Spaced training induces normal long-term memory in CREBmutant miceJeffrey H. Kogan*, Paul W. Frankland*, Julie A. Blendy†, Jodi Coblentz*,Zachary Marowitz*, Günther Schütz† and Alcino J. Silva*

Background: The cAMP responsive element binding protein (CREB) is atranscription factor the activity of which is modulated by increases in theintracellular levels of cAMP and calcium. Results from studies with Aplysia,Drosophila and mice indicate that CREB-activated transcription is required forlong-term memory. Furthermore, a recent study found that long-term memory forolfactory conditioning can be induced with a single trial in transgenic Drosophilaexpressing a CREB activator, whereas in normal flies, with presumably lowerCREB-mediated transcription levels, conditioning requires multiple spaced trials.This suggests that CREB-mediated transcription is important in determining thetype of training required for long-term memory of olfactory conditioning inDrosophila. Interestingly, studies with cultured Aplysia neurons indicated thatremoving a CREB repressor promoted the formation of long-term facilitation, acellular model of non-associative memory.

Results: Here, we have confirmed that mice lacking the a and D CREB proteins(CREBaD–) have abnormal long-term, but not short-term, memory, as tested in anethologically meaningful task. Importantly, additional spaced training canovercome the profound memory deficits of CREBaD– mutants. Increasing theintertrial interval from 1 to 60 minutes overcame the memory deficits of theCREBaD– mice in three distinct behavioral tasks: contextual fear conditioning,spatial learning and socially transmitted food preferences.

Conclusions: Previous findings and results presented here demonstrate thatCREB mutant mice have profound long-term memory deficits. Importantly, ourfindings indicate that manipulations of CREB function can affect the number oftrials and the intertrial interval required for committing information to long-termmemory. Remarkably, this effect of CREB function is not restricted to simpleconditioning tasks, but also affects complex behaviors such as spatial memoryand memory for socially transmitted food preferences.

BackgroundGenetic and pharmacological studies have implicatedboth cAMP and calcium-dependent signaling pathwaysin the cellular events underlying learning and memory.Information flowing through neural networks is thoughtto leave behind an imprint of cellular and synapticchanges that depend on these signaling pathways (forreviews see [1,2]). Some of these changes are short-term,involving only the covalent modification of existingcomponents, such as the phosphorylation of ionchannels, but others are more long-term and require thesynthesis of new proteins. Studies with a variety ofspecies and paradigms have established that proteinsynthesis around the time of training is essential for theformation of long-term memory (for a review see [3]).The newly synthesized proteins may have special rolesin the cellular processes supporting long-term memory

formation, such as the growth and restructuring ofsynapses (for reviews see [4,5]).

The cAMP responsive element binding protein (CREB) isa transcription factor the activity of which is regulated byincreases in the intracellular levels of cAMP and calcium(for reviews see [6–8]). Results from studies with Aplysia[9–11], Drosophila [12,13] and mice [14] indicate thatCREB-mediated transcription is required for long-termmemory. For example, injection of oligonucleotides withcAMP responsive elements (CREs) into cultured Aplysianeurons selectively blocks long-term, but not short-termfacilitation [9], a neuronal model of non-associativememory. Additionally, the induced expression of adominant-negative CREB transgene prior to trainingdisrupts long-term, but not short-term, memory in anolfactory conditioning task in Drosophila [12].

Addresses: *Cold Spring Harbor Laboratory, ColdSpring Harbor, New York 11724, USA. †GermanCancer Research Center, University of Heidelberg,D–69120 Heidelberg, Germany.

Correspondence: Alcino J. Silva

Received: 3 October 1996Revised: 24 October 1996Accepted: 29 October 1996

Published:12 December 1996Electronic identifier: 0960-9822-007-00001

Current Biology 1996, 7:1–11

© Current Biology Ltd ISSN 0960-9822

Research Paper 1

A recent study found that long-term memory for olfactoryconditioning can be induced with a single trial in trans-genic Drosophila expressing a CREB activator, while innormal flies, with presumably lower CREB-dependenttranscription levels, conditioning requires multiple spacedtrials [13]. These results indicate that the levels of activeCREB are important in determining the training schedulerequired for the formation of long-term memory inDrosophila olfactory conditioning.

Studies with cultured neurons suggest that CREB mayhave a similar role in non-associative learning in Aplysia[11]. A single pulse of serotonin, when given with nuclearinjections of an antibody against a CREB repressor, resultsin long-term facilitation (LTF; lasts longer than 24 hours)of neurotransmitter release in synapses between sensoryand motor neurons. Without the antibody, which isassumed to sequester the CREB repressor, the inductionof LTF requires multiple spaced pulses of serotonin [11].The results in Drosophila and Aplysia suggest that limita-tions in CREB-mediated transcription may be one of thereasons why spaced training results in better memory thanmassed training [11,13,15].

Mice with a targeted disruption of the genes encoding the aand D isoforms of CREB (CREBaD–) are profoundly defi-cient in long-term, but not in short-term, memory [14].These mice have compensatory increases in the levels of aminor CREB isoform (b), as well as in the levels of the acti-vator (t) and repressor forms (a and b) of the cAMP-responsive element modulator (CREM) [16,17]. It is esti-mated that these mutants still retain 10–20% of residualCREB activity [16,18], which may explain their surprisinglyrestricted behavioral abnormalities [14,18,19]. CREBaD–

mice appear healthy and have no obvious defects in growthor development. Strikingly, long-term potentiation (LTP),a synaptic mechanism that may have a role in memory, isalso unstable in these mice, demonstrating that theCREBaD– mutation affects both LTP and memory [14].Consistent with these results, studies in transgenic animalswith a reporter gene driven by a promoter sensitive toCREB function indicated that LTP-inducing stimuli canactivate CREB-dependent gene expression, reinforcing theconclusion that CREB function has a role in LTP [20].

The Aplysia and Drosophila studies summarized abovesuggest that CREB activity is crucial in determining thenumber and timing of training trials required for the for-mation of simple forms of long-term memory in inverte-brates. Here, we show that CREB-mediated transcriptionmay have a similar role in complex forms of memory inmammals. Although, in wild-type mice, single trainingtrials can induce robust long-term memory, in CREBaD-

mutant mice, with reportedly lower levels of CREB activ-ity, multiple spaced training is required to trigger normalmemory. Thus, manipulations of CREB function affect

the training schedule required for the formation of mam-malian long-term memory.

