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Behavioural Brain Research 223 (2011) 348–355
Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
Posttrial d-cycloserine enhances the emotional memory of an incentive
downshift event夽
Jacob N. Norris 1 , Leonardo A. Ortega, Mauricio R. Papini ∗
Department of Psychology, Texas Christian University, Fort Worth, TX 76129, USA
a r t i c l e
i n f o
Article history:
Received 20 March 2011
Received in revised form 28 April 2011
Accepted 2 May 2011
Available online 7 May 2011
Keywords:
Incentive downshift
d-Cycloserine
NMDARs
Frustration
Negative contrast
Emotional memory
a b s t r a c t
The present research was designed to determine whether an incentive downshift event induces an emotional memory that can be modulated by d-cycloserine (DCS), a partial agonist at the glycine site of
N-methyl-d-aspartate receptor (NMDAR). DCS has been reported to have memory-enhancing properties
in other training situations. Experiments 1 and 2 involved a consummatory successive negative contrast
(cSNC) situation in which animals are exposed to an incentive downshift involving sucrose solutions of
different concentrations. DCS administration (30 mg/kg, ip) immediately after the first 32-to-4% sucrose
downshift trial (Experiment 1) retarded recovery of consummatory behavior, but immediately after the
first 32-to-6% sucrose downshift trial (Experiment 2) did not affect recovery. There was no evidence that
DCS affected consummatory behavior in the absence of an incentive downshift in a manner analogous
to a conditioned taste aversion (Experiment 3). These results suggest that activation of NMDARs via the
glycine modulatory site enhances the emotional memory triggered by exposure to an incentive downshift
event.
© 2011 Elsevier B.V. All rights reserved.
Consummatory successive negative contrast (cSNC) is the
abrupt disruption of consummatory behavior after a downshift
from a large to a small incentive, relative to the consummatory
behavior of animals exposed only to the small incentive [1]. In a
typical cSNC experiment, the incentives are 32% and 4% sucrose
solutions, and the downshift occurs on Trial 11. The cSNC effect
indicates that the change in behavior is not an adjustment to the
new incentive conditions, but a reduction of consummatory behavior that may be the result of an increase in search behavior [2–4],
escape from a frustrating situation [5,6], or release from response
inhibition following a surprising devaluation of the incentive [7].
Assuming that the initial rejection of the devalued solution reflects
an aversive internal state [8,9], hypothesized that emotional conditioning takes place during the incentive downshift event. The
basis for this memory is postulated to be a Pavlovian association
between the taste of the downshifted, 4% sucrose solution and the
internal state of frustration induced by the downshift. As a result,
tasting the 4% sucrose solution would elicit an internal aversive
夽 Disclaimer: The views expressed in this article are those of the authors and do
not necessarily reflect the official policy or position of the Department of the Navy,
Department of Defense, nor the U.S. Government.
∗ Corresponding author. Tel.: +1 817 257 6084.
E-mail address: [email protected] (M.R. Papini).
1
Current address: Naval Health Research Center, 140 Sylvester Road, San Diego,
CA 92106, USA.
0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2011.05.001
state that would tend to suppress consummatory behavior and
also induce a behavioral switch from consumption to searching or
escaping. The associative reactivation of this emotional memory is
assumed to retard recovery from cSNC beyond the first downshift
trial. Emotional memory reactivation is a source of consummatory
suppression because animals tend to avoid stimuli associated with
negative emotional content, such as the area where they have consumed the devalued sucrose solution (i.e., secondary frustration;
[5,10]). The research reported here is concerned with the encoding
of this aversive memory of the downshift.
The effects of drugs on memory consolidation can be best
assessed by posttrial drug administration, that is, by administering
the drug immediately after the relevant experience. Memory consolidation is a time-dependent process occurring after acquisition
and rendering the memory trace more stable and more resistant
to disruption [11]. Because training occurs before drug administration, behavior during training is not affected directly by the drug.
Because behavioral testing of drug effects usually occurs a day after
drug administration (as in the present experiments), and because
many psychoactive drugs are metabolized within hours (e.g., in
rats, the half-life of d-cycloserine, used in these experiments, was
reported to be 60–70 min; [12,13]), it can be assumed that the
drug has been metabolized by the time of testing. As a result, posttrial drug effects on behavior can be more safely attributed to the
drug’s effect on memory consolidation, rather than to direct drug
effects on motor, motivational, or attentional processes active during training and/or testing ([14–17]; but see Experiment 3, below,
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J.N. Norris et al. / Behavioural Brain Research 223 (2011) 348–355
for a potential alternative of the effects of posttraining drug administration).
