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Intermediate-term processes in memory formation
Intermediate-term processes in memory formation Shara Stough, Justin L Shobe and Thomas J Carew Neuroscientists have invested considerable effort in attempting to elucidate the molecular mechanisms that mediate shortterm and long-term forms of learning and memory. For instance, the discovery of long-term potentiation inspired a field that has produced hundreds of studies examining both early and late forms of long-term potentiation. And at the behavioral level, most neuroscientists investigate either shortor long-term forms of memory or some combination of the two. The general belief that plasticity was restricted to short- and long-term temporal domains lasted for many years because of the apparent continuity of memory and its molecular characterization from one domain to the other. In cellular studies of plasticity, the short-term stage typically lasts in the range of minutes, and requires modification of pre-existing proteins, whereas long-term changes, such as synaptic growth, last for hours to days and require transcription and translation. As both behavioral and cellular studies covered a wider range of temporal domains, from the initiation of brief memory to the expression of long-lasting memory, it was at least tacitly assumed that these studies also captured any intervening domains as well. However, between these two temporal extremes lies a unique form of intermediate-term synaptic plasticity and memory, which mechanistically is a blend of the early and late forms. Addresses Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California, Irvine, Irvine, California 92697, USA Corresponding author: Carew, Thomas J ([email protected])
required new protein synthesis but not transcription. Sutton et al. [2,3] then confirmed these observations, and extended them into the behavioral arena by describing an analogous form of ITM. These seminal observations gave rise to the search for underlying molecular mechanisms, and several key cascades have emerged as important, including mitogen activated protein kinase (MAPK), protein kinase A (PKA) and protein kinase C (PKC). This general field of inquiry is not limited to Aplysia. In fact, evidence from several other invertebrate systems has made a substantial contribution to our understanding of these forms of behavioral and synaptic plasticity. A comparative analysis of memory formation in Aplysia, Drosophila melanogaster and Apis mellifera reveals a striking conservation of molecular cascades responsible for intermediate-term memory formation across these species. For the purpose of focus, and space constraints, we limit our discussion here to three key invertebrates that have significantly advanced our understanding of intermediate-term processing. However, we should stress that important insights have also come from other invertebrate systems, such as Hermissenda crassicornis and Lymnaea stagnalis, in addition to studies of the mammalian hippocampus, all of which exhibit interesting forms of enduring plasticity that are transcriptionally independent.
Three key systems elucidate intermediate-term processes Aplysia
Current Opinion in Neurobiology 2006, 16:672–678 This review comes from a themed issue on Neurobiology of behaviour Edited by John H Byrne and Wendy Suzuki Available online 13th November 2006 0959-4388/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2006.10.009
In the field of learning and memory, the marine mollusk Aplysia is one of the most studied invertebrates owing to several key features. Perhaps the most important is that Aplysia displays simple forms of learning such as sensitization, a non-associative form of memory that is accompanied by an enhancement in the synaptic strength between sensory (SN) and motor (MN) neurons, known as synaptic facilitation [4]. This, coupled with the fact that the nervous system has been extensively characterized and is well organized, has facilitated the investigation of memory mechanisms at the three levels, behavioral, synaptic and molecular.
Introduction Intermediate-term memory (ITM) and intermediateterm synaptic facilitation (ITF) are transcriptionally independent and generally last for hours, not days. Intermediate-term synaptic plasticity was first identified in Aplysia californica by the pioneering work of Ghirardi et al. [1], who demonstrated that synaptic facilitation induced by exogenous application of serotonin (5HT) at an identified sensory–motor synapse lasted for several hours and Current Opinion in Neurobiology 2006, 16:672–678
Sensitization of defensive reflexes
Sensitization is a strengthening of the defensive reflexes of an animal in response to noxious stimuli (e.g. tail shock). The memory of sensitization training is reflected by an enhanced withdrawal reflex of an animal in response to an innocuous test stimulus delivered after the training [5–9]. Tail nerve shock induces release of 5HT in the CNS [10] and sensitization requires 5HT [11]. www.sciencedirect.com
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Moreover, direct application of 5HT to SN–MN synapses produces facilitation, the cellular analog of sensitization, demonstrating that 5HT is sufficient to induce the underlying plasticity [9,12–14]. Memory for sensitization has three distinct temporal phases characterized by unique molecular profiles (for reviews see [15,16]). Short-term facilitation (STF), induced by a single pulse of 5HT, and short-term memory for sensitization (STM), induced by a single tail shock, are independent of translation and transcription; thus, they only require modification of pre-existing substrates [13]. Intermediate-term facilitation (ITF), induced by multiple pulses of 5HT, and intermediateterm memory for sensitization (ITM), induced by repeated tail shocks, are independent of transcription but do require new protein synthesis [1–3]. Finally, long-term facilitation (LTF) and long-term memory for sensitization (LTM) have the same induction requirements as intermediate-term, but they rely on both transcription and translation [13,17]. Within the intermediate-term domain, we discuss two types of facilitation and memory: repeated-trial ITM (RT-ITM) (discussed above) and site-specific ITM (SS-ITM). Each has a unique induction protocol. Whereas RT-ITM requires multiple shocks [2], SSITM is induced by a single shock to the test site [18]. Thus, testing occurs in the same receptive field as the shock, which provides the opportunity for activity-dependent modulation. In a similar manner, there is a synaptic counterpart to SS-ITM called activity-dependent facilitation (AD-ITF), which is induced by pairing a single pulse of 5HT with direct activation of a SN [3]. In addition to having unique induction protocols, these two types of plasticity have distinct molecular requirements. The induction of SS-ITM does not require new protein synthesis [18], and, thus, relies exclusively on the modification of pre-existing proteins. By contrast, the induction of RT-ITM requires translation [2]. Moreover, each type requires a unique complement of kinase activity (see below). Molecular mechanisms of repeated trial and activitydependent memory
