HIPPOCAMPUS 14:557–569 (2004)

Consolidation of CS and US Representationsin Associative Fear Conditioning
Paul W. Frankland, Sheena A. Josselyn, Stephan G. Anagnostaras, Jeffrey H. Kogan,
Eiki Takahashi, and Alcino J. Silva*

ABSTRACT:
Much attention has been paid to the associative processes
that are necessary to fuse together representations of the various components of an episodic memory. In the present study, we focus on theprocesses involved in the formation of lasting representations of theindividual components that make up a fear-conditioning episode. In onetrial contextual fear conditioning experiments, weak conditioning to context occurs if the shock is delivered immediately following placement of
the animal in a novel conditioning apparatus, a phenomenon known as theimmediate shock deﬁcit. We show that the immediate shock deﬁcit inmice may be alleviated by pre-exposure to either the context or shock. In
using this approach to temporally dissect a contextual fear-conditioning
task into its constituent representational and associative processes, we areable to examine directly the processes that are important for formation oflasting representations of the context conditioned stimulus (CS) or unconditioned stimulus (US). Our data indicate that the formation of a lasting
representation of the context or shock engages protein synthesis-dependent processes. Furthermore, genetic disruption of cAMP-responsive element binding protein (CREB), a transcription factor that regulates thesynthesis of new proteins required for long-term memory, disrupts theformation of lasting context memories. We go on to show that the stresshormone epinephrine modulates the consolidation of a context memory,and reverses consolidation deﬁcits in the CREB-deﬁcient mice. Finally weshow that disrupting either NMDA or calcium/calmodulin-dependent
kinase II (CaMKII) function impairs consolidation of context memories.
Together, these data suggest that this approach is particularly suited forthe characterization of molecular and cellular processes underlying theformation of stimulus representations. © 2004 Wiley-Liss, Inc.
KEY WORDS:
epinephrine

protein synthesis; hippocampus; CREB; NMDA; CaMKII;INTRODUCTION
In contextual fear conditioning, an association is formed between a distinctive place (conditioned stimulus [CS]) and an aversive event (uncondi-

Departments of Neurobiology, Psychology and Psychiatry, University ofCalifornia at Los Angeles, Los Angeles, California
Paul W, Frankland and Sheena A. Josselyn are currently at the Programs in
Integrative Biology and Brain and Behaviour, Hospital for Sick Children
Research Institute, Toronto, Canada.
Stephan G. Anagnostaras is currently at the Department of Psychology,
UCSD, La Jolla, CA.
Jeffrey H. Kogan is currently at Memory Pharmaceuticals Corp., Montvale,
NJ.
Grant sponsor: SNRP/National Institutes of Health (NIH)*Correspondence to: Alcino J. Silva, Departments of Neurobiology, Psychology and Psychiatry, UCLA, 695 Charles E. Young Drive South, Los
Angeles, CA 90095-1761. E-mail: silvaa@mednet.ucla.edu
Accepted for publication 28 September 2003
DOI 10.1002/hipo.10208
Published online 1 March 2004 in Wiley InterScience (www.interscience.
wiley.com).
© 2004 WILEY-LISS, INC.

tioned stimulus [US]) (Fanselow, 2000). It is possible todemonstrate that an animal has formed a speciﬁc CS-US
association by showing that reexposure to the conditioned context, but not dissimilar contexts, evokes conditioned fear behavior such as freezing (Fanselow, 1980).
Besides demonstrating that an animal remembers anaversive event (or the CS-US association), it is also possible to show that the animal forms independent memories for each of the component parts of the aversive event
or episode. For example, prior experience with the context CS or the shock US may retard or facilitate subsequent conditioning (Fanselow, 1990; Fanselow et al.,
1993; Kiernan and Westbrook, 1993; Kiernan et al.,
1995; Rudy and O’Reilly, 1999; Wiltgen et al., 2001;
Lattal and Abel, 2001b; Rudy et al., 2002). These observations indicate that animals likely form independent
representations of each of the components of an event
memory—the CS and US—in addition to the CS-US
association itself (Pavlov, 1927; Guthrie, 1935; Konorski, 1967).
Most Pavlovian fear conditioning studies have focusedon identifying the associative processes underlying thefusion of the various features of an event into a uniﬁed
memory, i.e., the biological processes underlying the formation of lasting CS-US associations (LeDoux, 2000;
Anagnostaras et al., 2001; Maren, 2001). Recent work,
however, has begun to focus on the processes underlying
the formation of representations of each of its constituent
parts—the building blocks of an event memory(Fanselow, 1990; Rudy and O’Reilly, 1999, 2001; Rudy
et al., 2002; Barrientos et al., 2002). In one-trial contextual fear conditioning experiments, weak conditioning tocontext occurs if the shock is delivered immediately after
placement of the animal in a novel conditioning apparatus, a phenomenon known as the immediate shock deﬁcit(Fanselow, 1986, 1990). In the present study, we showthat the immediate shock deﬁcit in mice may be alleviated by pre-exposure to either the context or shock.
Therefore, the use of these pre-exposure procedures permits temporal isolation of the processes underlying theformation of independent CS and US representations. In
a series of experiments, we show that these processes areprotein synthesis dependent, since pretreatment withanisomycin blocks the facilitative effects of pre-exposure
to the context or shock. In addition, disrupting either
N-methyl-D-aspartate (NMDA) receptor, calcium/calmodulin-dependent kinase II (CaMKII) or cAMP-re-

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sponsive element binding protein (CREB) function blocks thefacilitative effects of context pre-exposure, indicating that the formation of context memories engages each of these processes. Furthermore, systemic treatment with the stress hormone, epinephrine, enhances memory for context, and similar treatment
alleviates context impairments in CREB-deﬁcient mice.

MATERIALS AND METHODS
Subjects
Unless otherwise speciﬁed, we used the progeny from a crossbetween C57Bl/6NTacfBr (B6; Taconic Farms) and 129Sv/J(129; Jackson Laboratory) inbred mouse strains. For the CREB␣⌬
mice used in Experiments 3 and 5, we used the F2 progeny derived
from a cross between CREB␣⌬ heterozygotes in the B6 background (⬎99%) and wild-type (WT) 129 mice. The ␣-CaMKIIT286 mice used in Experiment 7 were heterozygotes derived from8 –9 crosses into B6. Mice were weaned at 3 weeks of age and were
subsequently genotyped using polymerase chain reaction (PCR)
protocols as previously described (Bourtchuladze et al., 1994;
Giese et al., 1998). All mice were group housed (2–5 mice per cage)and had continuous access to food and water. The vivarium wasmaintained on a 12:12 light/dark schedule, and all testing was
carried out during the light phase of the cycle. At the commencement of testing mice were at least 8 weeks old. All experiments usedapproximately equal numbers of male and female mice. All animalcare and testing procedures were approved by the Animal ResearchCommittee at UCLA and were in accordance with the NIH Principles of Laboratory Animal Care.