Evidence consistent with the encoding of a downshift memory
is provided by the effects of corticosterone administered immediately after the first downshift trial. Based on extensive research
in a variety of learning paradigms, glucocorticoids can be safely
assumed to enhance memory consolidation [18–21]. When administered in the cSNC situation, immediately after the first downshift
trial, corticosterone retards recovery from cSNC [22]. Posttraining
corticosterone has no effects on consummatory behavior under
the following conditions [22,23]: (1) In the absence of a downshift
event, as in unshifted controls or when administered after a single
exposure to 4% sucrose; thus, corticosterone’s effects in the cSNC
situation cannot be attributed to a conditioned taste aversion to 4%
sucrose. (2) When corticosterone is administered 3 h after the first
downshift trial, rather than immediately after, thus demonstrating
the time-dependent nature of its effects. (3) When corticosterone
is administered in an anticipatory contrast situation involving the
same solutions used in the cSNC situation, but in a procedure known
to activate different brain mechanisms from those involved in cSNC
[2]. Therefore, the selectivity of these corticosterone effects to the
cSNC situation is consistent with the enhancement of the aversive
memory of the downshift event.
Other drugs known from other learning paradigms to modulate memory consolidation and tested in the cSNC situation under
similar conditions do not affect the course of recovery from the
incentive downshift as corticosterone does. These include cholinergic drugs (e.g., atropine and physostigmine), known to modulate
consolidation of conditioned fear [24–26], but having no effect on
cSNC [27], and opioids (naloxone, DPDPE, and naltrindole), known
to modulate consolidation of conditioned fear [17,28,29], but having no detectable effect on recovery from cSNC [30]. Therefore,
memory consolidation of the incentive downshift experience is
modulated by some, but not all of the neurochemical systems
known to be involved in other types of emotional memory.
The present experiments were designed to determine whether
the N-methyl-d-aspartate glutamate receptor (NMDAR) is involved
in memory consolidation in the cSNC situation. There is a well
established connection between glucocorticoids and NMDARs,
although the relationship is complex. For example, glucocorticoids
enhance NMDAR activation in hippocampal tissue extracted from
neonatal rats [31]. Furthermore, Nair and Bonneau reported that
acute restraint stress induced glucocorticoid elevation that, in turn,
increased microglia proliferation (a proinflammatory response);
this effect was blocked by administration of the NMDA receptor antagonist MK-801. Thus, we hypothesized that the enhancing
effects of posttrial corticosterone on cSNC described above were
mediated by activation of NMDARs. In these experiments, animals
received administration of d-cycloserine (DCS) immediately after
the first downshift trial following a 32-to-4% sucrose downshift
(Experiment 1) or a 32-to-6% sucrose downshift (Experiment 2).
Finally, Experiment 3 explored the possibility that DCS’s effects
acted as an unconditioned stimulus supporting the development
of a conditioned taste aversion to the 4% sucrose. DCS is a partial agonist at the glycine binding site (NR1B) of the NMDAR; the
endogenous ligand, glycine, has greater affinity to this binding
site than DCS, which is thus characterized as a partial agonist.
As a result, DCS may act either as an agonist or an antagonist depending on the extracellular concentration of glycine [33].
DCS was selected because there is strong evidence from a variety of learning paradigms that this drug functions as a memory
enhancer. For example, in the fear conditioning situation, DCS
administration either before or after training sessions has been
shown to facilitate extinction [34–37], reduced reinstatement following extinction [38], and increased generalization of extinction
to nonextinguished stimuli [35]. In spatial learning situations,
349
DCS improves performance in rats made spatially deficient either
by hippocampal lesions [39] or scopolamine-induced amnesia
[40,41]. In flavor learning situations, pretrial DCS administration
enhanced LiCl-induced conditioned taste aversions [42] and also
enhanced fructose-induced conditioned taste preferences [43]. DCS
also reversed deficits on eyeblink conditioning in rabbits induced
by exposure to inescapable shocks [44] and restored episodic-like
memory in an object-recognition task with rats [45]. These references are but a subset of the growing literature on the effects of DCS
on learning, all suggesting that, within some constraints (e.g., there
is apparently an optimal dose for some of these effects; [46,47]),
DCS enhances learning (i.e., the acquisition of new information).
Because of the memory enhancing effects reported for both
corticosterone and DCS, and because of the connection between
glucocorticoid levels and NMDAR activation, we predicted that DCS
would have effects in the cSNC situation similar to those described
above for corticosterone. Furthermore, we assumed that DCS would
not affect well-established memories, such as the memory of the
preshift solution. Thus, if DCS retards recovery from cSNC, this
result would be interpreted as an enhancement of the emotional
memory of the downshift.
1. Experiment 1
It was hypothesized that posttrial DCS administration would
have the same effect on cSNC than corticosterone did in previous
experiments [22,23]. Therefore, Experiment 1 tested the hypothesis that DCS administration prolongs the cSNC effect. A 30 mg/kg
dose was chosen based on prior research showing that this dose
was effective with rats trained in other tasks [40,47,48].