ResultsSpaced training can trigger long-term memory forcontextual conditioning in CREBaD– mutantsContextual conditioning is a form of associative learning inwhich animals learn to fear the context — the conditionedstimulus or CS — in which they receive a foot shock — theunconditioned stimulus or US (for reviews see [21,22]).Conditioned animals, when re-exposed to the context inwhich they were shocked, tend to refrain from all but res-piratory movements by freezing [23,24]. Previous studiesindicate that CREBaD– mutants show little evidence ofcontextual memory when trained with a single trial andtested either 1 or 24 hours later [14]. However, mutantstested 30 minutes after training show normal levels offreezing [14]. Hence, short-term memory is intact, butlong-term memory is disrupted in CREBaD– mutant mice.

The nature of the memory deficits of CREBaD– mutantswas further investigated by testing the impact of additionaltraining. Higher levels of freezing 24 hours after trainingcan be elicited in wild-type controls with five training trials(with a 1 minute intertrial interval, ITI) than with eitherone, or two training trials. Training with either ten or fifteentrials, however, does not result in higher levels of freezingthan training with only five trials (24 hour test; data notshown). Thus, CREBaD– mutants (n = 7) and controls (n =7) were trained with five trials delivered one minute apart,as this protocol induces maximal levels of freezing. In con-trast to wild-type controls, which showed very high levels offreezing (70 ± 5 %), CREBaD– mutants displayed littlefreezing (19 ± 3 %; p <0.001) when tested 24 hours aftertraining (Fig. 1a). This result confirms the long-termmemory deficit of CREBaD– mutants, and demonstratesthat extended massed training cannot overcome this deficit.

Figure 1b shows the results of another group of CREBaD–

mutant (n = 15) and wild-type (n = 16) mice trained withonly two trials, given 1 hour apart. Compared to themutants trained with a 1 minute ITI, CREBaD– mutantstrained with a 1 hour ITI showed a clear improvement incontextual conditioning (p <0.001). In striking contrast tothe group trained with a 1 minute ITI, CREBaD– mutantsand wild-type mice trained with a 60 minute ITI showsimilar levels of contextual conditioning 24 hours aftertraining (39 ± 4 % and 50 ± 4 % for mutants and controls,respectively; p > 0.05). Planned comparisons showed thatthe levels of freezing expressed by the mutants and wild-type controls significantly differed only during thelast minute of testing. These results demonstrate thatspaced training improves dramatically the performance ofCREBaD– mutants in the contextual conditioning task.Interestingly, the wild-type mice in this study, given twotraining trials with a 1 hour ITI, had similar degrees of

2 Current Biology, Vol 7 No 1

freezing to the wild-type mice we studied previously [14]which had received only one training trial (50 ± 2 % and 48± 7 % for two trials and one trial, respectively). Thus, anadditional trial given 1 hour after the first trial does nothave a dramatic impact in the performance of controls, butit overcomes the performance deficits of the mutants (39 ±3 % and 12 ± 6 % for two trials and one trial, respectively).

The contextual conditioning studies described here weredone on a predominantly C57Bl/6 (> 87 %) genetic back-ground. However, we had difficulties obtaining CREBaD–

mice in this genetic background. We only obtained 6 % ofhomozygous mice from crosses of heterozygotes, which isconsiderably lower than the previously observed rate of15 % [17]. Nevertheless, the homozygotes that weobtained do not show any evidence of developmentalproblems: the animals are not runted, they have normallife expectancies, they show no hints of ataxia, stereotypy,seizures, aggression or any other overt behavioral abnor-mality; the results above even show that they exhibitnormal memory with spaced training. It is important tonote that neither recessive nor dominant mutations linkedto the CREB locus, and originating in the embryonic stemcells’ 129 genetic-background, could account for the con-textual conditioning phenotype of the CREBaD– mice,because both 129SVJ and C57Bl/6 mice show normalmemory for contextual fear conditioning.

Spaced training induces normal spatial memory inCREBaD– mutantsCREBaD– mutant and wild-type control mice were testedin the hidden-platform version of the Morris water maze

[25]. In this hippocampus-dependent, spatial learning task[26], mice have to locate a submerged platform in a pool ofopaque water. We have reported, and recently confirmedthat CREBaD– mice are profoundly impaired in this task[14]. When given one trial a day for 15 days, CREBaD–

mutants perform poorly both during training and in probetrials ([14] and data not shown).

To determine whether doubling the number of trainingtrials per day could compensate for the deficits of themutants, we trained CREBaD– mice (n = 14) and wild-typecontrols (n = 10) with two trials a day, with one minutebetween each of the two trials. Figure 2a shows thatCREBaD– mice still take significantly longer than controlsto reach the platform throughout training (p < 0.05). Theanimals were also tested in a probe trial on day 10. Inprobe trials, the platform is removed and the mice areallowed to search the pool for 60 seconds. Analysis of theprobe trial results showed that, whereas the wild-typemice searched selectively for the missing platform(49 ± 4 % of search time in the target quadrant, p < 0.001;4.0 ± 0.6 target crossings), the CREBaD– mice did not(35 ± 5 % of search time in the target quadrant, p > 0.05;2.3 ± 0.5 target crossings; Fig. 2b,c). These findings indi-cate that, just as in contextual conditioning, additionaltraining alone cannot induce normal spatial memory inCREBaD– mutants.

Mice require several days to learn the water maze, whichsuggests that information learned each day has to be com-mitted to memory. Perhaps the CREBaD– mutants forgetwhat they learn each day because the one minute ITI is

Research Paper Spaced training in mice Kogan et al. 3

Figure 1

Contextual conditioning with differentnumbers of training trials and intertrialintervals ITIs. (a) CREBaD– mutants (n = 7)and wild-type (WT) controls (n = 7) weretrained with five conditioning trials per daywith a 1 min ITI. The mice were tested forcontextual conditioning 24 h after training.The percentage of time spent freezing eachminute was observed for 5 consecutiveminutes. A two-way ANOVA with repeatedmeasures showed that the percentage oftime spent freezing by CREBaD– mutantswas significantly less than that by wild-typemice (F[1,12] = 153.77, p < 0.001). (b)CREBaD– mutants (n = 15) and wild-type(WT) controls (n = 16) were trained with twotrials per day with a 1 h ITI. The mice weretested for contextual conditioning 24 h aftertraining. A two-way ANOVA with repeatedmeasures showed that there was nodifference in the percentage of time spentfreezing by the two groups of animals(F[1,29] = 3.75, p > 0.05). The error barsrepresent standard errors in all figures.

