Introductory Review Cellscience Reviews Vol 2 No.2 ISSN 1742-8130

CREB and the CREB-C/EBP-dependent Gene Expression Cascade in Long-term Memory

Cristina M. Alberini¹ , Stephen M. Taubenfeld and Ana Garcia-Osta

Introduction

From invertebrates to mammals, the formation of long-term memory requires the activation of the transcription factor cAMP-response-element-binding-protein (CREB) and a CREB-dependent gene expression cascade. Within this cascade, another family of transcription regulators, the CCAAT enhancer binding proteins (C/EBPs), plays a central role. Here we will summarize and discuss some of the major findings regarding the temporal and anatomical distribution of the learning-induced CREB-C/EBP expression and activation in the brain. In addition, the required role of both transcription factor families in long-term memory formation will be reviewed.

CREB and long-term memory

Fifteen years ago very little was known about the nature of the molecular changes occurring in the brain following learning and required for the stabilization of memories. It was known that memory, in order to become long lasting, requires transcription and translation (Agranoff, 1967; Squire and Barondes 1970; Barondes 1975; Davis and Squire 1984). Thus, one of the important questions that molecular neuroscientists focused on throughout the last 15 years has been: what classes of genes and molecular mechanisms are necessary to stabilize memories and how do these molecular changes store the learned information? This question received a great deal of attention beginning in the early 1990s, and since then a tremendous amount of progress has shed light on the nature of the molecular basis underlying long-term memory formation.

One of the molecular pathways found to play an essential and evolutionarily conserved role during memory formation is the CREB-C/EBP-dependent gene expression pathway. CREB is a family of transcription factors ubiquitously expressed and activated in response to the stimulation of the cAMP pathway. Their structure, functions and expression regulation have been extensively characterized and this body of knowledge has been summarized in several excellent reviews (Brindle and Montminy 1992; Hai and Hartman 2001; Mayr and Montminy 2001; Johannessen et al. 2004).

The first indication that proteins binding to cAMP response elements (CREs) are required for long-term memory came from studies in the invertebrate Aplysia californica. Dash et al. (1990) used an in vitro preparation that reproduces the changes occurring in vivo during a simple form of memory known as sensitization of the siphon and gill withdrawal reflex. They found that, when the endogenous CRE binding sequences competed with an excess of recombinant CRE oligodeoxynucleotides (ODNs), the long-term synaptic response associated with memory formation was disrupted. Subsequently, CREB isoforms were cloned from Aplysia central nervous system (CNS) and, indeed, were found to be critical for long-term synaptic plasticity associated with memory formation (Bartsch et al. 1995; Bartsch et al. 1998).

In parallel, in another invertebrate, Drosophila melanogaster, and in mice, it was demonstrated for the first time that CREB plays a critical role in vivo during memory formation. The induction of a dominant negative form of CREB in Drosophila blocked long-term memory formation (Yin et al. 1994) and the knock-out of the most abundant CREB isoforms expressed in the mouse brain (CREBα/Δ) resulted in deficits in several types of memories, including spatial, contextual and cued (Bourtchuladze et al. 1994). In addition, CREB knockout mice also showed impaired long-term potentiation (LTP), a long-lasting synaptic response believed to underlie memory formation. These results were then followed by many studies which confirmed the essential role of CREB in memory in numerous additional species, brain regions and learning systems (Guzowski and McGaugh 1997; Lamprecht et al. 1997; Falls et al. 2000; Kogan et al. 2000; Pittenger et al. 2002; Yuan et al. 2003; Zhang et al. 2003; Josselyn et al. 2004; Brightwell et al. 2005; Countryman et al. 2005; Honjo et al. 2005; Warburton et al. 2005). Several reports also described the overexpression of activated forms of CREB which resulted in the enhancement of long-term memory in animals (Yin et al. 1995, but see Perazzona et al. 2004; Josselyn et al. 2001; Jasnow et al. 2005). In conclusion, a large body of evidence was generated, indicating that CREB is an evolutionarily conserved molecule critical for the formation of long-term memory (Yin and Tully 1996; Silva et al. 1998; Alberini 1999; Kandel 2001).