2. Method
2.1. Subjects
The subjects were 41 male Long-Evans rats bred at the TCU vivarium from an
original group purchased from Harlan Laboratories (Indianapolis, IN), approximately
90 days old at the start of the experiment and experimentally naïve. The average
ad libitum weight of the entire sample was 405 g (range: 338–484 g). Rats were
housed in a vivarium under a 12:12-h light, dark cycle (lights on at 07:00 h) and
were deprived of food to an 81–84% of the free-food weight. Water was continuously
available in each individual cage. Animals were trained during the light phase of the
daily cycle
2.2. Apparatus
Training was conducted in 4 conditioning boxes (MED Associates, St. Albans, VT)
constructed of aluminum and Plexiglas, and measuring 29.4 cm × 28.9 cm × 24.7 cm
(L × H × W). The floor was made of steel rods 0.5 cm in diameter and 1.2 cm apart
running perpendicular to the feeder wall. A bedding tray filled with corncob bedding
was placed below the floor to collect fecal pellets and urine. Against the feeder wall
was an elliptical hole 1 cm × 2 cm (W × H), 3.5 cm from the floor. A sipper tube, 1 cm
in diameter, was inserted through this hole. When fully inserted, the sipper tube
was flush against the wall. A computer located in an adjacent room controlled the
presentation and retraction of the sipper tube, and detected contact with the sipper
tube via a circuit involving the steel rods in the floor. Each conditioning box was
placed in a sound-attenuating chamber that contained a house light, a speaker to
deliver white noise, and a fan for ventilation. Together, the speaker and fan produced
noise with an intensity of 80.1 dB (SPL, Scale C)
2.3. Procedure
Training lasted 15 trials, administered 1 trial per day. Each rat was randomly
assigned to one of the conditioning boxes and always trained in that box. The order
of training of the 4-rat squads varied across days. After each trial, conditioning
boxes were cleaned with a damp paper towel, feces removed, and bedding material replaced as needed. During trials, the houselight, white noise, and fan were
on continuously. The 15 trials were divided into a preshift phase (Trials 1–10) and
a postshift phase (Trials 11–15). Prior to Trial 1, rats were matched by ad libitum
weight and then randomly assigned to the downshifted or unshifted condition. After
Trial 10, rats exposed to 32% sucrose were matched in terms of overall preshift performance and randomly assigned to one of the two different drug conditions, Saline
or 30 mg/kg DCS. The unshifted controls were assigned likewise. This drug dose is
frequently reported as effective in prior research (e.g., [37])
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For the two 32-to-4 groups (32/Sal, 32/DCS), the 10 preshift trials involved access
to a 32% sucrose solution (w/w, prepared by mixing 32 g of commercial sugar for
every 68 g of distilled water); the 5 postshift trials involved access to a 4% solution
(w/w, 4 g of sugar for every 96 g of distilled water). The two 4-to-4 groups (4/Sal,
4/DCS) received the 4% sucrose solution in all 15 trials. Each trial started with a
variable interval of 30 s (range: 15–45 s). At the end of this interval, the sipper tube
was automatically presented. The first recorded contact with the sipper tube initiated a 5-min interval during which the sucrose solution was freely available. At
the end of this interval, the sipper tube was retracted and a final variable interval
of 30 s (range: 15–45 s) ended the trial. The dependent variable was the cumulative
amount of time in contact with the sipper tube, measured in 0.05-s units and labeled
goal-tracking time. Goal-tracking times were recorded for each entire 5-min trial
and also in 5-s bins to analyze within-trial performance. Goal-tracking times were
subjected to conventional analysis of variance (ANOVA). The alpha value was set to
p < 0.05 for all statistical tests.
DCS or saline was administered immediately following the first downshift trial
(Trial 11). Saline animals received an equal-volume injection of isotonic saline. Drug
concentrations were adjusted to a 1 ml/kg volume. At the end of the trial, animals
were placed back into the transport rack and moved into the holding room where
they received the appropriate injection. This took approximately 30–90 s. The following groups were included: 32/Sal (n = 11), 32/DCS (n = 10), 4/Sal (n = 10), and
4/DCS (n = 10). It was assumed that DCS had no direct effects on Trials 12–15, as
DCS’s levels in brain and plasma are in the order of 60–70 min [12,13]. DCS was purchased from Sigma-Aldrich Chemicals (Saint Louis, MO). It was freshly dissolved in
isotonic saline solution (30 mg/ml).
