Molecular Enhancement of Memory Formation
Authors: Thomas J Carew
Journal: Neuron (1996)

A mathematician friend of mine tells the story of a colleague at her university, an ichthyologist, who proclaimed that he actively avoided remembering the names of the students he taught. When she admonished him for this point of view, he explained it simply: "Remember a name, forget a fish." This is admittedly a rather simplified view of memory, but it highlights the important point that committing an event to long-term memory is serious business. Typically, the encoding of a lasting memory entails considerable practice and rehearsal, but we are all also aware of those rare events in our lives that are so powerful as to be instantly encoded and remembered for a lifetime. Quite remarkably, insights into these fundamental properties of memory are beginning to emerge from an analysis of their underlying molecular substrates. A critical step in our understanding of the neuronal mechanisms of memory was the appreciation that the formation of long-lasting memories requires protein synthesis (reviewed in8Davis H.P Squire L.R Psychol. Bull. 1984; 96: 518-559Crossref PubMed Scopus (1262) Google Scholar). Recent work in the marine mollusc, Aplysia, and the fruit fly, Drosophila, have advanced the field another step. In these animals, it has been possible to identify some of the genes and gene products that contribute importantly to induction of the long-lasting neuronal and behavioral changes underlying long-term memory. Moreover, in two recent papers, which are the primary focus of this review23Yin J.C.P Del Vecchio M Zhou H Tully T Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (563) Google Scholar in Drosophila and 4Bartsch D Ghirardi M Skehel P.A Karl K.A Herder S.P Chen M Bailey C.H Kandel E.R Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (496) Google Scholar in Aplysia have shown that it is now possible, using new molecular tools, not only to impair long-term memory formation, but also to enhance it. These papers thus represent an important paradigm shift in the molecular analysis of memory. The reciprocal demonstration of loss of function and gain of function, which has been a hallmark of progress in understanding developmental genetics in Drosophila (15Greenspan R.J Neuron. 1995; 15: 747-750Abstract Full Text PDF PubMed Scopus (41) Google Scholar), can now be brought to bear on the fundamental question of the molecular regulation of memory formation. By way of background, considerable evidence has been amassed implicating the second messenger, cAMP, and a specific class of transcription factors, cAMP response element–binding proteins (CREBs), in the induction of long-term cellular and behavioral modifications in both Aplysia and Drosophila. For example, in Aplysia spaced behavioral training stimuli (electric shocks) delivered to the tail produce both long-term memory for sensitization and long-term facilitation of the synaptic connections between the sensory neurons and motor neurons of the tail-withdrawal reflex. Comparable long-term facilitation can be produced in the intact CNS and in isolated coculture of sensory and motor neurons by repeated spaced pulses of serotonin (5-hydroxytryptamine; 5-HT), a transmitter released by tail stimuli (18Montarolo P.G Goelet P Castelucci V.F Morgan J Kandel E.R Schacher S Science. 1986; 234: 1249-1254Crossref PubMed Scopus (604) Google Scholar, 11Emptage N.J Carew T.J Science. 1993; 262: 253-256Crossref PubMed Scopus (173) Google Scholar); this facilitation critically depends on protein synthesis (18Montarolo P.G Goelet P Castelucci V.F Morgan J Kandel E.R Schacher S Science. 1986; 234: 1249-1254Crossref PubMed Scopus (604) Google Scholar). Repeated 5-HT pulses also cause an increase in cAMP in the sensory neurons and give rise to translocation of the catalytic subunit of the cAMP-dependent protein kinase (PKA) to the sensory neuron nucleus (2Bacskai B.J Hochner B Mahut-Smith M Adams S.R Kaang B.K Kandel E.R Tsien R.Y Science. 1993; 260: 222-226Crossref PubMed Scopus (435) Google Scholar), where it appears to activate cAMP-inducible gene expression (16Kaang B.-K Kandel E.R Grant S.G.N Neuron. 1993; 10: 427-435Abstract Full Text PDF PubMed Scopus (262) Google Scholar). Finally, repeated 5-HT pulses and cAMP also induce protein synthesis–dependent, long-lasting structural changes in sensory neurons (reviewed in3Bailey C.H Kandel E.R Annu. Rev. Physiol. 1993; 55: 397-426Crossref PubMed Scopus (723) Google Scholar). In Drosophila, cAMP has been implicated in an important role in long-term memory produced in a form of olfactory avoidance learning in which flies are presented with two novel odors, one of which is paired with an electric shock. After training, flies express memory for the task by avoiding the odor that was paired with shock (19Tully T Quinn W.G J. Comp. Physiol. 