This conclusion still holds true despite some controversial issues that have been recently raised. Balschun et al. (2003) have found that the conditional disruption of all CREB isoforms in the hippocampal CA1 sub-region and other forebrain regions is not sufficient to impair hippocampal LTP and long-term depression (LTD), or contextual fear conditioning, and causes only a modest impairment in the early stages of water maze learning. Moreover, the same authors also reported that conditional mutants lacking all CREB isoforms throughout the brain showed no deficits either in hippocampal LTP and LTD or in contextual fear conditioning, but both mutants were significantly impaired in conditioned taste aversion. However Pittenger et al. (2002), using another conditional mouse line in which the function of all CREB subfamilies was blocked in CA1 neurons of the dorsal hippocampus, found that, while several forms of late-phase LTP were normal, forskolin-induced and dopamine-regulated potentiation were disrupted. The same animals were impaired in water maze learning, but performed normally in contextual fear conditioning. Finally, other interesting findings indicated that the genetic background and gene dosage may influence the phenotype of CREBα/Δ mutants (Gass et al. 1998). Given the large amount of evidence showing that CREB is essential for memory formation, a reasonable explanation for the contrasting results described above is that, under knockout conditions, compensatory mechanisms can overcome the CREB requirement for memory formation, and that different types of memories involve anatomically distinct CREB expression patterns. Despite these controversies, a large consensus of data supports the conclusion that CREB family members play a fundamental role in memory formation.

The discovery of such an important role of CREB gave rise to many questions: How does CRE-dependent gene expression allow memories to become stabilized? Which are the CREB target genes? What is the functional result of the learning-induced CRE-dependent gene expression? In order to address these issues, it was important to understand the anatomical and cellular distribution, as well as the temporal evolution of CREB-dependent gene expression induced by learning. Characterizing these molecular changes within the brains of living animals following learning is a challenging endeavor and, until now, has only been partially accomplished.

The long-lasting activation of CREB during memory formation

Guzowski and McGaugh (1997) used an antisense ODN approach to gain insight into the anatomical and temporal dynamics of CREB activity during consolidation. Their experiments were the first to demonstrate that CREB-mediated transcription in the rat dorsal hippocampus was a necessary component for learning and memory of a spatial task. Rats injected with CREB antisense ODN just prior to training had impaired retention 48 hours later, whereas treatments given 1 day after training had no effect on long-term memory. This argued for a time-limited CREB-dependent consolidation phase and an anatomical “hot-spot” within the hippocampus. Concurrently, other works delineated the time course of CREB activation following other types of hippocampal-dependent learning in the same hippocampal sub-region, thus elucidating the “on/off” dynamics of CREB activation itself. CREB activation is easily measured by detecting its phosphorylation at Ser-133 (pCREB), which is a prerequisite for CREB-mediated transcription of downstream target genes (Montminy et al. 1990).

In different laboratories, the use of an inhibitory avoidance (IA) or contextual fear conditioning (CFC) task coupled with the quantification of pCREB levels over time following learning was instrumental for uncovering the temporal dynamics of CREB activation. Both IA and CFC are ideal paradigms for these kinetic experiments since animals learn the hippocampal-dependent tasks after a single training session, and therefore, molecular changes following learning can be assayed accordingly. Using a step-down version of IA, Bernabeau et al. (1997) witnessed a learning-specific pCREB increase in the hippocampus with a biphasic profile. Their analysis showed two peaks of activation in CA1 hippocampal neurons, one immediately after training that returned to baseline at 30 minutes, and the other between 3 and 6 hours post-training. By 9 hours, pCREB returned to levels comparable to naïve animals. This trend of pCREB increase coincided with hippocampal PKA activity in rats trained in the same task, which argued that the CREB activation necessary for consolidation of the memory is mediated by PKA phosphorylation. Stanciu et al. (2001) also observed a biphasic pattern in pCREB activation in the hippocampus, parietal cortex and amygdala of mice trained in CFC. The first peak arose between 0 and 30 minutes and the second and highest peak occurred between 3 and 6 hours following fear conditioning.

Using the classical IA task, we asked just how long the pCREB time window remains open (Taubenfeld et al. 1999; Taubenfeld et al. 2001a). PCREB levels were determined using quantitative western blot analyses in the rat hippocampus at 0 (immediately after), 3, 6, 9 and 20 hours following IA training and found to be significantly increased throughout the entire time course compared to untrained controls (Fig. 1). The pCREB increase was due to the phosphorylation of pre-existing CREB. Moreover, it was learning-specific, as CREB phosphorylation was significantly higher than in animals which received uncoupled footshock or context exposure alone.