First 100 s
100
Goal-tracking Time (s)
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75
50
32/Sal
4/Sal
32/DCS
4/DCS
25
0
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10
11
12
13
14
15
Trials
Last 100 s
100
3. Results
Goal-tracking Time (s)
300
250
200
150
32/Sal
100
4/Sal
32/DCS
50
4/DCS
0
0
5
10
15
Trial
Fig. 1. Results of Experiment 1. Mean goal-tracking time (±SEM) is plotted as a
function of trial, contrast condition, and drug treatment. On Trials 1–10, Groups
32/Sal and 32/DCS received 32% sucrose solution, whereas Groups 4/Sal and 4/DCS
received access to 4% sucrose solution. All animals had access to 4% sucrose on
Trials 11–15. Animals were administered either saline (Sal) or d-cycloserine (DCS,
30 mg/kg, ip) immediately after Trial 11, the first downshift trial for Groups 32/Sal
and 32/DCS.
75
Goal-tracking Time (s)
The results are shown in Fig. 1. A Drug × Sucrose × Preshift Trial
analysis revealed a significant change across trials, F(9, 333) = 81.95,
p < 0.001, a main effect of sucrose, F(1, 37) = 10.81, p < 0.01, and a
significant trial by sucrose interaction, F(9, 333) = 3.18, p < 0.01, but
not significant main effect of drug or of drug-related interactions,
Fs < 1. A Drug × Sucrose × Postshift Trial analysis indicated a significant main effect of trial, F(4, 148) = 16.6, p < 0.001, a significant
trial by sucrose interaction, F(4, 148) = 7.14, p < 0.001, and a trial
by sucrose by drug interaction, F(4, 148) = 2.57, p < 0.001. All other
effects were not significant, Fs < 1.
To clarify the source of the three-way interaction, one-way
ANOVAs for Trials 11–15 were calculated for pairs of groups involving the same drug condition, but different behavioral treatment:
32/DCS vs. 4/DCS and 32/Sal vs. 4/Sal. For the saline groups, the
cSNC effect was significant on Trial 11, F(1, 19) = 8.34, p < 0.01,
marginal for Trial 12, F(1, 19) = 4.23, p = 0.054, and not significant for Trials 13–15, Fs < 1.49, ps > 0.23. However, the cSNC effect
was extended for DCS-treated groups throughout the entire postshift phase, Trials 11–15. Group 32/DCS contacted the sipper tube
50
32/Sal
25
4/Sal
32/DCS
4/DCS
0
9
10
11
12
13
14
15
Trials
Fig. 2. Performance during the first (top panel) and last (bottom panel) 100 s of
the last preshift trial (Trial 10) and each of the downshift trials (Trials 11–15) in
Experiment 1. Mean goal-tracking time (±SEM) plotted as a function of trial, contrast
condition, and drug treatment. Contrast treatments were as described in Fig. 1.
significantly less than Group 4/DCS on Trials 11–15, Fs (1, 18) > 4.86,
ps < 0.05.
Further pairwise comparisons were computed, also for Trials
11–15, between groups with the same behavioral condition, but
different drug condition: 32/DCS vs. 32/Sal and 4/DCS vs. 4/Sal.
Thus, Group 32/DCS contacted the sipper tube significantly less
than Group 32/Sal on Trial 13, F(1, 19) = 10.90, p < 0.005, marginally
on Trial 12, F(1, 19) = 3.79, p = 0.067, and not significantly on Trials 11, 14, and 15, Fs (1, 19) < 1.32, ps > 0.26. However, DCS had no
effect on the unshifted controls. The comparisons between Groups
4/DCS and 4/Sal were all not significant for Trials 11–15, Fs < 2.05,
ps > 0.16.
Previous research indicated that consummatory suppression is
not evident during the initial 100 s of postshift trials, but it emerges
fully during the last 100 s of such trials [49]. Consequently, the
within-trial results for the initial and last 100 s of Trials 10–15
are presented in Fig. 2. In both cases, a Drug (DCS, Sal) × Sucrose
(32%, 4%) × Trial (11–15) analysis yielded a triple interaction that
fell short of significance: F(4, 124) = 2.10, p = 0.085 for the first 100 s,
and F(4, 124) = 1.55, p = 0.191 for the last 100 s. Given the results for
the entire session (Fig. 1) and the graphical depiction of the withintrial data (Fig. 2), a second analysis was calculated only for Trials
11–13. The results were as follows.
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3.1. Experiment 2
In the present experiment, the same DCS dose and administration used previously occurred after a 32-to-6% sucrose downshift.
The 32-to-6 downshift was used for two reasons. First, a reduction in incentive disparity attenuates cSNC [50], thus reducing
the opportunity for floor effects in DCS-treated animals. Second,
Ruetti et al. [23] showed that posttrial administration of corticosterone enhanced cSNC following a 32-to-4% sucrose downshift, but
not an 8-to-4% sucrose downshift, concluding that corticosterone’s
enhancing effects were possible only when an animal experienced
a large incentive disparity. Similarly, Daniel et al. [30] showed that
naloxone administered prior to the first downshift trial reduced
consummatory behavior after a 32-to-6% sucrose downshift, but
not after a 16-to-6% sucrose downshift. Likewise, using a less severe
downshift will help determine whether the effects of DCS depend
on a minimum incentive disparity.
