Paul W. Frankland and Sheena A. Josselyn Abstract

The cAMP Responsive Element Binding Protein (CREB) is an activity regulated transcription factor that modulates the transcription of genes with cAMP responsive elements (CRE) located in their promoter regions. A variety of signaling pathways converge to phosphorylate CREB at Ser133 and induce transcription. Here, we review the key features of CREB-dependent transcription and evaluate evidence suggesting that CREB plays a key role in different forms of plasticity in a wide range of species. The unifying feature of these studies is that manipulations of CREB function affect long-term, but not short-term, memory. This suggests that CREB-dependent transcription is required for the cellular events underlying long-term memory.


Current psychological theories of memory propose that memory is not a unitary phenomenon. Rather, memory can be subdivided into qualitatively different memory systems, each of which is served by anatomically distinct brain regions.104 A second distinction can be made within these systems. That is, memory can be subdivided into different phases, each of which can be distinguished from one another in terms of their temporal and biochemical properties.32,78

Using animal studies, at least two phases of memory have been identified: Short-Term Memory (STM) and Long-term Memory (LTM). Short-Term memory is induced rapidly and does not persist beyond a few hours. It involves transient changes in synaptic strength. These are mediated by covalent modifications of preexisting synaptic molecules, such as the phosphorylation or dephosphorylation of enzymes, receptors and/or ion channels. These post-transla-tional modifications produce short-lasting changes in the efficacy of synaptic transmission.106

In contrast, Long-Term Memory lasts days or longer, and is thought to involve the growth and restructuring of synapses. There is extensive evidence from a wide variety of species that enduring changes underlying long-term memory require the synthesis of new proteins.30,76 An essential feature of all of these studies is that administration of protein synthesis inhibitors at the time of learning specifically disrupts the formation of LTM, without affecting learning or STM.

The synthesis of most proteins is mediated by activity-regulated transcription factors. Studies in a wide variety of species have shown that the synthesis of proteins necessary for LTM formation is regulated, at least in part, by cAMP (cyclic adenosine 3',5-monophosphate) responsive element binding protein (CREB). CREB is a transcription factor that modulates the transcription of genes with cAMP responsive elements (CRE) located in their promoter regions. 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 LTM, and not STM. The first studies showing that CREB plays a critical role in LTM

formation were conducted in Aplysia and Drosophila. Subsequent studies in vertebrate species (including, in particular, mice and rats) using a variety of molecular-genetic tools suggest that CREB has a highly conserved role in LTM formation.


CREB is a member of a family (CREB/ATF) of structurally similar, activity-regulated transcription factors. In mammals, at least three genes encode the CREB-like proteins, CREB, CREM (cAMP Response Element Modulator) and ATF-1 (Activating Transcription Factor).38'55'94 The mammalian CREB gene comprises 11 exons, ' 5 and alternative splicing generates the three major activator isoforms of CREB: a, A, and p.11,47,118 Each of these is highly expressed in all tissues. In addition to these transcriptional activators, the CREB family also includes repressors of transcription. For example, the CREM gene codes at least four isoforms that repress CRE-dependent transcription: the CREM a, P and Y proteins as well as the inducible cyclic AMP early repressor (ICER).37,82

CREB regulates gene expression in response to a wide array of extracellular signals. In its inactive state, CREB is prebound as a dimer to CRE sites in the promoter regions of target genes. Neuronal stimulation may lead to the activation of CREB (via activation of various CREB kinases). In its activated form CREB binds CREB-binding protein (CBP); the recruitment of CBP links CREB directly and indirectly to other components of the basal transcription machinery, thus promoting transcription.24


A large number of signaling pathways converge on CREB, indicating that the transcriptional activity of CREB is regulated by a wide variety of extracellular signals.31,77,100 Each of these pathways activate CREB via CREB kinases that phosphorylate CREB at serine 133 (Ser133). This is the critical residue for the transcriptional activity46 since mutation of this residue to a nonphosphorylatable alanine (Ala) residue abolishes the transcriptional response to elevated cAMP levels.4 ,83 Although CREB was initially identified as a transcription factor that responds to elevated levels of cAMP, it is now clear that CREB may be activated by three major signaling pathways (Fig. 1): 1) cAMP, 2) Ca2+, and 3) growth factors.