Figure 1. pCREB induction is sustained in the rat hippocampus for at least 20 hours after IA training. Hippocampal extracts of trained animals and controls (“no shock” and “shock only”) at various timepoints after training were assayed with either anti-Ser133-pCREB or anti-CREB antisera. Quantitative densitometric analyses of western blots are shown. Total CREB protein is unchanged after training (taken from Taubenfeld et al., 1999 and 2001a).

A similar temporal profile with a biphasic profile of CREB phosphorylation was also revealed following non-decremental late LTP (L-LTP) in vivo (Schulz et al. 1999). This phase of LTP requires new gene expression and its mechanisms are believed to resemble the molecular events of memory consolidation (Frey 2001). Schulz et al. showed that CREB phosphorylation first peaked at 30 min, and a second long-lasting peak began at 2 hours after tetanic stimulation and lasted for at least 24 hr. Moreover, similar results were found using in vitro models of LTP. Ahmed and Frey (2005) examined the temporal dynamics of pCREB in hippocampal slices during early- and late-LTP. Their data revealed a delayed onset of pCREB increase with two peaks of phosphorylation at 45 min and 6 hours, returning to baseline levels at 8 hours after LTP induction. When the reversible translational inhibitor anisomycin was applied to slices, only the late-LTP pCREB enhancement was blocked between 2 and 8 hours, indicating that the sustained phosphorylation of CREB is likely mediated by a kinase(s) activated during both an early and a late phase, but only the late phase is mediated by protein synthesis-dependent phosphorylation events. In agreement, Leutgeb et al. (2005) reported that the induction of LTP in CA1 was accompanied by a local increase in pCREB in rat organotypic hippocampal slices. This increase was sustained for at least 4 hours, supporting the hypothesis that CREB plays an important role in the late phase of LTP.

CREB regulates a cascade of gene expression in long-term memory: C/EBP is an evolutionarily conserved target gene

Which are the CREB target genes that are regulated during memory consolidation? When are they induced, and are they required for the stabilization of a new memory? We turned to one potential CREB target gene known to be induced downstream of CREB activation and required for long-term synaptic plasticity underlying memory in Aplysia, the transcription factor C/EBP (Alberini et al. 1994). C/EBP family members are structurally similar to CREB, as they are b-zip proteins and, like CREB, are induced/activated via the cAMP-dependent pathway (Takiguchi 1998; Wilson and Roesler 2002). Interestingly, their expression is often induced and regulated in a manner typical of immediate early genes.

We focused on two mammalian C/EBP family members that were known to respond to cAMP activation, C/EBPβ and C/EBPδ (Taubenfeld et al. 2001a). We analyzed endogenous C/EBPβ and δ expression in the hippocampus following IA learning and found that the induction of both isoforms was delayed relative to the CREB phosphorylation that occurred immediately after training. C/EBPβ mRNA expression remained significantly elevated between 9 and 20 hours after IA learning, while C/EBPδ mRNA showed a peak of induction at 20 hours. In agreement with the mRNA induction profile, C/EBPβ protein levels were increased between 9 and 28 hours. The argument that learning-related induction of C/EBPβ and δ was under the regulatory control of CREB was also strengthened since their expression was found to follow CREB phosphorylation in the same subset of hippocampal neurons. That is, the initial burst of pCREB seen in CA1 neurons immediately after training did not correlate with an increase of either C/EBPβ or δ, but when measured at 24 hours the induction of all three overlapped perfectly (Fig. 2). This characteristic temporal profile further supports the existence of a CREB-C/EBP-mediated gene expression phase underlying memory consolidation that lasts for many hours or days.

Figure 2. Learning-induced increase of C/EBPβ and δ co-localizes with CREB phosphorylation in the same subset of hippocampal neurons at 24 hours but not immediately after IA training. Examples of CA1 immunohistochemical staining of adjacent brain sections (modified from Taubenfeld et al., 2001a).

