Effects of D-cycloserine on extinction of learned fear to an olfactory cue.

Abstract

D-cycloserine (DCS), a partial NMDA receptor agonist, facilitates extinction of learned fear in rats and has been used to treat anxiety disorders in clinical populations. However, research into the effects of DCS on extinction is still in its infancy, with visual cues being the primary fear-eliciting stimuli under investigation. In both human and animal subjects odors have been found to associate strongly with aversive events. Therefore, this study examined the generality of the effects of DCS on extinction by testing odor cues. Sprague-Dawley rats were conditioned and extinguished to an odor using varying parameters, injected with either saline or DCS (15 mg/kg) following extinction, and then tested for a freezing response 24 h later. Experiment 1 demonstrated that after 3 odor-shock pairings, rats did not display short-term extinction and DCS had no effect on long-term extinction. Experiment 2 demonstrated that after 3 odor-noise pairings, rats displayed significant short-term extinction and DCS significantly facilitated long-term extinction. Following 2 odor-shock pairings in Experiment 3, half the rats displayed short-term extinction ("extinguishers") and half did not ("non-extinguishers"). DCS facilitated long-term extinction in the "extinguishers" condition but not in the "non-extinguishers" condition. In Experiment 4, following 2 odor-shock pairings and an extra extinction session, DCS had a significant facilitatory effect on long-term extinction. Thus, extinction of freezing to an odor cue was facilitated by systemic injections of DCS, but only when some amount of within-session extinction occurred prior to injection.

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Neurobiology of Learning and Memory 87 (2007) 476–482
www.elsevier.com/locate/ynlme
1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nlm.2006.12.010
EVects of D-cycloserine on extinction of learned fear to an olfactory cue
Marianne Weber, Joe Hart, Rick Richardson¤
School of Psychology, University of New South Wales, Sydney 2052, Australia
Received 11 July 2006; revised 28 November 2006; accepted 21 December 2006
Available online 1 February 2007
Abstract
D-cycloserine (DCS), a partial NMDA receptor agonist, facilitates extinction of learned fear in rats and has been used to treat anxiety
disorders in clinical populations. However, research into the eVects of DCS on extinction is still in its infancy, with visual cues being the
primary fear-eliciting stimuli under investigation. In both human and animal subjects odors have been found to associate strongly with
aversive events. Therefore, this study examined the generality of the eVects of DCS on extinction by testing odor cues. Sprague–Dawley
rats were conditioned and extinguished to an odor using varying parameters, injected with either saline or DCS (15mg/kg) following
extinction, and then tested for a freezing response 24h later. Experiment 1 demonstrated that after 3 odor-shock pairings, rats did not dis-
play short-term extinction and DCS had no eVect on long-term extinction. Experiment 2 demonstrated that after 3 odor-noise pairings,
rats displayed signiWcant short-term extinction and DCS signiWcantly facilitated long-term extinction. Following 2 odor-shock pairings in
Experiment 3, half the rats displayed short-term extinction (“extinguishers”) and half did not (“non-extinguishers”). DCS facilitated long-
term extinction in the “extinguishers” condition but not in the “non-extinguishers” condition. In Experiment 4, following 2 odor-shock
pairings and an extra extinction session, DCS had a signiWcant facilitatory eVect on long-term extinction. Thus, extinction of freezing to
an odor cue was facilitated by systemic injections of DCS, but only when some amount of within-session extinction occurred prior to
injection.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Rats; D-Cycloserine; Fear extinction; Odors; Freezing
1. Introduction
Anxiety disorders such as Phobia, post-traumatic stress
disorder (PTSD), and Obsessive–Compulsive Disorder are
often treated with exposure-based cognitive behavior ther-
apy (Andrews, Grino, Hunt, & Page, 1994; Foa & Kozak,
1986; Thyer, Baum, & Reid, 1988; Zarate & Agras, 1994).
This type of therapy involves exposing patients to a fearful,
or anxiety-provoking, stimulus within a calm and safe envi-
ronment. Gradually, over successive presentations of the
stimulus within this environment, patients learn to respond
without fear or anxiety. This type of treatment is procedur-
ally very similar to ‘extinction’ training in animal models of
emotional learning. For example, learned fear responses to
a conditioned stimulus (CS; e.g., a light that had previously
been paired with shock) can be reduced by repeatedly pre-
senting the CS in the absence of shock. Eventually the ani-
mal learns that the CS no longer predicts the occurrence of
shock and fear reactions decrease in amplitude and/or
frequency.
