Positive and negative regulatory mechanisms that mediate long-term memory storage 1

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1 r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž. Brain Research Reviews Positive and negative regulatory mechanisms that mediate long-term memory storage 1 Ted Abel, Eric Kandel ) Howard Hughes Medical Institute, Center for Neurobiology and BehaÕior, Columbia UniÕersity, 722 West 168th Street, New York, NY 10032, USA Abstract Ž. The protein kinase A pathway and the cyclic AMP-response element binding protein CREB appear to play a critical role in the consolidation of short-term changes in neuronal activity into long-term memory storage in a variety of systems ranging from the gill and siphon withdrawal reflex in Aplysia to olfactory conditioning in Drosophila to spatial and contextual learning in mice. In this review we describe the molecular machinery that mediates memory consolidation in each of these systems. One of the surprising findings to emerge, particularly from studies of long-term facilitation in Aplysia, is that memory storage is mediated by not only positive but also negative regulatory mechanisms, in much the same way as cell division is controlled by the proteins encoded by oncogenes and tumor suppressor genes. This suggests the interesting possibility that there are memory suppressor genes whose protein products impede memory storage. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Protein kinase A; Memory consolidation; Memory suppression; Behavior Contents 1. Introduction Long-term facilitation in Aplysia Molecular cascades during memory consolidation Spatial memory, long-term potentiation and the mammalian hippocampus Memory suppressor genes? CREB2: a repressor of long-term memory storage Cell adhesion molecules and structural changes The regulatory subunit of PKA: an inhibitor removed by proteolysis Memory storage involves positive and negative regulatory mechanisms References Introduction ) Corresponding author. 1 Published on the World Wide Web on 13 January One of the first distinguishing features in the biochemistry of long-term memory storage to be identified was the fact that inhibitors of transcription and translation selec-

2 ( ) T. Abel, E. KandelrBrain Research ReÕiews tively impaired long-term memory storage without affecting learning or short-term memory. This realization, first observed in studies of conditioned avoidance in rodents and goldfish w36 x, provided an important conceptual basis for much of the current research on the molecular basis of memory storage. In most of the species and behavioral paradigms studied, this requirement for ongoing RNA and protein synthesis had a strict time window, with long-term memory being most sensitive to disruption around the time of training. Animals experience no significant deficit in long-term memory if exposure to the protein synthesis inhibitor is delayed by as little as one hour after training. This distinctive requirement of long-term memory for protein and RNA synthesis during the consolidation period is evolutionarily conserved and is evident not only in vertebrates, but also in invertebrates such as Drosophila and Aplysia. Moreover, it holds for both explicit memory, the conscious memory for people, places and things, and implicit memory, the unconscious learning of perceptual and motor skills wx 2. The finding of this evolutionarily conserved time window for memory consolidation suggested that the transition from short-term to long-term memory storage requires gene induction. Specifically, the time window may correspond to a cascade of gene induction in which immediateearly genes, activated by constitutively expressed transcription factors, lead to the expression of downstream effector genes w47 x. To explore this idea, cellular correlates of this time window were sought and found in both hippocampal long-term potentiation in mammals w42,79x and long-term facilitation in Aplysia w72 x. At the cellular level, these processes share this requirement for protein and RNA synthesis during or immediately following induction, suggesting that training leads to the turning on of new genes in the appropriate cells in response to stimulation. Indeed, many immediate-early genes are induced in response to stimuli that lead to long-term potentiation in the mamw56 x. The importance of gene induction to memory consolida- malian hippocampus tion led to a variety of molecular models by which neuronal signals could initiate nuclear events, turning on new genes whose products are critical for facilitation w47,56 x. These models were guided in many ways by the ideas emerging from the study of development, oncogenesis and growth control. In these fields, transcriptional regulation plays a central role in the control of cell division and cell fate, with cascades of gene expression set into motion by the action of growth factors on receptors at the cell surface w19 x. The study of cell growth and differentiation, as exemplified by the identification of a wide variety of cellular oncoproteins, has defined a number of signal transduction pathways spanning from the cell membrane Ž e.g., growth factor receptors. through the cytoplasm Že.g., PKC, MAP kinase. to the nucleus Ž e.g., Jun, Fos.. Oncoproteins activate these pathways, as the result of loss of regulation or increased activity, leading to cell growth. The molecular analysis of the transition from short- to long-term memory storage has, in part, focused on analyzing the function of these signal transduction pathways in neurons. Three experimental systems have proven particularly useful: the gill and siphon withdrawal reflex in Aplysia, olfactory learning in Drosophila and spatial and contextual learning in mice. In each of these systems, the cyclic AMP-dependent protein kinase Ž PKA. pathway and the cyclic AMP-response element binding protein Ž CREB. appear to play a critical role in the consolidation of short-term changes in neuronal activity into long-term memory storage. In this review we will describe the molecular machinery that mediates this memory consolidation by focusing on these three systems. In the course of these studies, the surprising finding has emerged that long-term memory storage is mediated by not only positive but also negative regulatory mechanisms, in much the same way as cell division is controlled by the proteins encoded by oncogenes and tumor suppressor genes. This has suggested the interesting idea that there are memory suppressor genes. 