Kinases play a crucial role in regulating the induction and expression of RT-ITM and ITF. For example, PKA activity is required for the expression of ITM and ITF, whereas PKC activity is not involved [2,3]. Moreover, MAPK activation is required for the induction but not the expression of ITM. Consistent with these observations, ITF also requires MAPK activity [19]. Finally, synaptically and behaviorally relevant stimuli, such as temporally spaced multiple pulses of 5HT or repeated tail shocks, produce robust MAPK activation [19–22]. Taken together, these findings suggest that MAPK regulates the induction of ITF www.sciencedirect.com
and ITM, whereas PKA is required for the expression of ITF and ITM. What is the relationship between these kinases and the requirement for translation in RT-ITM? There are at least two distinct possibilities that are not mutually exclusive: first, translation could be required for the maintenance of persistent kinase activity, and/or second, kinases could directly upregulate the translational machinery. In support of the idea that translation regulates kinase activity, persistent PKA and MAPK activity induced by multiple training trials requires new protein synthesis [23,24]. Conversely, there is evidence that 5HT-induced phosphorylation of S6 kinase, an important regulator of protein synthesis, requires PKA activity [25]. Also, in the hippocampus, MAPK regulates translation in the induction of LTP [26]. Finally, what are the downstream molecular changes that are responsible for repeated-trial ITM and ITF? This remains an open question; however, several clues hint at some interesting possibilities. We know that plasticity in the intermediate domain does not require long-term structural changes; however, a recent study demonstrated that following RT training, empty synapses became filled with neurotransmitter vesicles in a translationally sensitive manner [27]. There is also an opportunity for PKA and MAPK to interact directly with the release machinery. For instance, there are putative phosphorylation sites on synapsin for both these kinases, and in Aplysia, 5HTinduced synapsin phosphorylation is blocked by inhibitors of either PKA or MAPK [28]. Recent work has also focused on the molecular mechanisms that govern SS-ITM. MAPK activity is required for the induction, but not expression, of SS-ITM [29]. To examine the mechanisms of MAPK activation, a molecular analog of SS training was developed: depolarization of tail sensory neurons was coupled with a single pulse of 5HT. This analog produces robust activation of MAPK that requires cAMP, possibly through guanine exchange factor (GEF) activity [29]. Taken together, these behavioral and molecular results suggest that 5HT and activity in the SNs combine to activate MAPK in the induction of site-specific ITM for sensitization. These findings, in combination with an earlier study demonstrating that PKM, a constitutively active form of PKC, is required for the expression of SS-ITM [18], begin to suggest a model in which these two kinases, PKC and MAPK, are crucial players for the induction and expression of SS-ITM. As in repeated-trial ITM, it remains unclear which downstream targets mediate the effects of PKC and MAPK in SS-ITM. However, it was recently shown that the Ca2+dependent PKC isoform, which is required for AD-ITF in cultured synapses, translocates to the plasma membrane Current Opinion in Neurobiology 2006, 16:672–678
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of SNs only in response to a combination of activity and 5HT stimulation [30]. Thus, it seems likely that the PKC at the membrane might phosphorylate specific ion channels. Moreover, new evidence also implicates SNAP-25 (synaptosoma-associated protein of 25 kDa), a protein involved in synaptic vesicle docking and fusion, as a direct target for PKC [31]. Drosophila
Drosophila offer considerable power for the study of learning as a model organism, because they enable the establishment of causal relationships between individual genes and behavior. A variety of techniques are used to produce genetic manipulations, such as forward genetics, chemical mutagenesis, P-element mutagenesis and enhancer detection (for review, see [32]). Flies that harbor mutations in learning and memory genes are typically identified as poor performers in associative olfactory learning. Associative olfactory learning
Olfactory classical conditioning is perhaps the most commonly used technique to assess behavioral learning in Drosophila. In this paradigm, flies are placed in a tube in which odors are sequentially drawn in through an air current, and an electric shock (unconditioned stimulus [US]) is delivered in the presence of one of the odors (conditioned stimulus [CS]). The flies are then placed in a T-maze in which they must to choose to avoid one of the odors. This procedure generates robust learning; on average 70–90% of the flies will make the correct odor–shock association [33]. Using this conditioning paradigm, the phenotype of specific mutants has revealed four temporal phases of memory formation, each with a unique molecular signature: short-term memory (STM) lasting about an hour; middleterm memory (MTM), which peaks at 1 h and decays by 5 h; anesthesia-resistant memory (ARM), which lasts for at least a day; and long-term memory (LTM), which starts at about 5 h post-training and lasts for several days (for reviews see [32,34,35] and Liu and Davis, this issue). Thus, acquisition initially produces STM, which quickly leads to MTM and ARM. By contrast, LTM requires several hours of consolidation. In many cases these phases are induced independently; however, there are also shared mechanisms. For instance, STM, MTM and ARM only rely on modification of pre-existing proteins, whereas LTM requires both transcription and translation [36,37]. These phases are further defined by distinct patterning requirements. Single trial and massed training induces STM, MTM and ARM, presumably by regulating the activity of plasticity-related proteins, such as kinases and phosphatases, and the translational machinery. Spaced training, however, uniquely induces LTM Current Opinion in Neurobiology 2006, 16:672–678
through a cAMP response element binding protein (CREB)-dependent upregulation of genes. Mechanisms of STM, MTM and ARM
