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A. SPECIFIC AIMS
Taste 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.
A1To
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 rodents
How 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 twotube 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 (1020) 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 once 51.
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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.
d-Phenylalanine, mM
Aspart ame, mM
75
As c ending c onc entrations
Preference, %
I nt ake, ml / 30 g B W
Sucrose, %
20
B6
12 9
10
Des c ending c onc entrations
50
25
0
0
1
1
2
2
4
3
8
4
16
5
32
6
3
1
10
2
30
3
100
4
0. 03 0. 1 0. 3 1
1
2
3
4
3
5
10
6
37.5
75
150
300
450
600
NaCl Concentration (mM)
Sweet ener concent rat ion
FIG. 2. Results of 48-h two-bottle choice tests using 8 B6 and 129 mice given ascending
concentrations of three compounds humans consider to be sweet (from 18 tested; see text).
Note the "taste blindness" of 129 mice for d-phenylalanine. Note also that for most solutions,
the standard errors were too small to display. Water intake (not shown) was <5 ml for all tests.
26
FIG. 1. NaCl preference of B6 mice
given 6 concentrations of NaCl in
either ascending or descending
order. (48-h 2-bottle choice tests).
Data from Ref7(in Appendix).
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4%
Su c r o s e
2. Order and concentration effects in long-term taste tests
Several 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.
Sa c c h a r in
30 m M
10%
d - P h e n y la la n in e
Et h a n o l
Cit r ic Ac id
of
m ic e
0. 1 m M
Nu m b e r
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
2 m M
6 0
50 m M
Cit r ic Ac id
4 0
2 0
6 0
250 m M
Na Cl
4 0
2 0
6 0
0. 03 m M
Q u in in e HCl
4 0
2 0
6 0
0. 3 m M
Q u in in e HCl
4 0
2 0
6 0
1 m g / l Ca p s a ic in
4 0
2 0
0
We have also conducted studies involving giving mice brief exposures to taste
0
50
100
Pr e f e r e n c e , %
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
FIG. 3. Frequency distributions
of 180 mutagenized mice given
rates during the first 15 min of fluid access each day. During the first week, the
48- or 96-h two-bottle choice
mice received water. During the second, they received 10% ethanol during the
tests with 10 taste solutions.
A2In
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".
27
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Week 1
Tuesday
Wednesday
Thur sday
Fr iday
Aver age
300
200
100
Week 2
Licks - 10% et hanol
0
B6
300
129
200
100
Week 4
0
Licks - 10% et hanol
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.
Licks - Wat er
M onday
300
200
100
0
0
2
4
6
8 10
0
2
4
6
8 10
0
2
4
6
8 10
0
2
4
6
8 10
0
2
4
6
8 10
0
2
4
6
8 10
Tim e ( m in)
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
Table 1. Comparison of the advantages and disadvantages of long-term, two-bottle choice tests and briefexposure, lickometer tests
Feature
Long-term, two-bottle choice test
Equipment required
Drinking tubes
Dependent variables
Taste solution and water intake, total fluid
intake, taste solution preference
Typically 48 h per taste solution, with
additional "wash-out" days
>300 mice/day. Limited only by time
required to collect data.
Test duration
Test capacity
Existing literature
Performance
Scope
Interpretation
Independence
Strong
Fairly independent of mouse's physical
competence
Wide. Can detect aberrant intakes due
to postingestive factors as well as
chemosensory ones.
Complex because oral and postingestive
factors are confounded
Carry-over effects can confound intake
on later tests
28
Brief-exposure, lickometer test
Drinking tube, lickometer amplifier,
computer interface, computer
Lick rate and number
Typically 2 min per taste solution with
24 h between tests
8-24 simultaneous tests, limited by
equipment but many replications can be
conducted each day
Small, particularly for mouse
Physical competence can affect licking
rate
Narrow. Detects aberrant intakes due to
chemosensory factors only.
No postingestive factors to worry about
No carry-over effects
Tordoff
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 briefexposure 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 METHODS
General 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 wedgeshaped 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
sucrose
glucose
saccharin
120 mM
120 mM
2 mM
3,4,6,16,59,60,75
Unpublished
results
amino acid-like
d-phenylalanine
aspartame-like
aspartame
amiloride-blockable
sodium channels
NaCl
nonspecific/mineral
KCl
CaCl2
Sour
none
HCl
citric acid
Bitter
quinine-like
quinine HCl
SOA-like
SOA
1 mM
Umami
none
MSG
300 mM
Starch or
maltodextrin
unclear
Polycose
maltodextrin
cornstarch
60 g/l
60 g/l
60 g/l
Unpublished
results
Fat
unknown
corn oil
10 g/l
Unpublished
results
Texture and
irritation
touch/texture
temperature
pain
capsaicin
ethanol
1 mg/l
10 g/l
4,6,16,93
Unpublished
results
Salt
30 mM
1 mM
75 mM
300 mM
400 mM
100 mM

30 mM
50 mM
0.3 mM
4-6,9,12,
Unpublished
results
4 Unpublished
results
5,18,19,42,47,57,
58,126
7 Unpublished
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.