Apparatus

Shock pre-exposure (PE) context
The shock PE context was housed in a different room from thatused for the conditioning context. The ﬂoor of the shock PE context was triangular in shape, with vertical Plexiglas walls. The sidesof the triangle were 24 cm long, and the height of the context was
20 cm. The total ﬂoor area was 250 cm2, similar to the conditioning context. An opaque blue material covered the exterior of twowalls of the shock PE context. The other wall was left transparent
to allow observation of the mice. The ﬂoor of the cage comprised a
shock grid. Between tests, the cage ﬂoor and interior of the conditioning context were cleaned with a 1% acetic acid solution.

Automated freezing apparatus
In a subset of experiments, we used an automated freezing apparatus to examine contextual fear conditioning in mice. With thisexperimental setup, freezing is measured automatically using a National Institutes of Health (NIH) Image-based algorithm. For afull description of apparatus and methods, see Anagnostaras et al.,(2000).

Behavior Measurement
Conditioning was assessed by “freezing” behavior. An animal
was determined to be freezing when it adopted a motionless posture, refraining from all but respiratory movement (Fanselow,1990). Freezing was assessed using a sampling method; 2-s observations were taken every 5 s. For an animal to be scored as freezing,it had to remain motionless for the entire 2-s observation. Theseobservations were made by an experimenter who was blind to theexperimental treatment and/or genotype of each mouse. Freezing
data is presented as the percent time spent freezing. That is, thenumber of observations when freezing was observed divided by the
total number of observations and multiplied by 100.

Conditioning context
The conditioning context was located in a windowless room. All
mice were tested individually. For each test, the mouse was transported to the test apparatus in a cage containing a mix of freshwood shavings and wood shavings from its home cage. The conditioning context was housed in a sound-attenuated box (interior
dimensions: 56 ⫻ 42 ⫻ 37 cm; length ⫻ width ⫻ height). Threeof the four interior walls of the sound-attenuated chamber werepainted white. The other wall consisted of black and yellow vertical
striped pattern. A clear Plexiglas window allowed the mice to becontinually observed. Background noise (68 dB) was provided by a
fan located in one of the walls of the sound-attenuated chamber.
The conditioning context (16 cm ⫻ 16 cm ⫻ 19 cm; length ⫻width ⫻ height) was rectangular in shape and its walls were madeof clear Plexiglas. The total ﬂoor area was 256 cm2. On one of thewalls there was a 24 V house light. The ﬂoor of the context consisted of a shock grid. Bars were 3 mm in diameter and 0.9 cm
apart. Each bar was connected to a Master Shocker (model
82402SS), a device that delivers scrambled shocks. Between tests,
the cage ﬂoor and interior of the conditioning context were cleaned
with a 75% ethanol solution.

Drugs
All drugs were administered intraperitioneally (i.p.) in a volumeof 10 ml/kg. Anisomycin (ANI; Sigma) was dissolved in phosphate-buffered saline (PBS) and pH-adjusted to 7.4. ANI injections (150 mg/kg) were given 30 min before pre-exposure or training. [⫾]-3-[2-Carboxypiperazin-4-yl]propanephosphonic acid
(CPP; Sigma) was dissolved in PBS, and given 20 min before
pre-exposure or training.
Epinephrine (EPI; Sigma) was dissolved in PBS. EPI injections(0, 0.05, 0.5 mg/kg) were given immediately following context
pre-exposure.

General Behavioral Procedures
Immediate shock training
Each mouse was placed in the conditioning context and 5 s later,a 2-s, 0.75-mA shock was delivered via the cage ﬂoor bars. Following this shock, the mice remained in the context for a further 60 s.
Each mouse was then removed and returned to its home cage.

_____________________________CS AND US REPRESENTATIONS IN ASSOCIATIVE CONDITIONING

Testing
For testing, each mouse was placed back in the context, andfreezing was assessed over a 3-min period. During this period, no
shocks were presented.

Speciﬁc Experimental Procedures

559

for generalization, 24 h following the completion of testing in theconditioning context, all groups of mice were tested in the original
shock pre-exposure context. The duration of this test was 3 min.

Experiment 2a: Effect of anisomycin treatment on
context pre-exposure in WT mice

Each mouse was placed in the conditioning context. Following
5 s (n ⫽ 10), 30 s (n ⫽ 9), 120 s (n ⫽ 7) or 300 s (n ⫽ 8) a 2-s,
0.75-mA shock was delivered via the cage ﬂoor bars. Following thisshock, the mouse remained in the context for a further 60 s. Mice
were then tested 30 min later.

In this experiment, we tested whether treatment with the protein
synthesis inhibitor ANI (150 mg/kg, i.p.) blocks the facilitative effectsof context pre-exposure on contextual fear conditioning with an immediate shock. Mice were pretreated with ANI (n ⫽ 7) or PBS (n ⫽7) before being pre-exposed to the conditioning context for 10 min. A
control group of mice was injected with ANI (n ⫽ 7) or PBS (n ⫽ 7),
but not pre-exposed to the conditioning context. Twenty-four h later,
both groups were fear conditioned with an immediate shock in theconditioning context, and tested following a 30-min delay.

Experiment 1b: Reactivity to an immediate versus
delayed shock

Experiment 2b: Effect of anisomycin treatment on
shock pre-exposure in WT miceMice (B6) were randomly assigned to an immediate shock (n ⫽10) or a delayed shock (n ⫽ 10) condition. Mice in either condition were placed in the conditioning context for a total of 90 s.
Mice in the immediate condition received a 2-s, 0.5-mA footshock
5 s after placement in the conditioning context. In contrast, micein the delayed condition received the footshock 60 s after placement in the conditioning context. Mice were then tested one day
later. In this experiment, both training and testing were conductedin the automated freezing apparatus, and freezing and shock reactivity were assessed using a computer-assisted automated scoring
system (Anagnostaras et al., 2000).

In this experiment, we tested whether treatment with ANI
blocks the facilitative effects of shock pre-exposure on contextual
fear conditioning with an immediate shock. ANI-treated (n ⫽ 7)
and PBS-treated (n ⫽ 7) mice were placed in the shock PE context
and received a 2-s, 0.75-mA shock following a 5 s delay. They
remained in this alternate context for a further 60 s and were thenremoved. A control group of mice were injected with ANI (n ⫽ 7)
or PBS (n ⫽ 8), but were not placed in the shock PE context anddid not receive a shock. Twenty-four h later, they were fear conditioned with an immediate shock in the conditioning context.
Thirty min following this they were tested.