1) cAMP: The activation of G-protein linked receptors (e.g., D1 receptors) leads to the increases in the second messenger cAMP via activation of adenylate cyclase.44 Rises in levels of cAMP lead to the activation of protein kinase A (PKA) by dissociating the regulatory (R) from the catalytic (C) subunits. The C subunits of PKA passively translocate to the nucleus where they may phosphorylate CREB at Ser133.5,29,50

2) Ca2+: Calcium is a pleiotropic second messenger that is activated via several different mechanisms following changes in membrane potential. Extracellular Ca2+ may enter the cytoplasm via ligand-gated ion channels of NMDA and AMPA receptors, or via voltage-gated calcium channels. In addition, Ca2+ may be released from intracellular stores.100 Calcium sig nals are then transduced via a large number of different CREB kinases which include: CamKII,

CaMKIV, RSK1-3 (via Ras-ERK), PKC and PKA.10,29,33,75,101,107 The different kinetics of each of these pathways provides a mechanism for sustained CREB activation and CRE-medi-ated transcription. For example, activation of CaMKIV produces a wave of CREB phosphorylation with rapid on- and offset (lasting only minutes), whereas activation of the Ras-ERK-RSK2 pathway promotes a slower phase of CREB phosphorylation.116 Furthermore, the distinct ki netic properties of these upstream regulatory pathways may allow CREB to compute information regarding the exact nature of the stimuli, perhaps allowing for specific stimuli (or patterns of stimulation) to be translated into specific patterns of gene expression. For example, recent data indicate that Ca2+ influx into neurons causes the phosphorylation of CREB at Ser142 and

Ser143 (in addition to Ser133), and that CREB-induced transcription induced by this triple phosphorylation may not require the participation of CBP.67 Therefore, Ca2+ influx promotes

CREB-mediated transcription via a set of mechanisms that are distinct from those produced by other extracellular activation.

Figure 1. Activation of CREB by a multiple signaling pathways. In the first pathway, a neurotransmitter may bind to a receptor (R) that is linked to a G-protein (G), which leads to the increases in the second messenger cAMP via activation of adenylate cyclase (AC). Rises in levels of cAMP leads to the activation of protein kinase A (PKA) by dissociating the regulatory (R) from the catalytic (C) subunits. The C subunits of PKA passively translocate to the nucleus where they may phosphorylate CREB at Ser133. In the second pathway growth factors (such as NGF or BDNF) bind to and activate a Trk receptor. This, in turn, activates Ras and the downstream kinases Raf, MEK and ERK. Activated ERKs stimulate the activity of MSKs and RSKs which may then phosphorylate CREB at Ser133. In the third pathway, intracellular increases in Ca2+ which binds to calmodulin (CaM) which activates CaM kinases (CaMKII, CaMKIV, CaMKK) which may also phosphorylate CREB at Ser133.

Figure 1. Activation of CREB by a multiple signaling pathways. In the first pathway, a neurotransmitter may bind to a receptor (R) that is linked to a G-protein (G), which leads to the increases in the second messenger cAMP via activation of adenylate cyclase (AC). Rises in levels of cAMP leads to the activation of protein kinase A (PKA) by dissociating the regulatory (R) from the catalytic (C) subunits. The C subunits of PKA passively translocate to the nucleus where they may phosphorylate CREB at Ser133. In the second pathway growth factors (such as NGF or BDNF) bind to and activate a Trk receptor. This, in turn, activates Ras and the downstream kinases Raf, MEK and ERK. Activated ERKs stimulate the activity of MSKs and RSKs which may then phosphorylate CREB at Ser133. In the third pathway, intracellular increases in Ca2+ which binds to calmodulin (CaM) which activates CaM kinases (CaMKII, CaMKIV, CaMKK) which may also phosphorylate CREB at Ser133.