STM, MTM and ARM do not require transcription, and, thus, they fall within our definition of intermediate-term processes. Genetic studies have identified numerous mutants that implicate several different signaling cascades, but cAMP signaling appears to be crucial (for reviews see [32,38]). In fact, the first learning and memory mutant identified was dunce [39], which expresses a mutated form of cAMP phosphodiesterase [40]. This discovery was soon followed by that of rutabaga [41], which expresses a mutated Ca2+–CaM-activated adenylate cyclase (AC) [42] and later DCO, in which the mutated gene encodes the catalytic subunit of PKA [43]. All three of these mutants have poor STM [33,43], presumably because of dysregulated cAMP signaling. Although STM only lasts for about an hour, flies display memory continuously for days. Thus, it was not clear that STM was a mechanistically distinct phase until the discovery of amnesiac [44], a mutant whose gene product is pituitary adenylyl cyclase-activating peptide (PACAP), a neuropeptide involved in adenylate cyclase activation [45,46]. After a single training trial, which produces STM, MTM, and ARM, amnesiac has normal STM; however, memory then decays and subsequently recovers by 7 h [33]. This illustrated that there was a unique middle phase of memory, MTM, flanked by the two other phases. The mutants DCO and rutabaga have impaired MTM, indicating that cAMP signaling is also required for MTM [33,47]. The phase that follows MTM has been identified as ARM, a form of memory that is resistant to anesthesia (by cooling) and translational inhibitors [36]. Rutabaga and amnesiac have normal memory in this phase [36,48] and expression of dominant-negative CREB does not affect ARM [37], demonstrating its independence from cAMP and the CREB-dependent transcriptional cascade. The initial genetic characterization of ARM came from the radish mutant, which had memory deficits [36,49]. The radish mutation is thought to reside in a phopholipase gene [50]. In addition, PKM, a constitutively active form of PKC, also seems to regulate ARM. Blocking PKC activity or expression of dominant-negative PKM blocks 24 h memory after massed training. Induction of a PKM transgene enhances memory following massed training. This enhancement is not blocked in radish, suggesting that PKM operates downstream of radish in ARM [51]. Several mutants have indicated the importance of the MAPK cascade. The leonardo mutant, which encodes a 14-3-3 protein, a regulator of Raf, compromises STM and MTM [52,53]. Also the neurofibromin mutant Nf1, which expresses a mutated form of a GTPase activating protein www.sciencedirect.com
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(GAP), is severely impaired in all phases of memory. Although Nf1 can be involved in Ras–MAPK signaling in Drosophila [54], it was demonstrated that at least some of the Nf1 effects on memory are mediated through the cAMP pathway [55]. It remains to be investigated whether disrupted MAPK signaling in Nf1 mutants is related to observed memory impairments. These two mutants indicate that the canonical Ras–Raf–Mek pathway leading to MAPK activation might have a role in memory formation.
(L-LTM) emerges. Although recent evidence demonstrates a requirement of new protein synthesis for induction of E-LTM [62] (for a review see [63]), several earlier studies found that E-LTM was independent of translation [64–66] (for review see [67]). Although it is clear that transcription is not required for E-LTM [64,68], the role of new protein synthesis remains to be resolved. As in previous systems, we would consider MTM and E-LTM to reflect intermediate-term processing. Mechanisms of MTM and E-LTM
Apis
Honeybees use olfactory cues to locate potential food sources in the environment. Moreover, in the presence of sucrose, a bee will reflexively extend its proboscis, a tubelike feeding organ. If an odor, such as geranium, is paired with sucrose presentation, the bee will form an association between the two stimuli: the odor alone comes to elicit proboscis extension. This form of learning is robust and, depending on the amount and pattern of training, can persist for a lifetime. Associative olfactory memory
The memory produced by single and multiple training trials differs in both duration and strength, in addition to mechanism. Single trial conditioning produces a memory that lasts for 1 day, and decays slowly until it is no longer apparent at 3 days. By contrast, three spaced conditioning trials result in maximal associative strength and a stable memory that typically lasts for the life of the bee (for reviews see [56,57]). One hallmark of single trial conditioning is its sensitivity to interference by amnestic agents, such as cooling or mild electrical shock. When applied immediately following training, these agents disrupt subsequent memory measured at 30 min or 3 h, but later applications are ineffective once the memory has been successfully consolidated [58,59–61]. Intriguingly, even if 3 h memory following a single trial is disrupted by cooling, 24 h memory remains intact [58]. These data show that a single CS–US pairing induces two forms of memory in parallel: one anesthesia-sensitive form that lasts for hours, and one anesthesia-insensitive form that is present at 24 h following training. Multiple training trials accelerate the transfer of memory into an anesthesia-resistant form [58,59]. Cooling or shock applied immediately after the third associative training trial has no effect on subsequent memory either at 3 h or at 24 h after conditioning. Multiple-trial memory has been characterized as three separate processes: first, middle-term memory (MTM) lasts for hours after training; second, beginning 1 day after training, a mechanistically distinct early-phase long-term memory (E-LTM) is observed; and third, by 3 days after training, a transcriptionally dependent late-phase LTM www.sciencedirect.com
Multiple training trials result in activation of PKC that begins 1 h after training and peaks at 3 h post training. This activation of persistent PKC activity requires the activity of calpain, a protease capable of cleaving PKC into a constitutively active catalytic fragment. If calpain is inhibited before training, 1 h memory is selectively blocked, whereas immediate memory formation and 24 h memory (E-LTM) are spared [68]. These studies, thus, clearly indicate the presence of a mechanistically distinct MTM phase of memory (for reviews see [69,70]). In addition to enhanced PKC activation, active PKA levels also show prolonged elevation in the antennal lobes following multiple, but not single, training trials [71]. Inhibition of PKA activation throughout training leads to a specific disruption of E-LTM at 24 h. However, MTM tested at 3 h remains intact. Moreover, the photo-release of caged cAMP can ‘substitute’ for additional training trials. When paired with a single training trial, which by itself does not result in prolonged PKA activation, released cAMP induces memory that is stable over 3 days [71]. Nitric oxide (NO) signaling is also required for the formation of E-LTM, but not for MTM [58]. The observation that E-LTM requires both NO signaling and PKA activation suggests that these molecules participate in a single signaling cascade. Indeed, NO synthase inhibitors block prolonged PKA activity induced by multiple training trials. Finally, cGMP inhibitors also blocked NO-induced PKA activation, suggesting that NO mediates PKA activation through cGMP [71]. As predicted by these molecular studies, inhibition of cGMP blocks ELTM formation, and a photo-releasable form of cGMP can also induce 3 day memory when paired with a single training trial.