The critical, evolutionarily conserved role of C/EBPβ was further investigated by infusing antisense ODN specific for C/EBPβ (β-ODN) into the dorsal hippocampus at various time points before and after IA training and evaluating its effect on the memory response. This knock-down manipulation is both reversible and time-limited allowing for the assessment of a particular gene’s functional role during a temporal window. When infused either 5 or 24 hours after training, the β-ODN strongly disrupted long-term memory, but attempts 1 hour before or 46 hours after training were ineffective. These results defined a limited temporal window - with an onset between 6 and 9 hours following training and a return to baseline 2 days post-training- during which C/EBPβ function is required in the hippocampus for IA memory consolidation. This transient requirement of C/EBPβ function closely approximates the time course of C/EBPβ induction following training. Thus, C/EBPβ is one of several evolutionarily conserved CREB-target genes essential for long-term memory consolidation (Taubenfeld et al. 2001b). In Fig. 3, we depict a schematic representation of the CREB-C/EBP-dependent gene expression cascade.

Figure 3. Schematic representation of the CREB-C/EBP pathway activated during memory formation.

Toward the discovery of a CREB-C/EBP-based etiology of temporal lobe lesion-induced amnesia

With the knowledge that sustained hippocampal CREB activation and C/EBP induction are essential events for the formation of several types of long-term memories, we turned to the expansive clinical literature of medial temporal lobe lesions and their associated amnesic symptomatology. Patients with bilateral lesions of the hippocampal system, like the famed H.M. (Scoville and Milner 1957), experience profound anterograde amnesia (Fig. 4). They are unable to store new information into an accessible long-term memory while older, pre-lesion memories remain fully intact and retrievable. The molecular extrapolation seemed obvious - if such lesions prevent consolidation, perhaps they do so by disabling the induction of the CREB response and the transcriptional activation of its target genes required for memory formation. We tested this hypothesis in the rat by severing the fornix bilaterally and assessing the molecular consequences following IA learning (Taubenfeld et al. 1999 and 2001a). A fornix lesion is equivalent to the deafferentation of the major neurotransmitter inputs to the hippocampus, and its effect on the consolidation of long-term memory closely approximates the consequences of temporal lobe damage on various forms of declarative memory. Rats with fornix lesions, trained in IA, demonstrated short-term memory up to 6 hours, but were impaired by 24 hours. These amnesic animals also failed to exhibit the characteristic induction and maintenance of the post-training hippocampal CREB response, as elevations in pCREB were undetectable compared to controls (Fig. 5). Moreover, the same animals also showed a complete lack of C/EBPβ and δ induction in their hippocampi. Therefore, inputs originating in the septum, hypothalamus and brain stem, traveling through the fornix, convey signals regulating CREB-C/EBP-mediated gene expression in the hippocampus, and this modulation is crucial for memory consolidation (Taubenfeld et al. 2001a).

Figure 4. Diagram showing the extent of the bilateral medial temporal lobe resection similar to the one carried out on the intractable epilepsy patient, H.M (one side is indicated here). The radical procedure likely destroyed most of the hippocampus, amygdala and entorhinal cortex bilaterally (taken from Scoville and Milner, 1957).

CREB-C/EBP-dependent target genes

An incomplete chapter of the CREB story remains: what is the nature and function of the CREB-C/EBP target genes? Identifying these genes is a challenging task because it is technically impractical to isolate target genes from the starting point of a single transcription factor. This is especially problematic when the starting material is a tissue such as the brain, which is composed of various cell populations. In fact, it is possible that, in distinct cell types (including glia and different subpopulations of neurons and brain areas involved in the memory process), unique patterns of target gene expression result from CREB-C/EBP activation.

Figure 5. IA learning results in a strong, region-specific increase in pCREB that is conspicuously absent in amnesic animals with fornix lesions. Groups of animals with either intact or resected fornices were trained and immediately sacrificed. Immunohistomchemical staining (examples shown) of hippocampal pCREB revealed a specific increase in CA1 and dentate gyrus (DG) subregions in intact animals. Rats with long-term memory impairment due to fornix lesions lacked the characteristic hippocampal pCREB response (taken from Taubenfeld et al., 1999).