Although very simple procedurally, extinction has
proven to be an extremely diYcult process to understand.
Nonetheless, considerable eVort has been directed at trying
to identify techniques that render extinction—and poten-
tially exposure therapy—more eVective. One signiWcant
advance in this area has been the demonstration that
D-cycloserine (DCS) can facilitate fear extinction. DCS was
used because of its indirect agonist properties at N-methyl-
D-aspartate receptors, which are critical for the neuronal
plasticity required for emotional memory (see, Davis, Ress-
ler, Rothbaum, & Richardson, 2006). Pre-clinical studies
have demonstrated that DCS is a potent facilitator of fear
*Corresponding author. Fax: +61 2 9385 3641.
E-mail address: rrichardson@psy.unsw.edu.au (R. Richardson).

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M. Weber et al. / Neurobiology of Learning and Memory 87 (2007) 476–482
477
extinction in rats (Ledgerwood, Richardson, & Cranney,
2003, 2004, 2005; Parnas, Weber, & Richardson, 2005;
Walker, Ressler, Lu, & Davis, 2002; Yang & Lu, 2005). In
addition, DCS has been found to enhance the eVectiveness
of exposure-based therapy in people treated for acrophobia
(Ressler et al., 2004) and Social Anxiety Disorder (Hof-
mann et al., 2006). However, research on the eVects of DCS
on extinction is still in its infancy, and it is still not known
whether DCS is eVective at facilitating extinction to any, or
all, types of feared stimuli, or whether its eVectiveness is
limited in some way.
The aim of the current study was to test the ‘generality’ of
the eVects of DCS on extinction. Both pre-clinically and
clinically, DCS has primarily been used in conjunction with
exposure to visual cues. However, fear-eliciting cues are not
always visually-based. Odors, in particular, have been found
to be especially eVective in cuing emotional memory in non-
clinical populations (Herz, 1998; Herz, Eliassen, Beland, &
Souza, 2004), and can evoke strong trauma-related memo-
ries in people with PTSD (Vermetten & Bremner, 2003).
Olfactory stimuli are also extremely salient and powerful
cues for memory retrieval in rats. For example, conditioned
fear associations using olfactory cues in rats are encoded
more rapidly (Paschall & Davis, 2002), and require more tri-
als to extinguish than do visual cues (Richardson, Tronson,
Bailey, & Parnas, 2002). These Wndings suggest that odor
cues may be processed by a diVerent memory system.
Indeed, in rats the neuroanatomical pathways subserving
olfactory information are slightly diVerent than for stimuli
of other sensory modalities. For example, visual and audi-
tory sensations are processed within the thalamus before
being sent to the amygdala, which is the brain region crucial
for recalling emotional associations. However, olfactory
information is transmitted directly to the amygdala (Price,
1973), suggesting that odors have ‘privileged’ access to the
amygdala and may prompt recall for emotional associations
more quickly and easily. Therefore, because (1) odor-evoked
memories can be powerful triggers for the emotional com-
ponent of memory, and (2) the neuroanatomical substrates
of odor-evoked emotional memories may diVer to that for
visually-evoked memories, the present study was designed to
determine whether DCS facilitates extinction of conditioned
fear to an odor CS in rats.
2. Methods and materials
2.1. Subjects
Experimentally-naive, male Sprague–Dawley rats (450–700g) obtained
from the breeding colony maintained by the School of Psychology at the
University of New South Wales were used. The rats were housed in groups
of eight in plastic boxes (67cm long£ 40cm wide£22cm high) in a col-
ony room with a natural light cycle. Food and water were continuously
available. All experimental procedures were approved by the Animal Care
and Ethics Committee of the University of New South Wales and adhered
to the ethical guidelines described in The Australian Code of Practice for
the Care and Use of Animals for ScientiWc Purposes (2004). Prior to exper-
imentation, all rats were handled for approximately 5min on 3 consecutive
days.