% F

reez

ing

Five trials (1 min ITI) Two trials (1 h ITI)90

80

70

60

50

40

30

20

10

0

90

80

70

60

50

40

30

20

10

01 2 3 4 5 1 2 3 4 5

WT

CREBα∆–

Time (min) Time (min)

(a) (b)

not sufficient to induce normal memory. As the contextualconditioning results described above suggest that spacedtraining can trigger normal memory in these mutants, wetested the impact of increasing the interval between eachof the two daily trials from 1 to 10 minutes. A 10 minuteITI is ideal for spaced training in Drosophila [13], andrecent results suggest that in the hippocampus, it maytake 3–8 minutes for maximal CREB phosphorylationafter synaptic activation [27].

Figure 3a shows that, when CREBaD– mutants (n = 16) andcontrols (n = 16) were given two trials a day with a 10minute ITI, there was still a significant difference betweenthe two groups of animals in the time they took to reachthe platform during the last day of training (p < 0.05). However, in a probe trial given after 10 days oftraining, both CREBaD– mutant and wild-type micefocused their searches for the platform in the target quad-rant (46 ± 4 %, p < 0.001 and 39 ± 4 %, p < 0.001 of searchtime in the target quadrant for wild-type and mutants,respectively; Fig. 3b). This result shows that increasing theinterval between daily trials from 1 to 10 minutes improvedthe performance of the CREBaD– mice. Nevertheless, thesearches of the wild-type mice were still more accuratethan those of CREBaD– mutants, as wild-type mice crossedthe exact location of the platform more often than the

mutants (3.9 ± 0.4 and 2.1 ± 0.5 for wild-type and mutants,respectively; p < 0.01; Fig. 3c). These data demonstratethat, even with a 10 minute ITI, CREBaD– mutants stillshow some evidence of spatial learning deficits.

Lengthening the interval between daily trials to 1 hourcompletely overcomes the deficits of the mutants. Figure4a shows that, with 1 hour ITI between daily trials, thereis no difference between the performances of CREBaD–

mutants (n = 13) and wild-type (n = 9) mice during train-ing (p > 0.05). Similar results were also obtained in aprobe trial given after 10 days of training. Figure 4b indi-cates that both mutants and wild-type mice searchedselectively for the platform (41 ± 5 %, p < 0.001 and 44 ±4 %, p < 0.001 of search time in the target quadrant formutants and wild-type, respectively). The two groupsalso crossed the target site an equal number of times (3.2± 0.8 and 3.1 ± 0.8 target crossings for mutant and wild-type mice, respectively; Fig. 4c). These data show thatthe performance of CREBaD– mutants in the water mazeprogressively improved as the interval between dailytrials was increased from 1 to 10 to 60 minutes.

Thus, even in a complex spatial learning task, longer ITIscan overcome the profound learning and memory impair-ments of the CREBaD– mice. It is important to note that

4 Current Biology, Vol 7 No 1

Figure 2

Training in the Morris water maze with a 1 minITI. (a) CREBaD– mutants (n = 14) and wild-type (WT) controls (n = 10) were trained for10 days with two trials per day with a 1 minITI. The average time to reach the hiddenplatform in the pool was plotted against thetraining day. A two-way ANOVA with repeatedmeasures showed that over the course oftraining, wild-type animals performedsignificantly better than CREBaD– mutants(F[1,22] = 6.13, p < 0.05). (b) Results of aprobe trial given after 10 days of training withthe schedule described above; the time spentsearching in each pool quadrant was plottedfor mutants and controls. An ANOVA withdependent measures revealed that wild-typeanimals searched very selectively in the areaof the pool where the platform was locatedduring training (F[3,36] = 35.214, p < 0.001).In contrast, CREBaD– mice did not (F[3, 52] =2.49; p > 0.05). There is a significantdifference in the amount of time that wild-typemice spent in the target quadrant comparedto the CREBaD– mice (F[1,22] = 4.76, p <0.05). (c) An ANOVA of the day 10 proberesults revealed that there is also a significantdifference in the number of target platformcrossings performed by wild-type (WT) micecompared to the CREBaD– mice (F[1,22] =4.78, p < 0.05). T, target quadrant; AR,adjacent right quadrant; AL, adjacent leftquadrant; O, opposite quadrant.

Tim

e to

pla

tform

(s)

% T

ime

No.

cro

ssin

gs

WT

CREBα∆–

WT

CREBα∆–

(a)

(b) (c)

60

40

20

0

60

40

20

0

5

3

4

2

1

0

1 2 3 4 5 6 7 8 9 10

Trial day

Quadrants Quadrants

1 min ITI

T A R A L OT A R A L OT A R A L OT A R A L O

previous results [14] indicated that CREBaD– mice trainedwith one trial per day for either 10 or 15 days show deficitsin the water maze. Therefore, the CREBaD– mice not onlyneed large ITIs, but they also require more than one train-ing trial each day to show long-term memory. A previousstudy demonstrated the effects of space training wild-typeanimals in the water maze [28]. We have found similarresults by comparing wild-type mice trained with 10 trialsin either 5 or 10 days: they performed randomly whentrained in 5 days (two trials per day), but learned to searchselectively for the platform when trained in 10 days (onetrial per day; data not shown). Therefore, ceiling effectsmost likely account for the lack of improvement of thewild-type mice under the spaced training conditions usedin the studies with the CREBaD– mutant mice.

As the viability of mice carrying the CREBaD– mutation onthe C57Bl/6 background is low (6 %), we used mice of adifferent genetic background in the water maze and foodpreference studies described here. We used F2 homozy-gotes derived from a cross between CREBaD– heterozy-gotes in the C57Bl/6 background (> 87 %) and wild-type129SVJ mice. The viability of mice carrying the CREBaD–

mutation on this background increases dramatically (to17 %). This new background could result in large experi-mental variability. However, our results demonstrate littleexperimental variability amongst the F2 animals tested,

indicating that phenotypic variability could not account forthe differences between CREBaD– mice and controls. It isalso important to note that neither 129SVJ nor C57Bl/6wild-type mice show an ITI-dependent deficit in thewater maze.

CREBaD– mice show normal short-term but deficient long-term memory in a socially transmitted food preference taskTo extend the studies of the CREBaD– mutants, we alsotested them in the social transmission of food preferencestest [29,30]. This task takes advantage of the fact thatrodents develop a natural preference for foods that theyhave recently smelled on the breath of other rodents [31].This task allows us to test whether CREBaD– mutantshave memory deficits in a hippocampus-dependent testthat involves neither aversive stimuli nor spatial learning[32,33]. Importantly, this task exploits ethologicallymeaningful behaviors: the animals’ ability to learn quicklyand remember information pertaining to social olfactorycues [29,30].