Experiment 1c: Effect of context pre-exposure on
the immediate shock deficit

Experiment 2c: Effect of anisomycin treatment on
context-shock learning in WT mice

In this experiment, we tested whether pre-exposure to the conditioning context can protect mice against the immediate shock deﬁcit.
Mice received various amounts of context pre-exposure (PE groups):
30 s (n ⫽ 8), 120 s (n ⫽ 9), 300 s (n ⫽ 8), or 600 s (n ⫽ 7). Control
groups of mice did not receive context pre-exposure (NPE groups).
Rather, they were taken to the room adjacent to the room housing theconditioning context in transport cages for an equivalent time period:
30 s (n ⫽ 7), 120 s (n ⫽ 11), 300 s (n ⫽ 11), or 600 s (n ⫽ 7). One
day following this both PE and NPE groups were trained with animmediate shock. Mice were tested 30 min later.

In this experiment, we examined the effects of blocking protein
synthesis during the training, rather than the pre-exposure, phase.
Mice were pre-exposed to the context for 10 min, and then 24 hlater trained with an immediate shock. Thirty min prior to training
mice were pretreated with either the ANI or PBS. Separate groupsof mice were tested 30 min (PBS ⫽ 11; ANI ⫽ 9) or 24 h (PBS ⫽9; ANI ⫽ 9) later.

Experiment 1a: Effect of placement-to-shock
interval on contextual conditioning

Experiment 1d: Effect of shock pre-exposure on theimmediate shock deficit
In this experiment, we tested whether pre-exposure to shock canprotect mice against the immediate shock deﬁcit. Mice were placed in
the shock PE context. Following a 5 s delay mice received either a0.25-mA (n ⫽ 14) or 0.75-mA (n ⫽ 6) shock, or no shock (n ⫽ 12).
Shocks were 2 s in duration. Following the delivery of the shock miceremained in shock PE context for a further 60 s, and they were thenremoved. Twenty-four h later, all groups of mice were fear conditioned with an immediate shock in the conditioning context. Thirty
min after this, they were tested in the conditioning context. To control

Experiment 3: Effect of context pre-exposure in
CREB␣⌬ⴚ/ⴚ mice
In this experiment, we tested whether context pre-exposure
would protect CREB␣⌬⫺/⫺ mice against the immediate shock deficit. Separate groups of WT and CREB␣⌬⫺/⫺ mice were pre-exposed to the conditioning context for 10 min (PE ⫹ Immediategroup: WT ⫽ 10, CREB␣⌬⫺/⫺ ⫽ 10) or not (Immediate group:
WT ⫽ 10, CREB␣⌬⫺/⫺ ⫽ 9). Twenty-four h later, they were fear
conditioned with an immediate shock. Thirty min later, they were
tested. An additional group of WT (n ⫽ 16) and CREB␣⌬⫺/⫺(n ⫽ 16) mice was trained with a delayed shock. Each mouse wasplaced in the conditioning context. After 2 min they received a 2-s,
0.75-mA shock, and 60 s later were removed from the context.
Thirty min later, they were tested.

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Experiment 4: Effect of epinephrine treatment on
context learning in WT mice
In this experiment, we examined the effect of systemic epinephrine
treatment on contextual learning. Immediately after a 2 min context
pre-exposure (PE group), WT mice were injected with PBS (n ⫽ 8),

0.05 mg/kg EPI (n ⫽ 9) or 0.5 mg/kg EPI (n ⫽ 8). Control groups ofmice, not pre-exposed to the context (NPE group), were given PBS(n ⫽ 7), 0.05 mg/kg EPI (n ⫽ 7) or 0.5 mg/kg EPI (n ⫽ 7). Twentyfour h later, all groups of mice were fear conditioned with an immediate shock. Thirty min following this they were tested.

FIGURE 1

_____________________________CS AND US REPRESENTATIONS IN ASSOCIATIVE CONDITIONING

Experiment 5: Effect of epinephrine treatment on
context learning in CREB␣⌬ⴚ/ⴚ mice561

In one-trial contextual conditioning, weak conditioning to context occurs if the shock is delivered immediately following placement of the animal in a novel conditioning apparatus. In thisexperiment, we varied the timing of the shock presentation during

training to obtain a placement-shock function for mice, as has
previously been described in rats (Fanselow, 1990). Mice wereplaced into the context and received a 2-s, 0.75-mA footshock at
the following delays: 5, 30, 120, and 300 s. Following the shock,
mice remained in the context for a further 60 s, and were tested 30
min later. The placement-shock interval during training inﬂuenced subsequent contextual fear (F(3,30) ⫽ 4.28, P ⬍ 0.05). Post
hoc analyses indicated that freezing levels in mice trained with theshortest delay (5 s) were signiﬁcantly lower than mice trained witheither the 120 s or 300 s delay (Newman-Keuls; P ⬍ 0.05) (Fig.
1a). These data show that mice exhibit an immediate shock deﬁcit
as previously reported (Paylor et al., 1994; Kiyama et al., 1998;
Milanovic et al., 1998; Lattal and Abel, 2001b; Stanciu et al.,
2001; Wiltgen et al., 2001).
We next tested whether reduced levels of freezing in the micetrained with the shortest delay were due to reduced reactivity to theshock. Two groups of mice were trained. Mice received a single
footshock either 5 s (immediate) or 60 s (delayed) following placement in the training context. Both groups of mice spent a total of
90 s in the training context, and were subsequently tested 24 hlater. Shock reactivity was similar regardless of whether the shock
was immediate or delayed (F(1,18) ⬍ 1) (Fig. 1b; left). Importantly, on subsequent testing, mice in the immediate shock group
showed signiﬁcantly lower levels of freezing compared to mice inthe delayed shock group (F(1,18) ⫽ 9.97, P ⬍ 0.05) (Fig. 1b;right). This result indicates that the immediate shock deﬁcit is not
related to reduced reactivity to the delivery of an immediate shock.
Furthermore, because mice in the immediate and delayed groups
spent equivalent amounts of time in the context during training,these data show that it is the timing of the shock during training,rather than the total amount of time spent in the conditioning
context, that determines subsequent levels of conditioned fear.
Context pre-exposure alleviates the immediate shock deﬁcit inrats (Fanselow, 1990; Kiernan et al., 1995; Rudy and O’Reilly,
1999; Wiltgen et al., 2001; Barrientos et al., 2002; Rudy et al.,2002). We systematically varied the duration of context pre-exposure to determine the minimal amount of time required to alleviate
the immediate shock deﬁcit in mice. Mice were pre-exposed to thecontext for different durations (30, 120, 300, or 600 s) (pre-exposure groups; PE). One day following context pre-exposure, mice
were trained with an immediate shock, and tested 30 min later.