2.2. Apparatus
In all experiments conditioning occurred in one context and extinc-
tion training and testing occurred in a diVerent context. All experimental
cages were located within a sound- and light-attenuating wood cabinet
where a ventilation fan provided a 60-dB ambient noise level in each
chamber. Conditioning occurred in one of 4 identical cages (20 cm
long £ 12 cm wide £ 12 cm high). The front wall, rear wall, and ceiling of
each cage were made of Plexiglas, and the side walls and Xoor consisted
of stainless steel bars, with 1.3 cm between each bar. Each cage was sus-
pended 13 cm above the Xoor of the wood cabinet and a tray of bedding
was placed under each cage, which was changed between each rat. Dur-
ing conditioning, the doors to the wood cabinets remained open and the
room was illuminated by a red light.
The context used for extinction training and testing in Experiments 1
and 2 diVered slightly from that used in Experiments 3 and 4. In Experi-
ments 1 and 2, extinction training and testing occurred in one of 2 identical
cages (30cm long£ 22.5cm wide£ 30cm high) with a clear Perspex front,
black and white striped side and rear walls, and a wood hinged lid. The
Xoor consisted of stainless steel bars, with 1.0cm between each bar, and
was suspended 8.5cm above the Xoor of the wood cabinet. In Experiments
3 and 4, extinction training and testing occurred in one of 2 identical clear
Perspex cages (29cm long£ 28cm wide£ 29cm high). The Xoor consisted
of stainless steel bars, with 1.5cm between each bar, and was suspended
10cm above the Xoor of the wood cabinets.
A white light bulb in each cabinet provided illumination at all times
during extinction and testing. Between sessions, these chambers were
wiped with 0.5% eucalyptus solution and the bedding below the grid Xoor
was changed. Extinction and test sessions were recorded for later assess-
ment.
2.3. Procedure
In all experiments the CS was a grape odor (0.1ml in a plastic jar;
#182380019 from Wild Flavours, Heidelberg) and the method of presenta-
tion involved the odor jar being placed under the experimental cage for the
required time period and then being removed and capped with an air-tight
lid. The unconditioned stimulus (US) varied for each experiment.
The procedures for Experiments 1 and 2 were identical except that a
diVerent US was used. In Experiment 1 the US was a 1 s, 0.6 mA foot-
shock and in Experiment 2 the US was a 100 ms, 120 dB white noise
stimulus (rise-fall time <5 ms). In each experiment a 2 £ 2 factorial
design was employed, where the Wrst factor was drug condition (DCS or
saline) and the second factor was condition (extinction or no extinction).
This produced 4 groups: extinction + DCS (E-DCS), extinction + saline
(E-Sal), no-extinction + DCS, and no-extinction + saline; the latter two
groups were subsequently pooled into a single group (NE-Pooled).
On Day 1, all rats were pre-exposed to the conditioning context for
20 min. Three hours later they were returned to the conditioning cages
and received 3 pairings of the CS and US. That is, 120 s after being
placed in the conditioning cages, the odor-jar was placed beneath the
cage. Ten seconds later, the US was presented and then the odor-jar was
removed. The 3 CS–US pairings occurred 120 s apart and rats were
removed from the conditioning cages 120 s after the last pairing. On
Day 2, rats allocated to the extinction groups were placed in the extinc-
tion cages. After 2 min in the cage, rats were presented with the jar con-
taining the odor 6 times (each exposure was 2-min long, with a 2-min
ITI). The rats were removed from the extinction cages 120 s after the
last extinction trial, and half were injected with saline (E-Sal) and half
were injected with DCS (E-DCS). Rats that did not receive extinction
training were removed from their home cage and injected with either
saline or DCS (NE-pooled). After injection all rats were returned to
their home cage. On Day 3, all rats were tested for odor-elicited freez-
ing. That is, 2 min after being placed in the cage, the odor was presented
for 3 min.
The procedure for Experiments 3 and 4 was identical to Experiments 1
and 2 except that rats received 2 CS–US pairings and the US was a 0.5s,
0.6mA footshock. After extinction training in Experiment 3 rats were

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M. Weber et al. / Neurobiology of Learning and Memory 87 (2007) 476–482
divided into 2 groups where half received an injection of DCS and half
received saline before being returned to the home cage. All rats were tested
for odor-elicited freezing the following day. For the purpose of analysis,
these groups were further divided equally into ‘extinguishers’ and ‘non-
extinguishers’. Those rats that had the lowest levels of freezing at the end
of extinction were put into the “extinguishers” condition while those with
the highest levels of freezing at the end of extinction were placed in the
“non-extinguishers” condition.
