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Department of Psychology, University of Florida, Gainesville, FL 32611 and * Department of Physiological Sciences, School of Veterinary Medicine and Food Intake Laboratory, University of California-Davis, Davis, CA 95616
4To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • lysine • threonine • specific appetite • 23-h intake • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Rozin 1965
). The best
example of deficiency-induced appetite occurs after sodium
depletion and is manifested by an avid responsiveness to salts
possessing this cation. To make general conclusions about the possible
behavioral strategies animals apply in the face of a nutritional
challenge, we drew from the sodium appetite literature and applied what
is known there to the study of other nutrient deficiencies. Because it
has been known for some time that lowering the level of or eliminating
an essential amino acid
(EAA)5
from the diet impairs physiologic function and feeding in rats (for
reviews, see Gietzen 1993
, Harper et al. 1970
), we
elected to examine deficiency of two such amino acids, i.e., lysine
(LYS) and threonine (THR).
EAA deficiency may prove to be a useful model to study recovery from a
specific nutritional challenge for several reasons. First, a relatively
small proportion of the extensive amino acid literature has focused on
behavioral questions, particularly those that emphasize
"strategies" used by the animal to produce recovery. Second,
ingestive behavior is indeed altered in response to the deficiency.
Although most of these behavioral effects have been demonstrated using
complex food intake paradigms (for reviews, see Gietzen 1993
, Harper et al. 1970
), at least a few
experiments support increased consumption of the missing amino acid
when it was presented in solution. For example, Rogers and Harper (1970)
found that histidine-deficient rats increased
their intake of histidine solutions, and Torii et al. (1986)
revealed that rats fed a diet lacking in lysine
increased their intake of 400 mmol/L lysine-HCl. Third, amino
acids presented in solution appear to be salient gustatory cues
(Grill et al. 1987
, Pritchard and Scott 1982
). Potentially, behavioral responsiveness to the taste of
the stimulus might be influenced by nutritional state; indeed, this has
been shown to occur in the sodium-deficient rat. Fourth, EAA
deficiency can be produced relatively easily through dietary depletion.
Finally, EAA are the building blocks of protein and are absolutely
necessary for growth and survival. Accordingly, there would appear to
be a selective advantage favoring behavioral mechanisms that defended
appropriate levels of such nutrients.
To determine whether EAA deficiency produces an appetite with features
emulating sodium appetite (i.e., innate, specific and taste-guided), we
used a paradigm similar to that used by Breslin et al. (1993 and 1995)
and Markison et al. (1995)
. In those
experiments, immediate licking (a taste-guided behavior) to an
array of salts and water (indicative of specificity) was measured in
naive (innate responsiveness) sodium-deficient rats. Deficient rats
with intact gustatory nerves showed consistently higher licking rates
to NaCl relative to the other stimuli in the array. Using an analogous
procedure, we presented naive LYS-deficient (LYS-DEF; Experiment 1)
and THR-deficient (THR-DEF; Experiment 2) rats with
brief-access (10 s) trials of a stimulus array of amino acids and
water. If EAA deficiency produces behavioral outcomes in any way
similar to that for sodium deficiency, one would expect these naive
rats to show elevated licking to the deficient EAA relative to the
other stimuli in the array as well as in comparison to the behavior of
nondeficient controls. Such an outcome would support the existence of
an innate, specific EAA appetite. Furthermore, these studies examined
whether increased responsiveness to the needed amino acid would occur
in long-term, 23-h intake tests conducted over a series of days.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague-Dawley rats (Charles River, Wilmington, MA) served as subjects. Experiment 1 used 17 rats weighing an average of 374 g (± 13 g) at the start of the experiment; Experiment 2 used 18 rats weighing an average of 203 g (± 12 g). Rats were housed singly in hanging, wire-mesh cages in a colony room in which temperature was automatically maintained. All experimental manipulations were performed during the light phase of the 12-h light:dark cycle (lights on 0600–1800 h). Rats had free access to tap water and pelleted feed (Purina Chow 5001; Ralston-Purina, St. Louis, MO) before the start of the experiments. When the experiments began, rats were given distilled water and powdered feed (Purina Chow) except where specified otherwise. The protocols described herein were approved by the Institutional Animal Care and Use Committee of the University of Florida.
Experimental diets.
Essential amino acid deficiency was accomplished by limiting the level
of the amino acid in the diet (Table 1
). Body weight was measured daily and used as an index of deficiency.
