Chem. Senses 27: 133-142,
2002
© Oxford University Press 2002
A Brief-access Test for Bitter Taste in Mice
Department of Anatomy and Neurobiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD 21201 - 1509, USA 1 Present address: Department of Psychology, Reed College, 3203 SE Woodstock Blvd, Portland, OR 97202, USA
Correspondence to be sent to: John D. Boughter Jr, Department of Anatomy & Neurobiology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD, USA. e-mail: jboughte{at}umaryland.edu
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Abstract |
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Inbred mouse strains vary in their response to bitter-tasting compounds as assessed by 48 h preference tests. These differences are generally assumed to result from altered gustatory function, although such long-term tests could easily reflect additional factors. We developed a brief-access taste test and tested the responses of two inbred strains, as well as C3. SW congenic mice, to the bitter stimulus sucrose octaacetate (SOA). Water-deprived trained mice were tested with five concentrations of SOA (0.00018-0.18 mM) and distilled water in a Davis MS- 160 apparatus. Trials were 5 s in duration and stimuli were presented randomly within blocks; each stimulus trial was preceded by a water rinse trial. Each concentration was presented twice in a session and mice were repeatedly tested across consecutive days. SOA-taster mice, including the SWR/J (SW) inbred and C3. SW congenic taster (T) mice, avoided licking SOA at concentrations >0.003 mM. In comparison, C3HeB/FeJ (C3) and C3. SW demitaster mice (D) licked all concentrations at the same rate as water. Concentration—response functions were similar across strains for both the brief-access test and a parallel 48 h preference test run on separate groups of mice. Furthermore, concentration—response functions were similar whether or not the brief-access test was preceded by a 4 day, single concentration pretest with SOA. The brief-access test is a suitable assay for bitter taste function in mice because it minimizes possible post-ingestive influences on taste.
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Introduction |
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Genetic variation exists among various strains of laboratory mice for solution preference or aversion, as measured by fluid intake tests (Fuller, 1974
;
Hoshishima et al.,
1961; Lush, 1991;
Whitney and Harder, 1994).
Such natural variation has fostered physiological, biochemical and molecular
approaches aimed at elucidating taste transduction mechanisms and the identity
of genes underlying these mechanisms
(Spielman et al.,
1996; Bachmanov et
al., 1997; Frank and
Blizard, 1999; Miyamoto et
al., 1999; Chandrashekar
et al., 2000; Inoue
et al., 2001). Furthermore, taste transduction components
and putative sweet and bitter taste receptors have been cloned
(McLaughlin et al.,
1992; Adler et al.,
2000; Matsunami et
al., 2000; Kitagawa
et al., 2001; Max
et al., 2001;
Montmayeur et al.,
2001; Sainz et al.,
2001), and mice with taste-related targeted gene deletions and
insertions have begun to appear (Wong
et al., 1996). These advancements make necessary the
development of taste salient behavioral assays for examining taste phenotypes
among mice.
Genetic studies have generally depended on intake in single bottle or
two-bottle tests as the dependent measure. These procedures are particularly
amenable for testing large numbers of mice required for quantitative analysis.
However, it is questionable whether these tests provide valid indicators of an
animal's ability to recognize or discriminate a substance based on gustatory
cues. This limitation has been recognized in studies with rats, and
differences between intake tests and brief-access tests have been
demonstrated. For example, the concentration—response function from a 24
h intake test for sucrose in rats has an inverted U shape, with intake peaking
at intermediate concentrations. In a brief-access experiment (30 s trials),
however, rats will increase licking with increased sucrose concentration in a
monotonic fashion (Smith,
1988). Further, the lick rate for sucrose corresponds closely to
the physiological response in the greater superficial petrosal nerve
(Nejad, 1986), which is the
peripheral taste nerve most responsive to sweet-tasting stimuli. This
correspondence supports the notion that lick rate in a brief-access test
reflects gustatory processing, independent of other controls of appetitive
behavior. To date, only a few studies have been published that examine licking
behavior in mice (Horowitz et
al., 1977; Harder et
al., 1984; Ninomiya and
Funakoshi, 1989).
The salient feature of a brief-access taste test is the presentation of a
taste stimulus for brief-duration trials (typically 5-30 s), and the dependent
measure is the number of licks an animal makes in a trial
(Grill et al., 1987).