Several studies have focused on the identification and characterization of individual putative CREB target genes, but the results have not been very conclusive. Indeed, during the last 15 years, numerous genes have been found to be regulated as a consequence of training in different systems and kinds of memories, but to a certain extent only C/EBP has been shown to be regulated by CREB activation in memory systems. Several genes, including c-fos, zif 268, bdnf and nurr1 are putative targets of CREB, because they contain CREs in their promoter regions and have been found to be regulated by CREB in other cell systems. However, it is still unknown whether these genes are regulated by CREB following training in a learning paradigm. The identification of C/EBPβ as a CRE-dependent target gene has been shown by Athos et al. (2002), who found that pre-training infusion of a CRE decoy in the CA1 region of the dorsal hippocampus of mice completely blocked both contextual fear conditioning retention at 24 hours and the training-induced increase in C/EBPβ expression. These results provided the first evidence that C/EBPβ is a (directly or indirectly) CREB-regulated target gene during memory formation.

In discussing the specificity of the molecular events underlying memory formation, it should be stressed that CREB is not activated only during memory consolidation. CREB is a ubiquitous transcription factor activated by cAMP in a variety of cells and found to be implicated in many functions, including cell survival, cell cycle, liver processes, circadian rhythm, hormonal cell transcription, cell development, depression, addiction, inflammation, spermatogenesis, hematopoiesis, and immunoresponse. In fact, the CREB family of transcription factors has been proposed to play a critical role in maintaining cellular homeostasis (Hai and Hartman 2001). Since it is clear that CREB is not a memory-specific gene, what is it that makes CREB essential for memory formation? Very likely, as for any other transcription factor involved in a variety of cellular functions, it is the combination of the transcripts that, together with CREB and its downstream genes, generates a unique pattern of gene expression that serves a specific function - which, in this case, happens to be memory.

One of the techniques that attempt to identify unique patterns of gene expression associated with specific functions is based on the use of differential hybridizations of DNA microarrays. Thus, in order to identify the memory-associated CREB-C/EBP-dependent target genes, transcripts isolated from the brains of animals that underwent training are subtracted from those of control animals at time points following learning-induced CREB activation. We used DNA arrays to analyze the differential profile of gene expression in the hippocampi of rats trained in IA versus those of untrained controls. We carried out our analysis at 20 hours after IA training, a time point at which both CREB and C/EBPβ are activated (Taubenfeld et al. 2001a). We found that several functional classes of genes are up- or down-regulated 20 hours following IA training. Interestingly, many of these genes play a role in synaptic functions or participate in functions that are likely to take place within synaptic compartments. For example, they include membrane receptors, kinases, phosphatases, synaptic vesicle proteins, transporters, channels and structural compartment proteins (Garcia-Osta et al. unpublished). These data support the idea that the target genes regulated as a result of the CREB-C/EBP-dependent cascade activation are implicated in long-term synaptic modifications.

These results are in line with the findings that both CREB and C/EBP are required for the synaptic structural changes occurring during long-term synaptic plasticity in Aplysia (Bartsch et al. 1995), and that CREB phosphorylation coincides with transient synapse formation in the rat hippocampus following IA training (O'Connell et al. 2000). Therefore, we propose that the contribution of the CREB-C/EBP-regulated gene expression to memory formation is to stabilize and maintain the connectivity among synapses whose activity generates a functional network that represents the trace of the memory. In other words, learning creates a trace of synaptic activity which, in order to create a long-lasting memory, needs to be stabilized and maintained through cellular homeostasis. This process of homeostatic synaptic stabilization is mediated by the CREB-C/EBP-dependent gene cascade. If this is the case, we can expect that CREB-C/EBP-dependent gene expression becomes activated anytime there is a need to stabilize the strength of the synaptic activity of the trace. This hypothesis remains to be tested. Nevertheless, several observations support this idea: for example, both CREB and C/EBP are also required during memory reconsolidation. Reconsolidation is the process by which an established memory is re-stabilized or maintained following a reactivation event causing the memory to become labile again (Alberini, 2005). Thus, as reconsolidation requires the re-stabilization of the memory trace, if the CREB-C/EBP response is required for an increase in synaptic strength, it should also be critical for reconsolidation. Indeed, this was found to be the case in several studies. Kida et al. (2002) using transgenic mice with an inducible and reversible CREB repressor showed that CREB is required for the reconsolidation of fear memories. Countryman and colleagues (Countryman et al. 2005) reported that pCREB levels increase in the hippocampi of rats following recall of a socially transmitted food preference. In addition, Hall et al. (2001) noticed an elevation of pCREB in the amygdala following the retrieval of a cued fear memory, while our own group demonstrated a requirement for C/EBPβ in the the same brain region during the reconsolidation of IA memory (Tronel et al. 2005). Finally, CREB was found to be critically involved in the formation of long-lasting representations in associative contextual fear conditioning (Frankland et al. 2004).

