers of mice. Administrative issues. Dissemination of research methods and results

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The methods that are generated by the proposed research will be useful to many investigators, including but not limited to those involved in phenotyping mutagenized mice. It is important that the methods and results are made easily and rapidly available to any interested party. This task is greatly simplified in our case because none of the proposed methods will produce inventions requiring patent protection. We have nothing to hide and lots to gain by having our methods widely used and accepted. We plan to take the following steps to do this.

Research protocols. We will develop a series of research protocols giving detailed information about the methods used for phenotyping. This will describe all aspects of the experiments, including but not limited to: animal sources and characteristics (age, sex, body weight), cage sizes (including vendors and description of materials), general animal maintenance conditions (temperature, bedding materials, light cycle), diet (including vendor, detailed formulation and composition), water source and purity, drinking tubes (including vendors for parts, construction methods and tools required), sources of errors and their solutions, and details about the lickometer (parts vendors, construction methods, wiring diagrams, executable files, software source code, description of software, troubleshooting guide, etc). These protocols will be produced by the combined effort of the investigators and project technician. Ms. Diane Pilchak, the technician, has had many years experience writing protocols used for training other technicians, and is thus well-suited to this task.

The protocols will be disseminated in the following manner: (1) They will be posted on our Institute's web site for as long as they remain peritinent (see letter of collaboration from Dr. Gary Beauchamp). (2) Paper copies will be mailed to any interested parties. (3) The methods will be published in Ingestive Behavior Protocols. This unique resource is maintained and published by the Society for the Study of Ingestive Behavior (SSIB) and is currently edited by Drs. P. Wellman and B.G. Hoebel. It is a collection of research protocols similar to Current Protocols in Neuroscience and will be updated yearly. (4) The protocols will be submitted to a central site containing phenotyping methods if/when such a site is established. We will also consult NIH staff about other appropriate methods of disseminating protocols. In particular, we anticipate consulting closely with investigators using our protocols, and this would include the PI visiting their facilities to ensure there are no errors or ambiguities in the test procedures being adopted.

Reference data. The experiments proposed above will yield many results of interest to researchers in the fields of "taste" and "ingestive behavior" and so it will not be difficult to publish them in top-notch journals with widespread distributions (e.g., American Journal of Physiology). However, it is recognized that for some purposes, investigators may require all the data collected for an experiment, particularly the reference data collected in Specific Aim 3. By "all the data" we mean the number of licks or milliliters ingested of each taste solution for each mouse and each test, as well as all ancillary and derived measures (e.g., body weights, preference ratios, group means, group variances, frequency distributions). To fulfill this, we will (1) Post on Monell's web site all data, along with detailed explanations of how it was collected, and (2) mail hard copies and/or send electronic media to any interested investigator requesting the information, and (3) submit these data to any repository of phenotype information. We will work with NIH program staff and other investigators involved with the mutagenesis project to disseminate the results wherever appropriate.

It is anticipated that protocols and reference data will be made available as soon as they have been established or collected. Although we anticipate a much faster rate of dissemination, all information will be submitted for publication, in press, or otherwise publicly available within 1 month after the project ends.

E. HUMAN SUBJECTSNot applicable

F. VERTEBRATE ANIMALS1. Description of proposed use of animals. This is a proposal to refine methods of measuring the taste responses of mice. Table 4 lists information about the mice to be tested, including their strain, age, and sex. In all experiments, mice will receive taste solutions to consume. In most, the solutions are available, along with water, for 48 h. In some, one taste solution will be available for only 2 min. The experiments involve benign manipulations of test conditions, such as the order the taste solutions are presented, the position of the drinking spouts, the age of the mice, etc.

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2. Justification of use of animals and species. The experiments are designed to optimize methods for testing mice with potential mutations. It is not ethical to induce mutations in humans. Mice are good subjects for this kind of research because they breed easily, are small, and can be housed easily, yet have many aspects of taste and physiology in common with humans.