3) Growth Factors: CREB mediates gene expression in response to a wide variety of growth factors, including nerve growth factor (NGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) and brain-derived neurotrophic factor (BDNF) (see Brandner, this book). Signal ing is then mediated by a large number of growth-factor-induced kinases. For example, NGF stimulation activates NGF receptors (tyrosine kinase receptor, Trk receptors) that stimulates guanine-nucleotide release factors (GRFs) that activate Ras, a small G protein. Activated Ras, in turn, stimulates the serine/threonine kinase, Raf, that triggers activation of MEK, and its targets, the ERK 1 /2 members of the MAPK family.12 One downstream substrate of the Ras/ ERK pathway is a 90 kDa ribosomal S-6 kinase-2 (RSK-2). Upon activation, both ERKs and

RSKs translocate to the nucleus where they may phosphorylate CREB at Ser133.23,36,117

Just as phosphorylation of Ser133 seems to be critical for activation of CREB, dephospho-

rylation of this residue is important for inactivation of CREB. As with all other phosphopro-teins, therefore, the level of CREB phosphorylation at Ser133 reflects a balance between the oppositional actions of kinases and phosphatases, such as protein phosphatase 1 and 2 (PP-1 and PP-2).49 For example, dephosphorylation of CREB at Ser 133 may be initiated by the activation of calcineurin (PP-2B) by the Ca2+-CaM pathway.10 The transcriptional activity of phosphorylated CREB may also be actively suppressed by transcriptional repressors, such as

CREM a, P and y isoforms or ICER.3770,82

The complexity of the pathways upstream from CREB may permit tight, fine-tuned regulation of CRE-mediated transcription, allowing it to produce distinct patterns of gene expression in response to different patterns of stimulation. For example, CREB activation may be moderated by phosphorylation events at sites other than Ser133 (e.g., Ser142 and/or Ser143), and also indirectly by phosphorylation or dephosphorylation of other components of the transcription machinery that CREB interacts with (e.g., CBP, POL II etc).

CREB and Electrophysiological Studies of Long-Term Plasticity in Aplysia

The withdrawal of the gill—an external respiratory organ— in the marine mollusk Aplysia can be produced by mechanical stimulation of either the siphon or mantle shelf. The reflex serves a defensive purpose: the retraction of the gill protects it from potential damage. This reflex exhibits a number of forms of plasticity. In particular, the sensitization of the withdrawal reflex—that is its enhancement following noncontingent shock applied to the tail of the animal—has been instrumental in the identification of many of the cellular and molecular mechanisms mediating synaptic and behavioral plasticity. The persistence of the reflex sensitization is related to the number of shocks applied to the tail: one shock produces a transient sensitiza-tion, lasting minutes, whereas 5 or more shocks produce a LTM lasting days or longer.6,18,25,39,45,62,91 Long-term sensitization at the synaptic level can be studied in reduced preparations containing the sensory-motor synapse: short- and long-term facilitation of this synapse mediates the behavioral sensitization of the reflex.

The role of CREB in memory and plasticity has been studied in cocultured Aplysia sensory and motor neurons.28 Injection of oligonucleotides with CRE sequences into cultured sensory neurons blocks long-term facilitation (LTF).28 Presumably, these CRE-oligonucleotides act as competitive antagonists, trapping the CREB proteins needed for the transcriptional activation of genes that ultimately mediate LTF.4,61 Moreover, a similar injection of a reporter gene driven by a CRE-containing promoter shows that stimuli that produce LTF also trigger CREB activation, while stimuli that do not produce LTF similarly do not trigger CREB activation.61