The social transmission of food preferences test takes placein three phases (Fig. 5). First, ‘demonstrator’ mice are givena distinctively scented food (ground mouse chow with cin-namon or cocoa). Second, the demonstrator mice areallowed to interact with ‘observer’ mice, during which theobserver mice have the opportunity to smell the scented

Research Paper Spaced training in mice Kogan et al. 5

Figure 3

Training in the Morris water maze, with a 10min ITI. (a) CREBaD– mutants (n = 16) andwild-type (WT) controls (n = 16) were trainedfor 10 days with two trials/day with a 10 min ITI.A two-way ANOVA with repeated measuresshowed that over the course of training, wild-type animals performed significantly better thanCREBaD– mutants (F[1,30] = 5.13, p < 0.05).Post-hoc analysis showed a significantdifference at day 10 between wild-type andmutants (p < 0.05). (b) Results of a probe trialgiven after 10 days of training with theschedule described above. An ANOVA withdependent measures revealed that both wild-type animals (F[3,60] = 25.14, p < 0.001) andCREBaD– mutant mice (F[3,60] = 5.18, p < 0.005) searched selectively in the area ofthe pool where the platform was located duringtraining. There is no difference in the amount oftime that wild-type mice spent in the targetquadrant compared to the CREBaD– mice(F[1,30] = 1.47, p > 0.05). (c) In contrast tothe percentage time in the target quadrant, anANOVA revealed that there is a significantdifference in the number of target platformcrossings performed by wild-type (WT) micecompared to the CREBaD– mice (F[1,30] =8.12, p < 0.01). T, target quadrant; AR,adjacent right quadrant; AL, adjacent leftquadrant; O, opposite quadrant.

00

1

2

3

4

560

40

20

01 2 3 4 5 6 7 8 9 10

60 10 min ITI

Trial day

40

20

No.

cro

ssin

gs

% T

ime

Tim

e to

pla

tform

(s)

Quadrants Quadrants

WT

CREBα∆–

WT

CREBα∆–

(a)

(b) (c)

T A R A L O T A R A L O T A R A L O T A R A L O

food on the breath of the demonstrator mice. To test theeffects of ITI on memory in CREBaD– mutants and wild-type controls, two demonstrators were used for each experi-ment. Each demonstrator was allowed to interact with theobservers in two separate 5 minute sessions. These twointeractions were separated by either 1 minute or 1 hour. Inthe third phase, either immediately or 24 hours later, food-deprived observer mice were given a choice between twoscented foods: either the same scented food that thedemonstrators had eaten (cued) or another distinctivelyscented food (non-cued).

When tested immediately after social interaction withdemonstrators, both CREBaD– (n = 11) and wild-typecontrol (n = 11) observers showed a strong preference forthe cued food compared to non-cued food (p < 0.05 forboth mutants and controls; Fig. 6a). CREBaD– observersate 0.46 ± 0.07 g of cued food versus 0.22 ± 0.06 g of non-cued food. Similarly, controls ate 0.56±0.12 g of cued foodversus 0.25 ± 0.06 g of non-cued food. Figure 6b showsthat, 24 hours after the interactions with the demonstrators,a second group of wild-type observers (n = 10) still showeda strong preference for the cued food (0.63 ± 0.13 g of cued

6 Current Biology, Vol 7 No 1

Figure 5

Part 1— Demonstrator eats cued food

DemonstratorCued food

Non-cued food

Part 3—Observer is tested for food preference

Observer

Part 2—Demonstrator interacts with observer

ObserverDemonstrator

Cued food

The social transmission of food preferences task. This behavioral taskcomprises three phases. First, ‘demonstrator’ mice are given adistinctively scented (cued) food. Second, the demonstrator mice areallowed to interact with ‘observer’ mice, during which the observermice have the opportunity to smell the scented food on the breath of

the demonstrator mice. Third, immediately or 24 h after interaction withdemonstrators, food-deprived observer mice are given a choicebetween two scented foods: either the same scented food that thedemonstrators had eaten (cued) or another distinctively scented food(non-cued).

Figure 4

Training in the Morris water maze with a 60min ITI. (a) CREBaD– mutants (n = 13) andwild-type (WT) controls (n = 9) were trainedfor 10 days with two trials/day with a one hourITI. A two-way ANOVA with repeatedmeasures showed that over the course oftraining, wild-type animals did not performsignificantly differently than CREBaD– mutants(F[1,20] = 2.47, p > 0.05). (b) Results of aprobe trial given after 10 days of training withthe schedule described above. An ANOVAwith dependent measures revealed that bothwild-type (F[3,32] = 15.99, p < 0.001)animals and CREBaD– mutant mice (F[3,48] =9.98, p < 0.001) searched selectively in thearea of the pool where the platform waslocated during training. There is no differencein the amount of time that wild-type micespent in the target quadrant compared to theCREBaD– mice (F[1,20] = 0.19, p > 0.05). (c) There is also no difference in the numberof target platform crossings performed bywild-type mice compared to the CREBaD–

mice (F[1,20] = 7.33, p > 0.05).

0

1

2

3

4

5

0

20

40

60

01 2 3 4 5 6 7 8 9 10

20

40

60

No.

cro

ssin

gs

Tim

e to

pla

tform

(s)

% T

ime

WT

CREBα∆–

WT

CREBα∆–

Trial day

60 min ITI(a)

(b) (c)

T A R A L O T A R A L O T A R A L O T A R A L O

Quadrants Quadrants

food versus 0.19 ± 0.06 g of non-cued food consumed, p <0.01). The CREBaD– observers (n = 10), however, were notselective, as they ate similar amounts of both foods(0.38 ± 0.09 g of cued food versus 0.32 ± 0.12 g of non-cuedfood consumed, p > 0.05).

It is important to note that, despite their loss of food pref-erence 24 hours after training, CREBaD– mutants con-sumed similar amounts of food to their wild-type controls(0.70 ± 0.08 g for mutants and 0.82 ± 0.11 g for controls;p > 0.05). Differences in either olfaction or taste percep-tion could not account for the 24 hour deficit observedwith the mutants, because they showed selective foodpreference when tested immediately after interaction withthe demonstrators. Importantly, in each experiment half ofthe mutants and controls were tested with cocoa as thecued food, and the others with cinnamon. Additionally,naive groups did not show a preference for either of thetwo scented foods used in these experiments (data notshown). These results confirm previous findings indicat-ing that the CREBaD– mice have normal short-term, butabnormal long-term memory [14].

Spaced training can trigger normal memory for sociallytransmitted food preference in CREBaD– miceTo determine whether a longer ITI could ameliorate theperformance of CREBaD– mice, as it did in the water mazeand in the contextual conditioning tasks, mutants (n = 12)and controls (n = 13) were trained with two interactionswith 1 hour, instead of 1 minute, between interactions.