Conclusions: common features shared between systems Each of the three species we have discussed provides a unique perspective into the dynamics and mechanisms of learning and memory. The value of this comparative approach lies in the appreciation that organisms with vastly different selection pressures use common molecular cascades in the service of memory formation. From this review, three kinases, PKA, MAPK and PKC, have emerged as crucial regulators of transcriptionally independent memory formation. Current Opinion in Neurobiology 2006, 16:672–678
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The cAMP signaling cascade is required for memory formation in Aplysia, Apis and Drosophila. In Aplysia, PKA activity is required for STF and RT-ITM. In Drosophila, the cAMP signaling mutants dunce and rutabaga are defective in STM and amnesiac is unable to form MTM. In Apis, a NO–cGMP–PKA pathway mediates E-LTM. In both Aplysia and Drosophila, MAPK signaling is crucial for memory formation. For instance, in Aplysia, two forms of ITM, RT-ITM and SS-ITM, require MAPK activity. In Drosophila, the importance of MAPK signaling appears especially pervasive because several mutants disrupt all memory phases. Finally, all three species have a PKC-dependent phase of memory. Perhaps even more striking is the fact that they all require some form of PKM during the intermediate phase of memory. Both Aplysia and Apis generate PKM from the cleavage of PKC by calpain. In Drosophila, however, it appears that ARM requires the activity of a unique PKM gene. The relationship between these three kinases and translation is especially interesting. In all instances, phases of memory that rely on PKA and MAPK cascades also require translation. By contrast, those that require PKC activity are independent of new protein synthesis. These observations are consistent with the known roles of these kinases in downstream signaling. Both PKA and MAPK have been extensively linked to the control of translational machinery [25,26,72] (for reviews see [73,74]). However, PKC typically phosphorylates downstream effectors, such as release machinery or ion channels, that directly regulate synaptic plasticity [30,31] (for a review see [75]). The fact that these cascades, that have very different temporal profiles, are often triggered by the same training procedures supports the view that multiple memory mechanisms are required for the continuity of memory expression over diverse temporal domains.
Acknowledgements This work was supported by National Institute of Mental Health Grant R01 M414-10183 and National Science Foundation Grant IBN-0049013 (to TJ Carew)
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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38. Davis RL: Physiology and biochemistry of Drosophila learning mutants. Physiol Rev 1996, 76:299-317. 39. Dudai Y, Jan Y-N, Byers D, Quinn WG, Benzer S: Dunce, a mutant of Drosophila deficient in learning. Proc Natl Acad Sci USA 1976, 73:1684-1688. 40. Chen CN, Denome S, Davis RL: Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce+ gene, the structural gene for cAMP phosphodiesterase. Proc Natl Acad Sci USA 1986, 83:9313-9317. This study conclusively demonstrates that the dunce gene encodes a cAMP phosphodiesterase, thereby strongly implying that cAMP signaling is crucial for memory formation. 41. Livingstone MS, Sziber PP, Quinn WG: Loss of calcium/ calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. Cell 1984, 37:205-215. 42. Levin LR, Han PL, Hwang PM, Feinstein PG, Davis RL, Reed RR: The Drosophila learning and memory gene rutabaga encodes a Ca2+/Calmodulin-responsive adenylyl cyclase. Cell 1992, 68:479-489. The demonstration that rutabaga is a adenylate cyclase gene, in combination with the earlier characterization of dunce, firmly established that cAMP signaling is crucially involved in the memory for classical conditioning. 43. Skoulakis EM, Kalderon D, Davis RL: Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory. Neuron 1993, 11:197-208. Although it was previously well established that cAMP signaling is required for memory formation, this was the first direct demonstration that PKA activity is required. 44. Quinn WG, Sziber PP, Booker R: The Drosophila memory mutant amnesiac. Nature 1979, 277:212-214. 45. Feany MB, Quinn WG: A neuropeptide gene defined by the Drosophila memory mutant amnesiac. Science 1995, 268:869-873. 46. DeZazzo J, Xia S, Christensen J, Velinzon K, Tully T: Developmental expression of an amn(+) transgene rescues the mutant memory defect of amnesiac adults. J Neurosci 1999, 19:8740-8746. 47. Li W, Tully T, Kalderon D: Effects of a conditional Drosophila PKA mutant on olfactory learning and memory. Learn Mem 1996, 2:320-333. 48. Tamura T, Chiang AS, Ito N, Liu HP, Horiuchi J, Tully T, Saitoe M: Aging specifically impairs amnesiac-dependent memory in Drosophila. Neuron 2003, 40:1003-1011. 49. Folkers E, Drain P, Quinn WG: Radish, a Drosophila mutant deficient in consolidated memory. Proc Natl Acad Sci USA 1993, 90:8123-8127. 