1985; 157: 263-277Crossref PubMed Scopus (898) Google Scholar). With repeated, temporally spaced training trials, an enduring memory that requires protein synthesis is formed (20Tully T Preat T Boynton S.C Del Vecchio M Cell. 1994; 79: 35-47Abstract Full Text PDF PubMed Scopus (721) Google Scholar). Using a forward genetic approach, several fly mutants that exhibit memory deficits for this type of learning have been shown to be impaired in the cAMP signaling cascade. These include animals with a disrupted phosphodiesterase gene (dunce), a defective adenylate cyclase gene (rutabaga), an impaired gene for the catalytic subunit of PKA (reviewed in9Davis R.L Neuron. 1993; 11: 1-14Abstract Full Text PDF PubMed Scopus (274) Google Scholar), and a defective gene that codes for a neuropeptide thought to engage the cAMP pathway (12Feany M.B Quinn W.G Science. 1995; 268: 869-873Crossref PubMed Scopus (233) Google Scholar). In addition, using a reverse genetic approach in which inducible transgenes can be made to express a peptide inhibitor of PKA, memory in the olfactory learning paradigm is also disrupted (10Drain P Folkers E Quinn W.G Neuron. 1991; 6: 71-82Abstract Full Text PDF PubMed Scopus (151) Google Scholar). Thus far, we have considered a wide range of studies implicating cAMP in long-term memory in Aplysia and Drosophila. But where does CREB fit into the picture? A thumbnail sketch of CREB activation will help to set the stage to explore this question. Under conditions of sufficient PKA activation, the catalytic subunit of PKA can translocate to the nucleus and phosphorylate CREB (17Meinkoth J.L Ji Y Taylor S.S Feramisco J.R Proc. Natl. Acad. Sci. USA. 1990; 87: 9595-9599Crossref PubMed Scopus (131) Google Scholar) at a specific amino acid, Ser-133 (14Gonzalez G.A Montminy M.R Cell. 1989; 59: 675-686Abstract Full Text PDF PubMed Scopus (2051) Google Scholar). Phosphorylated CREB leads to activation of transcription from an enhancer element (cAMP response element, or CRE) located in the upstream control region of many cAMP-responsive mammalian genes (21Yamamoto K.K Gonzalez G.A Biggs W.H.I Montminy M.R Nature. 1988; 334: 494-498Crossref PubMed Scopus (969) Google Scholar). In this fashion, some CREBs become activators that promote the transcription of immediate-early genes (IEGs). Some of the protein products of IEGs are themselves transcription factors that ultimately give rise to the activation of late response genes, whose products in turn are thought to subserve the encoding of structural and functional changes responsible for long-term synaptic alteration (reviewed in13Frank D.A Greenberg M.E Cell. 1994; 79: 5-8Abstract Full Text PDF PubMed Scopus (342) Google Scholar). From this brief overview, it is easy to appreciate that there are several sites where it is possible to interfere either with the induction of CREB or with its downstream effects. In Aplysia, interference with CREB activation has been accomplished by injecting into the nucleus of the sensory neuron either oligonucleotides containing CRE sites (7Dash P.K Hochner B Kandel E.R Nature. 1990; 345: 718-721Crossref PubMed Scopus (592) Google Scholar) or an antibody against the product of ApC/EBP (an IEG activated by cAMP and presumably by CREB; 1Albernini C.M Ghirardi M Metz R Kandel E.R Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (497) Google Scholar). In both cases, long-term synaptic facilitation was blocked while short-term facilitation was unchanged. In Drosophila, CREB function has been disrupted using a reverse genetic approach. A Drosophila CREB gene (dCREB2) that produces several isoforms has recently been cloned: dCREB2-a is an activator of PKA response transcription, whereas dCREB2-b is a blocker (24Yin J.C.P Wallach J.S Wilder E.L Klingensmith J Dang D Perrimon N Zhou H Tully T Quinn W.G Mol. Cell. Biol. 1995; 15: 5123-5130Crossref PubMed Scopus (91) Google Scholar). When expression of a dominant negative CREB transgene was transiently induced prior to training22Yin J.C.P Wallach J.S Del Vecchio M Wilder E.L Zhou H Quinn W.G Tully T Cell. 1994; 79: 49-58Abstract Full Text PDF PubMed Scopus (830) Google Scholar found that only long-term memory for olfactory learning was blocked; other forms of memory were unaffected. As mentioned at the outset, two recent reports have moved the molecular analysis of memory in Drosophila and Aplysia to the next level, for in both of these papers gain of function has been demonstrated: long-term memory or its cellular analog has been enhanced by functional activation of CREB. The first of these two papers appeared