In brief-access tests with aversive stimuli, water deprivation is commonly
used to motivate licking behavior, and to provide a baseline of licking from
which a concentration-dependent decrease can be measured. Depending on the
testing apparatus, multiple concentrations of a taste stimulus may be
presented, and a concentration—response function for an individual mouse
can be constructed with the data from just one or a few test sessions. In
comparison, concentration series in 24- or 48 h intake tests may take a week
or more. Because the trials are brief, and because immediate responses are
measured, intake of taste stimuli during a test session is limited and
post-ingestive factors such as those associated with satiety or toxicity are
greatly reduced or avoided. Thus, brief-access tests may be especially useful
for measuring taste behavior to bitter-tasting compounds, for which toxicity
often increases with concentration.
Based in part on methods from previous short-term taste tests with rats, we
developed a brief-access procedure for mice using a commercially available
taste-testing apparatus, the Davis MS-160
(Smith, 2001). We sought to
compare results from this procedure with results from a two-bottle intake
assay for sucrose octaacetate (SOA), a relatively common and non-toxic bitter
taste stimulus. SOA is avoided in two-bottle tests by some strains of mice;
this sensitivity is determined by allelic variation at a single genetic locus
(Warren and Lewis, 1970;
Lush, 1981). This locus,
Soa, was mapped to a position on mouse chromosome 6 that was later
determined to be the region where the T2R family of receptors is located
(Capeless et al.,
1992; Adler et al.,
2000; Matsunami et
al., 2000). Despite this concordance, the exact sequence and
gene product of Soa has yet to be determined. A congenic strain, C3.
SW-Soaa, was developed from SWR/J (SW: SOA taster) and C3
HeB/FeJ (C3: SOA demitaster) inbred strains (Boughter and Whitney,
1995,
1998). In two-bottle tests, SW
mice avoid concentrations of SOA as low as 0.001 mM. In contrast, C3 mice
respond indifferently to all concentrations except a near-saturated 1 mM, for
which they show a relative avoidance (demitasters are still significantly less
sensitive to 1 mM SOA than tasters; the third phenotype, non-taster, is
indifferent at this concentration). The C3. SW-Soaa
congenic taster mouse strain contains the SOA taster allele transposed on a
99% C3 genomic background, and the SOA taster allele causes SW-like
behavioral aversion to SOA. Significantly, these congenic mice have been
maintained as heterozygotes for Soa by repeating generations of
backcrossing phenotypic C3. SW tasters with C3 inbred mice. This means that
each new generation of C3. SW backcross mice must be behaviorally tested with
SOA in order to determine phenotype; each backcross mouse stands a 50:50
chance of being a phenotypic taster (T) or demitaster (D).
We took advantage of this previously studied SOA congenic system to test our brief-access procedure. We were interested in the ability of this procedure to produce in mice reliable concentration—response functions in response to the aversive taste of SOA. Furthermore, we compared the brief-access functions directly with those produced by two-bottle tests. Finally, because the C3. SW backcross mice must be tested to determine phenotype, we examined the ability of our assay to be used as a classifying procedure.
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Methods |
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Animals
A total of 93 adult inbred (C3 and SW) and C3. SW
N29-N32 congenic taster (T) and demitaster (D) mice
(Mus domesticus) were used in all experiments. All mice were
naïve at the start of each experiment. The total
numbers of mice per strain, per experiment are listed in
Table 1 along with information
about gender, age and weight. Overall, equal numbers of males (n =
46) and females (n = 47) were tested, and the age of the mice at the
start of each experiment ranged from 49 to 158 days, with a median age of 72
days. Care was taken to age-match the strains within each experiment as well
as possible. Previous studies have not indicated effects of gender on SOA or
quinine aversion in mice (Lush,
1984; Whitney et al.,
1991; Harder et al.,
1992). Inbred mice were either laboratory bred or obtained from
the Jackson Laboratory (Bar Harbor, ME). All T or D mice were laboratory bred
(see below). Food (Harlan teklab, 7012) and water were available ad
libitum, except where noted below.