The temporal evolution of learning-induced gene expression is one of the predominant themes that one is left with when reflecting on the investigation of the molecular basis of memory formation. As additional CREB-C/EBP-dependent target genes are discovered and characterized, we may further understand this dynamic transcriptional cascade underlying memory formation.

Summary

A CREB-C/EBP-dependent cascade of gene expression is an evolutionarily conserved molecular mechanism essential for long-term memory formation. In hippocampal-dependent memories, the formation of new memories is accompanied by the activation of CREB in the hippocampus. This activation occurs in a biphasic manner with an initial, relatively short peak that resolves within the first hour and a second, long-lasting phase that is sustained for days. During this second phase, an induction of the transcription factor C/EBP takes place. The temporal profile of the second phase suggests that the CREB-C/EBP-regulated target genes may be involved in (i) stabilizing the initial synaptic activation (trace) induced by learning and (ii) maintaining the new cellular homeostasis.

References

Agranoff, B.W., Ed. (1967). "Agents that block memory, in The Neurosciences: A study program." New York Rockefeller University Press.

Ahmed, T., Frey, J.U. (2005). "Plasticity-specific phosphorylation of CaMKII, MAP-kinases and CREB during late-LTP in rat hippocampal slices in vitro." Neuropharmacology 49(4): 477-492.

Alberini, C.M., Ghirardi, M., Metz, R., Kandel, E.R. (1994). "C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia." Cell 76: 1099-1114.

Alberini, C.M. (1999). "Genes to remember." J. Exp. Biol. 202: 2887-2891.

Alberini, C.M. (2005) "Mechanisms of memory stabilization: are consolidation and reconsolidation similar or distinct processes?" Trends Neurosci. 28: 51-56.

Athos, J., Impey, S., Pineda, V.V., Chen, X., Storm, D.R. (2002). "Hippocampal CRE-mediated gene expression is required for contextual memory formation." Nat. Neurosci. 5: 1119-1120.

Balschun, D., Wolfer, D.P., Gass, P., Mantamadiotis, T., Welzl, H., Gunther, S., Frey, J.U. Lipp, H.P. (2003). "Does cAMP response element-binding protein have a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent memory?" J. Neurosci. 23(15): 6304-6314.

Barondes, S.H. (1975). "Protein synthesis dependent and protein synthesis independent memory storage processes." Short-Term Memory. D. D. Deutsch, J. A. New York, Academic Press: 379-390.

Bartsch, D., Ghirardi, M., Skehel, P., Karl, K.A., Herder, S.P., Chen, M., Bailey, C.H. Kandel, E.R. (1995). "Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural changes." Cell 83: 979-992.

Bartsch, D., Casadio, A., Karl, K.A., Serodio, P., Kandel, E.R. (1998). "CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic modulator that form a regulatory unit critical for long-term facilitation." Cell 95: 211-223.

Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I., Medina, J.H. (1997). "Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats." Proc. Natl. Acad. Sci. USA 94: 7041-7046.

Bourtchuladze, R., Frenguelli, B., Cioffi, D., Blendy, J., Schultz, G., Silva, A. J. (1994). "Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element binding protein." Cell 79: 59-68.

Brightwell, J.J., Smith, C.A., Countryman, R.A., Neve, R.L., Colombo, P.J. (2005). "Hippocampal overexpression of mutant creb blocks long-term, but not short-term memory for a socially transmitted food preference." Learn. Mem. 12: 12-17.

Brindle, P.K., Montminy, M.R. (1992). "The CREB family of transcription activators." Curr. Opin. Genet. Dev. 2: 199-204.

Countryman, R.A., Orlowski, J.D., Brightwell, J.J., Oskowitz, A.Z. Colombo P.J. (2005). "CREB phosphorylation and c-Fos expression in the hippocampus of rats during acquisition and recall of a socially transmitted food preference." Hippocampus 15: 56-67.