In Experiment 4, rats received a second extinction training session that
occurred 45–90min after the Wrst. After this second extinction session, half
the rats received DCS and half received saline. All rats were tested for
odor-elicited freezing the following day.
2.4. Data analysis
Each rat was scored for freezing during the 6 extinction trials (or the
12 trials in Experiment 4) and during the test on Day 3. Freezing was
characterized by an absence of all bodily movements except those
required for breathing (Fanselow, 1980), and was measured using a time
sampling method. An observation was made every 5 s and a positive or
negative count was made. A percentage score was then calculated for the
proportion of the total observation period spent freezing. For the test
session on Day 3, freezing during the 120 s prior to the Wrst odor presen-
tation (pre-CS) was scored as an indication of baseline levels of fear.
Analysis of variance (ANOVA) was the primary statistical approach,
and post hoc comparisons were made with Tukey’s honestly signiWcant
diVerence (HSD) test.
2.5. Drugs
D-cycloserine (Sigma–Aldrich, Castle Hill, New South Wales) was
freshly dissolved in sterile saline at a concentration of 15mg/ml and
injected subcutaneously in a volume of 1ml/kg.
3. Results
3.1. Experiment 1
Rats that received extinction training displayed high lev-
els of conditioned freezing to each of the 6 odor presenta-
tions during the extinction session (Fig. 1a). Overall, levels
of freezing did not signiWcantly decline across the extinction
session, as conWrmed by a mixed-design ANOVA with 6
time points, F<1. In addition, rats allocated to the saline
condition (nD8) displayed similar levels of freezing to rats
that were to receive DCS (nD8).
At test, rats that received DCS but no extinction training
did not display a diVerent level of freezing to rats that
received saline and no extinction [t (6) <1]. Therefore, these
groups were pooled (NE-Pooled). Overall, there were
no group diVerences in the amount of pre-CS freezing
[F (2,23)D2.49, pD.11]. Additionally, there were no group
diVerences in the amount of freezing to the odor CS
[F (2,23)D1.85, pD.18] (Fig. 1b).
Thus, rats that received DCS immediately after extinc-
tion training to an odor CS previously paired with a
shock US did not freeze less at test than rats that received
saline, indicating that DCS did not facilitate extinction.
However, no decline in the fear response was observed in
either extinction group across the six 2-minute extinction
trials. It may be the case that DCS does not facilitate
extinction retention if no short-term extinction learning
Fig. 1. (a) Mean freezing during extinction trials in Experiment 1 where rats had received odor–shock pairings. Either saline or DCS (15mg/kg) was
injected immediately after extinction. (b) Mean freezing during test in Experiment 1. Rats had either been extinguished (E) or not (NE), and injected with
saline (SAL) or DCS. Half of the rats in the NE group were injected with saline while the others were injected with DCS; these rats were collapsed into a
single control group (pooled). (c) Mean freezing during extinction trials in Experiment 2 where rats had received odor–noise pairings. Either saline or DCS
(15mg/kg) was injected immediately after extinction. (d) Mean freezing during test in Experiment 2. Rats had been extinguished (E) or not (NE), and
injected with saline (SAL) or DCS. Half of the rats in the NE group were injected with saline while the others were injected with DCS; these rats were
collapsed into a single control group (pooled).
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M. Weber et al. / Neurobiology of Learning and Memory 87 (2007) 476–482
479
is observed. Alternatively, it may be the case that DCS
does not aVect extinction of conditioned fear when odor
cues are used.
The aim of Experiment 2 was to condition rats to an
odor cue in a manner in which short-term extinction was
observed. That is, rats were conditioned with a US that is
less aversive than a shock, namely a loud noise.
3.2. Experiment 2
Two rats did not display a robust conditioned response
to the odor (mean levels of freezing were less than 10%
across the 6 extinction trials) and were excluded from all
statistical analysis. Rats that received extinction training
displayed high levels of conditioned freezing to the odor
during the Wrst extinction trial and displayed signiWcantly
lower levels of freezing across trials [mixed-design ANOVA
with 6 time points, F (5,120)D39.0, pD.001] (Fig. 1c). Rats
that were to receive saline (nD12) did not diVer on the
amount of freezing during each extinction trial to rats that
were to receive DCS (nD14) [non-signiWcant interaction,
F<1 and a non-signiWcant between groups eVect, F
(1,24)D1.17, pD.29].