There are several CREB-like proteins in Aplysia. The CREB1 gene encodes three proteins (ApCREB1a, ApCREB1b and ApCREB1c) by alternative splicing.7 The ApCREB1a shares structural and functional homology with CREB transactivators in mammals, while ApCREB1b resembles mammalian ICER, a repressor of CREB transcription. Injection of antibodies or antisense against CREB1a blocks LTF (but not short-term facilitation) while injection of phos-phorylated ApCREB1a protein alone induces LTF.7 Application of ApCREB1b blocks LTF while decreasing ApCREB1b function lowers the threshold for producing LTF.7 ApCREB1c is a cytoplasmic protein that lacks a nuclear localization signal. Injection of unphosphorylated CREB1c followed by a single pulse of serotonin enhances STF and induces LTF. Therefore, this cytoplasmic form of CREB may play an important role not only in the modulation of CREB-mediated transcription necessary for LTF but also in STF.7 Aplysia CREB2 is structurally unrelated to Aplysia CREB1 but shares some homology with mouse ATF4.51 Decreasing ApCREB2 function decreases the threshold for producing LTF.7 However, the precise mechanism underlying the effects that ApCREB2 exerts on LTF is unclear.

One neuron may participate in the storage of multiple memories. Therefore, activity-dependent changes must be synapse-specific so that the same neuron can encode multiple patterns of stimulation. Experiments using a single sensory neuron composed of two branches that contact two spatially separated motor neurons show that local application of serotonin onto a single synapse induces LTF that is specific to that branch.22,73 This branch-specific LTF requires local protein synthesis (presumably at the synapse to be modified) as well as CREB activation in the nucleus of the presynaptic neuron. Repeated application of serotonin onto the cell body of the sensory neuron (rather than the branch) induces a transient, cell-wide LTF that does not persist beyond 48 hours. This transient LTF is CREB-dependent, but is not accompanied by synaptic growth. A similar pattern of transient LTF and no synaptic growth is pro-

duced by injection of phospho-CREB1 into the sensory neuron. In order for this transient LTF to become stable and for synaptic growth to appear, a single pulse of serotonin at either synapse is required. Thus, CREB-mediated transcription cooperatively induces synaptic changes in concert with local stimulation by serotonin, representing a mechanism by which individual synapses may be strengthened.

CREB and Memory in Drosophila

The molecular mechanisms underlying LTM have been successfully studied in Drosophila (or fruit flies). Learning in flies has been studied using an associative olfactory conditioning paradigm. Flies will learn to avoid a previously neutral odor that was paired with shock in favor of another odor that was not paired with shock in a T-maze.112 Both forward and reverse genetic approaches have been used to study the involvement of CREB in memory in Droso-phila.112 Using a forward genetic approach, the progeny of flies that were treated with a mu-tagen were screened for learning and memory impairments. Two mutants identified by this screen were subsequently determined to have disruptions in Ca2+/CaM-stimulated adenylate cyclase (rutabaga) and in cAMP-specific phosphodiesterase (dunce), both key enzymes in the regulation of intracellular levels of cAMP16,71,

Just as in other species, LTM (produced by multiple training trials) is dependent on protein synthesis.113 Using a reverse genetics approach, Yin and colleagues120 showed that disrupting CREB function in Drosophila blocks LTM produced by multiple training trials, suggesting that protein synthesis required for LTM is mediated, at least in part, by CREB. They found that transgenic over-expression of a CREB transcriptional repressor (dCREB2b) impairs LTM, but not STM, in this task. The finding that STM is intact indicates that the over-expression of this CREB repressor does not disrupt acquisition, and furthermore suggests that basic perceptual, motor, and motivational processes required for the task are intact in these flies.12

In species ranging from Aplysia to human, spaced training (training trials presented with intervening rest intervals) is more effective than massed training (the same number of training trials presented shorter intervening rest intervals) in producing LTM. The same is true in flies: multiple spaced training produces maximal LTM, whereas the same number of trials presented in a massed fashion produces strong STM but weak or no LTM. However, massed training alone is sufficient to produce maximal LTM if a CREB activator (dCREB2a) is over-expressed in transgenic flies prior to training. The over-expression of this CREB activator produces robust LTM following even just one training trial,1 1 perhaps the fly equivalent of 'photographic' or 'flashbulb' memory.119 Transgenic flies over-expressing a mutant activator, where Ser231 (similar to Ser133 of the mammalian CREB gene) was replaced by an Ala, do not show LTM after one training trial, indicating that phosphorylation of CREB at this residue is required for the enhancement of LTM.121 Together, these results show the importance of CREB in LTM formation in Drosophila and, furthermore, suggest that CREB may be a rate-limiting component of this process.