Dash, P.K., Hochner, B. Kandel, E.R. (1990). " Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation." Nature 345: 718-721.

Davis, H. P., Squire, L. R. (1984). "Protein synthesis and memory: a review." Psychol. Bull. 96: 518-559.

Falls, W.A., Kogan, J.H., Silva, A.J., Willott, J.F., Carlson, S., Turner, J.G. (2000). "Fear-potentiated startle, but not prepulse inhibition of startle, is impaired in CREBalphadelta-/- mutant mice." Behav. Neurosci. 114: 998-1004.

Frankland, P.W., Josselyn, S.A, Anagnostaras, S.G., Kogan, J.H., Takahashi, E., Silva, A.J. (2004). " Consolidation of CS and US representations in associative fear conditioning." Hippocampus 14: 557-569.

Frey, J.U. (2001). "Long-lasting hippocampal plasticity: cellular model from memory consolidation?" Cell polarity and subcellular RNA localization. Results and problems in cell differentiation. D. Richter. Berlin, Heidelberg, Springer-Verlag. 34: 27-40.

Gass, P., Wolfer, D.P., Balschun, D., Rudolph, D., Frey, U., Lipp, H.P., Schutz, G. (1998). "Deficits in memory tasks of mice with CREB mutations depend on gene dosage." Learn. Mem. 5: 274-288.

Guzowski, J.F., McGaugh, J.L. (1997). "Anti-sense oligodeoxynucleotide-mediated disruption of hippocampal CREB protein levels impairs memory of a spatial task." Proc. Natl. Acad. Sci. USA 94: 2693-2698.

Hai, T., Hartman, M.G. (2001). "The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis." Gene 273: 1-11.

Hall, J., Thomas, K.L., Everitt, B.J. (2001). "Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala." Eur. J. Neurosci. 13(7): 1453-1458.

Honjo, K., Furukubo-Tokunagua, K. (2005). "Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae." J. Neurosci. 25: 7905-7913.

Jasnow, A.M., Shi, C., Israel, J.E., Davis, M., Huhman, K.L. (2005). "Memory of social defeat is facilitated by cAMP response element-binding protein overexpression in the amygdala." Behav. Neurosci. 119: 1125-1130.

Johannessen, M., Delghandi, M.P., Moens, U. (2004). "What turns CREB on?" Cell Signal. 16: 1211-1227.

Josselyn, S.A., Kida, S., Silva, A.J. (2004). "Inducible repression of CREB function disrupts amygdala-dependent memory." Neurobiol. Learn. Mem. 82: 159-163.

Josselyn, S.A., Shi, C., Carlezon, W.A. Jr., Neve, R.L., Nestler, E.J., Davis, M. (2001). "Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala." J. Neurosci. 21: 2404-2412.

Kandel, E.R. (2001). "The molecular biology of memory storage: a dialogue between genes and synapses." Science 294: 1030-1038.

Kida, S., Josselyn, S.A., de Ortiz, S.P., Kogan, J.H., Chevere, I., Masushige, S. & Silva, A.J. (2002). "CREB required for the stability of new and reactivated fear memories." Nat Neurosci. 5: 348-355.

Kogan, J.H., Frankland, P.W., Silva, A.J. (2000). "Long-term memory underlying hippocampus-dependent social recognition in mice." Hippocampus 10: 47-56.

Lamprecht, R., Hazvi, S., Dudai, Y. (1997). "cAMP response element-binding protein in the amygdala is required for long-but not short-term conditioned taste aversion memory." J. Neurosci. 17: 8443-8450.

Leutgeb, J.K., Frey, J.U., Behnisch, T. (2005) "Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures." Neuroscience 131: 601-610.

Mayr, B., Montminy, M. (2001). "Transcriptional regulation by the phosphorylation-dependent factor CREB." Nat. Rev. Mol. Cell. Biol. 2: 599-609.

Montminy, M. R., Gonzalez, G.A., Yamamoto, K.K. (1990). "Regulation of cAMP-inducible genes by CREB." Trends Neurosci. 13: 184-188.

O'Connell, C., Gallagher, H.C., O'Malley, A., Bourke, M., Regan, C.M. (2000). "CREB phosphorylation coincides with transient synapse formation in the rat hippocampal dentate gyrus following avoidance learning." Neural, Plast. 7: 279-289.