At test, rats that received no extinction training and
DCS did not display a diVerent level of freezing to rats
that received saline and no extinction [t (10) <1]. There-
fore, these groups were pooled (NE-Pooled, nD12). Pre-
CS freezing was signiWcantly higher for rats that received
extinction training and saline injections (E-SAL) than rats
in the E-DCS and NE-Pooled conditions [F (2,37)D4.01,
pD.03, with post hoc comparisons signiWcant at p<.05].
Thus, freezing to the CS was analyzed with an ANCOVA,
with pre-CS freezing as a covariate. During the 3min odor
CS presentation, rats that received DCS following extinc-
tion (E-DCS) froze signiWcantly less than rats that
received saline following extinction (E-Sal), and signiW-
cantly less than rats that were not given extinction train-
ing (NE-Pooled; F (2,37)D5.32, pD.01, with post hoc
comparisons signiWcant at p<.05). Further, rats that
received extinction training and saline injections (E-SAL)
did not display signiWcantly diVerent levels of freezing to
rats that did not receive extinction training (NE-Pooled)
(Fig. 1d).
Experiment 2 demonstrated facilitated extinction to an
odor CS when DCS was injected after an extinction train-
ing session in which short-term extinction was observed.
This experiment demonstrates that long-term extinction of
odor-elicited fear can be facilitated by DCS, and suggests
that the failure to observe facilitation of extinction reten-
tion in Experiment 1 was due to the high level of freezing
maintained during extinction training. However, the diVer-
ence in US modality between the two experiments could
also account for the diVerence in the eVectiveness of DCS.
Therefore, the aim of Experiment 3 was to demonstrate
within-session extinction after odor–shock pairings by giv-
ing fewer odor–shock pairings than in Experiment 1, and
also by reducing the duration of the US.
3.3. Experiment 3
As noted in Section 2, rats in this experiment were
divided into two groups: “non-extinguishers” and “extin-
guishers”. Overall, levels of freezing did not signiWcantly
decline across the extinction session for rats in the “non-
extinguishers” condition, as conWrmed by a mixed-design
ANOVA with 6 time points, F<1 (Fig. 2a). In addition,
rats allocated to the saline condition (nD6) displayed simi-
lar levels of freezing to rats that were to receive DCS (nD6)
[non-signiWcant interaction F<1, and non-signiWcant
between groups eVect, F (1,10)D1.11, pD0.32]. At test,
there were no group diVerences in the amount of pre-CS
freezing [t (10) <1], and no group diVerences in the amount
of freezing to the odor CS [t (10), <1] (Fig. 2b).
Conversely, rats in the “extinguishers” condition dis-
played high levels of conditioned freezing to the odor during
the Wrst extinction trial and signiWcantly lower levels of freez-
ing across trials [mixed-design ANOVA with 6 time points, F
(5,50)D2.65, pD0.03] (Fig. 2c). Rats that were to receive
saline (nD6) did not diVer on the amount of freezing during
each extinction trial to rats that were to receive DCS (nD6)
[non-signiWcant interaction and non-signiWcant between
groups eVect, both Fs<1]. At test, there were no group diVer-
ences in the amount of pre-CS freezing [t (10)D2.09,
pD0.08], however, during the 3min odor CS presentation,
rats that received DCS froze signiWcantly less than rats that
received saline [t (10)D2.31, pD0.04] (Fig. 2d). In this
experiment DCS facilitated extinction in rats that displayed
within-session extinction (“extinguishers”) but not in rats
that maintained high levels of freezing before the DCS injec-
tion (“non-extinguishers”). Experiment 4 aimed to further
explore the idea that within-session extinction is necessary in
order for DCS to facilitate long-term extinction.