Table 4. Use of animals in this project.Experiment Procedures Strain Age Sex N

1a. Spout position Two-bottle taste tests (5 spout positions)

C57BL/6J129/SvJ

8 wk8 wk

malemale

1616

1b. Two vs three- bottle tests

Two-bottle or three-bottle tests (3 conditions)

C57BL/6J129/SvJ

8 wk8 wk

malemale

1616

1c. Test duration Two-bottle tests (4 durations) C57BL/6J129/SvJ

8 wk8 wk

malemale

1616

1d. Diet effects on solution acceptance

Two-bottle tests (6 maintenance diets)

C57BL/6J129/SvJ

8 wk8 wk

malemale

9696

1e. Age effects on solution acceptance

Two-bottle tests (12 ages) C57BL/6J and129/SvJ

3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 75, 100 wk

male female

384 384

1f. Carry-over effects Two-bottle taste tests (20 solutions) C57BL/6J 8 wk male 640

1f. Follow up Two-bottle taste tests with water on some days (5 solutions)

C57BL/6J 8 wk male 60

2a. Lickometer taste solution concentration

Brief-exposure (2 min) taste solution tests after 6 h water deprivation (20 taste solutions)

C57BL/6J129/SvJ

8 wk8 wk

malemale

64 64

2a additional studies Brief-exposure (2 min) taste solution tests after 6 h water deprivation (5 taste solutions)

C57BL/6J129/SvJ

8 wk8 wk

malemale

80 80

3. Establishing reference data

Brief exposure and two-bottle tests in the same animals (20 solutions)

C57BL/6J

129/SvJ

24 reference strains (16 of each sex)7 strains with known taste deficits (16 of each sex)8 groups of B6 mice with surgical or dietary manipulations (16 of each sex)

8 wk8 wk8 wk8 wk8 wk8 wk8 wk8 wk

8 wk8 wk

malefemalemale

femalemale

femalemale

female

femalemale

50 50 50 50400400112112

128 128

TOTAL* 3524

*An additional 226 mice are requested for unanticipated experiments, caused by replications where results are not clear, technical errors, equipment failures, and new ideas arising from the listed experiments.

3. Veterinary care. Dr. Moshe Shalev is employed by the Monell Center as a veterinarian. He has extensive experience with animal facilities and with mice, the only species used in this project. Dr. A. Bachmanov, the project co-PI, also has a degree in Veterinary Science and several years experience with mice.4. Procedures for reducing stress and pain. Nearly all the procedures proposed in this project are all noninvasive. Intake of test solutions is voluntary. Some animals will be deprived of water for 6 h, but this is very mild deprivation and unlikely to produce more than minimal stress. A small number of mice will receive selective surgical deafferentation of the tongue. These animals will be anesthetized during the surgery and given postoperative analgesics. We have experience with these procedures. 5. Method of euthanasia. Euthanasia will be achieved by CO2 asphyxiation or anesthetic overdose, as recommended by the American Veterinary Medical Association.

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G. LITERATURE CITED1. Ackroff, K., Manza, L., and Sclafani, A. The rat's preference for sucrose, Polycose and their mixtures.

Appetite 21: 69-80, 1993.2. Ad hoc committee on standards for nutritional studies. Second report of the ad hoc committee on

standards for nutritional studies. J. Nutr. 110: 1726, 1980.3. Bachmanov, A.A., Reed, D.R., Ninomiya, Y., Inoue, M., Tordoff, M.G., Price, R.A., and Beauchamp,

G.K. Sucrose consumption in mice: major influence of two genetic loci affecting peripheral sensory responses. Mammal. Genome 8: 545-548, 1997.

4. Bachmanov, A.A., Reed, D.R., Tordoff, M.G., Price, R.A., and Beauchamp, G.K. Intake of ethanol, sodium chloride, sucrose, citric acid, and quinine hydrochloride solutions by mice: a genetic analysis. Behav. Genetics 26: 563-573, 1996.