300
Goal-tracking Time (s)
For the first 100 s (Fig. 2, top panel), the Drug × Sucrose × Trial
(11–13) analysis indicated a significant triple interaction, F(2,
62) = 3.61, p < 0.04, as well as a significant contrast effect, with
downshifted scoring below unshifted controls, F(1, 31) = 9.24,
p < 0.01, and significant change across trials, F(2, 62) = 3.21, p < 0.05.
All other effects were not significant, Fs < 2.43, ps > 0.09. Pairwise
analyses for groups matched by drug condition showed that Groups
32/Sal vs. 4/Sal did not differ on Trials 11–13, Fs(1, 16) < 2.26,
ps > 0.15, whereas Groups 32/DCS vs. 4/DCS also failed to differ on
Trial 11, before DCS administration, F(1, 15) = 3.08, p > 0.09, but the
cSNC was significant for Trials 12–13, Fs(1, 15) > 14.61, ps < 0.003,
after DCS administration. Comparisons for groups matched in terms
of the behavioral treatment showed that Group 32/Sal contacted
the sipper tube significantly more than 32/DCS on Trial 13, F(1,
15) = 7.49, p < 0.02. On Trials 11 the difference was not significant,
F < 1, and on Trial 12 was also not significant, but marginal, F(1,
15) = 3.88, p = 0.068. However, DCS had no detectable effect on the
unshifted controls, as indicated by a comparison between Groups
4/Sal vs. 4/DCS for Trials 11–13, Fs < 1.
Fig. 2(bottom panel) also shows the results for the last 100 s
of Trials 10–15. The Drug × Sucrose × Trial (11–13) analysis provided a significant triple interaction, F(2, 62) = 3.71, p < 0.04, as
well as a significant contrast effect, F(1, 31) = 31.88, p < 0.001, and
significant change across trials, F(2, 62) = 4.97, p < 0.02. All other
effects were not significant, Fs < 3.16, ps > 0.08. Pairwise analyses
for groups matched by drug condition showed that Groups 32/Sal
vs. 4/Sal differed significantly on Trials 11–12, Fs(1, 16) > 8.01,
ps > 0.02, but not on Trial 13, F < 1, whereas Groups 32/DCS vs. 4/DCS
exhibited significant differences in all three postshift trials, Fs(1,
15) > 9.59, p > 0.006. Comparisons for groups matched for behavioral treatment showed that Group 32/Sal contacted the sipper tube
significantly more than 32/DCS on Trial 13, F(1, 15) = 8.92, p < 0.01.
On Trials 11 the difference was not significant, but marginal, F(1,
15) 4.07, p = 0.062, but on Trial 12 it was not significant, F < 1. DCS
had no detectable effect on the unshifted controls on Trials 11–13,
Fs(1, 15) < 1.37, ps > 0.25.
In summary, DCS administration after the first downshift trial
(Trial 11) enhanced suppression of consummatory behavior in subsequent trials. As shown by within-trial analyses, such suppression
was detectable during the initial 100 s of subsequent trials (Trials
12–13), as well as during the last 100 s of the same trials. The fact
that DCS-treated animals show consummatory suppression earlier
than saline-treated animals is consistent with facilitated retrieval
of the emotional memory of the incentive downshift event experienced a day earlier. Such facilitated retrieval seems to have modulated behavior throughout the trial, as assessed by facilitated cSNC
effect during the last 100 s, relative to saline groups. This result is
thus consistent with DCS-induced facilitation of memory retrieval.
351
250
200
150
32/Sal
6/Sal
100
32/DCS
50
0
6/DCS
0
5
10
15
Trials
Fig. 3. Results of Experiment 2. Goal-tracking time (s) is plotted as a function of trial,
contrast condition, and drug treatment. Contrast treatments were as described in
Fig. 1, except that the low solution was 6% sucrose.
4. Method
4.1. Subjects and apparatus
The subjects were 37 male Long-Evans hooded rats from the
TCU vivarium approximately 90 days old at the start of the experiment and experimentally naïve. The average ad libitum weight
was 423 g (range: 352–526 g). Housing, training procedure, apparatus, drug preparation, and route of administration were similar
to Experiment 1
4.2. Procedure
The only difference with respect to the previous experiment was
the use of 6% sucrose as the downshifted solution (w/w, prepared by
mixing 6 g of sucrose for every 94 g of distilled water). The following
groups were included: 32/Sal (n = 9), 32/DCS (n = 9), 6/Sal (n = 9), and
6/DCS (n = 10). All other procedural aspects were as described in the
previous experiment.