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. Maltodextrin, cornstarch, and
corn oil will be held in
suspension by homogenization 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, twobottle tests will involve a
choice between the taste
"solution" and its vehicle
(either 0.1% xanthan gum
or 0.1% ethanol). For briefexposure studies, a subthreshold 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.
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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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
Table 3. Solutions to be used in
qualities (sweet, salty, sour, and bitter). The concentrations were
the short test sequence
chosen based on our previous work and existing literature involving
4,6,12,42
tests of hundreds of B6 and 129 mice and their hybrids
. This
water
work indicates the two strains find the chosen concentrations of
2 mM saccharin
saccharin and NaCl moderately palatable and those of citric acid and
50 mM citric acid
0.3 mM quinine hydrochloride
quinine moderately unpalatable. The B6 and 129 mice respond
75 mM NaCl
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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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 apart
In 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
33
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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
representatives of all the taste qualities listed in Table 122,111. We emphasize that the dietary manipulations
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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 signalto-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
require careful coordination as we will have to maintain younger mice in our own colony for several months
35
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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.,
causes malaise) the mouse will learn to avoid that taste solution and solutions with similar orosensory
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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
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
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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
Wire to ground
Wire to lic k ometer c irc uitry
Tas te s olution
Metal drink ing s pout
(ins ulated from c age top)
Metal c age top
Mous e
Plas tic c age
Attac hment bolts
Metal plate (8 c m wide)
made, the modified cage lids can be washed using
automatic cage or tunnel washers without problems and FIG. 5. Diagram of cage modified for detection of individual
licks by a mouse (lickometer). Note that the mouse must
the steel sheet can be removed without damage to the
stand on a metal plate to reach the drinking spout.
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 LI157) 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
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
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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 them 101.
Moreover, as carry-over effects are most likely the result of the association of orosensory cues with their
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
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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 identicallytreated 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,
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
40
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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 soaa [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 soaa [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.
75 mM (0.5%) NaCl
Finally, we intend to test B6 animals given manipulations that are
Water
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
Control
Glossopharyngea
Chorda tympani
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
FIG. 3. Example of our work with gustatory nerve
postmortem. Other procedures to be used include (ii)
transections in B6 mice. The chorda tympani nerves
adrenalectomy, which interferes with sodium metabolism and
were accessed through the tympanic membrane (n =
5). The glossopharyngeal nerves were accessed by
induces a specific sodium appetite (see MS8 in Appendix), (iii)
tunneling under the digastric muscle (n = 3). Control
streptozotocin-induced diabetes, which interferes with blood
mice received sham surgery (n = 4). At 13 days
after the surgery, the mice were presented with 75
glucose regulation and alters sweetness acceptance (in rats)109 (iii)
mM for two days, given together with water. Note
maintenance on a high-fat, low-carbohydrate diet, which
that bilateral transection of the chorda tympani nerve
facilitates fat metabolism and increases preferences for fat-like taste eliminated the normal aversion of this strain to NaCl
solution.
solutions81,82. The surgically compromised groups will have their
41
5
Fluid intake, ml
4
3
2
1
0
Tordoff
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.
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
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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 briefexposure 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 longterm, 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
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
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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
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
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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 SUBJECTS
Not applicable
F. VERTEBRATE ANIMALS
1. 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.
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.
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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/6J
129/SvJ
8 wk
8 wk
male
male
16
16
1b. Two vs three- bottle
tests
Two-bottle or three-bottle tests (3
conditions)
C57BL/6J
129/SvJ
8 wk
8 wk
male
male
16
16
1c. Test duration
Two-bottle tests (4 durations)
C57BL/6J
129/SvJ
8 wk
8 wk
male
male
16
16
1d. Diet effects on
solution acceptance
Two-bottle tests (6 maintenance
diets)
C57BL/6J
129/SvJ
8 wk
8 wk
male
male
96
96
1e. Age effects on
solution acceptance
Two-bottle tests (12 ages)
C57BL/6J and
129/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/6J
129/SvJ
8 wk
8 wk
male
male
64
64
2a additional studies
Brief-exposure (2 min) taste solution
tests after 6 h water deprivation (5
taste solutions)
C57BL/6J
129/SvJ
8 wk
8 wk
male
male
80
80
3. Establishing reference
data
Brief exposure and two-bottle tests
in the same animals (20 solutions)
C57BL/6J
8 wk
8 wk
8 wk
8 wk
8 wk
8 wk
8 wk
8 wk
male
female
male
female
male
female
male
female
50
50
50
50
400
400
112
112
8 wk
8 wk
female
male
128
128
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)
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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CONSULTANTS
Letters from Drs. Beauchamp and Bucan follow.
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