Rats first had free access to a basal diet for 7 d. Basal diets
for both Experiments 1 and 2 consisted of ~11% protein and contained
all of the EAA, but were slightly limiting in either LYS (Experiment 1)
or THR (Experiment 2). The purpose of prefeeding the basal diet was to
reduce endogenous stores of circulating free amino acids and proteins
so that deficiency could be induced rapidly upon feeding the
EAA-deficient diet (see Gietzen and Beverly 1992
).
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The CON diet used in Experiment 1 contained 20.4% protein, consisting of a total of 2% LYS. The EAA-deficient group was fed a LYS-DEF diet that consisted of 19.4% protein, containing a total of only 0.1% LYS. Nitrogen levels were equalized between the two diets by adding the nonessential amino acid glycine (GLY) to the LYS-DEF diet.
In Experiment 2, the CON diet contained 20.8% protein, consisting of a
total of 0.6% THR. The EAA-deficient group was fed a THR-DEF diet
that consisted of 19.4% protein (a total of 0.2% THR). The two diets
were equalized in nitrogen content by adding the nonessential amino
acid proline to the THR-DEF diet. The ingredients for all six diets
used in the studies are presented in Table 1
. Diets were obtained from
Harlan Teklad (Madison, WI) in powdered form and kept refrigerated.
Chemical stimuli.
Taste stimuli were prepared daily using reagent grade chemicals and
room-temperature distilled water. Sucrose, used during training,
was obtained from Fisher Scientific (Fair Lawn, NJ). The amino acid
stimuli (i.e., L-lysine, L-threonine, glycine)
were obtained from Sigma Chemical (St. Louis, MO). Stimulus
concentrations were selected according to Pritchard and Scott (1982)
and were within the dynamic behavioral range described
by long-term, two-bottle preference-aversion functions. That
is, a preferred (or neutral in the case of LYS) and an avoided
concentration were chosen for these experiments. Furthermore,
electrophysiologic recordings (Pritchard and Scott 1982
)
confirmed that these concentrations were above the neural thresholds
for these stimuli as measured by chorda tympani nerve responsiveness.
Apparatus.
The gustometer (fully described by Spector et al. 1990
)
is a specially designed computer-automated taste-testing apparatus
that allows the delivery of small volumes (~5 µL per
lick) of fluid and precisely measures the number of licks elicited by a
given stimulus. Up to 12 liquid stimuli can be placed in separate
reservoirs and delivered through solenoid valves to a spout located
behind a 1-cm slit in one wall of the chamber. Upon tongue contact with
the spout, a low current (<50 nA) electrical circuit is completed and
a lick is recorded. At the termination of a stimulus trial (trial
length is determined by experimenter), the spout is rotated out of the
rat's reach, rinsed with distilled water, cleared by pressurized air
and then rotated back into place for the animal to initiate another
trial. This cleaning procedure requires ~6 s.
General procedure.
The procedures for Experiments 1 and 2 are summarized in Table 2
. Before training, the rats were habituated to the laboratory
environment for at least 9 d. Water bottles from the home cage
were then removed. The following day, pelleted feed was replaced with
powdered feed (Purina 5001) and gustometer training was begun. For
6 d, the rats were trained to lick in the gustometer (see below).
During the first 4 d of training, the rats were deprived of water.
On d 4, after the session, home-cage water bottles were replaced;
training on d 5–6 was conducted without water deprivation. The day
after gustometer training was completed, rats were given the basal diet
for 7 d. Rats were then divided into two groups, matched as
closely as possible for total number of trials taken in the gustometer
during the last 2 d of training, the average number of licks taken
to water and sucrose during those days, body weight and nonpurified
diet intake on the last day of basal diet feeding. One group was then
fed the CON diet and the other group was fed the EAA-deficient diet
for the next 10 d. After dietary depletion, the rats were tested
in the gustometer for their licking responses to an array of taste
stimuli including LYS, THR, GLY and distilled water. Two days after
Gustometer Test 1, two-bottle preference for the limiting EAA
(i.e., either LYS or THR) vs. distilled water was measured for 23-h
periods. Preference for the limiting amino acid was measured for a
total of 6 d in Experiment 1 (LYS deficiency) and 5 d in
Experiment 2 (THR deficiency). Solution intake was measured for only
5 d in Experiment 2 due to limited amounts of the diet; we were
unable to obtain more from our commercial source in sufficient time to
extend testing. On d 2 after bottles were removed from the cage,
EAA-deficient rats and their controls were tested again in the
gustometer as described (Gustometer Test 2). That test concluded the
THR deficiency experiment. In the LYS deficiency experiment, however,
rats were fed their respective diets for 10 more days before a third
gustometer test (Gustometer Test 3). Two days later, 23-h,
three-bottle intake was measured for 6 d. Body weight and food
intake were measured daily throughout both experiments. Liquid intake
was measured only during 23-h two- and three-bottle testing.