2. Long-term facilitation in Aplysia In the marine snail Aplysia californica, the defensive withdrawal of the gill in response to stimulation of the siphon has been used to explore the cellular and molecular basis of behavioral sensitization, a nonassociative form of implicit memory w24,26x Ž Fig. 1.. As a result of sensitization, an animal learns to strengthen its reflex responses to a previously neutral stimulus, such as a weak stimulus to the siphon, following the presentation of an aversive stimulus, such as a shock to the tail. When Aplysia is presented with a noxious stimulus to the tail, the animal recognizes the stimulus as aversive and learns to enhance its gillwithdrawal reflex response to a weak stimulus applied to the siphon. The duration of the consequent memory is a function of the number of sensitizing stimuli applied to the tail w44 x. A single noxious stimulus to the tail produces short-term memory that lasts for minutes and does not require new protein synthesis, whereas four or five noxious stimuli produce long-term memory that lasts for days and requires new protein synthesis. A number of key cellular components of the neural circuit for the gill-withdrawal reflex have been identified Ž Fig. 1.. The tail stimuli that produce sensitization strengthen the reflex by activating at least three classes of facilitating interneurons, one of which is serotonergic w22,44,46 x. These interneurons act on the sensory neuron to enhance transmitter release at the sensory motor neuron synapse. Although this reflex has both monosynaptic and polysynaptic components, we have focused on the monosynaptic portion of the reflex circuit and used it to probe the mechanistic basis of short-term and long-term

3 362 ( ) T. Abel, E. KandelrBrain Research ReÕiews Fig. 1. The circuitry underlying the gill-withdrawal reflex in the marine snail Aplysia. Ž A.. A tactile stimulus to the siphon leads to the reflex withdrawal of the gill. Sensitization of this reflex is produced by a noxious stimulus applied to the tail. Ž B.. Sensory neurons, which are activated by stimuli to the tail, stimulate excitatory interneurons, some of which are serotonergic. These interneurons synapse on terminals of sensory neurons innervating the siphon skin, enhancing transmitter release by presynaptic facilitation. Adapted from Ref. w59 x. memory because it can be reconstituted in culture w72,82 x. Both short-term and long-term sensitization share this locus, and the changes in this synapse track behavioral memory. In culture, long-term facilitation is produced by applying serotonin Ž 5-hydroxytryptamine, 5-HT., which would normally be released in vivo by interneurons activated by stimulation of the tail. Like behavioral sensitization, long-term facilitation at the sensory motor neuron synapse in culture varies with the number of 5-HT treatments. One pulse of 5-HT produces a synaptic enhancement that is transient and not affected by inhibitors of protein and RNA synthesis, but five pulses produce a long-term Ž 24 h. increase in synaptic strength that is blocked by inhibitors of protein and RNA synthesis w72 x. Thus, long-term facilitation, much like long-term memory, requires proteins and genes not crucial for the short-term process. How is the short-term process set up, and how is it converted to the long-term process with repeated application of 5-HT? A single shock to the tail, or one pulse of 5-HT, activates 5-HT receptors, some of which engage adenylyl cyclase, leading to an increase in camp and the activation of PKA w25 x. Other 5-HT receptors activate PKC w25 x. Both PKA and PKC enhance transmitter release by closing potassium channels, increasing the calcium influx which is important for transmitter release and acting directly on the release machinery itself. The first clue to the role of PKA in the long-term process came from the demonstration that long-term storage is sensitive to inw72 x, suggesting that hibitors of RNA and protein synthesis a PKA-responsive transcription factor may be crucial for this process. Indeed, treatment with five pulses of 5-HT leads to the induced expression of a variety of genes including the immediate-response genes C r EBP Ž ApC r EBP. wx 3 and ubiquitin carboxyl-terminal hydrolase Ž Apuch. w50 x, as well as the late effector genes BiP w64x and calreticulin w62 x. Experiments using fluorescent ratio imaging to track the catalytic subunit of PKA demonstrated that

4 ( ) T. Abel, E. KandelrBrain Research ReÕiews the catalytic subunit translocates to the nucleus of the sensory neurons in response to repeated pulses of 5-HT wx 4. In addition, MAP kinase also translocates to the nucleus during long-term facilitation w68 x. In eukaryotic cells, CREB is a transcription factor thought to be critical for the induction of gene expression in response to the activation of PKA w86 x. PKA phosphorylation of CREB1 on serine 119 converts this protein into a transcriptional activator, although other kinases, including MAP kinases and CaM kinases, can also modulate the activity of CREB1 w38,86,96 x. Studies of CREB proteins in Aplysia provided the initial evidence that transcriptional regulation by PKA is a key event in the induction of long-term memory storage. This evidence for a role for CREB proteins in long-term facilitation came from Dash et al. w35 x, who injected an oligonucleotide containing the camp-response element from the somatostatin gene into the nucleus of sensory neurons and demonstrated that this treatment blocked long-term facilitation in culture without affecting short-term facilitation. Further, the injection of oligonucleotides containing binding sites for other transcription factors, including NF-kB and HSF, did not affect long-term facilitation. Although experiments using the kinase-inducible domain Ž KID domain or P box. from mammalian CREB has shown that CREB is indeed phosphorylated on serine 119 during long-term facilitation w58 x, the study of the mechanistic role of CREB has required the cloning of Aplysia CREB1 Ž ApCREB1. w13,14 x. Importantly, the injection of antibodies to ApCREB1 or antisense