in the April, 1995, issue of Cell (23Yin J.C.P Del Vecchio M Zhou H Tully T Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (563) Google Scholar). In this report, Yin and colleagues first describe behavioral experiments examining the effects of multiple training sessions and different rest intervals in producing long-term memory in the olfactory conditioning paradigm. Confirming and extending previous work (20Tully T Preat T Boynton S.C Del Vecchio M Cell. 1994; 79: 35-47Abstract Full Text PDF PubMed Scopus (721) Google Scholar), they found that for the protein synthesis–dependent long-term memory (7 day retention) to become asymptotic, a minimum of 10 training sessions and a rest interval of about 10 min between sessions was required. No number of massed training trials produced long-term memory. They next examined memory in transgenic flies carrying a heat shock–inducible activator CREB isoform (hs-d CREB2-a). In the absence of heat shock, 1, 2, or 10 massed sessions failed to produce long-term memory in wild-type or transgenic flies, whereas 10 spaced sessions produced maximal memory in both groups. Three hours after heat shock, 10 spaced sessions still produced normal long-term memory in wild-type and transgenic animals; however, 1, 2, or 10 massed sessions now produced maximal memory in transgenic animals, but not in wild-type. These results show that heat shock itself has no adverse effects on memory (with a 3 hr recovery period), that memory after spaced training was normal in transgenic flies, and that long-term memory formation was enhanced (i.e., was induced more rapidly) in transgenic animals after heat shock induction of the CREB activator transgene. Finally, the authors showed that enhanced memory depends upon phosphorylation of the CREB activator isoform by generating transgenic dCREB2-a flies with a mutation at a PKA phosphorylation site. These animals failed to show heat shock–induced memory enhancement with a single training session, while in the same experiment the expected enhancement was again observed in CREB activator transgenic animals. As will be discussed below, based on these findings and their previous work, Yin et al. proposed a model suggesting that opposing functions of CREB activators and repressors ultimately govern the kinds of training parameters that will give rise to long-term memory (Figure 1). The second report appeared in a December, 1995, issue of Cell (4Bartsch D Ghirardi M Skehel P.A Karl K.A Herder S.P Chen M Bailey C.H Kandel E.R Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (496) Google Scholar). In this paper, Bartsch and colleagues first describe experiments in which they cloned an inhibitory form of CREB in Aplysia. They used a yeast two-hybrid system with the C-terminal portion of ApC/EBP as bait to screen an Aplysia CNS-specific cDNA library, and they landed a big fish: an inhibitory CREB form (ApCREB2) that resembles human CREB2 and mouse ATF4 but is not homologous to Drosophila dCREB2-b. They found that ApCREB2 is constituitively expressed in Aplysia sensory neurons and is a substrate for a variety of protein kinases, including PKA, PKC, MAP kinase, and CaM kinase. They also found that ApCREB2 is a repressor of ApCREB1, a transcriptional activator in mouse F9 cells; their data were consistent with the possibility that the repressor (ApCREB2) and activator (ApCREB1) may interact directly on a CRE. Having characterized these molecular features of ApCREB2, Bartsch and colleagues (1995) then asked the important question of whether interfering with the actions of this repressor form of CREB could actually enhance long-term synaptic facilitation. To examine this question, they injected anti-ApCREB2 antibodies into the nucleus of sensory neurons 1 hr before single or multiple exposures to 5-HT. Recall that, normally, a single 5-HT exposure produces only short-term facilitation lasting minutes; long-term facilitation requires multiple exposures (18Montarolo P.G Goelet P Castelucci V.F Morgan J Kandel E.R Schacher S Science. 1986; 234: 1249-1254Crossref PubMed Scopus (604) Google Scholar, 11Emptage N.J Carew T.J Science. 