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C3. SW Congenic mice
The development of C3. SW-Soaa heterozygous congenic
taster mice has been described in detail previously (Boughter and Whitney,
1995,
1998). Through a protracted
process of phenotypic selection (two-bottle tests with SOA) combined with
lineal backcrossing across 11 generations, the dominant
Soaa (taster) allele was transferred from the SW donor
strain on to the genomic background of the C3 inbred partner strain. In each
backcross generation, 50% of C3. SW mice were T and 50% were D mice,
consistent with expectations from a one-locus model with an autosomal allele
conferring a dominant taster phenotype. By generation N12, each T
mouse carried one copy of the Soa taster allele and was in theory
98.9% genetically identical to the C3 inbred strain for genetic material not
linked to the Soa locus (Flaherty,
1981). Further generations of C3. SW mice were developed,
including generations N29—N32 for use in the
present study. We used 52 C3. SW mice, and as previously
(Boughter and Whitney, 1998),
the phenotype of these mice (T or D) was determined or confirmed within each
experiment via two-bottle, 48 h tests with SOA (see Results). Twenty-five mice
were classified as T and 27 as D mice; this 50:50 ratio was consistent with
previous generations.
Two-bottle tests
Two-bottle preference tests were conducted in plastic cages (28 x 17.5 x 13 cm) with corn-cob bedding and stainless steel wire lids. Mice were housed individually, and food was available ad libitum. Two inverted graduated cylinders were placed on either side of a cage lid, and each cylinder had a neoprene stopper with a straight stainless-steel sipper tube that protruded into the cage about 6 cm from the cage floor. One cylinder contained 0.1 mM SOA, whereas the other contained distilled water. After 24 h the amount consumed from each cylinder was recorded and the position of the cylinders was switched. Amounts consumed were again recorded after 24 h. A preference ratio (PR) (amount of solution consumed/total amount consumed) was calculated for each day and the two were averaged to obtain a 48 h PR for each mouse.
Brief-access tests
Brief-access tests were conducted in a Davis MS-160, a commercially available taste test apparatus (DiLog Instruments, Inc., Tallahassee, FL). The mouse was placed in a rectangular test cage (30 x 14.5 x 16 cm) with a stainless steel mesh floor, and could access taste solutions or water via a small opening in the front wall of the chamber. Access to stimuli was computer controlled. A given trial began when a shutter was opened to allow access to a stainless steel sipper tube (2.5 cm from floor), and ended after a defined time period when the shutter closed. In between trials, the computer drove a stepping motor to position one of up to 16 drinking tubes in front of the stimulus-access opening in preparation for ensuing trials. Licks were counted with a high-frequency AC contact circuit.
For each brief-access experiment, water-deprived mice were first trained to lick water in the Davis apparatus, then tested in the apparatus with a concentration series of SOA plus water. Approximately 24 h prior to training, water bottles were removed from the home cages of individually housed mice. On the first training day, a mouse was placed in the test chamber and given access to distilled water for 30 min. The amount consumed was recorded and mice were returned to the home cage. On the second day, the procedure was repeated, and the number of licks in the 30-min session was recorded.
Testing occurred on days 3-5. During test sessions, the mouse initiated a 5 s trial with a single lick on the sipper tube. The time between the opening of the shutter and the first lick was recorded as the `latency to lick'. In the event that the mouse did not make a lick within 300 s of the shutter opening, the shutter was closed and the next trial begun. There were two types of trials: water rinse trials and test trials. Each test trial was preceded by a water rinse trial. The rationale for using water rinse trials was to minimize carryover effects from one trial to the next (e.g. after-taste or contrast effects). Test trials consisted of the presentation of either water (water test trial) or one of five concentrations of SOA (3/4 log steps: 0.00018, 0.001, 0.006, 0.03 and 0.18 mM), which were prepared daily from a reagent grade source (Sigma, St Louis, MO) and distilled water. The presentation order of test stimuli was randomized in two blocks of six; with rinse trials, the mouse could initiate up to 24 trials a day. A 15 s intertrial interval separated these 24 trials.
Data analysis
Number of licks for each SOA trial, plus water test trials, were averaged
across the three test sessions for each individual mouse. These data were then
reported as lick ratios (average number of licks to SOA/average number of
licks during water test trial). This served to control for potential
differences in lick rate that were non-gustatory in origin; i.e. the speed of
the central pattern generator (CPG) for licking or the overall activity of the
strain or individual. It is important to note that the water rinse trials were
not considered in the calculation of lick ratios for SOA, although these
trials were considered later in the analysis of water licking (e.g.