oligonucleotides to ApCREB1 into the sensory neuron selectively blocked long-term facilitation. Like mammalian CREB1, the ApCREB1 gene gives rise to several isoforms. The sensory motor neuron culture system provides an excellent physiological system in which to explore the specific functional role of each of these isoforms. How general is the CREB-triggered cascade of gene activation? The first evidence has come from Drosophila. Earlier work in Drosophila had shown that, as in Aplysia, learning and short-term memory involve camp and PKA. Three memory mutations that have been analyzed in molecular detail involve a step in the camp cascade, and the induced expression of an inhibitor of PKA using a heat shock promoter blocks learning w37 x. The mutation dunce involves a defect in the camp phosphodiesterase, rutabaga is defective in a calciumrcalmodulin-dependent adenylyl cyclase and amnesiac is a defect in a neuropeptide whose receptor stimulates adenylyl cyclase. More recently, Jerry Yin, Tim Tully, and Chip Quinn and their colleagues have shown that spaced training gives rise to memory that lasts at least seven days and is blocked by inhibitors of protein synthesis w94 x. This long-term memory is selectively blocked by the heat shock-induced expression of a dominant negative inhibitor of CREB w98 x. Thus, two forms of long-term implicit memory require CREB- and camp-induced gene expression w39 x. 3. Molecular cascades during memory consolidation In order to explore the changes in protein levels that occur in Aplysia sensory neurons in response to repeated exposure to 5-HT, Barzilai et al. w15x used quantitative two-dimensional gel electrophoresis to visualize 35 S- methionine labeled proteins from Aplysia sensory neurons in culture before and after 5-HT treatment. They found three temporally distinct sets of changes in the levels of specific proteins in response to 5-HT. A group of 10 proteins are rapidly and transiently induced within the first hour after repeated exposure to 5-HT, or after treatment with camp. These early changes are followed by two waves of change in other proteins. Levels of a second group of proteins peak transiently at three hours, while levels of a third group of proteins increase more persistently and over a slower time course. The induction of each of these sets of proteins requires ongoing RNA synthesis, suggesting that these changes occur at the level of transcriptional regulation. Thus, long-term facilitation in Aplysia may involve a cascade of gene activation in much the same way as immediate-early gene products are inw47,56 x. To identify the immediate-early genes that are recruited duced by growth factors during these waves of protein synthesis, Alberini et al. wx 3 and Hedge et al. w50x carried out two separate screens. Alberini et al. wx 3 focused on the CCAATrenhancer-binding protein Ž CrEBP. family of transcription regulatory proteins. Members of the CrEBP family of transcription factors contain a bipartite DNA binding domain, the basic leucine zipper Ž bzip. domain, which mediates DNA bindwx 1. CrEBPs are ing and homo- or hetero-dimerization involved in the terminal differentiation of a variety of cells and some members of the CrEBP family are responsive to camp whereas others act to regulate transcription from CREs w65 x. Aplysia CrEBP Ž ApCrEBP., which was isolated by expression cloning, contains a bzip domain that is 46% identical to the bzip domain of rat CrEBP. Importantly, a nonpalindromic CRE site is located in the promoter region just upstream from the TATA box of ApCr EBP gene, suggesting that ApC r EBP is an immediate-response gene. Indeed, ApCrEBP mrna levels increase in response to treatment with 5-HT or elevating camp levels by treatment with forskolin and IBMX. The induction of this mrna occurs even in the presence of protein synthesis inhibitors, an important characteristic feature shared by other immediate-early genes like fos, jun and zif268 w56 x. Thus, some of the genes that appear to be activated by CREB are themselves transcription factors implicating a cascade of transcription factors. Differential screens carried out to identify genes induced during long-term facilitation have identified a neuron-specific form of ubiquitin carboxyl-terminal hydrolase Ž Ap-uch. that is rapidly induced within 2 h following 5-HT treatment w50 x. This enzyme, which removes ubiquitin from multiubiquitinated substrates during proteolysis

5 364 ( ) T. Abel, E. KandelrBrain Research ReÕiews by the proteasome, appears to be a rate-limiting component of the ubiquitin-proteasome pathway. It associates with the proteasome and enhances its proteolytic activity. One might therefore predict, by analogy to the protein synthesis inhibition experiments, that blocking the action of the immediate-early gene products ApCrEBP or Ap-uch should block the long-term process. Indeed, inhibiting the function or expression of the hydrolase by injecting antibody or antisense oligonucleotide into the sensory neuron in the coculture system blocks the induction of long-term facilitation without affecting short-term processes w50x Ž Fig. 2.. Blocking the expression, activity, or function of ApCrEBP also selectively inhibits the long-term process wx 3. This cascade of immediate-response genes is interesting because it marks ApCrEBP and Ap-uch as part of the first protein synthesis-dependent steps in the initiation of the long-term process. By injecting an oligonucleotide containing an ApCrEBP binding site into sensory neurons at various times after 5-HT treatment, Alberini et al. wx 3 determined how long ApCrEBP activity is required. When injected 1, 6, or 9 h after 5-HT treatment, the oligonucleotide containing the ApCrEBP binding site reduced the observed facilitation measured 24 h after 5-HT treatment. When injected into the sensory neuron at 12 h after 5-HT application, however, the oligonucleotide no longer affected facilitation. Fig. 2. Injection of an antibody or antisense oligonucleotides to Aplysia ubiquitin carboxyl-terminal hydrolase into the presynaptic sensory neuron blocks long-term facilitation Ž A., but does not affect short-term facilitation Ž B.. Ab, immune antiserum; Pre, preimmune antiserum; As, antisense oligonucleotide; ScrAs, control scrambled antisense oligonucleotide. From Ref. w50 x.