Figure 6c shows that this longer interval allowed the trig-gering of normal memory for food preference in themutants. CREBaD– mice ate 0.57 ± 0.11 g of the cuedfood, and 0.20 ± 0.06 g of the non-cued food. Similarly,controls ate 0.59 ± 0.11 g of the cued food, and0.21 ± 0.05 g of the non-cued food.

DiscussionOur results show that CREB-mediated transcription has adramatic impact on memory formation. First, we con-firmed that the CREBaD– mutation affects long-term, butnot short-term memory. Second, our results indicate thatCREB-mediated transcription may be an important deter-mining factor of the training schedule — number of trialsand ITI — required for committing information to long-term memory. Remarkably, this effect of CREB-depen-dent transcription is not restricted to simple conditioningtasks [13]. It also affects complex behaviors, such as spatiallearning and socially transmitted food preferences.Together with results from Aplysia and Drosophila, ourfindings indicate that limitations in the levels of CREB-mediated transcription may be one of the reasons whyspaced training results in better memory than massedtraining [11,13,15].

The impact of CREB on Pavlovian conditioning in mice andfliesMice with an intact CREB gene can remember the contextin which they received a foot shock after only a single trial.The memory of this experience is not only robust but also

Research Paper Spaced training in mice Kogan et al. 7

Figure 6

WT CREBα∆– WT CREBα∆– WT CREBα∆–0

0.2

0.4

0.6

0.8

0

0.2

0.4

0.6

0.8

0

0.2

0.4

0.6

0.8

Food

eat

en (g

)

Food

eat

en (g

)

Food

eat

en (g

)

Immediate memory (1 min ITI) 24 h memory (1 min ITI) 24 h memory (1 h ITI)(a) (b) (c)

Cued

Non-cued

Training with different ITIs in the social transmission of food preferencestask. (a) CREBaD– mutant (n = 11) and wild-type control (n = 11)observer mice were given two interactions with demonstrator mice (1min ITI) that had eaten the cued food. Each interaction lasted 5 min.Testing was done immediately after training. Both wild-type mice(F[1,20] = 5.02, p < 0.05) and CREBaD– mutant mice (F[1,20] = 5.37, p< 0.05) showed selective preference for the cued food. (b) CREBaD–

mutant (n = 10) and wild-type control (n = 10) observer mice were giventwo interactions with demonstrator mice (1 min ITI) for 5 min each.

Testing was done 24 h after training. wild-type mice showed a strongselective preference for the cued food (F[1,18] = 9.20, p < 0.01). Incontrast, the CREBaD– mutant mice did not selectively consume thecued food (F[1,18] = 0.18, p > 0.05). (c) CREBaD– mutant (n = 12) andwild-type control (n = 13) observer mice were given two interactions (5min each) with demonstrator mice. In this case a one hour ITI was used.Testing was done 24 h after training. Both wild-type (F[1,24] = 9.68, p <0.005) and CREBaD– mutant mice (F[1,22] = 9.36, p < 0.01) showed aselective preference for the cued food.

long-lasting. Despite normal nociception, the same singletrial can only trigger a transient memory that lasts no morethan 60 minutes in CREBaD– mutant mice [14]. Not evenfive trials given one minute apart, which in wild-type miceproduces maximal long-term memory, can compensate forthe profound contextual amnesia of these mutants. Sur-prisingly, as few as two spaced trials, which in control micedo not trigger higher levels of conditioning than a singletrial, can nevertheless induce nearly normal 24 hourmemory in CREBaD– mutants.

These results are consistent with the results fromDrosophila [12,13]. Flies and mice with presumably moreactive CREB require only a single trial for triggering long-term memory in a conditioning task. In contrast, flies andmice with less active CREB require multiple-spaced trials.It thus appears that CREB-transcription has a similarimpact on Pavlovian memory tasks in both mice and flies.The related electrophysiological results from Aplysia [11]also suggest that these effects apply to non-associativeforms of memory, such as habituation and sensitization.

Spaced training can induce normal spatial memory inCREBaD– mutantsOne trial is sufficient to condition mice to a particularcontext. In contrast, mice require several days of trainingto master the Morris water maze, presumably because ofthe greater demands of the task. Learning this taskappears to be gradual, and to improve their performancethe mice must remember what they learn each day.CREBaD– mutant mice are profoundly deficient in thewater maze when trained with one trial per day for 15 days[14]. Perhaps a single trial is not sufficient to triggermemory formation in the mutants, and consequentlyCREBaD– mice simply forget what they learn each day.

The data reported in this paper show that even two dailytrials, given with one minute intervals, cannot triggernormal memory in the CREBaD– mutants. Just as withcontextual conditioning, additional training with longerintervals between daily trials dramatically improves theperformance of the mutants in the water maze. It is impor-tant to note that performance in the water maze cannot bethoroughly evaluated with a single measure (that is, thepercentage of time spent searching for the platform in thetraining quadrant during the probe trial). Instead, it isbetter to use at least three measures: the time taken toreach the platform during training, the percentage of timespent searching for the platform in the training quadrantduring the probe trial, and the number of platform cross-ings registered during the probe trial.

While 60 minute intervals are completely effective atovercoming the deficits of the mutants in all three mea-sures mentioned above, 10 minute ITIs are only partiallyeffective: the CREBaD– mutant mice spend just as much

time as controls searching for the platform in the trainingquadrant, but they still take longer than wild-type mice tolocate it during training. Furthermore, in probe trials theCREBaD– mice do not cross the exact position of themissing platform as frequently as wild-type mice, as ifthey were unable to precisely locate it. A 10 minute ITImay thus be just below the threshold for normal memoryformation in the mutants.

Interestingly, recent results suggest that it may take 3–8minutes for synaptic activation to trigger maximal CREBactivation (by phosphorylation) [27]. Maximal CREBphosphorylation may be essential for the impaired CREBmachinery to function in the mutants. All together, theseresults indicate that CREB-mediated transcription has animpact on the number of trials and ITI required for theformation of spatial memories in mice. Our finding ofnormal memory after spaced training in the water mazedemonstrates that the profound deficit seen with shorterintervals is not due to sensory, motor or motivationalabnormalities.

Spaced training can be more effective than massed train-ing of wild-type animals in the water maze [28]. This doesnot appear to be the case in the results described here, aswith the shortest ITI used (one minute) the wild-typemice already perform optimally. However, different train-ing schedules can reveal the effects of spaced versusmassed training in wild-type mice. For example, with 10trials given over 10 days, the wild-type mice learn the task,whereas the same number of trials given over 5 days (twotrials per day with 1 minute ITI) results in no learning.