Table 2). For concentration
series, mean lick ratio and PR data were fitted with sigmoidal three-parameter
functions: f(x) = (1 - d)/(1 +
(x/c)b) + d, where x
represents SOA concentration, b represents the slope, c
represents the concentration of SOA that evoked the half-maximum response, and
d represents the asymptotic minimum. Parameter c was
compared between strains with t-tests. Additionally, mean data were
compared between strains using one- or two-way analyses of variance, with a
repeated measures design and post-hoc comparison tests
(Scheffé) where appropriate. The statistical
rejection criterion (
) for all tests was set at the 0.05 level.
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Experiment 1: A—B—A design
In this experiment, nine C3, nine SW and 13 C3. SW N29 congenic mice were given two consecutive 48 h two-bottle tests with 0.1 mM SOA during the first week. The purpose of these `pretests' was to classify the C3. SW mice as T or D prior to brief-access testing. At the conclusion of these tests, mice were placed in home cages with ad libitum access to water and chow for 2 days. Water bottles were then removed from the home cages, and the mice were trained (2 days) and tested (3 days) using the brief-access procedure. During this week, mice received their daily fluid intake during the training and testing sessions. After the last test session, water bottles were replaced on the home cages, and after two more days they were again given two consecutive 48 h two-bottle tests with 0.1 mM SOA.
Experiment 2: B—A design
In this experiment, three C3, four SW and 12 C3.SW N31 or N32 mice were given the brief-access tests in the first week, and two-bottle tests in the second week. There were two reasons for this design: first, we examined whether C3.SW T and D mice could be reliably classified on the basis of the brief-access test alone. The SOA phenotypes of these mice, as well as the inbred strains, were then verified based on the results of the two-bottle post-test. Second, this protocol allowed us to compare lick ratios in naïve mice versus those with prior experience with SOA (A—B—A design).
Experiment 3: two-bottle concentration series
Five C3, five SW and 17 C3.SW N30 mice were given consecutive 48 h two-bottle tests with the same concentration range (0.00018-0.18 mM, 3/4 log steps) of SOA that was used in the brief-access tests. This experiment was repeated with a second group of mice from all three strains (three C3, three SW, 10 C3.SW N32) and for analysis data were collapsed across both experiments. C3.SW mice were classified as T or D on the basis of their PRs to 0.13 mM SOA; this classification was verified with a single 48 h post-test with 0.1 mM SOA. This experiment was conducted in order to make a direct, concentration-by-concentration comparison of the brief-access and two-bottle procedures.
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Results |
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A—B—A design
Pre- and post-tests
Individual PRs and strain means for the two-bottle pretest are shown in
Table 3. For both test periods
all SW mice dramatically avoided SOA (PR < 0.05), whereas C3 mice were
indifferent (PR ranged from 0.31 to 0.72). Based on previous studies with
C3.SW congenic mice, we set a criterion PR to determine Soa phenotype
among C3.SW mice: mice were classified as T if they had a PR of <0.15, D if
0.15 (Boughter and Whitney,
1995). Using this criterion, 13 C3.SW mice were unambiguously
classified as T (seven) or D (six) mice
(Table 3). Across both test
periods, SW and T mice avoided 0.1 mM SOA in a similar fashion, whereas C3 and
D mice were indifferent; the strain difference was significant [two-way ANOVA,
F(3,27) = 175.27; P < 0.001]. During preference testing,
SW mice consumed more total fluid (mean = 13.1 ml) than the other three
strains (C3 = 8.9, T = 10.2, D = 9.0) during each 48 h test. This strain
difference for total fluid intake was significant [two-way ANOVA,
F(3,27) = 14.65; P < 0.001]. T mice differed from D mice
in terms of SOA aversion, but not in total fluid intake, indicating that the
Soa taster allele did not influence the latter measure.
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Table 3 also contains results from the post-test. In every case, PRs for individual mice were virtually identical to those in the pretest, with all mice easily meeting the criterion value. In addition, total fluid intake was similar to the pretest (data not shown).
Brief-access test
In the brief-access test, SW and T mice avoided SOA in a
concentration-dependent manner, whereas C3 and D mice were indifferent to SOA
(Figure 1). A two-way ANOVA
(strain x concentration) indicated significant effects for strain
[F(3,27) = 50.97; P < 0.001], concentration
[F(4,108) = 72.10.2; P < 0.001], and the strain x
concentration interaction [F(12,108) = 25.56; P < 0.001].