3.4. Experiment 4
One rat in the DCS group displayed a high level of freez-
ing during the pre-CS period at test (>50%) and was
excluded from all statistical analysis. By the end of the sec-
ond extinction training session, and before the drug injec-
tion, all rats displayed signiWcant levels of extinction
[mixed-design ANOVA with
(11,143)D13.62, pD.005] (Fig. 3a). Rats that were to
receive saline (nD8) did not diVer on the amount of freez-
ing during each extinction trial to rats that were to receive
DCS (nD7) [non-signiWcant interaction F (11,143)D1.03,
pD.42, and non-signiWcant between groups eVect, F<1]. At
test, the two groups did not diVer in the level of pre-CS
freezing [t (13) <1], however during the 3min odor presen-
tation, rats that received DCS froze signiWcantly less than
rats that received saline [t (13)D2.43, pD.03] (Fig. 3b).
4. Discussion
12 time points,
F
This study demonstrated that post-extinction, systemic
injections of DCS facilitated extinction retention with an

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M. Weber et al. / Neurobiology of Learning and Memory 87 (2007) 476–482
aversive odor CS only if short-term, or within-session,
extinction of conditioned freezing had been observed prior
to injection. This occurred whether short-term extinction
was observed following conditioning with a noise US
(Experiment 2), or fewer odor–shock pairings (Experiment
3—“extinguishers”), or when additional extinction training
was given after odor–shock pairings (Experiment 4). When
high levels of freezing were maintained throughout the
extinction session, DCS-treated rats displayed the same
level of extinction retention as did saline-treated rats (as
shown in Experiment 1 and the “non-extinguishers” in
Experiment 3). Together, these experiments demonstrate
that (1) DCS can be used eVectively to facilitate extinction
with odor cues, and (2) DCS is ineVective at facilitating
extinction retention if no short-term extinction learning
occurs prior to injection.
The dissociation between the eVects of DCS on extinc-
tion learning in the experiments in this study and the
hypothesized diVerences between the amounts of short- and
long-term learning across extinction training provide an
interesting perspective from which to speculate on the
mechanisms underlying the facilitatory eVects of DCS on
extinction retention. DCS exerts its eVects in the brain by
binding to the glycine regulatory site of the NMDA com-
plex. When the glycine site on the NMDA complex is acti-
vated, cell depolarization is ampliWed by an increased
frequency of channel openings, and therefore excitatory
neurotransmission through the NMDA receptor is facili-
tated (Kleckner & Dingledine, 1988). Normal activation of
NMDA receptors has been shown to be critical for the neu-
ral plasticity underlying learning (e.g., long-term potentia-
tion; Bear & Malenka, 1994) and is, more speciWcally, one
mechanism by which experiences are translated from short-
to long-term memory (Sweatt, 1999). Given that DCS aug-
ments neurotransmission through NMDA receptors, and
working under the assumption that long-term memories
Fig. 2. (a) Mean freezing during extinction trials in Experiment 3 for rats in the “non-extinguishers” condition. Either saline or DCS (15mg/kg) was
injected immediately after extinction. (b) Mean freezing of rats in the “non-extinguishers” condition during test in Experiment 3. (c) Mean freezing during
extinction trials in Experiment 3 for rats in the “extinguishers” condition. Either saline or DCS (15mg/kg) was injected immediately after extinction. (d)
Mean freezing of rats in the “extinguishers” condition during test in Experiment 3.
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100
DCS
SAL
Trial
% Freezing
DCS SAL
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25
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100
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% Freezing
a
c
b
d
Fig. 3. (a) Mean freezing during extinction trials in Experiment 4 across 12 trials that were separated into two groups of 6 by 45–90min. Either saline or
DCS (15mg/kg) was injected immediately after extinction. (b) Mean freezing at test in Experiment 4.
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M. Weber et al. / Neurobiology of Learning and Memory 87 (2007) 476–482
481
cannot be formed without short-term learning, it is possible
that DCS facilitates extinction retention by increasing the
amount of short-term extinction learning that is translated
into long-term memory. For example, in Experiments 2, 3
(“extinguishers”), and 4, there was a signiWcant amount of
short-term extinction exhibited across the extinction trials
and the translation of that learning into long-term memory
was increased in rats that received DCS. The saline-treated
rats that received extinction training in these experiments
displayed very little extinction retention at test (indeed in
Experiment 1, these rats responded similarly to the no-
extinction control groups) and therefore either did not
form a long-term extinction memory, or the memory was
not strong enough to reach threshold for behavioral expres-
sion. However, in the case where no short-term extinction
learning occurs, regardless of whether NMDA receptors
are activated or not, no translation into long-term memory
can occur. For example, there was no apparent short-term
extinction learning in Experiments 1 and 3 (“non-extin-
guishers”) because rats displayed a consistently high level
of conditioned freezing across all six extinction trials. In
this case, the absence of short-term learning meant there
could be no translation from short- to long-term memory
and therefore DCS could not facilitate this transfer. Hence,
at test there were no group diVerences.