Memory for socially transmitted food preferencesThe socially transmitted food preferences task has severalattractive characteristics that are well suited for memorystudies. First, similar to contextual conditioning, a singlebrief trial is sufficient to induce a memory that lasts manydays. Second, this task takes advantage of natural behav-iors (learning which foods are safe to eat) [29,30], and doesnot involve aversive stimuli. Third, memory for food pref-erence is triggered by a single brief interaction betweenmice, and consequently it is possible to differentiatebetween learning (as judged by performance immediatelyafter training) and memory (as judged by performancehours or days after training).

As previously shown for rats [29,30,32,33], our resultsdemonstrate that mice can show socially transmitted foodpreferences, and that the memory for food preference isboth robust and stable. A single 5 minute interactionbetween two mice is sufficient to induce a memory thatlasts for more than 6 days in controls (data not shown). InCREBaD– mutants, however, even two interactions (1minute ITI) cannot trigger a 24 hour memory. In contrast,short-term memory for food-preference is normal in

8 Current Biology, Vol 7 No 1

CREBaD– mutants, which indicates that the long-termmemory deficit is not due to abnormalities in behaviorsrequired for performance in this task, such as olfactory andtaste discrimination, social interaction, stress responses tofood deprivation, and so on.

In parallel with the results with contextual conditioningand with the water maze, increasing the interval betweentraining episodes from 1 to 60 minutes can completelyovercome the amnesia of the CREBaD– mice in thesocially transmitted food preferences task. It is noteworthythat controls show similar memory for food preferenceswhether one or 60 minute intervals are used, demonstrat-ing again that, although the interval has a dramatic impacton memory in CREBaD– mutants, it does not affectmemory of wild-type controls. Thus, the deficit in theCREBaD– mice in this task mirrors that for the contextualconditioning task. As with the water maze, hippocampallesions affect performance in the socially transmitted foodpreferences task [32,33], which suggests that thehippocampus may be an appropriate region in which toinvestigate the molecular and physiological basis of thememory deficits in CREBaD– mutants.

A model for the effects of the CREBaD– mutation onmemoryRecent insights into the mechanisms of CREB-mediatedtranscription suggest a possible explanation for the behav-ioral findings reported here. Synaptic stimuli can triggerintracellular signaling pathways that modulate CREB-dependent transcription [34,35]. Calcium signals, originat-ing predominantly through the synaptic activation ofN-methyl-D-aspartate (NMDA) receptors and L-typecalcium channels, are thought to activate a signal transduc-tion cascade that may ultimately result in the phosphoryla-tion and activation of CREB [34]. Phosphorylated CREB,in turn, may bind to the transcription adaptor known asCREB-binding protein (CBP), and initiate transcriptionfrom genes with CRE-containing promoters [36,37].

The net levels of CREB-mediated transcription are notonly affected by the phosphorylation status of several stim-ulatory and inhibitory sites in CREB activators [38], butthey depend also on the levels of CREB inhibitors, such asthe a and b isoforms of CREM [39], and the general avail-ability of other components of the transcriptional machin-ery (such as p300 and CBP) [37,40]. Besides a completeloss of the major a and D CREB activators, the CREBaD–

mutants have a compensatory increase in the levels of aminor CREB isoform (b), as well as in the levels of the acti-vator (t) and inhibitor forms (a and b) of CREM [16,17].

The decreased levels of activators and the increased levelsof inhibitors are thought to result in a 80–90 % decrease inCREB activity in the mutants [16,18]. Therefore, it is pos-sible that the residual CREB activity of the mutant mice

cannot respond normally to the synaptic activation result-ing from a single training trial. In turn, this may lead to ashortage of proteins required to support the growth andsynaptic remodeling thought to underlie memory consoli-dation (for reviews see [4,5]). A decrease in CREB func-tion could explain why the CREBaD– mutants haveabnormal long-term memory following single or massedtraining trials. That they have normal short-term memoryis not surprising, as this form of memory is protein synthe-sis independent (for a review see [3]).

Why does the formation of long-term memory requireadditional spaced training in the CREBaD– mutants? Addi-tional trials may provide more episodes of CREB activa-tion, and consequently further opportunities for synthesisof components needed for memory. Nevertheless, ourresults demonstrate that, if the interval between trials istoo small (1 min ITI), additional trials do not improve thememory of the CREBaD– mutants. We found that to beeffective, the trials must be spaced (between 10 and 60minutes). Interestingly, recent studies with an antibodythat detects phosphorylated (active) CREB protein, sug-gested that maximal CREB activation in the hippocampusand in the cortex may take place 3–8 minutes after synap-tic activation, and that it declines slowly thereafter [27].Therefore, it is possible that an episode of activationengages the residual CREB-machinery of the mutants fora limited time, during which further activation has no addi-tional effect. This would explain why additional massedtrials do not seem to benefit the performance of theCREBaD– mutants. Previous models suggested that, duringthe intervals of spaced training, there may be a decrease inthe activity of CREB inhibitors and a net increase in theactivity of activators, which could allow enhanced tran-scriptional responses to additional trials [11,13].

ConclusionsWe found that normal long-term memory can be inducedin CREBaD– mutant mice with spaced, but not withmassed training. This finding is consistent with otherresults from Aplysia [11] and Drosophila [13]. The signifi-cance of our results thus goes beyond a description of thememory deficits of CREBaD– mutants — they reflect auniversal role of CREB in memory formation. The resultspresented here, together with findings in Aplysia [11] andDrosophila [13], indicate that CREB is an important deter-minant of the number of trials, as well as of the ITI,required to commit information into long-term memory.

Materials and methodsRaising and genotyping the miceIn all experiments described, the experimenters were blind to the geno-type of the mice used. At 4–5 weeks postnatally, the animals wereweaned and their genotypes were determined with polymerase chainreaction (PCR) analysis of tail DNA samples. All experiments weredone with mice 3–7 months old, and a similar number of males andfemales were used. The mice were kept on a 12:12 light–dark cycle,

Research Paper Spaced training in mice Kogan et al. 9

and the experiments were always conducted during the light phase ofthe cycle. With the exception of testing times, the mice had ad libaccess to food and water. The Cold Spring Harbor Laboratory animalfacility is fully accredited by the American Association of Accreditationof Laboratory Animal Care. Animals are maintained in accordance withthe applicable portions of the Animal Welfare Act and the DHHS‘Guide to the Care and Use of Laboratory Animals’.