Post-hoc tests (Scheffé) confirmed SW and T
mice did not differ from one another but did differ from C3 and D mice. The
latter strains did not differ. The concentration of SOA that evoked the
half-maximum avoidance response, as calculated from the functions fitted to
the SW and T data, did not differ significantly.
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Latency to first lick. We also examined the latency to first lick
in each trial, because concentration-dependent changes in this measure have
been shown previously in the Davis apparatus to be indicative of olfactory
contributions to sucrose responsiveness
(Rhinehart-Doty et al.,
1994), and because another study suggested that animals may be
able to smell the bitter compound quinine
(Benjamin, 1960). Latency did
vary significantly with SOA concentration
[Figure 2A; F(5,135) =
2.47; P < 0.04], although there was not a significant effect of
strain. When latencies for each concentration were collapsed across strain, it
was evident that mice had a somewhat shorter latency to lick during the water
test trials, and during trials with 0.00018 mM, than during trials with
0.001-0.18 mM SOA (Figure 2B).
This possibly indicated that mice could smell or otherwise anticipate the
higher concentrations of SOA, or conversely could anticipate the water and the
most diluted SOA concentration. In either case, the latencies for the higher
four concentrations were roughly equal, indicating that the mice could not
distinguish SOA concentration with a non-taste cue. Mice of all strains took
about 10-20 s to make initial contact with the spout. However, this varied
considerably within each set of trials; mice tended to have a shorter latency
earlier in the test session when they were thirstier. Collapsed across both
strain and concentration, the mean latency for trials 1-12 was 8.27 s, whereas
the mean latency in trials 13-24 was 22.20 s.
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Differences in water lick rate. Although SW and T mice displayed similar responses to SOA (Figure 1), when water test trials and water rinse trials were examined, we found that SW mice actually licked water at a higher rate than the other strains [Table 2A; F(3,27) = 9.9, P < 0.001]. This suggested that SW mice might be capable of producing more licks in the 5 s taste trial than mice from other strains, which was further supported by comparing the maximum number of licks produced by mice from each strain (Table 2A).
Because of the difference in licks to water, we hypothesized that SW mice
may have a faster CPG for licking as compared with the other strains. To test
this hypothesis, we examined the inter-lick interval (ILI) distribution for
each strain during the 30 min training period with water (i.e. day 2). If the
SW mice had a faster CPG, the interval between licks should generally be
shorter than those of the other strains of mice
(Horowitz et al.,
1977). The mean ILIs (per strain) for each 5 ms bin, expressed as
a percentage of the total number of ILIs across all bins, is shown in
Figure 3. Perhaps surprisingly,
the strains did not differ in their ILI distributions: On average, each strain
possessed a bimodal distribution of ILIs, with the mode occurring at about
110-115 ms. The second, much smaller, peak in the distributions, occurring at
190-220 ms, reflects incidents where mice `missed' a lick in a given sequence,
thus resulting in an ILI about double that of the normal.
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A second possibility is that SW mice may be thirstier than mice from other strains. Body weight did not differ significantly as a function of strain (data not shown), although SW mice produced about twice as many licks as mice from other strains during the 30 min training session on day 2. Whatever the source for differences in lick rate to water, these differences in phenotype are not influenced by Soa, as T and D mice are far more similar to the C3 parental strain than the SW. These differences, while minor, underscore the appropriateness of expressing taste-responsivity as a ratio relative to each animal's average licking rate to water.
B—A design
As in the A—B—A design, SW mice decreased their licking as a function of concentration, whereas C3 mice licked all concentrations at the same rate as water (Figure 4). Of 12 C3.SW mice, three individuals had decreased lick ratios at the higher concentrations and were subsequently classified as tasters after two-bottle post-tests with 0.1 mM SOA (Table 4). The remaining nine C3.SW mice possessed a mean lick rate that was similar to water for all concentrations of SOA. These mice were subsequently confirmed as demitasters by post-test results (Table 4). In either case, lick ratio functions for individual C3.SW mice (not shown) were clearly indicative of phenotype. A two-way ANOVA indicated significant effects for strain [F(3,15) = 31.98; P < 0.001], concentration [F(4,60) = 30.08; P < 0.001], and the strain x concentration interaction [F(12,60) = 10.13; P < 0.001]. Post-hoc tests (Scheffé) confirmed that SW and T mice possessed lower lick ratios than C3 and D mice. Two-bottle post-test results for all strains were well within taster—demitaster criterion (Table 4; cf. Table 3). As in the A—B—A test, the half-max parameter did not differ between SW and T mice. Analysis of latency did not reveal significant effects, in contrast to the modest effects seen in the A—B—A design.