1993; 262: 253-256Crossref PubMed Scopus (173) Google Scholar). However, when a single pulse of 5-HT was paired with injection of the ApCREB2 antiserum, long-term (24 hr) synaptic facilitation was produced. Moreover, long-term facilitation produced in this fashion required both RNA and protein synthesis; injection of the anti-ApCREB2 antibody occluded the effects of five pulses of 5-HT, implying that maximum facilitation was already induced by the single 5-HT pulse in conjunction with injection of the antiserum. Finally, this form of long-term facilitation was accompanied by an increase in sensory neuron varicosities contacting motor neurons in culture. Collectively, these data show that relief of ApCREB2 repression converts a 5-HT-induced process that normally gives rise to short-term facilitation into long-term functional and structural changes in sensory neurons. Bartsch and colleagues do not yet know the specific mechanisms of functional repression by ApCREB2; however, a working model is shown in Figure 2. The authors suggest that under basal circumstances CREB2 maintains CREB1 in a repressed state, and that repeated pulses of 5-HT might activate kinases (or phosphatases) that phosphorylate (or dephosphorylate) CREB2, thereby relieving CREB2's repressor actions and allowing CREB1 to form homodimers and bind to adaptor proteins, ultimately inducing transcription of cAMP-inducible genes. Many behavioral experiments show that spaced training with intercalated rest intervals usually produces better long-term memory than equivalent (massed) training without rest (6Carew T.J Pinsker H.M Kandel E.R Science. 1972; 175: 451-454Crossref PubMed Scopus (255) Google Scholar, 23Yin J.C.P Del Vecchio M Zhou H Tully T Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (563) Google Scholar). The Drosophila model in Figure 1 provides a possible explanation for this fundamental property of memory. The idea proposed by 23Yin J.C.P Del Vecchio M Zhou H Tully T Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (563) Google Scholar is that associative training induces both activator and repressor isoforms of CREB. Right after training, enough repressor is active to block downstream CREB-induced events that are required for long-term memory. The key to the model is the differential activities of CREB repressors and activators during the rest interval. In the simplest case, CREB repressor isoforms are posited to inactivate more quickly than the activators, so a net amount of functional activator (ΔC = activators − repressors) accumulates over spaced trials, ultimately inducing long-term memory. Massed trials don't work because the rapidly occurring next trial (with no rest) reinstates repressor activation, thereby yielding a zero sum molecular game between repressors and activators. Although quite simple, this model yields several important predictions, all of which have been confirmed in Drosophila (23Yin J.C.P Del Vecchio M Zhou H Tully T Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (563) Google Scholar). Memories are particularly striking when they are induced after only a single event, usually one that is quite salient or emotionally charged. Bartsch and colleagues (1995) suggest a way to envision possible molecular events giving rise to these "flashbulb" memories. They point out that emotional stimuli are processed through recruitment of many modulatory transmitter systems in the brain, some of which could act to relieve CREB2-like repression. They suggest that this could prime the memory system for action so that a single event could have immediate access to the processing machinery involved in encoding long-term memory. As the work presented in this review attests, important progress has been made in Drosophila and Aplysia, implicating a particular class of transcription factors in memory formation. This progress takes on added significance in light of the recent demonstration that CREB-induced transcription may play an important role in long-term memory induction in mice as well (5Bourtchuladze R Frenguelli B Blendy J Cioffi D Schutz G Silva A.J Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1577) Google Scholar). Despite this exciting progress, it is still premature to order a CREB cocktail and sign up for Jeopardy. Important questions remain. For example, in Drosophila a major step will be identifying the neurons and circuits specifically activated in memory formation, while in Aplysia it will be important to relate the CREB-induced changes in sensory neurons (and perhaps other neural elements) directly to the behavioral expression of long-term memory. In addition, since multiple signal transduction pathways can modulate CREB activation and repression, the current insights gained from Drosophila and Aplysia are likely to be only the tip of the molecular iceberg in memory formation; greater complexities are sure to emerge. But the good news is that we can realistically expect rapid progress as the behavioral, cellular, and molecular tools of the trade continue to be refined and expanded in a variety of experimental systems. The recent work in Drosophila and Aplysia provides an excellent case study of the kind of progress that can be made when different preparations bring their unique strengths to bear on fundamental molecular questions in memory formation.