Genetic background of the CREBaD– mutationThe contextual conditioning studies described here were done on a pre-dominantly C57Bl/6 (> 87 %) genetic background. The mice used forthe water maze and social transmission of food preferences studieswere F2 progeny derived from a cross between CREBaD– heterozygotesin the C57Bl/6 background (> 87 %) and wild-type 129SVJ mice.

Contextual fear conditioningThe basic protocol and apparatus used for these experiments was asdescribed before [14]. Mice were placed in a conditioning chamber fortwo minutes before the onset of the CS (30 seconds, 2800 Hz, 85 dBsound). In the last two seconds of the CS, they were exposed to theUS (0.75 mA, 2 seconds continuous foot shock). After the CS/USpairing the mice were left in the conditioning chamber for another 30seconds, and then placed back in their home cages. Multiple trainingtrials (CS/US pairings) were given with intertrial intervals of either 1 or60 minutes. Animals were tested at 24 hours after training. Condition-ing was assessed by measuring ‘freezing’: the animals were judged aseither completely immobile or not (respiratory movements are notcounted) at 10 second intervals. Freezing was measured for 5 consec-utive minutes in the chamber where the mice were trained.

Water maze studiesThe basic protocol and apparatus used for these experiments was asdescribed before [14]. Prior to testing in the water maze the mice werehandled for 2 minutes each day for 10 days. On day one of training,before the first trial, the mice were placed on the platform for30 seconds, followed by a 30 seconds practice swim and three practiceclimbs onto the platform surface. Every training trial began with theanimal on the platform for 60 seconds. The mouse was then placed intothe water facing the wall of the pool and allowed to search for the plat-form. The trial ended either when an animal climbed onto the platform orwhen a maximum of 60 seconds elapsed. At the end of each trial themouse was allowed to rest on the platform for 60 seconds. In a block oftrials the starting position was varied pseudo-randomly among four posi-tions. The platform remained in the same pool position for a particularmouse for the duration of training, but groups of animals were trainedwith different platform positions to avoid quadrant biases. Mice weretrained with two trials per day, with groups of animals receiving either a1, 10 or 60 minutes intertrial interval between daily trials. Animals weretrained for 10 days at approximately the same time each day.

Probe tests were administered after the training trials on day 10.During the probe tests the platform was removed from the pool. Ananimal was started in a position opposite the location of the trainingplatform position, and allowed to swim for 60 seconds. For analysis,both the amount of time each mouse spent searching in each poolquadrant and the number of times the mouse crossed the former plat-form location was measured.

Social transmission of food preferencesMice used for this experiment were housed in cages in groups of threeto five. In their home cage, these mice had continuous access to foodand water. At least 2 days before olfactory training, mice were shapedto eat ground chow from a metal cup. All food pellets were removedfrom the cage, and a metal cup (9 cm diameter, 5 cm deep) wasplaced at one end of the cage on top of the bedding. The food cupremained in place until the day before the experiment.

Training and testing comprized three main stages. First, a demonstratormouse was removed from its home cage and food-deprived for

18 hours. Then it was allowed to eat from a food cup containing ascented food for 2 hours. Ground chow was scented either with cinna-mon (1 % per weight) or cocoa (2 % per weight). Cups were weighedbefore and after to determine the total amount of food consumed. Onlymice eating greater than 0.2 g were used as demonstrators. In theseexperiments two demonstrators were used for each cage. Pairs ofdemonstrators were given the same scented food, and the type ofscent (cinnamon or cocoa) was counterbalanced across groups ofmice to control for the possibility of food bias.

The second stage of the experiment involved an interaction sessionbetween the demonstrators and littermate observer mice. Immediatelyfollowing scented food consumption, one of the pair of demonstratorswas placed back into their home cage and allowed to associate freelywith observers. Each demonstrator interaction session lasted 5minutes, after which the demonstrator was removed from the cage. Theinterval between the first and second demonstrator interaction ses-sions was either 1 minute or 1 hour for different groups of observers.

In the third phase, observer mice were tested for food preference.Groups were tested either immediately following the interaction ses-sions, or 24 hours later. Prior to testing, observer mice were fooddeprived for 18 hours. During the test, mice were placed individually ina larger hamster cage that contained water and two weighed cups withscented-food, placed 10 cm apart, at one end of the cage. One ofthese cups contained food scented identically to that eaten by thedemonstrators. Observers were allowed to consume food from thecups for two hours, after which the cups were re-weighed to determinethe total amount of each food eaten.

Data analysisExperimentally naïve mice were used in all experiments. Approximatelyequal numbers of male and female mice were examined. Results frommale and female mice were combined, as we did not find any signifi-cant differences between them. wild-type controls were littermates ofCREBaD– mutants used in each study. The experimenter was alwayskept blind to the genotype of the animals. For the analysis of data weperformed two-way analysis of variance (ANOVA) with repeated mea-sures, and one-way ANOVA with dependent measures. All values in thetext and figure legends are expressed as mean ± SEM.

AcknowledgementsWe thank K.P. Giese and Y. Cho for invaluable discussions that helped toshape this manuscript, and H. Eichenbaum for advice concerning the socialtransmission of food preferences task. This work was funded by grants fromthe Whitehall Foundation, Beckman Foundation, Klingenstein Foundation,McKnight Foundation, Merck Foundation and the NIH (P01HD33098) toA.J.S., the Deutsche Forschungsgemeinschaft and the Fonds der Chemis-chen Industrie to G.S.

References1. Mayford M, Abel T, Kandel ER: Transgenic approaches to cognition.

Curr Opin Neurobiol 1995, 5:141–148.2. Byrne JH: Cellular analysis of associative learning. Physiol Rev

1987, 67:329–439.3. Davis HP, Squire LR: Protein synthesis and memory. Psychol Bull

1984, 96:518–559.4. Weiler IJ, Hawrylak N, Greenough: Morphogenesis in memory

formation: synaptic and cellular mechanisms. Behav Brain Res1995, 66:1–6.

5. Bailey CH, Kandel ER: Structural changes accompanying memorystorage. Annu Rev Physiol 1993, 55:397–426.

6. Brindle PK, Montminy MR: The CREB family of transcriptionactivators. Curr Opin Genet Dev 1992, 2:199–204.

7. Lee KA, Masson N: Transcriptional regulation by CREB and itsrelatives. Biochim Biophys Acta 1993, 1174:221–233.

8. Lalli E, Sassone-Corsi P: Signal transduction and gene regulation:the nuclear response to cAMP. J Biol Chem 1994,269:17359–17362.

9. Dash PK, Hochner B, Kandel ER: Injection of the cAMP-responsiveelement into the nucleus of Aplysia sensory neurons blocks long-term facilitation. Nature 1990, 345:718–721.