FIGURE 1.
Behavioral examination of the immediate shockdeﬁcit in mice. a: Effect of placement-shock interval on the development of contextual fear conditioning. During subsequent testing, freezing levels are higher in mice trained with longer placement-shock intervals. b: Shock reactivity is similar in mice trainedwith an immediate (placement-shock interval of 5 s) versus delayed(placement-shock interval of 60 s) shock (left). Despite this, in
subsequent testing mice trained with the delayed shock exhibit
signiﬁcantly greater levels of freezing compared to those trainedwith an immediate shock (right). c: Effect of context pre-exposure(PE) vs. no pre-exposure (NPE) on contextual conditioning with
an immediate shock. Extended pre-exposure to the context protects mice against the immediate shock deﬁcit. Mice pre-exposed tothe context for 10 min exhibited greater levels of freezing on subsequent tests compared to mice pre-exposed for shorter durations,or mice that were not pre-exposed. d: Distribution of freezing overtime in mice exhibiting the immediate shock deﬁcit. These test
data are from mice in the NPE conditions in Experiment 1c. Freezing is not concentrated at the start of testing—that is, at the time
the shock was delivered during training—as would be predicted bytiming accounts of the immediate shock deﬁcit. e: Prior experience
with shock (delivered in an alternate context) facilitates contextual
conditioning with an immediate shock. Mice receiving a 0.75-mA
shock 24 h prior to conditioning, exhibited higher levels of freezing on subsequent testing. f: One day following testing in theconditioning context (Experiment 1d), mice were also tested in theshock PE context. Freezing in this context was lower in all groups,
indicating that generalization from the shock PE context to theconditioning context cannot account for the facilitative effects ofshock pre-exposure.

In this experiment, we tested whether systemic epinephrine treatment would reverse contextual learning deﬁcits in CREB␣⌬⫺/⫺ mice.
All mice were pre-exposed to the conditioning context for 10 min.
Immediately following this they were injected with PBS (WT ⫽ 9;
CREB␣⌬⫺/⫺ ⫽ 7), 0.05 mg/kg EPI (WT ⫽ 11; CREB␣⌬⫺/⫺ ⫽ 7),
or 0.5 mg/kg EPI (WT ⫽ 8; CREB␣⌬⫺/⫺ ⫽ 7). Twenty-four h later,
all groups of mice were fear conditioned with an immediate shock.
Thirty min later, they were tested.

Experiment 6: Effect of CPP treatment on context
pre-exposure in WT mice
In this experiment, we tested whether treatment with CPP(0 –10 mg/kg, i.p.) blocks the facilitative effects of context preexposure on contextual fear conditioning with an immediateshock. Mice (B6) were pretreated with PBS (n ⫽ 10), 5 mg/kg
CPP (n ⫽ 9) or 10 mg/kg CPP (n ⫽ 6) before being pre-exposedto the conditioning context for 10 min. Twenty-four h later, mice
were fear conditioned with an immediate shock in the conditioning context, and tested the following day.

Experiment 7: Effect of genetic disruption of
CaMKII on context pre-exposure
In this experiment, we tested whether mice that are heterozygous for a point mutation at T286 (␣-CaMKII-T286⫹/⫺) exhibit
impaired contextual processing. WT (n ⫽ 10) and ␣-CaMKIIT286⫺/⫺ (n ⫽ 9) mice were trained and tested in an identicalfashion to the mice in Experiment 6.

RESULTS
Immediate Shock Deﬁcit: Effects of Context (CS)or Shock (US) Pre-exposure

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FRANKLAND ET AL.

Control groups of mice were removed from the vivarium, but notplaced in the context (no pre-exposure group; NPE). Only extended pre-exposure to the conditioning context alleviated theimmediate shock deﬁcit (exposure ⫻ time interaction; F(3,60) ⫽3.06, P ⬍ 0.05) (Fig. 1c). Post hoc analyses revealed that freezing
levels were higher in the mice that were pre-exposed for 600 s
compared to each of the other PE groups. In addition, freezing in
the 600-s PE group was signiﬁcantly higher than in the control
600-s NPE group (P ⬍ 0.05). Although not tested here, the facilitative effects of context pre-exposure are speciﬁc to the to-beconditioned context as pre-exposure to an alternate context fails toreverse the immediate shock deﬁcit in rats (Kiernan et al., 1995;
Rudy et al., 2002) and mice (Lattal and Abel, 2001b).
The reduction in freezing levels following immediate shock
training might be because mice tend to concentrate freezing bouts
towards the beginning of the test session—that is, at a time corresponding to shock delivery during training. Examination of freezing in the mice that were not pre-exposed to the context (NPEgroups) reveals that this is not the case. Collapsing across each ofthese control groups, freezing tended to increase, rather than decrease, as a function of time during testing (F(5,175) ⫽ 5.02, P ⬍0.05) (Fig. 1d). It is unclear why freezing increases over time, but
this analysis is nonetheless inconsistent with timing accounts of theimmediate shock deﬁcit (Bevins and Ayres, 1995; Gallistel andGibbon, 2000).
Since extended context pre-exposure alleviated the immediateshock deﬁcit, we next tested whether pre-exposure to the shock US
produces similar effects. Mice received a shock (2-s duration;
0.25-mA or 0.75 mA) 5 s following placement in an alternate
distinctive (shock PE) context. One day later, mice were trained in
the conditioning context using immediate shock procedures, andtested 30 min later. Pre-exposure to shock in the alternate context
facilitated contextual conditioning following training with immediate shock procedures (F(2,29) ⫽ 4.38, P ⬍ 0.05) (Fig. 1e).
Freezing during subsequent testing was signiﬁcantly higher in micethat were pre-exposed to the high shock (0.75 mA) compared tomice not receiving shock pre-exposure (0 mA) (P ⬍ 0.05).
These data indicate that pre-exposure to the shock US facilitatessubsequent fear conditioning, as does pre-exposure to the context
CS. The pre-exposure shock was delivered immediately to minimize conditioning to the shock pre-exposure context. Nevertheless, it is possible that generalization from the shock pre-exposure
context to the conditioning context might account for the facilitated conditioning in the shock-pre-exposed mice. To examine
this, following testing in the conditioning context, mice were also
tested in the shock pre-exposure context. Mice froze less in shock
PE context compared to the conditioning context (F(1,29) ⫽38.3, P ⬍ 0.05) (Fig. 1f), a ﬁnding that makes it unlikely thatgeneralization occurred from the shock PE context to the conditioning context. Because mice were tested in the shock pre-exposure context after they were tested in the conditioning context, it
should be noted that extinction could account for reduced freezingin the shock pre-exposure context. However, multiple exposures tothe conditioning context following training do not produce thesame magnitude decrement in conditioned freezing (data notshown).