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Water lick rate
As in the A—B—A experiment, SW mice licked at a higher rate
than the other strains during the water test and water rinse trials
[Table 2B; F(3,15) =
26.8, P < 0.001]. This strain difference was also manifest in
maximum licks in a trial (Table
2B). Unlike the A—B—A experiment, however, there was a
modest strain difference with regards to weight loss during water deprivation
[F(3,15) = 4.66; P < 0.02]. Post-hoc tests indicated that
SW mice differed only from D mice.
Preference testing with concentration series of SOA
Figure 5 contains the
results of testing mice from each strain with an ascending series of SOA
(0.00018-0.18). C3.SW mice were classified (after testing) as T (n =
14) or D (n = 12) on the basis of their PR at the highest
concentration (0.18 mM), using the same criterion (PR <0.15 or 0.15) as
the previous experiments. This classification was confirmed for all T and D
mice with a single, 48 h post-test with 0.1 mM SOA (data not shown). Avoidance
levels for the SOA concentration series were very similar to those obtained in
brief-access testing (Figure 5;
cf. Figures 1 and
3). Results from a two-way
ANOVA showed significant effects of strain [F(3,34) = 120.97;
P < 0.001] and concentration [F(4,136) = 26.17;
P < 0.001]. There was also a statistically significant interaction
[F(12,136) = 19.12; P < 0.001]. Post-hoc tests
(Scheffé) confirmed that SW and T mice
possessed lower PRs than C3 and D mice. As in the brief-access tests, the
half-max parameter did not differ significantly between SW and T mice. In
fact, this parameter did not differ significantly among either SW or T mice
when the brief-access functions from the A—B—A and B—A tests
were compared with the functions from the preference test, or with each other.
This further illustrates the point that the level of aversion was similar for
these two strains in either type of test.
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Discussion |
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Lick ratio functions for SOA
In the first experiment (A—B—A), inbred and backcross mice were
first `screened' with consecutive 48 h SOA two-bottle preference tests,
brief-access tested with a SOA concentration series, and then preference
tested again. The initial screen confirmed that SW inbred mice avoided 0.1 mM
SOA, whereas C3 inbred mice were indifferent. Additionally, the screen
unambiguously revealed that seven of 13 C3.SW congenic backcross mice were
phenotypic tasters (T); the remaining six mice were classified as demitasters
(D). Concentration—response functions for each mouse, and mean functions
for each strain, were then generated from 3 days of brief-access testing. SW
and T mice avoided SOA in a concentration-dependent manner, whereas C3 and D
mice licked all concentrations of SOA at the same rate as water. These results
were consistent with expectations that variation at the Soa allele
influences taste sensitivity to SOA itself, i.e. the T mice displayed a
similar level of taste avoidance as the SW mice. Because the trials with SOA
were only 5 s in duration, post-ingestive cues could not specifically affect
an ongoing trial. Furthermore, because all stimuli (water and SOA
concentrations) were presented intermixed in a single test session,
post-ingestive feedback could not differentially affect lick rate across
stimuli. The results of the brief-access test can therefore be ascribed to
gustatory processing with considerably more confidence than can the two-bottle
tests. While this test offers these advantages over intake tests, it is
important to acknowledge that other orosensory factors, such as
somatosensation, could contribute to the behavioral differences observed. The
hypothesis that the brief-access results are based solely on gustatory input
could be examined with further studies combining behavior with nerve
transection as has been done in the rat
(St. John et al.,
1994). After gustatory nerve transection, somatosensory input
would persist via the lingual nerve. If the concentration-based rejection in
SW and T were reduced by the nerve cuts, a role for gustatory input in the
observed behavior would be clarified.
The lick ratio functions for each strain were extremely similar to
preference functions using the same concentration range (cf. Figures
1 and
5). Perhaps this is not
surprising, given that SOA is a non-toxic aversive compound. We expect that
the brief-access test will prove especially useful in testing those
bitter-tasting stimuli that are toxic, such as the commonly used bitter taste
stimuli caffeine and cycloheximide. There are few behavioral studies that have
examined sensitivity to these compounds, either in rats or mice. However, one
example is potentially instructive: Strains of mice differentially avoid
phenylthiocarbamide (PTC) in two-bottle intake tests, but typically only after
a few days of consumption (Whitney and
Harder, 1986). It is thought that this aversion is due to a form
of taste conditioning associated with the mild toxic effects of PTC intake.