50. Chiang AS, Blum A, Barditch J, Chen YH, Chiu SL, Regulski M, Armstrong JD, Tully T, Dubnau J: Radish encodes a phospholipase-A2 and defines a neural circuit involved in anesthesia-resistant memory. Curr Biol 2004, 14:263-272. 51. Drier EA, Tello MK, Cowan M, Wu P, Blace N, Sacktor TC, Yin JC: Memory enhancement and formation by atypical PKM activity in Drosophila melanogaster. Nat Neurosci 2002, 5:316-324. This study demonstrates that PKM is sufficient for memory induction and suggests that continued PKC activity is required for memory maintenance but not learning. Interestingly, unlike in many other systems, the PKM is not generated by proteolytic cleavage but rather is expressed in a constitutively active form. 52. Philip N, Acevedo SF, Skoulakis EM: Conditional rescue of olfactory learning and memory defects in mutants of the 14-3-3z gene leonardo. J Neurosci 2001, 21:8417-8425. 53. Skoulakis EM, Davis RL: Olfactory learning deficits in mutants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron 1996, 17:931-944. 54. Williams JA, Su HS, Bernards A, Field J, Sehgal A: A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK. Science 2001, 293:2251-2256. Current Opinion in Neurobiology 2006, 16:672–678
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55. Guo HF, Tong J, Hannan F, Luo L, Zhong Y: A neurofibromatosis1-regulated pathway is required for learning in Drosophila. Nature 2000, 403:895-898. 56. Hammer M, Menzel R: Learning and memory in the honeybee. J Neurosci 1995, 15:1617-1630. 57. Menzel R, Hammer M, Braun G, Mauelshagen J, Sugawa M: Neurobiology of learning and memory in honeybees. In The Behaviour and Physiology of Bees. Edited by Goodman LJ, Fisher RC. CAB International; 1991:323-353. 58. Mu¨ller U: Inhibition of nitric oxide synthase impairs a distinct form of long-term memory in the honeybee, Apis mellifera. Neuron 1996, 16:541-549. This study identified nitric oxide signaling as an important mechanism underlying long-term memory formation. NOS inhibitors selectively inhibited 24-hr memory, but not 3-hr memory, demonstrating that memory in different time domains is mediated by different molecular mechanisms. 59. Menzel R, Sugawa M: Time course of short-term memory depends on associative events. Naturwissenschaften 1986, 73:564-565. 60. Erber J, Masuhr TH, Menzel R: Localization of short-term memory in the brain of the bee, Apis mellifera. Physiol Entomol 1980, 5:343-358. 61. Menzel R, Erber J, Masuhr T: Learning and memory in the honeybee. In Experimental Analysis of Insect Behaviour. Edited by Browne L. Springer-Verlag; 1974:195-217. 62. Friedrich A, Thomas U, Mu¨ller U: Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory. J Neurosci 2004, 24:4460-4468. 63. Schwa¨rzel M, Mu¨ller U: Dynamic memory networks: dissecting molecular mechanisms underlying associative memory in the temporal domain. Cell Mol Life Sci 2006, 63:989-998. 64. Wu¨stenberg D, Gerber B, Menzel R: Long- but not medium-term retention of olfactory memories in honeybees is impaired by actinomycin D and anisomycin. Eur J Neurosci 1998, 10:2742-2745. 65. Wittstock S, Kaatz HH, Menzel R: Inhibition of brain protein synthesis by cycloheximide does not affect formation
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of long-term memory in honeybees after olfactory conditioning. J Neurosci 1993, 13:1379-1386. 66. Menzel R, Gaio UC, Gerberding M, Nemrava EA, Wittstock S: Formation of long-term olfactory memory in honeybees does not require protein synthesis. Naturwissenschaften 1993, 80:380-382. 67. Menzel R: Searching for the memory trace in a mini-brain, the honeybee. Learn Mem 2001, 8:53-62. 68. Gru¨nbaum L, Mu¨ller U: Induction of a specific olfactory memory leads to a long-lasting activation of protein kinase C in the antennal lobe of the honeybee. J Neurosci 1998, 18:4384-4392. Using an in vitro phosphorylation assay, the authors mapped a precise time course of PKC activation resulting from multiple conditioning trials. Importantly, they found that different mechanisms were responsible for PKC activation at various times following training, and that PKM was required for expression of MTM. 69. Menzel R: Memory dynamics in the honeybee. J Comp Physiol [A] 1999, 185:323-340. 70. Mu¨ller U: Learning in honeybees: from molecules to behavior. Zoology 2002, 105:313-320. 71. Mu¨ller U: Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees. Neuron 2000, 27:159-168. This study demonstrated the importance of PKA in the formation of long-term memory. In addition, it extended previous observations, by determining that NO signaling caused PKA activation via cGMP. 72. Banko JL, Hou L, Klann E: NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1. J Neurochem 2004, 91:462-470. 73. Sutton MA, Schuman EM: Local translational control in dendrites and its role in long-term synaptic plasticity. J Neurobiol 2005, 64:116-131. 74. Kelleher RJ III, Govindarajan A, Tonegawa S: Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron 2004, 44:59-73. 75. Majewski H, Iannazzo L: Protein kinase C: a physiological mediator of enhanced transmitter output. Prog Neurobiol 1998, 55:463-475.