Perazzona, B., Isabel, G., Preat, T., Davis, R.L. (2004). "The role of cAMP response element binding protein in Drosophila long-term memory." J. Neurosci. 24: 8823-8828.

Pittenger, C., Huang, Y.Y., Paletzki, R.F., Bourtchouladze, R., Scanlin, H., Vronskaya, S., Kandel, E.R. (2002). "Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory." Neuron 34: 447-462.

Schulz, S., Siemer, H., Krug, M., Hollt, V. (1999) "Direct evidence for biphasic cAMP responsive element-binding protein phosphorylation during long-term potentiation in the rat dentate gyrus in vivo." J. Neurosci. 19: 5683-5692.

Scoville, W.B., Milner, B. (1957). "Loss of recent memory after bilateral hippocampal lesions." J. Neurol. Neurosurg. Psychiat. 20: 11-21.

Silva, A.J., Kogan, J.H., Frankland, P.W. & Kida, S. (1998). "CREB and Memory." Ann. Rev. Neurosci. 21: 127-148.

Squire, L., Barondes, S. (1970). "Actinomycin-D:effects on memory at different times after training." Nature 225: 649-650.

Stanciu, M., Radulovic, J., Spiess, J. (2001). "Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production." Brain. Res. Mol. Brain. Res. 94: 15-24.

Takiguchi, M. (1998). "The C/EBP family of transcription factors in the liver and other organs." Int. J. Exp. Pathol. 79: 369-391.

Taubenfeld, S.M., Wiig, K.A., Bear, M.F., Alberini, C.M. (1999). "A molecular correlate of memory and amnesia in the hippocampus." Nat. Neurosci. 2: 309-310.

Taubenfeld, S.M., Wiig, K.A., Monti, B., Dolan, B., Pollonini, G., Alberini, C.M. (2001a). "Fornix-dependent induction of hippocampal CCAAT enhancer-binding protein b and d co-localizes with phosphorylated cAMP response element binding protein and accompanies long-term memory consolidation." J. Neurosci. 21: 84-91.

Taubenfeld. S.M., Milekic, M.H., Monti, B., Alberini, C.M. (2001b). "The consolidation of new but not reactivated memory requires hippocampal C/EBPbeta." Nat. Neurosci. 4: 813-818.

Tronel, S., Milekic, M.H., Alberini, C.M. (2005). "Linking new information to a reactivated memory requires consolidation and not reconsolidation mechanisms." PLoS Biol. 3: 1630-1638.

Warburton, E.C., Glover, C.P., Massey, P.V., Wan, H., Johnson, B., Bienemann, A., Deuschle, U., Kew, J.N., Aggleton, J.P., Bashir, Z.I., Uney, J., Brown, M.W. (2005). "cAMP responsive element-binding protein phosphorylation is necessary for perirhinal long-term potentiation and recognition memory." J. Neurosci. 27: 6296-6303.

Wilson, H.L., Roesler, W.J. (2002). "CCAAT/enhancer binding proteins: do they possess intrinsic cAMP-inducible activity?" Mol. Cell. Endocrinol. 188: 15-20.

Yin, J. C. P., Wallach, J.S., Del Vecchio, M., Wilder, E.L., Zhou, H., Quinn, W. G. & Tully, T. (1994). "Induction of a dominant-negative CREB transgene specifically blocks long-term memory in Drosophila melanogaster." Cell 79: 49-58.

Yin, J.C.P., Del Vecchio, M., Zhou, H., Tully, T. (1995). "CREB as a memory modulator: Induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila." Cell 81: 107-115.

Yin, J.C.P., Tully, T. (1996). "CREB and the formation of long-term memory." Curr. Opin. Neurobiol. 6: 264-268.

Yuan, Q., Harley, C.W., Darby-King, A., Neve, R.L., McLean, J.H. (2003). "Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB." J. Neurosci. 23(11): 4760-4765.

Zhang, J.J., Okutani, F., Inoue, S., Kaba, H. (2003). "Activation of the cyclic AMP response element-binding protein signaling pathway in the olfactory bulb is required for the acquisition of olfactory aversive learning in young rats." Neuroscience 117: 707-713.