5. Bachmanov, A.A., Schlager, G., Tordoff, M.G., and Beauchamp, G.K. Consumption of electrolytes and quinine by mouse strains with different blood pressures. Physiol. Behav. 64, 1998.

6. Bachmanov, A.A., Tordoff, M.G., and Beauchamp, G.K. Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol Clin. Exp. Res. 20: 201-206, 1996.

7. Bachmanov, A.A., Tordoff, M.G., and Beauchamp, G.K. Intake of umami-tasting solutions by mice: a genetic analysis. J. Nutr. , in press.

8. Bachmanov, A.A., Tordoff, M.G., and Beauchamp, G.K. NaCl preferences in 13 inbred mouse strains. Annual Meeting of the Association for Chemoreception Sciences, Sarasota, FL, 1999.

9. Bachmanov, A.A., Tordoff, M.G., and Beauchamp, G.K. Voluntary sodium chloride consumption by mice: differences among five inbred strains. Behav. Genetics 28: 117-124, 1998.

10. Bartoshuk, L.M. Is sweetness unitary? An evaluation of the evidence for multiple sweets. In: Sweetness, edited by Dobbing, J. London: Springer-Verlag, 1987, p. 33-47.

11. Beauchamp, G. Kirin international symposium on bitter taste. In: Physiol. Behav. Pergamon, 1994.12. Beauchamp, G.K., and Fisher, A.S. Strain differences in consumption of saline solutions by mice.

Physiol Behav 54: 179-84, 1993.13. Beidler, L.M., Fishman, I.Y., and Hardiman, C.W. Species differences in taste responses. Am. J.

Physiol. 181: 235-239, 1955.14. Biddle, F.G., Coffaro, C.M., Ziehr, J.E., and Eales, B.A. Genetic variation in paw preference

(handedness) in the mouse. Genome 36: 935-43, 1993.15. Biddle, F.G., and Eales, B.A. The degree of lateralization of paw usage (handedness) in the mouse is

defined by three major phenotypes. Behav Genet 26: 391-406, 1996.16. Blizard, D., and McClearn, G. Sweet genes and ethanol intake in mice. Alcohol Clin. Exp. Res. 20:

25A, 1996.17. Boudreau, J.C., Hoang, N.K., Oravec, J., and Do, L.T. Rat neurophysiological taste responses to salt

solutions. Chem. Senses 8: 131-150, 1983.18. Boughter, J.D., Jr., and Whitney, G. Behavioral specificity of the bitter taste gene Soa. Physiol Behav

63: 101-8, 1997.19. Boughter, J.D., Jr., and Whitney, G. C3.SW-Soaa heterozygous congenic taster mice. Behav Genet

25: 233-7, 1995.20. Brosvic, G.M., and Slotnick, B.M. Absolute and intensity-difference taste thresholds in the rat:

evaluation of an automated multi-channel gustometer. Physiol Behav 38: 711-7, 1986.21. Bruning, J.L., and Kintz, B.L. Computational Handbook of Statistics. Dallas: Scott, Foresman and Co.,

1977.22. Coldwell, S.E., and Tordoff, M.G. Acceptance of minerals and other compounds by calcium-deprived

rats: 24-h tests. Am. J. Physiol. 271: R1-R10, 1996.23. Coldwell, S.E., and Tordoff, M.G. Immediate acceptance of minerals and HCl by calcium-deprived rats:

brief exposure tests. Am. J. Physiol. 271: R11-R17, 1996.24. Contreras, R.J. Changes in gustatory nerve discharges with sodium deficiency: A single unit analysis.

Brain Res. 121: 373-378, 1977.25. Contreras, R.J., and Frank, M. Sodium deprivation alters neural responses to gustatory stimuli. J. Gen.

Physiol. 73: 569-594, 1979.26. Coons, M., Adler, E., Lindemeier, J., Battey, J., Ryba, N., and Zuker, C. Putative mammalian taste

receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell 96: 541-551, 1999.