Facilitative Effects of Context (CS) or Shock (US)Pre-exposure Are Blocked by Protein Synthesis
Inhibition
Contextual fear conditioning is thought to require an animal toform a representation of the training context (CS), the shock (US)as well as a context-shock (CS-US) association (Fanselow, 2000;
Rudy et al., 2002). The above experiments dissociated CS and US
representational processes underlying Pavlovian fear conditioning,and suggest that these procedures can be used to directly examine
mechanisms underlying the formation of CS and US representations, independent of CS-US associations.
Protein synthesis is essential for the formation of long-term
memories (Davis and Squire, 1984). We therefore asked whether
the formation of either lasting CS (context) or US (shock) representations depends on protein synthesis. To test whether the formation of a context representation is protein synthesis dependent,
we pre-exposed mice to the training context for 10 min. Mice werepretreated with either the protein synthesis inhibitor ANI or PBS.
One day later, they were trained using immediate shock procedures, and tested 30 min later. A group of control mice weretreated identically, except that they were not pre-exposed to thetraining context. Disrupting protein synthesis speciﬁcally attenuated the effects of context pre-exposure on contextual conditioning(exposure ⫻ drug interaction; F(1,24) ⫽ 26.8, P ⬍ 0.05) (Fig. 2a).
In mice pre-exposed to the context, ANI pretreatment signiﬁcantly
reduced freezing levels compared to PBS-treated controls (P ⬍0.05).
To test whether the formation of a shock representation is protein synthesis dependent, we pre-exposed mice to a 0.75-mA shockin the alternate (shock PE) context. Mice were pretreated with ANI
or PBS. One day later, they were trained using immediate shockprocedures, and tested 30 min later. A group of control mice weretreated identically, except that they were not pre-exposed to shock.
Disrupting protein synthesis speciﬁcally blocked the facilitative
effects of shock pre-exposure on contextual conditioning (exposure ⫻ drug interaction; F(1,25) ⫽ 6.66, P ⬍ 0.05) (Fig. 2b). In
mice pre-exposed to the shock, ANI pretreatment signiﬁcantly
reduced freezing levels in the subsequent test compared to PBStreated controls (P ⬍ 0.05).
Therefore, the facilitative effects of pre-exposure to the context
CS or shock US are blocked by protein synthesis inhibition. In thenext experiment we examined the effects of blocking protein synthesis during the training, rather than the pre-exposure, phase.
Mice were pre-exposed to the context and 24 h later trained withimmediate shock procedures. Prior to training, mice were treatedwith either ANI or PBS. To examine the effects of ANI treatment
on both short- and long-term memory, separate groups of mice
were then tested 30 min or 24 h later. As expected, mice pretreatedwith PBS exhibited robust conditioning whether tested 30 min or
24 h following training. In contrast, mice pretreated with ANIprior to training exhibited normal memory 30 min following training, but impaired memory when tested 24 h following training(drug ⫻ test delay interaction; F(1,34) ⫽ 6.43, P ⬍ 0.05) (Fig. 2c).
Post hoc analyses showed that conditioned freezing levels werereduced in ANI-treated mice compared to the PBS-treated mice in_____________________________CS AND US REPRESENTATIONS IN ASSOCIATIVE CONDITIONING

563

the test 24 h following training (P ⬍ 0.05). Consistent with a large
literature, these data indicate that disruption of protein synthesis
blocks the formation of long-term (but not short-term) memory(Davis and Squire, 1984). While it is tempting to conclude thatthese data show that protein synthesis inhibition blocks the formation of lasting CS-US associations, this experiment does not ruleout the possibility that protein synthesis inhibition blocks the formation of a lasting US memory at the time of training. For example, an intact US memory may be necessary for the retrieval of the
CS-US association during subsequent testing.

Facilitative Effects of Context Pre-Exposure Are
Blocked in Mice With a Targeted Disruption of
CREB Function
Studies in a wide variety of species have shown that the synthesisof proteins necessary for long-term memory formation is regulated,
at least in part, at the transcriptional level by CREB (Yin and Tully,
1996; Silva et al., 1998; Alberini, 1999; Kandel and Pittenger,
1999). Just as in studies examining the effects of protein synthesis
inhibition on memory formation, a unifying feature of these studies is that manipulating CREB function affects only long-term
memory (i.e., tested at 24 h), and not short-term memory (i.e.,tested at 1 h or less). Accordingly, we have previously shown thatmice with a targeted disruption of the ␣ and ⌬ CREB isoforms(CREB␣⌬⫺/⫺ mice) have normal short-term, but impaired longterm, memory for contextual fear conditioning (Bourtchuladze etal., 1994; Kogan et al., 1997).
Disruptions of processes underlying the formation of lasting
context (CS), shock (US) representations, or stable CS-US associations, may account for these deﬁcits in long-term contextual fear
memory. In the next experiment we examined whether normal
CREB function is required for the formation of lasting context
representations (Fig. 3). We ﬁrst showed that CREB␣⌬⫺/⫺ mice
exhibit normal short-term memory (30 min) when trained with adelayed shock (F(1,30) ⬍ 1, P ⬎ 0.05). These data indicate thatshort-lived processes required for the expression of contextual fear
memories 30 min following training—for example, the formationof CS (context), US (shock) representations, and CS-US (contextshock) associations—are unaffected by the CREB␣⌬⫺/⫺ mutation.
When trained with an immediate shock, however, both CREB␣⌬⫺/⫺FIGURE 2.
Effect of protein synthesis inhibition on the formation of lasting conditioned stimulus (CS) and unconditioned stimulus(US) representations, and on lasting CS-US associations. a: Mice were
either pre-exposed to the conditioning context (PE) or not pre-exposed (NPE). ANI-treatment (open bars) speciﬁcally blocked the facilitative effects of context pre-exposure compared to phosphate-buffered saline (PBS)-treated controls (closed bars). b: Mice received
shock pre-exposure (shock) or not (no shock). ANI-treatment (openbars) speciﬁcally blocked the facilitative effects of shock pre-exposurecompared to PBS-treated controls (closed bars). c: Mice were preexposed to the conditioning context for 10 min. One day later, they
were trained with immediate shock procedures. Pre-training ANItreatment (open bars) blocked long-term memory (tested 24 h following training), but not short-term memory (tested 30 min following
training). PBS-treated mice (closed bars) exhibited similar levels of
freezing at 30 min and 24 h.