6 ( ) T. Abel, E. KandelrBrain Research ReÕiews Thus, by 12 h after 5-HT treatment, synaptic facilitation has become independent of the action of the immediateearly transcription factor ApCrEBP. By characterizing individual genes, Alberini et al. wx 3 and Hedge et al. w50x have begun to define the consolidation period in molecular terms, showing that blocking one protein can substitute for a general inhibitor of protein synthesis. That ApC r EBP and AP-uch are induced during the time window for protein synthesis and that their expression is essential for the establishment of a self-maintained long-term process suggests that ApCrEBP, Ap-uch and other immediate-response genes represent the molecular components of the consolidation phase, leading to the expression of effector genes which are important for the stabilization of long-term memory storage. This stabilization appears to be achieved by the growth of new synaptic connections, and some of these effector genes may play an important role in the formation of new synapses w11,53 x. 4. Spatial memory, long-term potentiation and the mammalian hippocampus We have so far considered only a simple, reflexive, implicit form of learning found in the invertebrate Aplysia. What about more complex, explicit forms of learning found in mammals? The sort of explicit memory storage we associate with our memories for people, places, and objects requires the hippocampus and the medial temporal lobe system w32 x. With the development of techniques such as targeted gene ablation and transgenesis, it has become clear that mice offer a superb genetic system for determining the role of individual gene products in synaptic plasticity and memory storage. In rodents, spatial and contextual learning is particularly well documented and these forms of explicit learning are sensitive to lesions of the hippocampal formation. Several physiological properties of the rodent hippocampus are also of potential importance as cellular mechanisms underlying memory storage. First, the hippocampus has a cellular learning mechanism longterm potentiation Ž LTP. that is thought to be involved in at least some aspects of spatial memory w20 x. The hippocampus also contains a cellular representation of space. Individual hippocampal-pyramidal cells have place fields, and they fire action potentials only when the animal is in a certain location in space w77 x. Hippocampal LTP is a long-lasting increase in synaptic strength that occurs in response to brief, repetitive stimulation. LTP exhibits synapse specificity and associativity because it occurs only when presynaptic activity is paired with postsynaptic depolarization, a characteristic that can be explained on the molecular level by the fact that the NMDA subtype of glutamate receptor is both ligand-gated and voltage-sensitive. Combined with its longevity Žup to 14 h in hippocampal slices and weeks in vivo., this synapse specificity and associativity of LTP have made it an attractive model for memory storage. Importantly, many forms of hippocampal LTP share a dependence on NMDA receptor function with many forms of spatial memory. Thus, the NMDA receptor antagonist APV blocks Schaffer collateral LTP in hippocampal slices and impairs spatial memory when injected into the hippocampus w74,75 x. In addition to the pharmacological dependence on the NMDA receptor, there are now a number of suggestive correlations between LTP and memory storage from gene knockout experiments. The strongest correlation between LTP and spatial memory comes from the study of mice in which the R1 subunit of the NMDA receptor was knocked out in a regionally restricted fashion, only in hippocampal area CA1 w93 x. These mice have impaired Schaffer collateral LTP and deficits in spatial memory, providing evidence supporting an important role for hippocampal area CA1, as suggested earlier by the study of the patient R. B. by Squire and colleagues w84 x. However, these mutant mice also have impaired long-term depression Ž LTD. w93 x, and thus the nature of the synaptic plasticity deficit underlying their behavioral abnormality is unclear. In addition, it will be interesting to test these animals in nonspatial forms of hippocampus-dependent learning such as contextual fear conditioning w63,81x and olfactory-based tasks such as social transmission of food preferences w23,95 x. Thus, the early steps of LTP are initiated postsynaptically by Ca 2q influx through the NMDA receptor and the subsequent activation of several protein kinases. The study of other genetically modified mice has focused on this early, transient phase of LTP Ž E-LTP. in hippocampal area CA1 that lasts about an hour, and these studies have shown that genetic manipulation of any one of several kinases interferes with not only E-LTP, but also short-term memw29,69 x. The study of amnestic patients and experimen- ory tal animals has revealed, however, that the role of the hippocampus in memory storage extends from weeks to months w90 x, suggesting that longer lasting forms of hippocampal synaptic plasticity may be required. We have therefore examined the relation between LTP and spatial learning by looking specifically at long-lasting forms of LTP and exploring the relationship of these forms of LTP to long-term memory for complex spatial and contextual tasks. There are three major pathways in the hippocampal trisynaptic circuit: the medial perforant pathway between the entorhinal cortex and dentate gyrus granule cells, the mossy fiber pathway between dentate gyrus granule cells and CA3 pyramidal cells and the Schaffer collateral pathway between CA3 and CA1 pyramidal cells Ž Fig. 3.. LTP in each of these pathways has phases w55x and the late phase of LTP Ž L-LTP. can be distinguished from E-LTP by varying the number of stimulus trains. Although the early phase of LTP differs in each of these pathways, in every case the late phase requires repeated bursts of tetanic stimulation, ongoing RNA and protein synthesis and PKA activity.