47

Tordoff

27. Cowart, B.J. Development of taste perception in humans: sensitivity and preference throughout the life span. Psychol Bull 90: 43-73, 1981.

28. Davis, J., and Levine, M. A model for the control of ingestion. Psych. Rev. 84: 379-412, 1977.29. Davis, J.D., and Smith, G.P. Analysis of lick rate measures the positive and negative feedback effects

of carbohydrates on eating. Appetite 11: 229-38, 1988.30. Denton, D. The hunger for salt: An anthropological, physiological and medical analysis. Berlin: Springer-

Verlag, 1984.31. DeSimone, J., and Ferrell, F. Analysis of amiloride inhibition of chorda tympani raste response of rat to

NaCl. Am. J. Physiol. 249: R52-R61, 1985.32. Dole, V., and Gentry, T. An improved technique for monitoring the drinking behavior of mice.

Physiology and Behavior 30: 971-974, 1983.33. Elizalde, G., and Sclafani, A. Starch-based conditioned flavor preferences in rats: influence of taste,

calories and CS-US delay. Appetite 11: 179-200, 1988.34. Faurion, A., Saito, S., and MacLeod, P. Sweet taste involves several distinct receptor mechanisms.

Chem. Senses 5: 317-330, 1980.35. Ferrell, F., and Gray, S.D. Longitudinal study of salt preferences in normotensive and hypertensive rats.

Hypertension 7: 326-32, 1985.36. Fishman, I.Y. Single fiber gustatory impulses in rat and hamster. J. Cell. Comp. Physiol. 49: 319-334,

1957.37. Flaherty, C.F., and Rowan, G.A. Successive, simultaneous, and anticipatory contrast in the

consumption of saccharin solutions. J Exp Psychol Anim Behav Process 12: 381-93, 1986.38. Formaker, B.K., and Hill, D.L. Alterations of salt taste perception in the developing rat. Behav Neurosci

104: 356-64, 1990.39. Fregly, M.J., and Rowland, N.E. Role of the renin-angiotensin-aldosterone system in NaCl appetite of

rats. Am. J. Physiol. 248: R1-R11, 1985.40. Gibson, E.L., and Booth, D.A. Acquired protein appetite in rats: dependence on a protein-specific need

state. Experientia 42: 1003-4, 1986.41. Green, D., and Swets, J. Signal detection theory and psychophysics. NY: John Wiley, 1966.42. Harder, D.B., and Whitney, G. A common polygenic basis for quinine and PROP avoidance in mice.

Chem Senses 23: 327-32, 1998.43. Heck, G.L., Mierson, S., and DeSimone, J.A. Salt taste transduction occurs through an amiloride-

sensitive sodium transport pathway. Science 223: 403-405, 1984.44. Holman, E.W. Immediate and delayed reinforcers for flavor preferences in rats. Learn. Motiv. 6: 91-100,

1975.45. Hoon, M.A., Northup, J.K., Margolskee, R.F., and Ryba, N.J. Functional expression of the taste

specific G-protein, alpha-gustducin. Biochem J 309: 629-36, 1995.46. Horowitz, G.P., Stephan, F.K., Smith, J.C., and Whitney, G. Genetic and environmental variability in

lick rates of mice. Physiol Behav 19: 493-6, 1977.47. Hoshishima, K., Yokoyama, S., and Seto, K. Taste sensitivity in various strains of mice. Am. J.

Physiol. 202: 1200-1204, 1962.48. Jacobs, K.M., Mark, G.P., and Scott, T.R. Taste responses in the nucleus tractus solitarius of sodium-

deprived rats. J. Physiol. (Lond). 406: 393-410, 1988.49. Kanaka, R., Dua-Sharma, S., and Sharma, K.N. Gustatory preferences during estrus cycle in rats.