10 Current Biology, Vol 7 No 1

10. Alberini CM, Ghirardi M, Metz R, Kandel ER: C/EBP is animmediate-early gene required for the consolidation of long-termfacilitation in Aplysia. Cell 1994, 76:1099–1114.

11. Bartsch D, Ghirardi M, Skehel PA, Karl KA, Herder SP, Chen M,Bailey CH, Kandel ER: Aplysia CREB2 represses long-termfacilitation: relief of repression converts transient facilitation intolong-term functional and structural change. Cell 1995, 83:979–92.

12. Yin J, Wallach JS, Vecchio MD, Wilder EL, Zhou H, Quinn WG, TullyT: Induction of a dominant-negative CREB transgene specificallyblocks long-term memory in Drosophila melanogaster. Cell 1994,79:49–58.

13. Yin J, Del Vecchio M, Zhou H, Tully T: CREB as a memorymodulator: Induced expression of a dCREB2 activator isoformenhances long-term memory in Drosophila. Cell 1995,81:107–115.

14. Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva A:Deficient long-term memory in mice with a targeted mutation ofthe cAMP-responsive element binding protein. Cell 1994,79:59–68.

15. Carew TJ, Pinsker HM, Kandel ER: Long-term habituation of adefensive withdrawal reflex in Aplysia. Science 1972,175:451–454.

16. Blendy JA, Kaestner KH, Schmid W, Gass P, Schutz G: Targeting ofthe CREB gene leads to up-regulation of a novel CREB mRNAisoform. EMBO J 1996, 15:1098–1106.

17. Hummler E, Cole TJ, Blendy JA, Ganss R, Aguzzi A, Schmid W,Beermann F, Schutz G: Targeted mutation of the cAMP responseelement binding protein (CREB) gene: compensation within theCREB/ATF family of transcription factors. Proc Natl Acad Sci USA1994, 91:5647–5651.

18. Maldonado R, Blendy J, Tzavara E, Gass P, Roques BP, Hanoune J,Schutz G: Reduction of morphine abstinence in mice with amutation in the gene encoding CREB. Science 1996,273:657–659.

19. Blendy JA, Schmid W, Kiessling M, Schutz G, Gass P: Effects ofkainic acid induced seizures on immediate early gene expressionin mice with a targeted mutation of the CREB gene. Brain Res1995, 681:8–14.

20. Impey S, Mark M, Villacres EC, Poser S, Chavkin C, Storm DR:Induction of CRE-mediated gene expression by stimuli thatgenerate long-lasting LTP in area CA1 of the hippocampus.Neuron 1996, 16:973–982.

21. Dickinson A, Mackintosh NJ: Classical conditioning in animals.Annu Rev Psychol 1978, 29:587–612.

22. Fanselow MS, The adaptive function of conditioned defensivebehavior: An ecological approach to Pavlovian stimulussubstitution theory. In Ethoexperimental approaches to the study ofbehavior. Edited by Blanchard PFB RJ, Blanchard DC, Parmigiani S.Dordrecht: Kluwer; 1989:151–166.

23. Fanselow MS, Bolles RC: Naloxone and shock-elicited freezing. J Comp Physiol Psychol 1979, 93:736–744.

24. Miller NE, Weiss JM: Effects of the somatic or visceral responsesto punishment. In Punishment and aversive behavior. Edited byCampbel BA, Church RM. New York: Appleton-Century Crofts; 1969.

25. Morris RGM: Spatial localization does not require the presence oflocal cues. Learn Motiv 1981, 12:239–260.

26. Morris RGM, Garrud P, Rawlins JNP, O’Keefe J: Place navigationimpaired in rats with hippocampal lesions. Nature 1982,297:681–683.

27. Moore AN, Waxham MN, Dash PK: Neuronal activity increases thephosphorylation of the transcription factor cAMP responseelement-binding protein (CREB) in rat hippocampus and cortex.J Biol Chem 1996, 271:14214–14220.

28. Mandel RJ, Gage FH, Thal LJ: Enhanced detection of nucleusbasalis magnocellularis lesion-induced spatial learning deficit inrats by modification of training regimen. Behav Brain Res 1989,31:221–229.

29. Galef Jr BG, Wigmore SW: Transfer of information concerningdistant foods: a laboratory investigation of the ‘information-centre’ hypothesis. An Behav 1983, 31:748–758.

30. Strupp BJ, Levitsky DA: Social transmission of food preferences inadult Hooded rats (Rattus norvegicus). J Comp Psychol 1984,98:257–266.

31. Galef Jr BG, Mason JR, Preti G, Bean NJ: Carbon disulfide: asemiochemical mediating socially-induced diet choice in rats.Physiol Behav 1988, 42:119–124.

32. Winocur G: Anterograde and retrograde amnesia in rats withdorsal hippocampal or dorsomedial thalamic lesions. Behav BrainRes 1990, 38:145–154.

33. Bunsey M, Eichenbaum H: Selective damage to the hippocampalregion blocks long-term retention of a natural and nonspatialstimulus–stimulus association. Hippocampus 1995, 5:546–556.

34. Deisseroth K, Bito H, Tsien RW: Signaling from synapse tonucleus: postsynaptic CREB phosphorylation during multipleforms of hippocampal synaptic plasticity. Neuron 1996,16:89–101.

35. Kaang B-K, Kandel ER, Grant SGN: Acitivation of cAMP-responsivegenes by stimuli that produce long-term facilitation in Aplysiasensory neurons. Neuron 1993, 10:427–435.

36. Kwok RPS, Lundblad J, Chrivia J, Richards J, Bachinger H, Brennan R,Roberts S, et al.: Nuclear protein CBP is a coactivator for thetranscription factor CREB. Nature 1994, 370:223–226.

37. Chrivia JC, Kwok RPS, Lamb N, Hagiwara M, Montminy MR,Goodman RH: Phosphorylated CREB binds specifically to thenuclear protein CBP. Nature 1993, 365:855–859.

38. Yamamoto KK, Gonzalez GA, Biggs WH, Montminy MR:Phosphorylation-induced binding and transcriptional efficacy ofnuclear factor CREB. Nature 1988, 334:494–498.

39. Foulkes NS, Borrelli E, Sassone-Corsi P: CREM gene: use ofalternative DNA-binding domains generates multiple antagonistsof cAMP-induced transcription. Cell 1991, 64:739–749.

40. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC,Heyman RA, Rose DW, Glass CK, et al.: A CBP integrator complexmediates transcriptional activation and AP-1 inhibition by nuclearreceptors. Cell 1996, 85:403–414.

Research Paper Spaced training in mice Kogan et al. 11