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After ~23 h of water deprivation, rats were placed in the gustometer for 30 min where they had continuous access to distilled water from the drinking spout. This gustometer habituation phase lasted for 2 d. On d 3 and 4 of gustometer training, the rats were left in the gustometer for 40 min during which the drinking spout rotated in and out of the animal's reach according to a timed-trial structure. When the rat licked the drinking spout twice within 500 ms, a 10-s trial was initiated. During these sessions, the rat had access to water and 0.3 mol/L sucrose presented in random order. This preferred concentration of sucrose was included in the stimulus array during gustometer training to provoke stimulus sampling in conditions in which the animals would not be deprived of water before the session. After training on d 4, water bottles were replaced on the home cage. On d 5 and 6, rats were again placed in the gustometer for 40-min sessions in which they had 10-s trials of distilled water and 0.3 mol/L sucrose. The only difference between d 3–4 and 5–6 was that during the latter training days, the animals were not deprived of water. During the final 2 d, spout training was conducted without water deprivation because this more accurately simulated the subsequent test conditions.
Gustometer tests 1, 2 and 3.
The number of licks to an array of taste solutions, presented in 10-s trials was measured during one 40-min session for each rat. Rats were not deprived of water during testing. The stimulus array included distilled water and two concentrations each of LYS (0.2 and 1.0 mol/L), GLY (0.1 and 1.0 mol/L) and THR (0.1 and 0.7 mol/L). As during training, two licks on the drinking spout (within 500 ms) initiated a 10-s trial. In Experiment 1 (LYS deficiency), rats were always presented with 0.2 mol/L LYS for the first trial. In Experiment 2 (THR deficiency), the first trial for every rat was always 0.1 mol/L THR. After the first trial, the stimuli were presented in randomized blocks of seven. The limiting amino acid was presented first in an attempt to motivate the EAA-deficient rats to continue sampling the tastants from the spout. In Experiment 1, three identical gustometer tests were conducted on different days. In Experiment 2, two identical gustometer tests were conducted on different days.
Twenty-three–hour two-bottle intake testing.
Rats were presented with two graduated cylinders on the home cage. One bottle contained distilled water and the other bottle contained the limiting amino acid (0.2 mol/L LYS in Experiment 1 and 0.1 mol/L THR in Experiment 2). Intake was measured to the nearest 0.5 mL every 23 h for 6 d in Experiment 1 and for 5 d in Experiment 3. Fresh solutions were mixed and replaced daily at ~1000 h. Bottle positions were alternated daily.
Twenty-three–hour three-bottle intake testing.
The last measurement in Experiment 1 was a series of six, three-bottle 23-h intake tests. LYS-DEF rats and their nondepleted counterparts were presented with three graduated cylinders on the home cage. One bottle contained distilled water, the second bottle contained 0.2 mol/L LYS, and the third bottle contained 0.1 mol/L THR. For 6 d, intake to the nearest 0.5 mL was measured 23 h after the bottles were put on the cage the previous day. Solutions were mixed each day and replaced at ~1000 h. Bottle position was switched daily in a counterbalanced way.
Statistics.
Body weights were converted to percentage of initial body weight by dividing each rat's weight on a given day by its weight on the day before the basal diet was presented. These values were then compared using two-way group by day ANOVA for two phases of the experiment (the 7 d of basal diet feeding and the first 10 d of experimental diet feeding). Subsequent independent Student's t tests were performed between groups for each day of the experiment.