7 366 ( ) T. Abel, E. KandelrBrain Research ReÕiews Fig. 3. Long-term potentiation in the Schaffer collateral pathway of the mammalian hippocampus. Ž A.. The hippocampus contains three major pathways, the perforant pathway, the mossy fiber pathway and the Schaffer collateral pathway. Ž B. The early phase of LTP Ž E-LTP. is induced in the Schaffer collateral pathway by a single 1 s, 100 Hz tetanus. The late phase of LTP, which is induced by four 1 s, 100 Hz trains separated by 5 min differs from E-LTP in requiring PKA activity, as well as RNA and protein synthesis. Adapted from Ref. w59 x. With the repeated trains of stimulation necessary to induce L-LTP in area CA1, camp levels increase w30,41 x. This is mediated through the NMDA receptor and is thought to be secondary to the action of Ca 2q on a Ca 2q -sensitive adenylyl cyclase. The increase in camp is followed by the activation of PKA and CREB. Indeed, stimuli that induce L-LTP also induce the expression of a CRE-lacz reporter gene w57 x. Furthermore, knockout mice lacking the a and D isoforms of CREB have long-term memory deficits w21 x. These CREB knockouts, however, have deficits in LTP induced by a single stimulus train as well as altered basal synaptic transmission. To investigate genetically the relationship between this form of synaptic plasticity and behavioral memory, we have taken a transgenic approach to reduce PKA activity in the hippocampus by using RŽ AB., a dominant negative form of the RIa regulatory subunit of PKA wx 2. RŽ AB. carries mutations in both camp binding sites and acts as a dominant inhibitor of both types of PKA catalytic subunits w31 x. To limit expression of RŽ AB. to the postnatal forebrain, we have used the promoter from the CaMKIIa gene w70 x, a promoter that drives a high level of expression postnatally only in neurons within the forebrain. The RŽ AB. transgene functioned in an inhibitory manner, resulting in a 50% reduction in hippocampal PKA activity. The RŽ AB. transgenic mice did not significantly differ from wild-type animals in basal synaptic transmission or in short-term plasticity. E-LTP induced by applying one or two 100 Hz trains of stimulation to the Schaffer collateral pathway is independent of PKA activity w54x and was unchanged in RŽ AB. transgenic animals. By contrast, L- LTP induced by repeated tetanization Žfour 100 Hz trains, 1 s duration, spaced 5 min apart. of the Schaffer collateral pathway was impaired in slices obtained from RŽ AB. transgenic mice Ž Fig. 4.. The observation that E-LTP induced by one or two stimulus trains is unchanged in the

8 ( ) T. Abel, E. KandelrBrain Research ReÕiews Fig. 4. The late phase of LTP is selectively impaired in two different lines of transgenic mice expressing RŽ AB., an inhibitory form of the regulatory subunit of protein kinase A. LTP induced by one Ž A. or two Ž B. 1 s, 100 Hz trains is unchanged in the RŽ AB. transgenic mice. Ž C. The late phase of LTP induced following four 1 s, 100 Hz trains 5 min apart, was significantly impaired in both lines of transgenic animals. From Ref. wx 2. RŽ AB. transgenics whereas L-LTP is reduced suggests that L-LTP, unlike E-LTP, requires PKA and recruits distinct signalling pathways immediately following tetanization. In mammals, the hippocampus is essential for the initial consolidation of explicit, or declarative, memory w89 x. Our analysis of hippocampal function in the RŽ AB. transgenic animals initially focused on the hidden platform version of the Morris water maze task w76 x. Transgenic animals improved during training, but when tested for memory in a probe trial, they exhibited spatial memory deficits. The Morris maze task requires repeated training over several days and does not, therefore, provide the temporal resolution necessary to distinguish the different phases of memory storage. To define more precisely the time course of the memory deficit in RŽ AB. transgenics, we turned to contextual and cued fear conditioning tasks, in which learning can be accomplished by a single trial w63,81 x. This good temporal resolution that allows one to follow a memory over time. The RŽ AB. transgenics exhibited normal short-term memory, consistent with normal E-LTP, but deficient long-term memory for contextual fear conditioning Ž Fig. 5.. The time course of the memory deficit of RŽ AB. transgenics in contextual fear conditioning parallels that of wild-type animals treated with the protein synthesis

9 368 ( ) T. Abel, E. KandelrBrain Research ReÕiews Fig. 5. The genetic reduction of hippocampal PKA activity selectively impairs long-term memory for contextual fear conditioning Ž A., while not significantly impairing cued conditioning Ž B.. From Ref. wx 2. inhibitor anisomycin. By contrast, the long-term memory for cued conditioning, a task which is mediated by the amygdala, is not disrupted in RŽ AB. mice. Our findings strengthen the idea of multiple memory systems and show that genetic inhibition of PKA has major consequences for one memory system, but not for another. The long-term memory deficits in RŽ AB. transgenic mice demonstrate that PKA plays a role in the hippocampus in initiating the molecular events leading to the consolidation of short-term changes in neuronal activity into long-term memory. In the hippocampus, as in Aplysia, a late phase of synaptic plasticity has been characterized in physiological, molecular and genetic terms. Altering this late phase is reflected selectively in deficits in hippocampus-dependent long-term memory. In both implicit and explicit forms of learning, then, there is evidence for at least two stages of memory, short-term and a long-term storage. Further, these memory stages have a representation on the cellular level: there is a short-term form that involves a covalent modification of preexisting proteins, and a long-term phase that involves transcription. Thus, molecular studies of cognition have revealed that, although different learning mechanisms may recruit different signal transduction pathways for short-term memory storage, they recruit a common set of genetic mechanisms for the long-term process, which involves PKA and the activation of transcription by CREB1. 