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required new protein synthesis but not transcription. Sutton et al. [2,3] then confirmed these observations, and extended them into the behavioral arena by describing an analogous form of ITM. These seminal observations gave rise to the search for underlying molecular mechanisms, and several key cascades have emerged as important, including mitogen activated protein kinase (MAPK), protein kinase A (PKA) and protein kinase C (PKC). This general field of inquiry is not limited to Aplysia. In fact, evidence from several other invertebrate systems has made a substantial contribution to our understanding of these forms of behavioral and synaptic plasticity. A comparative analysis of memory formation in Aplysia, Drosophila melanogaster and Apis mellifera reveals a striking conservation of molecular cascades responsible for intermediate-term memory formation across these species. For the purpose of focus, and space constraints, we limit our discussion here to three key invertebrates that have significantly advanced our understanding of intermediate-term processing. However, we should stress that important insights have also come from other invertebrate systems, such as Hermissenda crassicornis and Lymnaea stagnalis, in addition to studies of the mammalian hippocampus, all of which exhibit interesting forms of enduring plasticity that are transcriptionally independent.
Three key systems elucidate intermediate-term processes Aplysia
Current Opinion in Neurobiology 2006, 16:672–678 This review comes from a themed issue on Neurobiology of behaviour Edited by John H Byrne and Wendy Suzuki Available online 13th November 2006 0959-4388/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2006.10.009
In the field of learning and memory, the marine mollusk Aplysia is one of the most studied invertebrates owing to several key features. Perhaps the most important is that Aplysia displays simple forms of learning such as sensitization, a non-associative form of memory that is accompanied by an enhancement in the synaptic strength between sensory (SN) and motor (MN) neurons, known as synaptic facilitation [4]. This, coupled with the fact that the nervous system has been extensively characterized and is well organized, has facilitated the investigation of memory mechanisms at the three levels, behavioral, synaptic and molecular.
Introduction Intermediate-term memory (ITM) and intermediateterm synaptic facilitation (ITF) are transcriptionally independent and generally last for hours, not days. Intermediate-term synaptic plasticity was first identified in Aplysia californica by the pioneering work of Ghirardi et al. [1], who demonstrated that synaptic facilitation induced by exogenous application of serotonin (5HT) at an identified sensory–motor synapse lasted for several hours and Current Opinion in Neurobiology 2006, 16:672–678
Sensitization of defensive reflexes
Sensitization is a strengthening of the defensive reflexes of an animal in response to noxious stimuli (e.g. tail shock). The memory of sensitization training is reflected by an enhanced withdrawal reflex of an animal in response to an innocuous test stimulus delivered after the training [5–9]. Tail nerve shock induces release of 5HT in the CNS [10] and sensitization requires 5HT [11]. www.sciencedirect.com
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Moreover, direct application of 5HT to SN–MN synapses produces facilitation, the cellular analog of sensitization, demonstrating that 5HT is sufficient to induce the underlying plasticity [9,12–14]. Memory for sensitization has three distinct temporal phases characterized by unique molecular profiles (for reviews see [15,16]). Short-term facilitation (STF), induced by a single pulse of 5HT, and short-term memory for sensitization (STM), induced by a single tail shock, are independent of translation and transcription; thus, they only require modification of pre-existing substrates [13]. Intermediate-term facilitation (ITF), induced by multiple pulses of 5HT, and intermediateterm memory for sensitization (ITM), induced by repeated tail shocks, are independent of transcription but do require new protein synthesis [1–3]. Finally, long-term facilitation (LTF) and long-term memory for sensitization (LTM) have the same induction requirements as intermediate-term, but they rely on both transcription and translation [13,17]. Within the intermediate-term domain, we discuss two types of facilitation and memory: repeated-trial ITM (RT-ITM) (discussed above) and site-specific ITM (SS-ITM). Each has a unique induction protocol. Whereas RT-ITM requires multiple shocks [2], SSITM is induced by a single shock to the test site [18]. Thus, testing occurs in the same receptive field as the shock, which provides the opportunity for activity-dependent modulation. In a similar manner, there is a synaptic counterpart to SS-ITM called activity-dependent facilitation (AD-ITF), which is induced by pairing a single pulse of 5HT with direct activation of a SN [3]. In addition to having unique induction protocols, these two types of plasticity have distinct molecular requirements. The induction of SS-ITM does not require new protein synthesis [18], and, thus, relies exclusively on the modification of pre-existing proteins. By contrast, the induction of RT-ITM requires translation [2]. Moreover, each type requires a unique complement of kinase activity (see below). Molecular mechanisms of repeated trial and activitydependent memory
Kinases play a crucial role in regulating the induction and expression of RT-ITM and ITF. For example, PKA activity is required for the expression of ITM and ITF, whereas PKC activity is not involved [2,3]. Moreover, MAPK activation is required for the induction but not the expression of ITM. Consistent with these observations, ITF also requires MAPK activity [19]. Finally, synaptically and behaviorally relevant stimuli, such as temporally spaced multiple pulses of 5HT or repeated tail shocks, produce robust MAPK activation [19–22]. Taken together, these findings suggest that MAPK regulates the induction of ITF www.sciencedirect.com
and ITM, whereas PKA is required for the expression of ITF and ITM. What is the relationship between these kinases and the requirement for translation in RT-ITM? There are at least two distinct possibilities that are not mutually exclusive: first, translation could be required for the maintenance of persistent kinase activity, and/or second, kinases could directly upregulate the translational machinery. In support of the idea that translation regulates kinase activity, persistent PKA and MAPK activity induced by multiple training trials requires new protein synthesis [23,24]. Conversely, there is evidence that 5HT-induced phosphorylation of S6 kinase, an important regulator of protein synthesis, requires PKA activity [25]. Also, in the hippocampus, MAPK regulates translation in the induction of LTP [26]. Finally, what are the downstream molecular changes that are responsible for repeated-trial ITM and ITF? This remains an open question; however, several clues hint at some interesting possibilities. We know that plasticity in the intermediate domain does not require long-term structural changes; however, a recent study demonstrated that following RT training, empty synapses became filled with neurotransmitter vesicles in a translationally sensitive manner [27]. There is also an opportunity for PKA and MAPK to interact directly with the release