Indian J Physiol Pharmacol 23: 277-84, 1979.50. Kawamura, Y., and Kare, M. Umami: a basic taste. New York: Marcel Dekker, 1987.51. Kiefer, S.W., Hill, K.G., and Kaczmarek, H.J. Taste reactivity to alcohol and basic tastes in outbred

mice. Alcohol Clin Exp Res 22: 1146-51, 1998.52. Kinnamon, S., and Cummings, T.A. Chemosensory transduction mechanisms in taste. Annu. Rev.

Physiol. 54: 715-731, 1992.53. Kotlus, B.S., and Blizard, D.A. Measuring gustatory variation in mice: a short-term fluid-intake test.

Physiol Behav 64: 37-47, 1998.54. Lawless, H.T., and Stevens, D.A. Cross adaptation of sucrose and intensive sweeteners. Chem.

Senses 7: 309-315, 1983.55. Lawrence, G.J., and Kiefer, S.W. Generalization of specific taste aversions to alcohol in the rat. Chem.

Senses 12: 591-599, 1987.48

Tordoff

56. Leshem, M., Neufeld, M., and Del Canho, S. Ontogeny of the ionic specificity of sodium appetite in the rat pup. Dev Psychobiol 27: 381-94, 1994.

57. Lush, I.E. The genetics of tasting in mice. III Quinine. Genetic. Res. 44: 151-160, 1984.58. Lush, I.E. The genetics of tasting in mice. IV. The acetates of raffinose, galactose and d-lactose.

Genetic. Res. 47: 117-123, 1986.59. Lush, I.E. The genetics of tasting in mice. VI. Saccharin, acesulfame, dulcin and sucrose. Genetic. Res.

53: 95-99, 1989.60. Lush, I.E., Hornigold, N., King, P., and Stoye, J.P. The genetics of tasting in mice. VII. Glycine

revisited, and the chromosomal location of Sac and Soa. Genet Res 66: 167-74, 1995.61. Margolskee, R.F. The biochemistry and molecular biology of taste transduction. Curr Opin Neurobiol 3:

526-31, 1993.62. Margolskee, R.F. The molecular biology of taste transduction. Bioessays 15: 645-50, 1993.63. Markison, S., St. John, S.J., and Spector, A.C. Glossopharyngeal nerve transection does not

compromise the specificity of taste-guided sodium appetite in rats. Am J Physiol 269: R215-21, 1995.64. McLaughlin, S.K., McKinnon, P.J., and Margolskee, R.F. Gustducin is a taste-cell-specific G protein

closely related to the transducins. Nature 357: 563-9, 1992.65. McLaughlin, S.K., McKinnon, P.J., Robichon, A., Spickofsky, N., and Margolskee, R.F. Gustducin

and transducin: a tale of two G proteins. Ciba Found Symp 179: 186-96, 1993.66. Midkiff, E.E., and Bernstein, I.L. The influence of age and experience on salt preference of the rat. Dev

Psychobiol 16: 385-94, 1983.67. Nachman, M. Taste preferences for sodium salts by adrenalectomized rats. J. Comp. Physiol. Psychol.

55: 1124-1129, 1962.68. Nance, D.M., Gorski, R.A., and Panksepp, J. Neural and hormonal determinants of sex differences in

food intake and body weight. In: Hunger: basic mechanisms and clinical implications, edited by Novin, D., Wyrwicka, W. and Bray, G. New York: Raven Press, 1976, p. 257-271.

69. Ninomiya, Y., and Funakoshi, M. Genetic and neurobehavioral approaches to taste receptor mechanism in mammals. In: Mechanisms of taste transduction, edited by Simon, S. and Roper, S. Boca Raton, FL: CRC Press, 1993, p. 254-272.

70. Ninomiya, Y., Higashi, T., Katsukawa, H., Mizukoshi, H., and Funakoshi, M. Qualitative discrimination of gustatory stimuli in three strains of mice. Brain Res. 922: 83-90, 1984.