5. Memory suppressor genes? Our discussion thus far has focused on the positive regulatory mechanisms involved in the switch from shortterm to long-term synaptic facilitation. This emphasis on positive regulatory mechanisms is also seen in many studies of growth regulation and development that have focused on oncogenes, such as myc and src, which encode proteins that act in a dominant fashion to lead to cell division w19 x. Oncogenes, which were originally identified as genes carried by viruses that cause the transformation of target cells, were subsequently found to result from a mutation of normal cellular genes. Tumorigenesis results from the increased or altered activity of a gene product, leading to misregulation in some component of the cellular pathways that regulate growth. The study of growth control, however, has also led to the discovery of a group of genes whose products act to suppress cell division. Certain cancers, such as a tumor of the retina known as retinoblastoma, are caused by the loss of both alleles at a locus. These recessive diseases, which can be inherited through the germline or occur as the result of somatic mutation, are caused by the inactivation of tumor suppressor genes, whose products normally prevent cell division. Tumor suppressor genes, such as retinoblastoma and p53, were identified as loss-of-function mutations that allow increased cell proliferation. Recent work has also revealed that negative regulatory mechanisms are also important during development, preventing cells from going down certain differentiation pathways. These findings have raised the question: Do such negative regulatory mechanisms, driven by memory suppressor genes, also play a role in modulating memory storage? Recent work has revealed that the switch from shortterm to long-term facilitation in Aplysia involves the inactivation of gene products that otherwise would act to inhibit memory consolidation. Long-term facilitation requires not only positive regulatory mechanisms driven by factors such as CREB1, but also the concomitant removal of several inhibitory constraints. Three major inhibitory constraints have been identified so far in Aplysia. These include the relief of repression by CREB2 w14 x, the persistent activation of PKA as a result of the regulated proteolyw18,48x and the sis of the regulatory subunits of PKA removal of an inhibition of synaptic growth as a result of

10 ( ) T. Abel, E. KandelrBrain Research ReÕiews the down regulation of the cell adhesion molecule apcam by endocytotic degradation w9,10 x. We consider each of these in turn. 6. CREB2: a repressor of long-term memory storage Experiments directed at the molecular characterization of CREB in Aplysia have provided the clearest evidence for the existence of repressive mechanisms that impede memory storage. The CREB family of transcriptional regulatory proteins belongs to the larger group of basic leucine zipper Ž bzip. DNA binding proteins. These proteins form homo- or heterodimers through the leucine zipper domain and interact in a sequence specific manner with the major groove of DNA through the basic region wx 1. Bartsch et al. w14x cloned ApCREB2, a bzip protein that differs from CREB1 in that it lacks the kinase-inducible domain ŽP box. which in CREB1 is phosphorylated by PKA, leading to transcriptional activation. Like CREB1, ApCREB2 is constitutively expressed and its levels are not altered by treatment with 5-HT. Within the CREB family, ApCREB2 is most closely related at the sequence level to mouse ATF4 and human CREB2. Human CREB2 has been shown to be a repressor of CREB1 transcriptional activity w60 x, and in cotransfection experiments in F9 cells, ApCREB2 represses the PKA-dependent activation of ApCREB1. In addition, ApCREB2 contains a transcriptional activation domain and it can activate transcription in the absence of ApCREB1 from a reporter containing multiple CRE binding sites. How, then, does ApCREB2 regulate transcription in Aplysia sensory neurons? The exact mechanism remains unclear, but recent experiments have suggested a model w13,14 x. ApCREB2 forms effective heterodimers with ApCREB1 on an asymmetrical CRE. By contrast, ApCREB2 homodimers have a much lower affinity for the CRE than ApCREB1 homodimers or ApCREB1r ApCREB2 heterodimers. It therefore appears that ApCREB2 does not compete with ApCREB1 for binding to the CRE, nor does the binding of ApCREB2 to ApCREB1 interfere with the binding of the resulting complex to the CRE. Rather, ApCREB2 may inhibit the activation of ApCREB1 by forming a heterodimer in which the activating domains on both proteins are masked. This suggests the possibility that the threshold for longterm facilitation is highly regulated at its earliest steps and implies that the long-term process is under dual control: it is activated by ApCREB1 and repressed by ApCREB2. Turning genes on during the consolidation period therefore involves at least two steps. First, ApCREB1 is activated by PKA. Second, repression by ApCREB2 is relieved by means of some other covalent modification, perhaps MAP kinase or protein kinase C, both of which phosphorylate ApCREB2 in vitro. If this is so, then relieving the repression might facilitate the activation process and lower the threshold for the long-term process. To test these ideas and to determine if ApCREB2 could act as a functional repressor of long-term facilitation in sensory motor neuron cocultures in parallel to its action as a transcriptional represw14x injected ApCREB2 antiserum into sor, Bartsch et al. sensory neurons one hour before exposure to single or multiple pulses of 5-HT. After injection of ApCREB2 antiserum, a single pulse of serotonin that normally produces only short-term now produced long-term facilitation Ž Fig. 6.. This long-term process produced by one pulse of Fig. 6. One pulse of 5-HT produces long-term facilitation when paired with the injection of ApCREB2 antiserum into the presynaptic sensory neuron. Ž A. Time course of changes in the L7 motor neuron EPSP amplitude after one and five treatments with 5-HT or after one pulse of 5-HT and injection of anti-apcreb2 antibodies. Long-term facilitation produced by this pairing is sensitive to the RNA synthesis inhibitor actinomycin and the protein synthesis inhibitor anisomycin. Ž B. Sample EPSPs recorded in the L7 motor neuron. Injection of preimmune serum or depleted immune serum does not induce long-term facilitation when paired with one 5-HT treatment. From Ref. w14 x.