CREB and LTM in Mammals

Targeted Disruption of CREB Function in a Mouse

The study of the role of CREB in mammalian memory was first made possible by the generation of a mouse in which the CREB gene was disrupted. A neomycin resistance (neo) gene was inserted into exon 2 of the CREB gene, which was believed to contain the translation initiation site for all CREB isoforms.56 This neo insertion resulted in the loss of two main isoforms of CREB (a and A). However, the translation of a previously unknown CREB isoform (CREBP) starts from exon 4. Therefore insertion of the neo gene into exon 2 did not disrupt the translation of this isoform; rather, in these CREBaA mice, expression of the CREBP isoform is elevated.11 The expression levels of CREM activator (t) and repressor (a and P) isoforms were also increased in these mice. However, importantly, CREB-dependent transcription is decreased in these CREBaA mutant mice.11 The homozygous deletion of all major CREB isoforms (a, P and A; CREBnul1) is lethal.96

Since the CREBaA mice were generated, they have been exhaustively characterized at the behavioral level. Consistent with the effects of protein synthesis inhibition,2'13'99 CREBaA mice exhibit normal STM but impaired LTM in several fear conditioning paradigms. For example, CREBaA mice show normal conditioned freezing to both tone and context when tested shortly (<1 hour) after training. However, both contextual and tone fear conditioning are impaired if these mice are tested 24 hours after training.14,66 A similar pattern of results has been observed using a different assay of conditioned fear—fear-potentiated startle.34

A parallel set of findings has been observed in studies examining two forms of social learning in CREBaA mice. Rodents develop a preference for foods recently smelled on the breath of other rodents.15,41,42 Memory for this socially transmitted food preference is normal in CREBaA mice when tested immediately following training. However, just as in fear conditioning, CREBaA mice are impaired when tested 24 hours following training.42,66 The ability of rodents to remember conspecifics can be assessed in a social recognition task. Recognition is inferred from a decrease in the amount of time spent investigating a familiar (vs. unfamiliar) conspecific. Again, LTM, but not STM, for social recognition is disrupted in CREBaA mice.65 Together with the fear conditioning data, these findings show that the CREBaA mutation specifically affects LTM, and not STM, in a variety of behavioral paradigms with widely varying performance demands. The extent of these impairments is influenced by gene dosage.42 Further disruption of CREB function can be achieved by combining the CREBaA and CREBdu11 mutations to produce mice carrying only a single allele for the CREBP isoform (CREBcomb). Memory impairments are more severe in these CREBcomb mice compared to the CREBaA mice carrying two alleles for the CREBP isoform.

Drawing an intriguing parallel with the fly experiments, Kogan et al66 showed that the LTM deficits in the CREBaA mice were rescued by increasing the spacing between training trials. This was true in three different forms of LTM: spatial (Morris water maze), contextual (fear conditioning) and social (socially transmitted food preference). These parallels suggest that the levels of activated CREB are rate-limiting for memory formation: The over-expression of the CREB activator (dCREB2a) in the transgenic flies removes the requirement for spaced training trials for LTM formation; Conversely, the reduced levels of CREB in the CREBaA mice necessitates multiple, spaced training, rather than fewer massed trials, to produced stable LTM.

One difficulty in the analysis of knockout mice in learning and memory studies is distinguishing between the effects of a given mutation on mnemonic vs. nonmnemonic processes. This problem is largely circumvented in the CREBaA and related mice since these mice show normal learning and STM. Therefore, compromising CREB function alone does not seem to have nonspecific effects on sensory, motor and motivational processes required for the acquisition and expression of learning. Rather, compromising CREB function appears to specifically affect the formation of LTM.