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FIGURE 3.
Context pre-exposed (PE) does not alleviate the immediate shock deﬁcit in cAMP-responsive element binding protein(CREB)␣⌬ⴚ/ⴚ mice. a: The three training conditions for wild-type(WT) and CREB␣⌬ⴚ/ⴚ mice. b: When trained with a delayed shock,
WT (closed bars) and CREB␣⌬ⴚ/ⴚ (open bars) mice showed normal
short-term (30-min) contextual fear memory. However, if the shock isdelivered immediately during training (rather than after a delay) both
WT and CREB␣⌬ⴚ/ⴚ mice exhibit reduced levels of conditioned
freezing on test, or an immediate shock deﬁcit. Pre-exposure to thecontext protected WT, but not CREB␣⌬ⴚ/ⴚ, mice against this immediate shock deﬁcit.

and WT control mice exhibited reduced levels of conditioned fear
when tested 30 min later. In WT mice, this immediate shock deﬁcit
was rescued by pre-exposure to the conditioning context 24 h prior totraining. In contrast, context pre-exposure failed to rescue the immediate shock deﬁcit in CREB␣⌬⫺/⫺ mice (genotype ⫻ exposure interaction: F(1,35) ⫽ 4.43, P ⬍ 0.05). Critically, there was no differencein levels of freezing between CREB␣⌬⫺/⫺ mice that were pre-exposed(PE ⫹ immediate group) and those that were not (immediate group)(P ⬎ 0.05). Furthermore, pre-exposed WT mice exhibited signiﬁcantly more freezing compared to pre-exposed CREB␣⌬⫺/⫺ mice
(P ⬍ 0.05).
In these experiments, we used only a short delay (30 min) between training and testing. Because memory is normal at theseshort delays in CREB␣⌬⫺/⫺ mice, this design allows us to examine
the impact of the CREB␣⌬⫺/⫺ mutation on contextual processing:that is, on processes necessary to form and maintain a context
memory during the 24 period between pre-exposure and training.
Therefore, these data indicate that the targeted disruption of
CREB function in the CREB␣⌬ mice impairs the formation of alasting representation of context.

(McGaugh, 2002). To test whether memory for context may be
modulated in a similar manner, we examined the impact of epinephrine treatment on the effectiveness of context pre-exposure.
Since we expected epinephrine treatment to enhance memory, weused a pre-exposure duration (2 min) that does not normally alleviate the immediate shock deﬁcit (see Fig. 1c). Consistent with ourearlier experiment, a 2 min PE was insufﬁcient to alleviate theimmediate shock deﬁcit: the PBS-treated mice, regardless ofwhether or not they had been pre-exposed to the context, showedsimilarly low levels of freezing when tested. However, mice treatedwith epinephrine immediately following the 2 min context preexposure showed increased contextual fear conditioning. Importantly, the facilitative effects of epinephrine were limited to micethat were pre-exposed to the context (dose ⫻ exposure interaction:
F(2,40) ⫽ 3.45, P ⬍ 0.05) (Fig. 4). This indicates that epinephrine
does not produce nonspeciﬁc facilitation of conditioning; rather
the facilitative effects are contingent on pre-exposure to the to-beconditioned context.

Context Memories Are Enhanced by Epinephrine
Memories for emotionally charged events tend to be stronger
and more persistent (Cahill and McGaugh, 1996; McGaugh andRoozendaal, 2002). A large number of studies have shown that theactivation of adrenal stress hormones, such as epinephrine, facilitates memory consolidation via central ␤-adrenergic mechanisms

Context Deﬁcits in CREB␣⌬ⴚ/ⴚ Mice Are
Reversed by Epinephrine Treatment
The memory-enhancing effects of epinephrine are mediatedcentrally by ␤-adrenergic receptors (Liang et al., 1986). Since theactivation of ␤-adrenergic receptors is coupled to cAMP/PKA signaling, and CREB-dependent transcription is reduced, but noteliminated, in the CREB␣⌬ mice (Blendy et al., 1996), we tested
whether epinephrine treatment would reverse deﬁcits in contextual

_____________________________CS AND US REPRESENTATIONS IN ASSOCIATIVE CONDITIONING

FIGURE 4.
Effect of epinephrine treatment on consolidation ofcontext memories. Wild-type (WT) mice were pre-exposed (PE) or
not pre-exposed (NPE) to the conditioning context for 2 min. Immediately following this, mice were treated with epinephrine. Twentyfour h later, they were trained with immediate shock procedures andthen tested after a 30 min delay. Epinephrine dose-dependently facilitated conditioning only in the pre-exposed mice.

␣⌬⫺/⫺processing in these mutants (see Fig. 3). To do this, CREB
mice and their WT controls were pre-exposed to the conditioning
context for 10 min. Immediately following this, mice were injectedwith epinephrine or PBS. One day later, they were trained using
immediate shock procedures, and tested 30 min later. Epinephrine
treatment given immediately following context pre-exposure facilitated subsequent conditioning in the CREB␣⌬⫺/⫺ mice (Fig. 5).
For the highest dose of epinephrine (0.5 mg/kg), WT and
CREB␣⌬⫺/⫺ mice exhibited equivalent levels of freezing on test
(Planned comparison; P ⫽ 0.49). However, for lower doses of
epinephrine (0 or 0.05 mg/kg) WT mice froze signiﬁcantly morethan CREB␣⌬⫺/⫺ mice on test (Planned comparisons; P ⬍ 0.05).
These data suggest deﬁcits in forming a lasting context representation in CREB␣⌬⫺/⫺ mice can be partially reversed by treatment
with epinephrine, most likely via activation of residual CREBdependent processes.