However, another interpretation of our results can be
derived from the notion that rather than having a “quanti-
tative” eVect on memory (i.e., the amount transferred), DCS
has a “qualitative” eVect on memory. Rescorla (2004) noted
that what is learnt during an extinction session is less stable
with time than what is learnt during initial acquisition. The
Wnding that extinguished responses often ‘spontaneously
recover’ after a passage of time following non-reinforce-
ment is a clear demonstration of this idea and can be used
to explain the current data. In other words, rather than
facilitating the neural process underlying the translation of
extinction learning from short- to long-term memory, such
that more is learnt, the results of the current study could
suggest that DCS fortiWes any learning that does occur dur-
ing extinction training and renders it more stable over time.
In this way, the eVects of DCS on extinction could be inter-
preted as facilitating extinction retention by impairing
spontaneous recovery. SpeciWcally, in Experiments 1 and 3
(“non-extinguishers”) rats had not shown a signiWcant
decrease in freezing by the end of the extinction session.
Thus, it was impossible to observe any spontaneous recov-
ery of the original learning because no inhibition of the
original response was observed. It follows then that it was
impossible to observe any impairment of spontaneous
recovery by DCS. Therefore, the following day, rats that
had received an injection of DCS displayed a similar level
of freezing to the level they exhibited on the Wnal trial of
extinction training, and similar levels of freezing to the
saline group. However, in the experiments where rats exhib-
ited a signiWcant decrease in freezing by the Wnal extinction
trial, the originally learned responses to the CS had been
inhibited (to diVerent degrees, depending on the condition-
ing and extinction parameters). The following day, saline-
treated rats displayed spontaneous recovery of conditioned
freezing and DCS-treated rats displayed an impairment in
spontaneous recovery. Indeed, in Experiments 3 (“extin-
guishers”) and 4, the level of freezing exhibited at test by
DCS-treated rats matched their level of freezing during the
last extinction trial (see Figs. 2c and d, and 3a and b). From
this perspective, DCS may facilitate extinction retention by
strengthening the long-term memory such that it is not sus-
ceptible to degradation during the retention interval. It will
be diYcult to design experiments to distinguish between
these two interpretations. Whichever account is eventually
supported, however, the present results suggest that in
order for DCS to have a facilitatory eVect on extinction
there needs to be at least some within-session extinction.
The current study demonstrated that in an animal prep-
aration, fear responses elicited by an odor can be extin-
guished, and further, that DCS facilitated the long-term
extinction of those responses. Most clinical and preclinical
studies to date have investigated the eVects of DCS on
extinction using visual cues. However, in both humans and
animals stimuli from other sensory senses can be equally, if
not more, eVective at retrieving traumatic memories. Odors,
in particular, can be powerful for triggering the emotional
component of memory. For example, Herz (1998) demon-
strated that odors were equally eVective at cuing recall for
emotionally arousing pictures as tactile, visual, or auditory
cues, however the emotional ‘potency’ of the memories was
signiWcantly greater for odor-evoked memories. In a clinical
setting, the emotional quality of memories is clearly an
important factor underlying the maintenance of anxiety
disorders. Our Wndings suggest that DCS can facilitate
extinction of learned fear to odor cues if there is some
within-session extinction to those cues during the extinction
training session. It would be of interest whether these Wnd-
ings are limited to odor cues, and indeed, to animal prepa-
rations. Previous experiments investigating the eVects of
DCS on fear extinction in human studies have not reported
any data on within-session loss of fear, but from the per-
spective of the present results, this may be a critical factor
in determining whether DCS is eVective at enhancing the
long-term loss of fear in clinical samples.
Acknowledgment
This research was supported by a grant from the
National Health and Medical Research Council (#300538).
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