Gaining Temporal and Spatial Control of CREB Function in Mammals

One of the problems with traditional knockout approaches is that the target protein is deleted throughout development and in all tissues. For example, compensatory upregulation of the CREBP and CREM isoforms complicates the analysis and interpretation of the CREBaA mice. Therefore, achieving both spatial and temporal control over gene expression has been one of the major goals, and three approaches have attempted to meet this challenge.

First, two studies have examined the effects of CREB antisense oligonucleotides on learning and memory in rats. Guzowski and McGaugh48 examined acquisition in the hidden version of the water maze following injections of antisense against CREB mRNA directly into the dorsal hippocampus of rats. These injections disrupted acquisition in the hidden version of the water maze, a form of learning known to be dependent upon the hippocampus. Similar injections 2 days post-training had no effect on subsequent performance in the water maze, indicating that decreasing CREB function does not affect the expression of a previously consolidated memory. Acutely disrupting CREB function in the amygdala has also been shown to disrupt the development of a conditioned taste aversion.69 Injections of antisense directed against CREB blocked long-term (3-5 days), but not short-term (2 hour), CTA memory. Sense control infusions, as well as infusions of antisense into brain regions (basal ganglia) not critical for plasticity underlying CTA, had no effect.

Second, a transgenic line of mice has been developed that inducibly expresses a CREB repressor (aCREB33A).63 The induciblity of the system is produced by fusing the CREB repressor to a ligand-binding domain (LBD) of a human estrogen receptor with a G521R mutation (LBDG521R). The activity of this mutated LBD is regulated not by estrogen but by the synthetic ligand, tamoxifen.27,35,72 In the absence of tamoxifen, the LBDG521R-CREBS133A fusion protein is bound to heat shock proteins and is therefore inactive.35 However, administration of tamoxifen activates this inducible CREB repressor (CREBIR) fusion protein, allowing it to compete with endogenous CREB and disrupt CRE-mediated transcription. This mouse has been used to dissect the role of CREB in potentially dissociable memory processes. By administering tamoxifen to activate the repressor in CREBir transgenic mice at key time points in a fear conditioning protocol, the effects of acutely disrupting CREB function on 1) encoding or STM, 2) consolidation into LTM, 3) storage or maintenance, 4) retrieval were assessed. CREB is crucial for the consolidation of long-term conditioned fear memories, but not for encoding, storage or retrieval of these memories. While acute over-expression of a CREB repressor disrupts LTM, chronic over-expression of the same transgene throughout development has much milder effects.93 The weaker effects associated with chronic over-expression of a CREB repressor (compared to conditional over-expression of this transgene in the CREBIR mice) may be due to upregulation or compensation through development. Alternatively, the weaker phenotype might be due to a milder disruption of CREB function in these mice: for example, transgene expression levels may not be sufficiently high to compete effectively with endogenous CREB.

A third approach has used viral vector-mediated gene transfer technology to manipulate CREB levels.19-21 Josselyn et al used herpes simplex viral vector-mediated gene transfer technology to specifically increase CREB expression in the amygdala of rats. This method exploits the natural ability of the herpes simplex virus to insert DNA into specific neuronal popula-tions.103 These rats were fear-conditioned using massed training that normally only produces STM but no or weak LTM for a light-shock pairing (Fig. 2). However, the over-expression of CREB in the amygdala neurons now results in normal LTM. These data are consistent with results in Drosophila showing that increasing CREB levels reduces the number of training trials required to produce LTM, or overcomes the requirement for trial spacing to produce LTM.121

Detecting CREB Activation During Learning

Complementary to approaches demonstrating that disruption of CREB function blocks the formation of LTM are those showing that CREB is activated following learning. These studies are invaluable since they provide a powerful synergy between systems and molecular approaches. They not only show that CREB-mediated transcription is critical for the formation of long-term memories, but they identify where and when these processes occur.