The number of trials initiated during the gustometer tests was analyzed using two-way (group by test session) ANOVA. Main effects were further investigated using independent Student's t tests. For the gustometer tests, the number of licks in response to a given stimulus was averaged over the session for each rat. From the lick data analysis, we excluded rats that took fewer than two trials of each of the seven stimuli presented during a gustometer test because less than two trials per stimulus would represent an unreliable sample of behavior. This was the case with 4 of the 8 CON rats in Experiment 1 and 6 of the 9 CON rats in Experiment 2. Thus, the average number of licks to the array of test stimuli was compared between the different gustometer tests within the same group using two-way (test session by stimulus) ANOVA. As a test of simple effects, matched t tests were conducted to determine differences in licking between tests for each stimulus. We also compared responsiveness during the first gustometer test between the two EAA-deficient groups using a group by stimulus ANOVA. Bonferroni-adjusted t tests were conduced to examine the stimulus licking profile across the seven-stimulus test array.
For the 23-h two-bottle intake tests, intake for the EAA was divided by total intake (water + EAA) and multiplied by 100 to obtain the percentage of intake that was the EAA. This was done for each day that intake was measured. The EAA preference scores were then analyzed by two-way (group by day) ANOVA. Significant group by day interactions were investigated using independent Student's t tests to compare preference scores between the groups on each of the test days.
For the 23-h three-bottle intake tests (Experiment 1 only), intake
for LYS was divided by total intake (water + LYS + THR) and multiplied
by 100 to obtain the percentage of intake that was LYS. This was done
for each of the 6 d on which intake was measured. The LYS
preference scores were then analyzed using a two-way (group by day)
ANOVA. Significant main effects of day were investigated using
independent Student's t tests collapsed over group.
Values presented in the text are means ± SEM
Differences were considered significant when the P-value
was 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean body weights of the EAA-deficient rats and their CON
groups for both Experiments 1 and 2 are depicted in Figure 1
. As expected, an ANOVA (group by day) over the 7 d of basal diet
feeding indicated that there were no differences between the dietary
groups in either experiment. There were significant main effects of day
in both experiments, indicating that all groups of rats gained weight
over the 7 d of basal diet feeding (Experiment 1: F(6,90) = 152.0, P < 0.001; Experiment 2: F(6,96) = 38.83,
P < 0.001). Clearly, the basal diet was sufficient to
support growth.
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for ANOVA results). Subsequent Bonferroni-adjusted t
tests revealed that the rats fed the LYS-DEF diet had significantly
lower body weights relative to their CON group by d 2 after the diet
was presented (all P < 0.05). Similarly,
Bonferroni-adjusted t tests indicated that the rats fed
the THR-DEF diet had significantly lower body weights relative to
their CON group by d 1 after the diet was presented (all P
< 0.001). In both experiments, the rats fed the EAA-deficient
diet had significantly lower body weights for all remaining days in the
experiments (all P < 0.001). These body weight results
demonstrate the effectiveness of these diets in inducing deficiency.
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The number of trials that the rats initiated varied as a function of
whether they were EAA-deficient (Fig. 2
). Clearly, EAA-deficient rats were more motivated (as reflected by
increased trial number) in this task compared with the nondepleted CON
rats. In Experiment 1, a group by test session ANOVA revealed a
significant main effect of group [F(1,15) = 9.06, P
< 0.01], indicating that the LYS-DEF rats took significantly
more trials than the CON rats during all three gustometer tests. In
Experiment 2, a group by test session ANOVA revealed a significant main
effect of group [F(1,16) = 19.85, P < 0.001]
and a significant main effect of test session, [F(1,16) = 31.82,
P < 0.001]. THR-DEF rats took more trials in both
gustometer tests relative to the CON rats. A paired t test
(collapsed over group) revealed that the rats took more trials during
Gustometer Test 1 than Gustometer Test 2 [T (17) = 5.62,
P < 0.001].
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As measured by the three gustometer tests, LYS-DEF rats (Experiment
1) did not show a specific, innate licking response profile to LYS
(Fig. 3
). A test session by stimulus ANOVA revealed a significant main effect
of session [F(2,14) = 4.87, P < 0.05] and of
stimulus [F(6,42) = 22.44, P < 0.001].
One-way ANOVA conducted for each stimulus revealed a significant
difference among sessions only at 0.7 mol/L THR [F(2,14) = 9.14,
P < 0.01]. Paired t tests indicated that
the LYS-DEF rats licked more to 0.7 mol/L THR during Test 1 and
Test 2 relative to Test 3. There were no other significant
differences among test sessions.
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. A session by stimulus ANOVA revealed significant main effects of
session [F(1,6) =42.61, P < 0.001] and stimulus
[F(6,36) = 32.28, P < 0.001]. Paired
t tests done at each stimulus revealed significant
differences between the two sessions at 0.2 mol/L LYS, 1.0 mol/L LYS
and 0.7 mol/L THR (all P < 0.01); licking was
significantly less during Test 2.