machinery. For instance, there are putative phosphorylation sites on synapsin for both these kinases, and in Aplysia, 5HTinduced synapsin phosphorylation is blocked by inhibitors of either PKA or MAPK [28]. Recent work has also focused on the molecular mechanisms that govern SS-ITM. MAPK activity is required for the induction, but not expression, of SS-ITM [29]. To examine the mechanisms of MAPK activation, a molecular analog of SS training was developed: depolarization of tail sensory neurons was coupled with a single pulse of 5HT. This analog produces robust activation of MAPK that requires cAMP, possibly through guanine exchange factor (GEF) activity [29]. Taken together, these behavioral and molecular results suggest that 5HT and activity in the SNs combine to activate MAPK in the induction of site-specific ITM for sensitization. These findings, in combination with an earlier study demonstrating that PKM, a constitutively active form of PKC, is required for the expression of SS-ITM [18], begin to suggest a model in which these two kinases, PKC and MAPK, are crucial players for the induction and expression of SS-ITM. As in repeated-trial ITM, it remains unclear which downstream targets mediate the effects of PKC and MAPK in SS-ITM. However, it was recently shown that the Ca2+dependent PKC isoform, which is required for AD-ITF in cultured synapses, translocates to the plasma membrane Current Opinion in Neurobiology 2006, 16:672–678
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of SNs only in response to a combination of activity and 5HT stimulation [30]. Thus, it seems likely that the PKC at the membrane might phosphorylate specific ion channels. Moreover, new evidence also implicates SNAP-25 (synaptosoma-associated protein of 25 kDa), a protein involved in synaptic vesicle docking and fusion, as a direct target for PKC [31]. Drosophila
Drosophila offer considerable power for the study of learning as a model organism, because they enable the establishment of causal relationships between individual genes and behavior. A variety of techniques are used to produce genetic manipulations, such as forward genetics, chemical mutagenesis, P-element mutagenesis and enhancer detection (for review, see [32]). Flies that harbor mutations in learning and memory genes are typically identified as poor performers in associative olfactory learning. Associative olfactory learning
Olfactory classical conditioning is perhaps the most commonly used technique to assess behavioral learning in Drosophila. In this paradigm, flies are placed in a tube in which odors are sequentially drawn in through an air current, and an electric shock (unconditioned stimulus [US]) is delivered in the presence of one of the odors (conditioned stimulus [CS]). The flies are then placed in a T-maze in which they must to choose to avoid one of the odors. This procedure generates robust learning; on average 70–90% of the flies will make the correct odor–shock association [33]. Using this conditioning paradigm, the phenotype of specific mutants has revealed four temporal phases of memory formation, each with a unique molecular signature: short-term memory (STM) lasting about an hour; middleterm memory (MTM), which peaks at 1 h and decays by 5 h; anesthesia-resistant memory (ARM), which lasts for at least a day; and long-term memory (LTM), which starts at about 5 h post-training and lasts for several days (for reviews see [32,34,35] and Liu and Davis, this issue). Thus, acquisition initially produces STM, which quickly leads to MTM and ARM. By contrast, LTM requires several hours of consolidation. In many cases these phases are induced independently; however, there are also shared mechanisms. For instance, STM, MTM and ARM only rely on modification of pre-existing proteins, whereas LTM requires both transcription and translation [36,37]. These phases are further defined by distinct patterning requirements. Single trial and massed training induces STM, MTM and ARM, presumably by regulating the activity of plasticity-related proteins, such as kinases and phosphatases, and the translational machinery. Spaced training, however, uniquely induces LTM Current Opinion in Neurobiology 2006, 16:672–678
through a cAMP response element binding protein (CREB)-dependent upregulation of genes. Mechanisms of STM, MTM and ARM
STM, MTM and ARM do not require transcription, and, thus, they fall within our definition of intermediate-term processes. Genetic studies have identified numerous mutants that implicate several different signaling cascades, but cAMP signaling appears to be crucial (for reviews see [32,38]). In fact, the first learning and memory mutant identified was dunce [39], which expresses a mutated form of cAMP phosphodiesterase [40]. This discovery was soon followed by that of rutabaga [41], which expresses a mutated Ca2+–CaM-activated adenylate cyclase (AC) [42] and later DCO, in which the mutated gene encodes the catalytic subunit of PKA [43]. All three of these mutants have poor STM [33,43], presumably because of dysregulated cAMP signaling. Although STM only lasts for about an hour, flies display memory continuously for days. Thus, it was not clear that STM was a mechanistically distinct phase until the discovery of amnesiac [44], a mutant whose gene product is pituitary adenylyl cyclase-activating peptide (PACAP), a neuropeptide involved in adenylate cyclase activation [45,46]. After a single training trial, which produces STM, MTM, and ARM, amnesiac has normal STM; however, memory then decays and subsequently recovers by 7 h [33]. This illustrated that there was a unique middle phase of memory, MTM, flanked by the two other phases. The mutants DCO and rutabaga have impaired MTM, indicating that cAMP signaling is also required for MTM [33,47]. The phase that follows MTM has been identified as ARM, a form of memory that is resistant to anesthesia (by cooling) and translational inhibitors [36]. Rutabaga and amnesiac have normal memory in this phase [36,48] and expression of dominant-negative CREB does not affect ARM [37], demonstrating its independence from cAMP and the CREB-dependent transcriptional cascade. The initial genetic characterization of ARM came from the radish mutant, which had memory deficits [36,49]. The radish mutation is thought to reside in a phopholipase gene [50]. In addition, PKM, a constitutively active form of PKC, also seems to regulate ARM. Blocking PKC activity or expression of dominant-negative PKM blocks 24 h memory after massed training. Induction of a PKM transgene enhances memory following massed training. This enhancement is not blocked in radish, suggesting that PKM operates downstream of radish in ARM [51]. Several mutants have indicated the importance of the MAPK cascade. The leonardo mutant, which encodes a 14-3-3 protein, a regulator of Raf, compromises STM and MTM [52,53]. Also the neurofibromin mutant Nf1, which expresses a mutated form of a GTPase activating protein www.sciencedirect.com
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(GAP), is severely impaired in all phases of memory. Although Nf1 can be involved in Ras–MAPK signaling in Drosophila [54], it was demonstrated that at least some of the Nf1 effects on memory are mediated through the cAMP pathway [55]. It remains to be investigated whether disrupted MAPK signaling in Nf1 mutants is related to observed memory impairments. These two mutants indicate that the canonical Ras–Raf–Mek pathway leading to MAPK activation might have a role in memory formation.