5. Results
The results are shown in Fig. 3. A Drug × Sucrose × Preshift Trial
(1–10) analysis revealed significantly higher goal-tracking scores
with 32% sucrose than 6% sucrose, F(1, 33) = 4.83, p < 0.04, and a
significant increase across trials, F(9, 297) = 83.64, p < 0.001; the
main effect for drug and the interactions were all not significant, Fs(9, 297) < 1.56, ps > 0.12. A Drug × Sucrose × Postshift Trial
(11–15) analysis yielded significant main effects for sucrose, F(1,
33) = 6.67, p < 0.02, trial, F(4, 132) = 16.45, p < 0.001, and the trial by
sucrose interaction, F(4, 132) = 18.4, p < 0.001. All other effects were
not significant, Fs < 1, including the triple interaction.
The results of the within-trial analysis are presented in Fig. 4.
Whether in the first or last 100 s, there was no hint of a DCS effect
in these data. Drug × Sucrose × Trial (11–15) analyses, whether for
the first or last 100 s, provided no evidence of a triple interaction, Fs < 1. The contrast effect was significant for both the first
and last 100 s sets, in terms of a sucrose by trial interaction, F(4,
132) = 6.70, p < 0.001. The main effect of sucrose as also significant
for the last 100 s data set, F(1, 33) = 11.43, p < 0.003, but not for the
first 100 s data set, F < 1. Changes across trials were significant for
both analyses, Fs(4, 132) > 3.47, ps < 0.02. Other factors were not
significant, Fs < 1. The same results were obtained with a similar
analysis restricted to Trials 11–13, as had been done in Experiment
1.
The results of Experiment 2 failed to uncover any effects of DCS
after a 32-to-6% sucrose downshift. One implication of these results
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First 100 s
Goal-tracking Time (s)
100
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50
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12
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Trials
Last 100 s
Goal-tracking Time (s)
100
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involving other drugs administered after the first downshift trial in
the cSNC preparation [23,30,51]. Briefly, the 4% sucrose solution is
relatively novel for downshifted animals and may act as an efficient
conditioned stimulus for the effects induced by DCS administration,
which would be the unconditioned stimulus. This factor would be
less prominent in unshifted controls because of their extensive preexposure to 4% sucrose during preshift trials (i.e., latent inhibition
reduces conditioned taste aversions; [52]).
Experiment 2 provided data inconsistent with the conditioned
taste aversion hypothesis because DCS had no measurable effect
on the subsequent consumption of 6% sucrose. However, because
of the different concentrations used in Experiments 1 and 2 (4%
vs. 6% sucrose) the role of this factor cannot be safely excluded. For
example, the lesser disparity between 6 and 32% sucrose, compared
to 4 and 32% sucrose, may have rendered the 6% solution relatively
less novel and, therefore, relatively less likely to enter into an association with the consequences of DCS administration. Experiment
3 evaluated the conditioned taste aversion hypothesis by pairing
4% sucrose to DCS in the absence of an incentive downshift, but in
a situation that was otherwise the same as that used in Experiment
1. Moreover, unlike in Nunnink et al.’s [42] experiment, the present
study included a nonassociative control group that received exposure to both 4% sucrose and DCS, but in an unpaired fashion. If the
effects of DCS observed in previous experiments are mediated by
a conditioned taste aversion, rather than memory consolidation,
then DCS should result in a similar suppression of consummatory
behavior in the absence of the downshift event.
50
6. Method
32/Sal
6/Sal
32/DCS
6/DCS
25
0
9
10
11
12
13
14
15
Trials
Fig. 4. Performance during the first (top panel) and last (bottom panel) 100 s of
the last preshift trial (Trial 10) and each of the downshift trials (Trials 11–15) in
Experiment 2. Mean goal-tracking time (±SEM) plotted as a function of trial, contrast condition, and drug treatment. Contrast treatments were as described in Fig.
1, except that the low solution was 6% sucrose.
is that a minimum disparity between preshift and postshift sucrose
concentrations is required for DCS administered immediately after
the first downshift trial to affect recovery from cSNC. Notice that the
failure to detect an effect of posttrial DCS administration occurred
despite the presence of a significant cSNC effect after a 32-to-6%
sucrose downshift.