Preliminary data from our laboratory (unpublished data) suggest that mice did
not avoid concentrations of PTC in a brief-access test that they will avoid in
an intake test.
Classification of mice using brief-access test
In the A—B—A design, mice had experience with SOA during the preference test that conceivably might have altered SOA sensitivity in the brief-access test. We directly tested this possibility in a second experiment in which mice were first screened with the brief-access procedure, only then `confirming' this classification with the traditionally used preference test. In fact, there was no evidence that the concentration—response functions differed substantially between the A—B—A and B—A conditions (Figures 1 and 4). The brief-access procedure allowed for discrimination of T from D mice. Because of recent advancements in molecular and genomic approaches to the taste system, there is great interest in taste-salient screening techniques for mice with taste-related targeted gene insertions and deletions. Taste behavior differences must be demonstrated in the absence of potential post-ingestive cues if the goal is to elucidate the molecular or physiological underpinnings of taste behavior. We believe that (at least for bitter stimuli) our brief-access procedure accomplishes this; the mere possibility that post-ingestive factors can influence the results begs for the use of the more taste-salient short-term test.
Multiple concentrations of a stimulus can be delivered in a single session using the Davis MS-160, and reliable concentration—response functions can be generated with a few days of testing. In contrast, it generally takes one or more weeks to collect a concentration—response function using a two-bottle assay. On the other hand, there is a practical limit to how many mice can be tested in a single day using the brief-access procedure, while two-bottle tests can be reasonably done with large groups of mice (i.e. 50-100), depending on the availability of testing equipment and space. Our procedure represents a more taste-salient approach, one that may be amenable for phenotypically screening smaller squads of mice (i.e. 10-20) in a single week. We suggest that the two-bottle testing paradigm be used as a screening tool only after it has been verified that, over a range of concentrations, the preference testing functions match those generated by a more taste-salient test, like the brief-access test shown here. This criterion is met for SOA in the present strains of mice.
Differences in water lick rate
Interestingly, there was a difference in the licks to water between strains: Inbred SW mice tended to lick water more than C3, T or D mice in either a 5 s trial or during a 30 min training session. This difference was not due simply to a faster lick rate, because the ILI distributions in a 30 min water trial were similar among strains (Figure 4). It was possibly due to a difference in thirst, although weight loss during water deprivation was similar between strains. Furthermore, SW mice differed from all other strains in a non-deprived state: SW mice had a greater total consumption of both SOA and water during consecutive 48 h two-bottle tests with 0.1 mM SOA. In any case, the Soa taster allele did not have an effect on water licking, or total consumption: T mice were similar to C3 and D mice, and dissimilar to SW mice. This finding also provides a rationale for presenting brief-access lick behavior in terms of lick ratios. The ratio corrects for any difference in a mouse's ability or tendency to make a certain number of licks in a given trial. After data are normalized in this fashion, SW and T mice have concentration—response functions that virtually overlap, reflecting the complete effect of the Soa taster allele on SOA taste sensitivity.
In summary, the brief-access test detailed here appears to be a useful tool for accurate assessment of taste function in mice. Because this test relies on water deprivation to motivate licking behavior, the test is appropriate for aversive stimuli, such as bitter-tasting stimuli or acids. Unlike two-bottle assays, the brief-access procedure allows a greater depth of analysis concerning taste behavior. In addition to the defining feature of lick ratio measurements in brief trials, this procedure offers the ability to examine microstructural aspects of licking patterns during water training (e.g. ILI distributions), examine latencies to discern possible olfactory contributions, to test a full range of concentrations in a single session, to test quickly trained mice on other stimuli of interest in the same or ensuing test sessions, and to make comparisons with intake tests in order to implicate post-ingestive influences.
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This manuscript was written with the support of NIDCD Grant DC00353 (D.S.), and a grant from the Bressler Foundation of the University of Maryland School of Medicine (J.B.). We appreciate the helpful comments of Dr Vanessa Anseloni and Dr Kevin Kelliher, and the technical help of Kate Jordan. A portion of these results was presented at the 2000 Annual Meeting of the Association for Chemoreception Sciences.
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Accepted October 23, 2001
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