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In addition to the comparisons made between test sessions in each
experiment, the two EAA-deficient groups were compared between
experiments on their first gustometer test (see Fig. 5
). A group by stimulus ANOVA revealed no significant main effect of
group or interaction. However, lick responsiveness did vary among
stimuli [F(6,78) = 46.71, P < 0.001].
Bonferroni-adjusted paired t tests revealed that rats
licked significantly more to every stimulus relative to water (all
P < 0.001).
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Figure 6
shows intake (panels A and B) and LYS preference
(panel C) resulting from the two-bottle tests in
Experiment 1. Separate group by day ANOVA revealed that LYS-DEF
rats drank more LYS [F(1,15) = 8.05, P < 0.05]
and less water [F(1,15) = 10.57, P < 0.001]
relative to CON rats, resulting in greater LYS preference [F(1,15)
= 13.13, P < 0.01]. There were no significant
main effects of test day or any interactions.
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). The THR-DEF group ingested more THR [F(1,15) = 19.15,
P < 0.01] and less water [F(1,15) = 14.57,
P < 0.01] compared with CON rats. Furthermore, the
deficient rats preferred THR more than CON rats [F(1,15) = 16.24,
P < 0.01]. For THR intake, there was also a
significant main effect of day [F(4,60) = 5.43, P
< 0.001], with THR intake decreasing over days. Paired
t tests, collapsed over group, revealed that rats drank more
THR on d 1 relative to d 3, 4 and 5 (all P < 0.05).
There were no significant interactions.
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Lysine preference was maintained even when LYS-DEF
rats were given access to LYS, THR and distilled water (Fig. 8
). Separate group by day ANOVA showed significant main effects of group
for LYS intake [F(1,15) = 18.82, P < 0.01],
water intake [F(1,15) = 9.52, P < 0.01)] and
LYS preference [F(1,15) = 12.29, P < 0.01],
indicating that the LYS-DEF rats drank more total LYS and less
water than CON rats, producing higher LYS preference scores. The groups
did not differ in responsiveness to THR. However, there were main
effects of day for LYS intake [F(5,75) = 11.60, P
< 0.01] and LYS preference [F(5,75) = 4.10, P
< 0.01], with both decreasing over days. Paired t
tests collapsed over group showed that LYS intake was significantly
decreased on d 2, 3, 4, 5 and 6 relative to d 1 and preference was
significantly lower on d 3 and 5 compared with d 1. There were
no significant interactions in any of the ANOVA results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Nachman 1962
). Second, sodium appetite is very specific
for salts containing the limiting mineral. When sodium-deficient
rats are presented with a choice of sodium and nonsodium salts, they
respond preferentially to the former (with the exception of LiCl;
Fregly 1958
). Sodium-specific appetite has been
demonstrated in both short-duration tests (i.e., brief-access; see
Breslin et al. 1993 and 1995
, Handal 1965
, Krieckhaus and Wolf 1968
,
Krieckhaus 1970
, Markison et al. 1995
,
Nachman 1962
) and long-duration testing paradigms
(i.e., 24-h intake; Richter and Eckert 1938
). A third
defining feature of sodium appetite is that it appears to be
taste-guided. The importance of gustatory signals in the expression
of sodium appetite is strongly supported by results generated from
brief-access tests that minimize the influence of
post-ingestive signals and experiments demonstrating impairment
after the following events: 1) transection of the chorda
tympani branch of cranial nerve VII (Breslin et al. 1993 and 1995
, Markison et al. 1995
) or 2)
adulteration of the NaCl with the epithelial sodium channel blocker,
amiloride, which suppresses activity in Na-specific taste afferents
(Bernstein and Hennessy 1987
, McCutcheon 1991
). The features defined for sodium deficiency, however, do
not seem to generalize to other specific nutrient deficiencies, or at
least not EAA deficiency involving LYS and THR.
Specifically, the initial brief-access licking test was conducted
to determine whether EAA-deficient rats would express an innate
(i.e., unconditioned), specific, taste-guided increase in licking
to a needed commodity. The pattern of licking to the array of amino
acids was not altered as a function of deficiency; LYS-DEF rats did
not specifically increase responsiveness to LYS, nor did THR-DEF
rats show elevated licking to only THR. In fact, the entire profile of
responsiveness across the stimulus array was nearly identical between
groups deficient in either LYS or THR (see Fig. 5
). Unfortunately, we
were unable to compare, with confidence, the responses during
brief-access trials in EAA-deficient rats to those in
nondepleted CON rats because the nondeficient, nonwater-deprived
CON rats did not initiate a sufficient number of trials to serve as a
meaningful comparison. This is apparent from the analysis of the number
of trials initiated by the groups in the gustometer tests (see Fig. 2
).