11 370 ( ) T. Abel, E. KandelrBrain Research ReÕiews HT after the injection of ApCREB2 antiserum into the sensory cell phenocopies the long-term process produced by five pulses. This long-term process requires translation, transcription, and occludes the effects of five pulses of 5-HT. CREB plays a critical role in another form of invertebrate learning, the conditioned odor avoidance response in Drosophila w97,98 x. Induced expression of an inhibitory form of CREB disrupts long-term memory for this task, without affecting other aspects of learning w98 x. The induced expression of an activator isoform of CREB in Drosophila enhances the formation of long-term memory w97 x. In much the same way as blocking the action of ApCREB2 converts the transient facilitation induced by one pulse of serotonin into long-term functional and structural changes, long-term memory is achieved in transgenic flies expressing an activator isoform of CREB after only a single training session. Importantly, this effect of CREB depends upon the phosphorylation of the transgene because it is not seen in transgenic flies expressing a mutant activator isoform in which the consensus PKA phosphorylation site is disabled. CREB, then, appears to be a critical switch for long-term memory formation, and increasing the activity of CREB, by overexpressing an activator isoform or inhibiting a repressor isoform, results in one-trial learning. Humans also exhibit photographic memory, often in association with historical events or important, often emotionally charged, personal experiences. Such flashbulb memories can be especially detailed and vivid recollections that are retained for a lifetime w33 x. It is intriguing to speculate that the CREB family of transcriptional regulatory molecules may play a role in these forms of photographic memory w14 x. 7. Cell adhesion molecules and structural changes How are these modulations of signal transduction pathways and the induction of new genes by ApCREB1 and ApCrEBP converted into stable long-term memory? Cellular studies of memory storage in both Aplysia and mammals suggest that what sustains memory is the growth of new synaptic connections w11 x. In the gill-withdrawal reflex of Aplysia, long-term sensitization involves structural changes at the sensory motor neuron synapse with five pulses of 5-HT resulting in a 50% increase in the number of varicosities w5 8 x. This growth of new synaptic connections between the sensory and motor neurons can be induced in the intact ganglion by the intracellular injection of camp w78x and by treatment with repeated pulses of 5-HT in coculture w45 x. The appearance of new synapses parallels the synaptic facilitation, and, like long-term facilitation and behavioral memory, these structural changes are blocked by inhibitors of RNA and protein synthesis w12 x. Interestingly, these same structural changes are observed during the long-term facilitation induced by one pulse of 5-HT after the injection of ApCREB2 antiserum. By fluorescently labelling sensory neuron varicosities, Bartsch et al. w14x demonstrated that the pairing of a single pulse of 5-HT with the injection of ApCREB2 antiserum induces a 60% increase in the number of presynaptic varicosities. Thus, ApCREB2 acts as a repressor of the morphological as well as the functional changes that accompany long-term facilitation. By combining selective intracellular labelling techniques with the analysis of serial sections, Bailey and Chen w5 8x reconstructed identified sensory neuron synapses from control animals and animals after behavioral sensitization. They described structural alterations induced by long-term sensitization at two distinct levels of synaptic organization, both of which occur in the presynaptic cell. First, sensitization leads to a larger number, size, and vesicle complement of sensory neuron active zones. Second, a more widespread alteration involves a twofold increase in the total number of presynaptic varicosities and an expansion of the axonal arbor of each sensory neuron. Although these structural changes share a requirement for ongoing macromolecular synthesis with behavioral senw12 x, which, if any, of these structural changes at sitization the sensory neuron synapse is important for long-term memory storage? As a first approach to answer this queswx 8 compared the time course for tion, Bailey and Chen each morphological change with the behavioral duration of sensitization. The increase in the size and vesicle complement of the sensory neuron active zone was more transient than the memory, returning to baseline levels within one week. Increases in the number of active zones and varicosities persisted for one week and were only partially reversed after three weeks, suggesting that an increase in synapse number is the most likely structural change to contribute to the maintenance of long-term sensitization. The importance of these structural changes to synaptic plasticity is further underscored by the observation that the neuropeptide FMRFamide, which causes long-term depresw73 x, leads to the loss of sion at sensory motor synapses neurites and synaptic varicosities on the presynaptic senw87 x. Changes in gene and protein expression set in motion sory neuron by the covalent modification of preexisting proteins, such as CREB, result in the growth of new synaptic connections. The first insight into the molecules that may contribute to these structural changes came from Barzilai et al. w15 x, who examined changes in specific proteins in Aplysia sensory neurons in response to repeated applications of 5-HT, and identified a group of five proteins whose levels decrease within the first hour after 5-HT treatment. Mayw71x used monoclonal antibodies produced against ford et al. membranes from the Aplysia central nervous system w61x to demonstrate that these down-regulated proteins correspond to apcams, Aplysia members of the immunoglobulin class of cell adhesion molecules that includes