5.1. Experiment 3
Experiment 1 showed that DCS administered immediately after
the first downshift trial prolonged the cSNC effect in subsequent
trials. Whereas these results provide support for the hypothesis
that activation of NMDA receptors using DCS enhances cSNC by
facilitating the consolidation and subsequent retrieval of the emotional memory of the incentive downshift event, one alternative
remains to be evaluated. Nunnink et al. [37] reported that pairing DCS at 30 mg/kg with access to saccharine reduced preference
against water in subsequent two-bottle tests, compared to a saline
control. Although the experiment lacked an unpaired control, it
is plausible that the effect of DCS on cSNC may be the result of a
conditioned taste aversion, rather than of emotional memory consolidation. This analysis is described in detail in similar experiments
6.1. Subjects and apparatus
The subjects were 36 male, experimentally naïve Long-Evans
hooded rats, approximately 90 days old at the start of the experiment. The average ad libitum weight for the entire sample was
397 g (range: 305–519 g). Rats were maintained as described in
Experiment 1. The training apparatus and drug preparation were
also the same described previously.
6.2. Procedure
Training lasted 3 daily trials, scheduled to be identical to trials in the cSNC experiments reported above. Subjects received 4%
sucrose solution in every trial. Prior to Trial 1, rats were matched
by ad libitum weight and then randomly assigned to one of three
conditions: Paired (n = 12), Unpaired (n = 12), or Saline (n = 12). All
groups received two injections; the first injection was administered immediately at the end of Trial 1 and the second injection
was administered 3 h after the end of Trial 1. The first injection
was saline for Groups Unpaired and Saline, and DCS (30 mg/kg,
ip) for Group Paired. The second injection was saline for Groups
Paired and Saline, but DCS (30 mg/kg, ip) for Group Unpaired. Thus,
groups were matched in terms of the number and temporal distribution of injections; moreover, Groups Paired and Unpaired were
also matched in terms of exposure to DCS.
7. Results
The results are shown in Fig. 5. A Group × Trial (1–3) analysis showed a significant increase of behavior across trials, F(2,
66) = 67.77, p < 0.001, but groups were not significantly different,
F(2, 33) = 1.14, p > 0.30, as was the interaction effect, F < 1. Oneway ANOVAs for each trial yielded not significant group effect
for Trials 1–3, Fs(2, 35) < 1.57, ps > 0.22. The not significant difference obtained on Trial 1 indicates that subjects were behaviorally
Author's personal copy
Goal-Tracking Time (s)
J.N. Norris et al. / Behavioural Brain Research 223 (2011) 348–355
300
250
Paired
Unpaired
Saline
200
150
100
50
0
0
1
2
3
Trials
Fig. 5. Results of Experiment 3. Goal-tracking time (s) is plotted as a function of trial,
and drug treatment. On Trials 1–3 Paired, Unpaired, and Saline groups received 4%
sucrose solution. Group Paired received DCS (30 mg/kg, ip) immediately after Trial
1 and saline 3 h after Trial 1. Group Unpaired received saline immediately after Trial
1 and DCS (30 mg/kg, ip) 3 h after Trial 1. Group Saline received saline injections
immediately after and 3 h after Trial 1. All groups were equated in terms of the
number and temporal distribution of injections; Groups Paired and Unpaired were
also equated in terms of DCS administration. Notice that none of the groups was
exposed to an incentive downshift in Experiment 3.
matched before the treatment. These results provide no evidence
that the effects of DCS reported in Experiment 1 were the result a
conditioned taste aversion.
8. General discussion
The current studies demonstrated that the partial NMDAR
agonist DCS retarded recovery from cSNC when administered
immediately following the first trial involving a 32-to-4% sucrose
incentive downshift. DCS failed to induce conditioned taste aversion, suggesting that its suppressive effects on consummatory
behavior were not the result of an acquired rejection of the
relatively novel 4% sucrose solution in downshifted animals. Furthermore, DCS had no detectable effect on the consummatory
behavior of nonshifted rats. These results suggest that the effects
of DCS on cSNC were not caused by performance factors (i.e., perceptual, motivational, or motor effects). Therefore, the effects of
DCS were restricted to animals that had some experience with
an incentive downshift event—an interpretation consistent with a
mnemonic effect of DCS. These results were interpreted to support
the hypothesis that NMDA receptor activation via the glycinemodulatory site enhances the consolidation, hence facilitating
retrieval of the emotional aversive memory induced by the unexpected incentive downshift event.
The effects of posttrial DCS administration were interpreted
as acting on mnemonic processes that presumably occur after
DCS administration, but conclude prior to next day’s trial. It is
implausible that DCS is still active a day after its administration
[12,13]. Behavioral evidence from the present experiments (e.g.,
unshifted groups in both experiments) and from fear conditioning experiments [48] suggests that the dose used here (30 mg/kg,
ip) does not have measurable behavioral effects 24 h after its
administration.
When DCS was selected for study (see Section 1 for rationale),
it was assumed that DCS facilitated memory consolidation based
mainly on results from the fear conditioning situation. Recent evidence suggests that the effects of DCS on fear extinction reflect the
erasure of the original fear acquisition, rather than an enhancement of fear extinction learning [53]. Mao et al. [53] reported that
DCS facilitated the reversal of postsynaptic changes caused as a
result of fear extinction, as assessed in terms of a decreased surface
expression of GluR1, a subunit of AMPA(␣-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid) receptors, a type of receptor
known to mediate conditioning [54]. Thus, DCS may act to facilitate
NMDA-mediated endocytosis of AMPA receptors, erasing learning.