In the absence of water deprivation, most CON rats did not sample the
drinking spout. In the future, tests that do not rely on appetitive
behaviors for stimulus delivery and measure only consummatory
responding (e.g., intraoral infusion, taste reactivity; see
Breslin et al. 1990
, Grill and Norgren 1978
) might be applied to circumvent this sampling problem.
There were, nonetheless, significant differences in the number of trials initiated in nondeficient vs. EAA-deficient rats. This suggests that, although deficient rats do not specifically ingest the nutrient lacking in their diet in short-duration tests, they do appear to be "motivated"; EAA deficiency seems to increase appetitive behavioral responding (i.e., approaching the spout to initiate licking).
Although deficiency did not influence immediate lick responsiveness in
brief-access tests, LYS- and THR-deficient rats did express a
preference during long-term intake tests for the amino acid that
was limited in their diet (see Figs. 6
, 7
and 8
). Differences in
long-duration tests occurred presumably because consumption is
under the control of both oral-sensory and post-ingestive cues.
These preference results support and extend the work of others who have
shown that rats can compensate for deficient diets by ingesting the
missing nutrient when it is offered to them in solution during longer
time intervals. This has been shown with vitamin B deficiency
(Richter et al. 1937
), protein deficiency
(Halsted and Gallagher 1962
), histidine deficiency
(Rogers and Harper 1970
), tryptophan deficiency
(Mori et al. 1991
) and LYS deficiency (Mori et al. 1991
, Torii et al. 1986
). The findings of
Torii et al. (1986)
and Mori et al. (1991)
are most intriguing because of the extensive stimulus
array offered. These authors reported that when rats were presented
with a LYS-deficient diet and had access to 5 (Mori et al. 1991
) or as many as 15 (Torii et al. 1986
)
solutions, they altered their intake, resulting in a preference for
LYS. One may ponder how a rat can differentially ingest one solution
when having access to so many at once. The mechanisms governing such
choice behavior certainly merit investigation.
The findings from the later gustometer tests were somewhat surprising. Although the deficient rats did not demonstrate an innate response to the limiting EAA in the initial gustometer tests, we expected to see evidence of a learned response in the later brief-access tests, those conducted after the two-bottle intake tests. The rationale here was that the two-bottle tests would allow the deficient rats to learn an association between the sensory cues (i.e., the taste) of the limiting EAA and repletion. We hypothesized that this learning would be apparent as a specific increase in licking to the deficient amino acid in the short-duration test. Even after this opportunity to associate the taste and positive post-ingestive consequences of the missing EAA, however, rats still did not increase licking to the EAA. One explanation is that Gustometer Test 2 was only 2 d after the intake testing and it is possible that the rats were LYS replete. If this were the case, physiologic state (i.e., repleted) could account for the lack of effect during the second test in Experiments 1 and 2. However, the results from the third gustometer test (Experiment 1), conducted after 10 more days of deficient diet feeding, revealed that rats still did not show enhanced responsiveness to LYS.
There are at least two possible explanations for the failure to see a
conditioned preference in the gustometer tests. First, it could be
argued that at the concentrations presented, these amino acids are not
detectable by the taste system and therefore rats are unable to
distinguish them from water in short-duration tests. This
explanation appears unlikely on the basis of both behavioral and
electrophysiologic findings. In short-duration tests, which are
postulated to reflect oral-sensory control, several investigations
have shown differential responsiveness to amino acids as a function of
concentration. Additionally, when licking during short-duration
trials was measured in the gustometer tests, rats took significantly
more licks to each and every amino acid stimulus relative to water. In
the case of GLY, Grill et al. (1987)
showed that
nondeprived rats increased ingestive taste reactivity as a function of
concentration (0.03 mol/L, 0.1 mol/L, 1.0 mol/L). Electrophysiologic
measures demonstrate that the gustatory system responds to the
concentrations of amino acids used in these experiments.