Activation of CREB leads to the transcription of genes with CRE sites in their promoter regions. Transgenic mice with a P-galactosidase reporter construct under the regulation of a CRE-containing promoter (CRE-LacZ) have been used to identify where in the brain learning-related CREB-mediated transcription occurs.58,59 Following fear conditioning significant increases in CRE-dependent gene expression are observed in both the hippocampus and the amygdala, consistent with the idea that plasticity in these structures is critical for learning context-US and tone-US associations. In a clever control study Impey and colleagues showed that CRE-dependent gene expression related to tone-US associations was limited to the amygdala

Figure 2. Effects of CREB over-expression in the amygdala on fear-potentiated startle. A) Massed training produces weak LTM, as assessed by mean fear-potentiated startle difference scores (difference between mean startle amplitude on light-tone (LT) trials from tone-alone (TA) trials). B) The same number of trials presented in a spaced fashion produces robust LTM. C) Infusion of HSV-LacZ herpes simplex viral vectors encoding LacZ (HSV-LacZ) into the basolateral amygdala produces high expression of |3-galactosidase that is restricted to the basolateral amygdala. D) A high-power image of the amygdala following infusion of HSV-CREB showing over-expression of CREB that is restricted to the lateral nucleus of the amygdala. E) Infusion of HSV-LacZ into the amygdala does not change the weak LTM normally induced by massed training. F) Infusion of HSV-CREB into the amygdala prior to massed training enhances LTM.

Figure 2. Effects of CREB over-expression in the amygdala on fear-potentiated startle. A) Massed training produces weak LTM, as assessed by mean fear-potentiated startle difference scores (difference between mean startle amplitude on light-tone (LT) trials from tone-alone (TA) trials). B) The same number of trials presented in a spaced fashion produces robust LTM. C) Infusion of HSV-LacZ herpes simplex viral vectors encoding LacZ (HSV-LacZ) into the basolateral amygdala produces high expression of |3-galactosidase that is restricted to the basolateral amygdala. D) A high-power image of the amygdala following infusion of HSV-CREB showing over-expression of CREB that is restricted to the lateral nucleus of the amygdala. E) Infusion of HSV-LacZ into the amygdala does not change the weak LTM normally induced by massed training. F) Infusion of HSV-CREB into the amygdala prior to massed training enhances LTM.

using a latent inhibition protocol. To minimize the likelihood of the context becoming associated with shock, mice were pre-exposed to the training context for 12 hours prior to training. These procedures produced significant increases in CRE-dependent gene expression in the amygdala, but not the hippocampus. Consistent with this, when these mice were tested they only showed conditioned freezing when re-exposed to the tone, but not the context.

A second approach has been to use immunocytochemical procedures to detect learning-induced changes in levels of phosphorylated CREB (pCREB). For example, levels of pCREB are elevated in the olfactory bulbs following olfactory conditioning in neonate rat pups.79 Consistent with the effects intra-amygdala infusions of CREB antisense oligonucleotides on the development of a conditioned taste aversion,69 increases in pCREB levels are observed in the lateral nucleus of the amygdala following pairing of saccharin (CS) and LiCl-induced illness (US). Similar increases are not observed if the rats are exposed to the CS (saccharin) or US (LiCl) alone, indicating that activation of CREB is related to associative learning.

Several studies have examined pCREB levels in fear-motivated learning paradigms. Inhibitory avoidance training, for example, induces phosphorylation of CREB in the CA1 and Dentate Gyrus regions of the hippocampus.9,17,88, 08-110,114 These immunocytochemical data confirm similar findings using the CRE-reporter mouse.59 Contextual fear conditioning increases pCREB levels in both the hippocampus and amygdala,105 again consistent with the observations of Impey.59