353
This memory-erasure hypothesis can be applied to the cSNC
situation as follows. With posttrial administration (Experiment
1), it could be argued that DCS erased the emotional memory of
the downshift event acquired during the first downshift trial. This
would predict a cSNC effect on the second downshift trial (Trial
12) that would be essentially identical to that of the first downshift trial (Trial 11). Instead, Experiment 1 showed that DCS-treated
animals exhibited greater early suppression on the second and
third downshift trials (Trials 12–13) than saline-treated animals,
an effect contradicting the memory erasure hypothesis. Moreover,
this hypothesis cannot adequately explain the effects of DCS on
maze learning, conditioned place preference, and conditioned taste
aversion reported by others [42,43,47].
The effects of DCS on cSNC reported here were assumed to
be mediated by an increase in the activity of NMDA receptors,
which would lead to the strengthening of the emotional memory of the downshift event. However, available evidence suggests
that DCS could act in some cases as an antagonist. For example,
Hood et al. [33] have shown that DCS’s binding to the NMDA receptor is only approximately 40–50% as effective as glycine. Thus,
under an appropriate combination of DCS dose and extracellular
glycine levels, DCS could actually reduce NMDA-receptor effectiveness. Along these lines, Saul’skaya and Solov’eva [55] reported
that glycine levels in the nucleus accumbens are transiently elevated following forced changes in feeding behavior in rats. This is
especially relevant to the current studies for two reasons. First, the
nucleus accumbens shows increased c-Fos transcription (a transcription factor for immediate early gene expression correlated
with synaptic changes such as growth and protein kinase activity)
and reduced dopamine efflux following 32-to-4% sucrose downshift [56,57]. Second, increased glycine levels in Saul’skaya and
Solov’eva’s [55] study were observed selectively when animals
were exposed to both feeding and a fear-inducing stimulus previously paired with shock-induced pain. Thus, it is plausible that
the forced rearrangement of feeding behavior caused by incentive
downshift in the cSNC situation acted to increase glycine levels,
thus converting DCS into an antagonist at the glycine site. Despite
these potential interactions of DCS and extracellular glycine levels, the present results do not support the hypothesis that DCS
acted as an antagonist. Experiments 1 and 2 showed that the
effectiveness of DCS scaled to the magnitude of the downshift.
DCS interfered with recovery from cSNC following a 32-to-4%
sucrose downshift, but had no detectable effect following a 32to-6% sucrose downshift. If 30 mg/kg DCS acted as an antagonist,
then the effects of posttrial DCS administration following these
two levels of incentive downshift should have yielded similar
results. However, if DCS at 30 mg/kg acted as an agonist, then
its effects should be directly related to the size of the disparity
between preshift and postshift incentives. Therefore, the results
reported here indicate that DCS functioned as an agonist in the cSNC
preparation.
The present results are consistent with the hypothesis that DCS
enhanced the consolidation of the emotional memory of the incentive downshift event. This is so because DCS retarded recovery
from cSNC, a consequence consistent with the strengthening of
an emotional memory. Because posttrial administration of corticosterone also retarded recovery from cSNC [22,23], it is tempting
to argue that these two effects are related. The memory enhancing effects of stress hormones like corticosterone, epinephrine,
and norepinephrine are well documented [15,17]. These mnemonic
effects of stress hormones are mediated by glutamatergic receptors
in the brain. For example, norepinephrine levels enhance contextual fear conditioning in mice by phosphorylating GluR1 subunits
of the AMPA receptor [54]. Similar interactions occur between
glucocorticoid administration and activation of NMDA receptors.
For example, restraint stress enhances allodynia (increased pain
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J.N. Norris et al. / Behavioural Brain Research 223 (2011) 348–355
sensitivity to nonpainful stimuli) induced by neuropathic pain
and this effect is mediated by glucocorticoid release. Corticosterone administration often has similar effects to those of restraint
stress and its effects on allodynia are eliminated by coadministration of memantine, an NMDA receptor antagonist [58]. Because
the effects of corticosterone in other situations are mediated
by glutamatergic activity [32], we hypothesize that the retardation of recovery from cSNC induced by corticosterone and DCS
reflects effects at two different stages of the same cascade leading to enhanced consolidation of the emotional memory of the
downshift.
Acknowledgements
The research reported here was partially supported by SERC
grant # 80301, TCU School of Science and Engineering. The authors
thank C. Torres, A. E. Mustaca, and M. F. Lopez-Seal for valuable
comments on an earlier version of the manuscript.
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