Pritchard and Scott (1982
) found chorda tympani
whole-nerve thresholds (the weakest concentration that evoked a
response in the whole nerve) to be 0.8 mmol/L for LYS, 20 mmol/L for
THR and 3.5 mmol/L for GLY. Thus, on the basis of previous work and the
fact that lick responsiveness to LYS and THR in our experiment was
significantly above that of water, we feel confident that the
concentrations employed in this experiment fall within the range that
stimulates the taste system.
The second possible reason that we did not observe a "conditioned
EAA preference" may be due to the methodology employed. Although we
expected that rats would eventually show higher lick rates to the EAA
in which they were deficient, it is possible that the experience (i.e.,
training) and testing paradigm used here were not appropriate to
produce and/or detect a conditioned preference. Conditioned preferences
can be produced using the oral-simultaneous conditioning method.
This paradigm has been nicely exemplified in the work of Sclafani and
colleagues (see Sclafani 1995
for review). With this
procedure, rats are allowed to drink a flavored (e.g., grape) nutritive
solution (e.g., glucose or polycose) and, on alternating training
sessions, drink a nonnutritive substance (e.g., water) mixed with a
different arbitrary flavor (e.g., cherry). In Pavlovian terms, the
nutrient is considered the unconditioned stimulus (UCS), the arbitrary
flavor paired with the nutrient acts as the conditioned stimulus or the
CS+ and the flavor presented with water or the nonnutritive solution is
termed the CS-. The ensuing conditioned preference for the CS+ flavor
is demonstrated during two-bottle intake tests (CS+ flavor vs. CS-
flavor). On the basis of this learning model, we hypothesized that
LYS-DEF rats, after ingesting LYS and presumably accruing
post-ingestive benefits, would demonstrate a preference for LYS
during 10-s trials. Our procedure lacks certain features relative to
the paradigm described above, however. Perhaps most important is that
rats did not have training with a CS- flavored stimulus. Additionally,
the measurement to detect the conditioned preference was not a
two-bottle intake test. Instead, rats were required to display high
lick rates when offered seven different stimuli presented during 10-s
trials in random order; choice behavior was not explicitly measured.
Whether rats will express a conditioned preference for a LYS solution
after more traditional preference training is an empirical question,
and subsequent experiments should be undertaken to address this key
issue. It should be noted, however, that a variation on the
"preference conditioning paradigm" has been used successfully
with isoleucine- and threonine-deficient diets
(Gietzen et al. 1992
, Naito-Hoopes et al. 1993
).
It is interesting to speculate that conditioned preferences are
expressed as a choice for the CS+, but do not drive appetitive behavior
itself. It appears that increased acceptance in one-bottle tests
does occur in some cases but typically is not as robust as might be
suggested by the results of choice tests (Drucker et al. 1994
) and certainly depends on the training paradigm
implemented (Perez et al. 1998
).
In sum, both LYS-DEF and THR-DEF rats showed elevated intake of the limiting amino acid as measured during long-duration (i.e., 23-h) intake tests. Additionally, this intake is somewhat specific, at least in the case of LYS deficiency because LYS-DEF rats preferred LYS even when it was presented simultaneously with LYS, THR and water. It is unlikely that EAA deficiency promotes an "innate" appetite; LYS-DEF rats do not increase intake of the deficient EAA immediately in short-duration tests. Furthermore, even after experience with the limiting amino acid, rats do not show elevated lick rates in brief-access tests. The question remains then, what mechanisms are responsible for the adaptive elevation in intake that occurs as a result of EAA deficiency? Do rats learn an association between the taste of the amino acid and the beneficial results of ingesting it when they are deficient? Does this association lead to a conditioned preference for the EAA that guides future behavior? Or, do EAA-deficient rats increase intake of the EAA based solely on need at the time of ingestion, responding to more immediate post-ingestive feedback? Further investigation is required to determine the mechanisms guiding the behavior.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Presented in part in dissertation form in
partial fulfillment of a Doctor of Philosophy degree from the
University of Florida, Gainesville, FL 32611.
3 Current address: Department of Psychology,
University of Pennsylvania, Philadelphia, PA 19104–6196.
5 Abbreviations used: CON, control; CS,
conditioned stimulus; EAA, essential amino acid(s); GLY, glycine; LYS,
lysine; LYS-DEF, lysine-deficient; THR, threonine; THR-DEF,
threonine-deficient; UCS, unconditioned stimulus.
Manuscript received December 3, 1998. Initial review completed January 25, 1999. Revision accepted May 10, 1999.
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