The contribution of these studies is that they show that CREB activation is restricted to the brain regions that have been shown to critically mediate learning in each of these tasks. Furthermore, they allow us to characterize the time course of CREB activation following a learning event. Indeed, both contextual fear conditioning and inhibitory avoidance training appear to produce two waves of CREB activation:8,105 pCREB levels are initially increased 0-30 minutes following training, and later 3-6 hours following training. These observations are consistent with the idea that LTM formation may involve multiple rounds of protein synthesis. For example, protein synthesis inhibition immediately following, or 4 hours following training, disrupts long-term contextual fear memories.13 It is speculated that these later waves may be mediated by sustained PKA activity: In Aplysia CREB activation leads to the induction of a number of immediate response genes, including carboxy-terminal ubiquitin hydrolase. This hydrolase removes the regulatory subunit of PKA, allowing the kinase to become persistently


CREB and Reconsolidation

Two studies showed that either CRE-dependent gene expression59 or CREB activation105 are detected in the amygdala following fear conditioning training. A third study has shown that pCREB levels are elevated in the amygdala following testing.5 Therefore retrieval, as well as encoding, of fear memories initiates signaling cascades that culminate in CREB activation and presumably gene expression. These findings support the idea that memories are dynamic and modifiable entities. 1,85-87,98 That is, memory retrieval may induce a state of plasticity in which memories become labile before becoming stable again. The process of re-stabilization of the trace, or reconsolidation, following retrieval has been shown to be protein-synthesis depen-dent.86 Consistent with the role for CREB in regulating gene expression required for initial consolidation of memories, recent data supports the role for CREB in regulating gene expression required for reconsolidation, implied by the Hall52 study. Using the inducible CREB repressor transgenic mice, Kida et al63 showed that acutely repressing CREB function following memory reactivation impairs the stability of memory. Although the exact molecular mechanisms mediating consolidation and reconsolidation may differ,1 8 CREB appears to be necessary for both.


Much effort, using a wide variety of tools, has been focused on identifying the molecular mechanisms underlying learning and memory. Establishing that a particular molecule participates critically in these processes relies, it might be argued, on presentation of at least two types of evidence.74,95 First, disruption of normal molecular function should interfere with memory formation. Second, activation of the molecule, in a predictable, region-specific manner, should be observed following learning. Reliance on evidence from one line of inquiry increases the potential for false-negative and false-positive results.43,64 For example, targeted deletion of a particular molecule may cause learning impairments not because that molecule directly participates in processes critical for plasticity; rather, the loss of that molecule may produce more global disruption of cellular processes that indirectly impair the neuron's ability to respond appropriately to extracellular signals.

The case for the critical involvement of CREB in LTM is compelling since both types of evidence have been brought to bear on the problem. That is, disrupting CREB function, be it via the generation and testing of genetically-engineered mice or via the infusion of oligonucle-otides, specifically disrupts LTM, but not learning. Secondly, studies using reporter mice or immunocytochemical approaches, have shown that CREB is activated following learning in a temporally- and region-restricted manner. In rodents, this conclusion is strengthened since these observations are drawn from a wide variety of tasks, each with widely varying stimulus properties and performance demands.

Similar manipulations of CREB function produce qualitatively similar effects in a wide variety of species including Aplysia, Drosophila, Chasmagnathus crab, honey bees, and song birds.1,3,57,68,84,92,97,102,119 In humans, it is particularly noteworthy that the cognitive disabilities in several disorders appear to be directly related to disruption of CREB-mediated transcription. Mutations in RSK2, a protein kinase that activates CREB by phosphorylation at Ser133, are associated with Coffin-Lowry syndrome,111 as well as nonspecific mental retardation.80 For example, in tissue from Coffin-Lowry patients, reductions in RSK2-mediated CREB phosphorylation (following EGF stimulation) are linearly related to severity of cognitive deficits.53 In addition, Rubinstein-Taybi syndrome, which is caused by a mutation of CBP—the cofactor that is essential for transcriptional activation of CREB—is associated with mental retardation.90 Consistent with this, mice that are heterozygous for CBP mutation exhibit learning impairments.89 These studies, along with those from sea slugs and flies, mice and rats, suggest an evolutionarily conserved role for CREB-transcription in role in long-term memory formation.


We thank Rui M. Costa for comments and discussion.


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