71. Nowlis, G.H., Frank, M.E., and Pfaffmann, C. Specificity of acquired aversions to taste qualities in hamsters and rats. J Comp Physiol Psychol 94: 932-42, 1980.

72. O'Mahony, M. Understanding discrimination tests: A user-friendly treatment of response bias, rating and ranking R-index tests and their relationship to signal detection. Journal of Sensory Studies 7: 1-47, 1992.

73. Perez, C., and Sclafani, A. Developmental changes in sugar and starch taste preferences in young rats. Physiol Behav 48: 7-12, 1990.

74. Pfaffmann, C. The sense of taste. In: Handbook of Physiology: Section 1: Neurophysiology, edited by Magoun, H.W. Washington: American Physiological Society, 1959, p. 507-533.

75. Phillips, T.J., Crabbe, J.C., Metten, P., and Belknap, J.K. Localization of genes affecting alcohol drinking in mice. Alcohol Clin Exp Res 18: 931-41, 1994.

76. Plattig, K.H., Dazert, S., and Maeyama, T. A new gustometer for computer evaluation of taste responses in men and animals. Acta Otolaryngol Suppl 458: 123-8, 1988.

77. Priehs, T.W., Mooney, K.J., and Bernard, R.A. High dietary sodium enhances gustatory nerve activity and behavioral responses to NaCl. Am. J. Physiol. 261: R52-R58, 1991.

78. Puchalski, R., Kelly, E., Bachmanov, A., Tordoff, M., Brazier, S., Kuang, J., Arrighi, I., and Barhanin, J. Salt appetite is abolished by null mutation of the Isk gene. Annual Meeting of the Association for Chemoreception Sciences, 1999.

79. Ramirez, I. Chemoreception for an insoluble novolatile substance: starch taste? Am. J. Physiol. 260: R192-R199, 1991.

80. Ramirez, I. Does starch taste like Polycose? Physiology and Behavior 50: 389-392, 1991.81. Reed, D.R., and Friedman, M.I. Diet composition alters the preference for fat by rats. Appetite 14: 219-

230, 1990.82. Reed, D.R., Tordoff, M.G., and Friedman, M.I. Enhanced acceptance and metabolism of fats by rats

fed a high-fat diet. Am. J. Physiol. 261: R1084-R1088, 1991.49

Tordoff

83. Reed, D.R., Tordoff, M.G., and Friedman, M.I. Sham feeding of corn oil by rats: sensory and postingestive factors. Physiology and Behavior 47: 779-781, 1990.

84. Reeves, P.G., Nielsen, F.H., and Fahey, G.C., Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123: 1939-51, 1993.

85. Reilly, S., Norgren, R., and Pritchard, T.C. A new gustometer for testing taste discrimination in the monkey. Physiol Behav 55: 401-6, 1994.

86. Richter, C., and Brailey, M. Water intake and its relation to the surface area of the body. Proc. Natl. Acad. Sci. USA 15: 570-578, 1929.

87. Richter, C.P. Salt appetite of mammals: its dependence on instinct and metabolism. In: L'instinct dans le comportement des animaux et de l'homme, edited by Cie, M.e. Paris: Librares de l'Academie de Medécine, 1956, p. 577-629.

88. Richter, C.P. Salt taste thresholds of normal and adrenalectomized rats. Endocrinol. 24: 367-371, 1939.89. Richter, C.P., and Barelare, B. Nutritional requirements of pregnant and lactating rats studied by the

self-selection method. Endocrinol. 23: 15-24, 1938.90. Richter, C.P., and Eckert, J.F. Mineral appetite of parathyroidectomized rats. Am. J. Med. Sci. 9: 9-16,

1932.91. Richter, C.P., and Eckert, J.F. Mineral metabolism of adrenalectomized rats studied by the appetite

method. Endocrinol. 22: 214-224, 1938.92. Riley, A.L., and Clarke, C.M. Conditioned taste aversions: a bibliography. In: Learning mechanisms in

food selection, edited by Barker, L.M., Best, M.R. and Domjan, M. Waco, TX: Baylor University Press, 1977, p. 593-615.