565

normal NMDA receptor function is required for the formation ofcontext representations, we tested whether the NMDA antagonist,
CPP, blocks the facilitative effect of context pre-exposure. Mice
were pre-exposed to the conditioning context for 10 min following
pretreatment with CPP (0 –10 mg/kg). Twenty-four h later, they
were trained using immediate shock procedures, and then tested
the next day. Pharmacological disruption of NMDA receptor
function blocked the facilitative effects of context pre-exposure oncontextual conditioning (F(2,22) ⫽ 3.89, P ⬍ 0.05) (Fig. 6a).
Similarly, a heterozygous point mutation at T286 (␣-CaMKIIT286⫹/⫺) blocked the facilitative effects of context pre-exposure
on contextual conditioning (F(1,17) ⫽ 6.92, P ⬍ 0.05) (Fig. 6b).
These data suggest that this approach can be broadly applied. Furthermore, since ␣-CaMKII-T286⫹/⫺ mice exhibit normal contextual fear conditioning using standard procedures (Ohno et al.,2001), context pre-exposure approaches may represent a more sensitive behavioral assay for the detection of contextual processing
deﬁcits in mice.

DISCUSSION
In this study, we used behavioral procedures to dissect a Pavlovian fear-conditioning task into its constituent representational andassociative components. Using this approach, we focused in particular on those processes that are important for the formation oflasting representations of the context CS or shock US. Our data
indicate that the formation of lasting context (and shock) representations requires the activation of NMDA receptors, autophosphorylation of CaMKII at T286, CREB-dependent transcription
and protein-synthesis. Furthermore, we show that stress hormones, such as epinephrine, modulate the consolidation of the

Facilitative Effects of Context Pre-exposure Are
Blocked by Pharmacological Blockade of NMDA
Receptors and Genetic Disruption of ␣-CaMKIIThe above series of experiments indicate that examination of the
facilitative effects context pre-exposure on the immediate shockdeﬁcit may be an effective approach for the identiﬁcation of molecular events underlying the formation of contextual memories.
To characterize further the utility of this approach, we extended
our analyses to examine the effects of two treatments (pharmacological and genetic) known to disrupt contextual fear conditioning
and hippocampal long-term potentiation.
The activation of NMDA receptors and subsequent autophosphorylation of ␣-CaMKII at T286 are known to play key roles inhippocampal-dependent behavioral and synaptic plasticity (Martin et al., 2000; Lisman et al., 2002). Therefore, to test whether

FIGURE 5.
Context deﬁcits in cAMP-responsive element binding protein (CREB)␣⌬ⴚ/ⴚ mice are reversed by epinephrine treatment. Wild-type (WT) and CREB␣⌬ⴚ/ⴚ mice were pre-exposed to theconditioning context for 10 min. Twenty-four h later, they weretrained with immediate shock procedures and then tested after a 30min delay. Epinephrine treatment immediately following context preexposure dose-dependently alleviated conditioning deﬁcits in the
CREB␣⌬ⴚ/ⴚ mice.

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FIGURE 6.
Disrupting either N-methyl-D-aspartate (NMDA) receptor or calcium/calmodulin-dependent kinase II (CaMKII) function
blocks the facilitative effects of context pre-exposure. a: Wild-type (WT) mice were pre-exposed to the conditioning context for 10 min. CPP
treatment dose-dependently blocked the facilitative effects of context pre-exposure compared to phosphate-buffered saline (PBS)-treatedcontrols. b: WT and ␣-CaMKII-T286ⴙ/ⴚ mice were pre-exposed to the conditioning context for 10 min. The facilitative effects of context
pre-exposure were attenuated in the ␣-CaMKII-T286ⴙ/ⴚ mice.

context memory. Finally, we show that the failure to successfully
consolidate context in CREB␣⌬⫺/⫺ mice is alleviated by epinephrine. Together, our data indicate that the mechanisms responsible
for forming lasting representations of various features of an event
overlap with the associative processes responsible for fusing together these individual representations into a uniﬁed episode. Furthermore, they suggest that these procedures may be especiallyeffective for identifying molecular and cellular processes underlying the formation of lasting stimulus representations.
In one-trial contextual fear conditioning experiments, weak
conditioning to context occurs if the shock is delivered immediately upon placement of the animal in the conditioning apparatus,
a phenomenon known as the immediate shock deﬁcit (Fanselow,
1986, 1990). We found that pre-exposing mice to either the context or the shock protected mice against the immediate shock deficit. Deﬁcits in contextual conditioning following training with animmediate shock have been attributed to failures of either CS(Fanselow, 1990) or US (Lattal and Abel, 2001b) processing. Thatis, either the short delay between placement in the context andshock delivery, or other factors such as stress-related deﬁcits insensory processing associated with handling, interfere with the animal’s ability to process effectively (1) the context, or (2) the shock.
Our data suggest that failures in either CS or US processing may
contribute to the effect since prior experience with either alleviates
the immediate shock deﬁcit. Regardless of the mechanism, theimpact of either context or shock pre-exposure on the immediateshock deﬁcit indicates that mice can readily form a lasting memoryof the context or the shock (Fanselow and Gale, 2003), independent of a pairing between the two. Therefore, we were able to use
these pre-exposure procedures to effectively isolate processes underlying the formation of context or shock memories.
Previous studies have shown that protein synthesis inhibition
blocks the formation of long-term contextual fear conditioning
memories (Abel et al., 1997; Bourtchouladze et al., 1998; Schafe etal., 1999; Stiedl et al., 1999). However, using standard contextual
conditioning procedures it is not possible to determine whether
these deﬁcits are due to a block of the formation of a lasting rep-resentation of the context CS or shock US, or a lasting memory for
the CS-US association. We found that protein synthesis is required
for the establishment of lasting memories for each of the to-beassociated elements: the context and the shock. These data areconsistent with a recent study that found that intra-hippocampal
infusions of anisomycin block the facilitative effects of context
pre-exposure on contextual conditioning (Barrientos et al., 2002).
Furthermore, our data parallel similar ﬁndings in other paradigms(e.g., conditioned taste aversion, latent inhibition) showing thatthe formation of lasting CS and US representations depend on
protein synthesis (Berman and Dudai, 2001; Rosenblum et al.,
1993; Schauz and Koch, 2000).
The synthesis of most proteins is mediated by activity-regulatedtranscription factors (Shaywitz and Greenberg, 1999). Studies in awide variety of species have shown that the synthesis of proteinsnecessary for long-term memory formation are regulated, at least inpart, by the transcription factor CREB (Alberini, 1999; Kandel
and Pittenger, 1999; Silva et al., 1998; Yin and Tully, 1996).
Previously we have found that CREB␣⌬ mutant mice exhibit impaired long-term memory (24 h), but normal short-term memory(ⱕ1 h) in contextual fear conditioning (Bourtchuladze et al., 1994;
Kogan et al., 1997; Falls et al., 2000). By using pre-exposure procedures to examine context memory in isolation, our current data
indicate that CREB␣⌬⫺/⫺ mice are unable to form a lasting representation of the context. Therefore, inhibiting protein synthesis
and disrupting CREB function produce similar effects: both manipulations block the development of lasting representations ofplace, demonstrating that CREB plays a critical role in the transcriptional activation required for these processes.
The hippocampus plays a central role in the representation ofcontexts or places (Frankland et al., 1998; Anagnostaras et al.,1999, 2001; Fanselow, 2000; O’Reilly and Rudy, 2001). Therefore, the inability to form lasting representations of place is consistent with observations that the stability of newly formed hippocampal place cells, as well as the formation of lasting spatial
memories, is disrupted by protein synthesis inhibitors (Meiri andRosenblum, 1998; Agnihotri et al., 2001; Lattal and Abel, 2001a)