12 ( ) T. Abel, E. KandelrBrain Research ReÕiews mammalian NCAM and Drosophila Fasciclin II Ž Fig. 7.. Although many studies have focused on the role of cell adhesion molecules during development, it is now clear from the work in Aplysia on apcam that this class of cell surface molecules can play a critical part in synapse formation and maintenance during memory storage. In a variety of other species, ranging from Drosophila w67x to mice w40 x, cell adhesion molecules play a role in synaptic plasticity. To examine the mechanisms by which 5-HT modulates levels of apcam, Bailey et al. wx 9 used a gold-labelled apcam-specific monoclonal antibody to visualize apcam in thin sections using electron microscopy. One hour after the application of 5-HT, levels of apcam on the surface of the sensory neuron decrease by 50%. This decrease, which is specific for the presynaptic sensory neuron and requires ongoing protein synthesis, is the result of the internalization of apcam via the endosomal pathway. This activation of endocytosis is accompanied by an increase in the number of coated pits and coated vesicles, and it may be mediated by the increase in the expression of the light chain of clathrin that is induced by 5-HT and camp w53 x. How is this down regulation of apcam achieved? Is each of the isoforms Ž see Fig. 7. internalized in response to 5-HT treatment, or is one of the isoforms selectively affected? The initial analysis of the amino acid sequence of apcam suggested candidate domains that might be important for internalization and degradation, including a PEST domain, which is often found in proteins with short half-lives w83 x, as well as putative MAP kinase phosphorylation sites within the intracellular domain of the transw10,71 x. By overexpressing membrane form of apcam epitope-tagged versions of each of the isoforms of apcam in cultured sensory neurons, Bailey et al. w10x found that only the transmembrane form was internalized in response to treatment with 5-HT Ž Fig. 8.. This suggests that the cytoplasmic tail of the transmembrane domain may be the target of the signal transduction pathways leading to endocytosis. Indeed, deletion of the entire cytoplasmic tail, removal of the PEST sequence, or mutations in the MAP kinase consensus sites blocks endocytosis w10 x. The MAP kinase pathway appears to be crucial for long-term facilita- tion w68x and it may play a particularly important role in removing inhibitory constraints on memory storage. In addition to being required for the down regulation of the transmembrane form of apcam, MAP kinase may also inactivate the transcriptional repressor ApCREB2 w14 x. What role, then, does the selective internalization of the transmembrane isoform of apcam play in synaptic facilitation? Down regulation of apcam on the surface of the sensory neuron leads to defasciculation w71,80 x. This defasciculation may act in a permissive way, now allowing neurites of the sensory neuron to interact with the motor neuron, leading to the formation of new synaptic connecw99 x. Interestingly, apcam also appears to be re- tions quired for the growth of new synaptic connections based on the observation that incubation with the anti-apcam monoclonal antibody blocks the synaptic facilitation as well as the increase in varicosities induced by repeated exposures to 5-HT w100 x. Indeed, in sensory motor cocultures, apcam levels at preexisting sensory neuron varicosities increase in response to repeated pulses of 5-HT w100 x. Alternatively, apcam on the surface of the target cell may mediate synapse formation because sensory neuron varicosities form primarily at the site of high apcam levels on the surface of the motor neuron w100 x. The observed enrichment of apcam at existing synapses may be the result of the selective down regulation of the transmembrane form of apcam at extrasynaptic sites, or apcam may be retained at synaptic sites as a result of the redistribution of the remaining molecules of apcam on the cell surface. Regardless of the molecular process, the selective change in apcam expression decreases the interaction of sensory neuron neurites with each other and increases the attractiveness of the surface of the motor neuron for the formation of new synaptic connections. In Drosophila, cell adhesion molecules also play an important role in the formation of new synapses. A Drosophila homolog of NCAM and apcam, Fasciclin II, plays a central role in activity-dependent synapse remodeling at the neuromuscular junction Žreviewed in Ref. w67 x.. At this synapse, the presynaptic down regulation of Fasciclin II is both necessary and sufficient for activity-dependent synaptic sprouting. Interestingly, the activation of the Fig. 7. Schematic diagram of the domain structure of the three forms of apcam: the transmembrane form and the long and short glycosylphosphoinositol Ž GPI. linked forms. Ig, immunoglobulin type c2 domain; Fn, fibronectin type III domain; TM, transmembrane domain; cyto, cytoplasmic domain; PEST, PEST sequence. Adapted from Ref. w71 x.

13 372 ( ) T. Abel, E. KandelrBrain Research ReÕiews Fig HT selectively induces the down regulation of the transmembrane isoform of apcam. Ž A. Surface density and Ž B. density inside the cell of each of the tagged GPI-linked and transmembrane forms of apcam in sensory neuron culture after 1 h exposure to 5-HT. From Ref. w10 x. PKArCREB pathway is necessary for changes in the functional strength of these connections. 8. The regulatory subunit of PKA: an inhibitor removed by proteolysis Bridging the transition from short-term to long-term facilitation in Aplysia is a persistent increase in the activity of PKA which continues even in the absence of camp or 5-HT w48,91 x. This is distinct from the case in the mammalian hippocampus, where increases in camp and PKA activity after LTP are transient and there is a time window around induction during which PKA is required w54,85 x. To assess the importance of this persistently active PKA for facilitation in the Aplysia sensory motor neuron culture system, Hedge et al. w50x applied the PKA inhibitor RpcAMPS at various times after treatment with five pulses