93. Rodriguez, L.A., Plomin, R., Blizard, D.A., Jones, B.C., and McClearn, G.E. Alcohol acceptance, preference, and sensitivity in mice. II. Quantitative trait loci mapping analysis using BXD recombinant inbred strains. Alcohol Clin Exp Res 19: 367-73, 1995.

94. Schiffman, S., Lockhead, E., and Mars, F. Amiloride reduces the taste intesnsity of Na+ and Li+ salts and sweeteners. Proc. Natl. Acad. Sci. USA 80: 6136-6140, 1983.

95. Schiffman, S.S., Frey, A.E., Suggs, M.S., Cragoe, E.J., and Erickson, R.P. The effect of amiloride analogs on taste responses in the gerbil. Physiology and Behavior 47: 435-441, 1990.

96. Sclafani, A. Carbohydrate taste, appetite and obesity: an overview. Neurosci. Biobeh. Rev. 11: 131-153, 1987.

97. Shimura, T., Norgren, R., and Grigson, P.S. Brainstem lesions and gustatory function: I. The role of the nucleus of the solitary tract during a brief intake test in rats. Behav Neurosci 111: 155-68, 1997.

98. Simpson, E., Linder, C., Sargent, E., Davisson, M., Mobraaten, L., and Sharp, J. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nature Genetics 16: 19-27, 1997.

99. Sinclair, J.D., Kampov-Polevoy, A., Stewart, R., and Li, T.-K. Taste preferences in rat lines selected for low and high alcohol consumption. Alcohol 9: 155-160, 1992.

100. Smith, D.V., and Frank, M. Cross adaptation between salts in the chorda tympani nerve of the rat. Physiol. Behav. 8: 213-220, 1972.

101. Smith, J.C., Davis, J.D., and O'Keefe, G.B. Lack of an order effect in brief contact taste tests with closely spaced test trials. Physiol Behav 52: 1107-11, 1992.

102. Spector, A., and Grill, H. Salt taste discrimination after bilateral section of the chorda tympani or glossopharangeal nerves. Am. J. Physiol. 263: R169-R176, 1992.

103. Spector, A., Klumpp, P., and Kaplan, J. Analytical issues in the evaluation of food deprivation and sucrose concentration effects on the microstructure of licking behavior in the rat. Behav. Neurosci. 112: 678-694, 1998.

104. Spector, A.C., Andrews-Labenski, J., and Letterio, F.C. A new gustometer for psychophysical taste testing in the rat. Physiol Behav 47: 795-803, 1990.

105. Spector, A.C., Guagliardo, N.A., and St. John, S.J. Amiloride disrupts NaCl versus KCl discrimination performance: implications for salt taste coding in rats. J Neurosci 16: 8115-22, 1996.

106. Spielman, A.I. Gustducin and its role in taste. J Dent Res 77: 539-44, 1998.107. St. John, S., Garcea, M., and Spector, A. Combined, but not single, gustatory nerve transection

substantially alters taste-guided licking behavior to quinine in rats. Behav. Neurosci. 108: 131-140, 1994.

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Tordoff

108. St. John, S., and Spector, A. Combined glossopharangeal and chorda tympani nerve transectionn elevates quinine detection threshods in rats (Rattus nervegicus). Behav. Neurosci. 110: 1456-1468, 1996.

109. Tepper, B.J., and Friedman, M.I. Altered acceptability of and preference for sugar solutions by diabetic rats is normalized by high-fat diet. Appetite 16: 25-38, 1991.

110. Tordoff, M., Hughes, R., and Pilchak, D. Calcium intake by the rat: Influence of parathyroid hormone, calcitonin, and 1,25-dihydroxyvitamin D. Am. J. Physiol. 274: R214-R231, 1998.