_____________________________CS AND US REPRESENTATIONS IN ASSOCIATIVE CONDITIONING

and compromised in CREB␣⌬⫺/⫺ mice (Bourtchuladze et al.,
1994; Kogan et al., 1997; Cho et al., 1998). Importantly, it has
recently been shown that either hippocampal lesions or intra-hippocampal infusions of anisomycin block the facilitative effects ofcontext pre-exposure on contextual conditioning (Barrientos et al.,
2002; Rudy et al., 2002), indicating that protein synthesis in the
hippocampus is required for the formation of stable representations of place. We extend these ﬁndings to show that the formationof lasting context memories also requires the activation of NMDA
receptors, ␣-CaMKII and CREB. These data, together with thoseof Rudy and colleagues (Rudy and O’Reilly, 1999; Rudy andO’Reilly, 2001; Barrientos et al., 2002; Rudy et al., 2002), suggestthat context pre-exposure procedures may be a particularly effective method for identifying hippocampal molecular and cellular
processes associated with the formation of lasting representationsof place. Indeed, studying context in isolation, rather than in aversively-motivated situations such as the water maze and contextual
fear conditioning, may be a more appropriate behavioral correlate
for place cell studies.
Using standard one-trial contextual fear conditioning procedures the optimal placement-shock interval was 150 s, althougha much shorter interval (i.e., 30 s) appeared to be sufﬁcient forconditioning to occur. In stark contrast, much longer context
pre-exposure durations were necessary to alleviate the immediate shock deﬁcit: only a pre-exposure lasting 600 s was sufﬁcient
to reverse the immediate shock deﬁcit. Therefore, in the absence of an aversive reinforcer, such as shock, longer duration
exposures are required to form a lasting memory for the context.
Emotionally arousing stimuli, such as shock delivery, facilitate
memory consolidation by activating adrenal stress hormones(McGaugh and Roozendaal, 2002). The activation of adrenal
stress hormones following shock delivery therefore allows animals to selectively remember more emotionally charged events,
at the expense of less important ones (Cahill and McGaugh,1996). We tested this idea by giving mice a 2 min pre-exposure
to the context 24 h before training them using immediate shockprocedures. Under normal conditions this 2 min pre-exposure
has no effect on subsequent conditioning. However, epinephrine treatment, given immediately following pre-exposure,makes this 2 min pre-exposure as effective as a 10 min preexposure in alleviating the immediate shock deﬁcit. This way, arelatively neutral experience— one that by itself would not elicit
conditioned responding—is made effective, presumably by activation of the adrenal stress hormone system. That is, mimicking the effects of shock with epinephrine treatment ensures thatthe context memory is fully consolidated even after a relatively
brief exposure. A previous study found that epinephrine administration following contextual fear conditioning (using standard
procedures) was ineffective (Lee et al., 2001). However, thepresent context pre-exposure procedures may provide a moresensitive method for detection of modulatory inﬂuences oncontextual memories since short duration pre-exposures (incombination with immediate shock training) produce nearbaseline levels of freezing on test.
Epinephrine also facilitated the consolidation of a context memory in CREB␣⌬⫺/⫺ mice. CREB function is reduced, rather than567

eliminated, in CREB␣⌬⫺/⫺ mice. Therefore, epinephrine may activate residual CREB via activation of ␤-adrenergic receptors thatare coupled to cAMP signaling (Kobayashi and Yasoshima, 2001).
This idea recalls an older literature showing that the amnestic
effects of protein synthesis inhibitors given prior to training may beattenuated by posttraining administration of stimulants, including
epinephrine (Martinez et al., 1981). Furthermore, the ﬁnding thatepinephrine was effective when given following context pre-exposure suggests the following. First, that the efﬁciency of CREBmediated consolidation may be regulated during a brief time-window following initial learning. Second, the context impairments in
CREB␣⌬⫺/⫺ mice are due to a failure to consolidate, rather than
encode, contextual information. This conclusion is consistent withrecent reports (Kida et al., 2002; Pittenger et al., 2002). To test
these ideas further it will be necessary to show that CREB activity
is elevated in trained CREB␣⌬⫺/⫺ mice following epinephrine
treatment. Previous studies suggested that the memory deﬁcits of
the CREB␣⌬⫺/⫺ mice can be partially alleviated when the mutation is crossed into some strains of mice (Gass et al., 1998). Ourdata suggest that one possibility is that the upregulation of genes(either controlling the synthesis or the effects of epinephrine) inthese mouse strains is responsible for the partial rescue of the memory deﬁcits caused by the CREB␣⌬⫺/⫺ mutation.
A CS or US representation must incorporate a large amountof information. Not only must the underlying neural networks
represent both the sensory and emotional features of a given
stimulus, but they must also encode complex relational information. For example, the temporal relationship of the stimuluswith other CS and US must also be encoded. Furthermore, thestimulus representation is presumably dynamic in nature, withnew instances or experience leading to the integration of relevant information into these networks (O’Reilly and Rudy,
2001). The formation of episodic memories is then thought toinvolve the rapid and automatic fusion of dynamic, information-laden representations into a uniﬁed memory (Martin et al.,
2000; Morris et al., 2003). The use of immediate shock approaches make it possible to examine the molecular and cellular
processes underlying the formation of CS and US representations in isolation. Our data show that the building of CS (andUS) representations requires NMDA receptor activation, autophosphorylation of CaMKII at T286, CREB-dependent transcription and protein synthesis. Previous molecular and cellularstudies of memory have focused on associative processes in fear
conditioning. The present data suggest similar approaches canbe used to understand the molecular and cellular bases of stimulus representations.

Acknowledgments
The authors thank Bernard Balleine, Tad Blair, Rui Costa, Steve
Kushner, Karim Nader, Jerry Rudy, and Brian Wiltgen for comments on earlier versions of this manuscript. This work was supported by an SNRP/National Institutes of Health (NIH) grant (toA.J.S.).

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