111. Tordoff, M., and Rabusa, S. Calcium-deprived rats avoid sweet compounds. J. Nutr. 128: 1232-1238, 1998.

112. Tordoff, M.G. Metabolic basis of learned food preferences. In: Chemical senses: appetite and nutrition, edited by Friedman, M.I., Kare, M.R. and Tordoff, M.G. New York: Marcel Dekker, 1991, p. 239-260.

113. Tordoff, M.G. Polyethylene glycol-induced calcium appetite. Am. J. Physiol. 273: R587-R596, 1997.114. Tordoff, M.G. Voluntary intake of calcium and other minerals by rats. Am. J. Physiol. 267: R470-R475,

1994.115. Tordoff, M.G., Bachmanov, A.A., Reed, D.R., Price, R.A., and Beauchamp, G.K. Influence of

saccharin preference (Sac) locus on ethanol preference in mice. Research Society on Alcoholism Annual Scientific Meeting, Hilton Head, NC, 1998.

116. Tordoff, M.G., and Friedman, M.I. Drinking saccharin increases food intake and preference: III. Sensory and associative factors. Appetite 12: 23-35, 1989.

117. Tordoff, M.G., and Okiyama, A. Daily rhythm of NaCl intake in rats fed low-Ca2+ diet: relation to plasma and urinary minerals and hormones. Am. J. Physiol. 270: R505-R517, 1996.

118. Torii, K., Mimura, T., and Yugari, Y. Effects of dietary protein on the taste preference for amino acids in rats. In: Interaction of the chemical senses with nutrition, edited by Kare, M.R. and Brand, J.G. Orlando, Florida: Academic Press, 1986, p. 45-69.

119. Vinogradova, E.P., and Zaichenko, I.N. The effect of the stage of the estrous cycle and of acute stress on sugar consumption by female white rats. Zh Vyssh Nerv Deiat Im I P Pavlova 46: 607-9, 1996.

120. Weber, H. [An electric gustometer and its use]. Z Med Labortech 9: 296-302, 1968.121. Weiffenbach, J.M., Cowart, B.J., and Baum, B.J. Taste intensity perception in aging. J Gerontol 41:

460-8, 1986.122. Weijnen, J., and Mendelson, J. Drinking behavior: oral stimulation, reinforcement, and preference.

New York: Plenum Press, 1977.123. Weijnen, J.A. Lick sensors as tools in behavioral and neuroscience research. Physiol Behav 46: 923-8,

1989.124. Weijnen, J.A. Licking behavior in the rat: measurement and situational control of licking frequency.

Neurosci Biobehav Rev 22: 751-60, 1998.125. Weijnen, J.A.W.M. The recording of licking behavior. In: Drinking behavior: oral stimulation,

reinforcement and preference, edited by Weijnen, J.A.W.M. and Mendelson, J. New York: Plenum, 1977, p. 93-114.

126. Whitney, G., and Harder, D.B. Genetics of bitter perception in mice. Physiol Behav 56: 1141-7, 1994.127. Wong, G.T., Ruiz-Avila, L., Ming, D., Gannon, K.S., and Margolskee, R.F. Biochemical and

transgenic analysis of gustducin's role in bitter and sweet transduction. Cold Spring Harb Symp Quant Biol 61: 173-84, 1996.

128. Yamamoto, T., Matsuo, R., Fujimoto, Y., Fukunaga, I., Miyasaka, A., and Imoto, T. Electrophysiological and behavioral studies on the taste of umami substances in the rat. Physiol. Behav. 49: 919-925, 1991.

129. Yamamoto, T., Matsuo, R., Kiyomitsu, Y., and Kitamura, R. Taste effects of 'umami' substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques. Brain Res 451: 147-62, 1988.

130. Young, P.T., and Falk, J.L. The relative acceptability of sodium chloride solutions as a function of concentration and water need. J. Comp. Physiol. Psychol. 49: 569-575, 1956.

CONSULTANTSLetters from Drs. Beauchamp and Bucan follow.

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