(L-LTM) emerges. Although recent evidence demonstrates a requirement of new protein synthesis for induction of E-LTM [62] (for a review see [63]), several earlier studies found that E-LTM was independent of translation [64–66] (for review see [67]). Although it is clear that transcription is not required for E-LTM [64,68], the role of new protein synthesis remains to be resolved. As in previous systems, we would consider MTM and E-LTM to reflect intermediate-term processing. Mechanisms of MTM and E-LTM
Apis
Honeybees use olfactory cues to locate potential food sources in the environment. Moreover, in the presence of sucrose, a bee will reflexively extend its proboscis, a tubelike feeding organ. If an odor, such as geranium, is paired with sucrose presentation, the bee will form an association between the two stimuli: the odor alone comes to elicit proboscis extension. This form of learning is robust and, depending on the amount and pattern of training, can persist for a lifetime. Associative olfactory memory
The memory produced by single and multiple training trials differs in both duration and strength, in addition to mechanism. Single trial conditioning produces a memory that lasts for 1 day, and decays slowly until it is no longer apparent at 3 days. By contrast, three spaced conditioning trials result in maximal associative strength and a stable memory that typically lasts for the life of the bee (for reviews see [56,57]). One hallmark of single trial conditioning is its sensitivity to interference by amnestic agents, such as cooling or mild electrical shock. When applied immediately following training, these agents disrupt subsequent memory measured at 30 min or 3 h, but later applications are ineffective once the memory has been successfully consolidated [58,59–61]. Intriguingly, even if 3 h memory following a single trial is disrupted by cooling, 24 h memory remains intact [58]. These data show that a single CS–US pairing induces two forms of memory in parallel: one anesthesia-sensitive form that lasts for hours, and one anesthesia-insensitive form that is present at 24 h following training. Multiple training trials accelerate the transfer of memory into an anesthesia-resistant form [58,59]. Cooling or shock applied immediately after the third associative training trial has no effect on subsequent memory either at 3 h or at 24 h after conditioning. Multiple-trial memory has been characterized as three separate processes: first, middle-term memory (MTM) lasts for hours after training; second, beginning 1 day after training, a mechanistically distinct early-phase long-term memory (E-LTM) is observed; and third, by 3 days after training, a transcriptionally dependent late-phase LTM www.sciencedirect.com
Multiple training trials result in activation of PKC that begins 1 h after training and peaks at 3 h post training. This activation of persistent PKC activity requires the activity of calpain, a protease capable of cleaving PKC into a constitutively active catalytic fragment. If calpain is inhibited before training, 1 h memory is selectively blocked, whereas immediate memory formation and 24 h memory (E-LTM) are spared [68]. These studies, thus, clearly indicate the presence of a mechanistically distinct MTM phase of memory (for reviews see [69,70]). In addition to enhanced PKC activation, active PKA levels also show prolonged elevation in the antennal lobes following multiple, but not single, training trials [71]. Inhibition of PKA activation throughout training leads to a specific disruption of E-LTM at 24 h. However, MTM tested at 3 h remains intact. Moreover, the photo-release of caged cAMP can ‘substitute’ for additional training trials. When paired with a single training trial, which by itself does not result in prolonged PKA activation, released cAMP induces memory that is stable over 3 days [71]. Nitric oxide (NO) signaling is also required for the formation of E-LTM, but not for MTM [58]. The observation that E-LTM requires both NO signaling and PKA activation suggests that these molecules participate in a single signaling cascade. Indeed, NO synthase inhibitors block prolonged PKA activity induced by multiple training trials. Finally, cGMP inhibitors also blocked NO-induced PKA activation, suggesting that NO mediates PKA activation through cGMP [71]. As predicted by these molecular studies, inhibition of cGMP blocks ELTM formation, and a photo-releasable form of cGMP can also induce 3 day memory when paired with a single training trial.
Conclusions: common features shared between systems Each of the three species we have discussed provides a unique perspective into the dynamics and mechanisms of learning and memory. The value of this comparative approach lies in the appreciation that organisms with vastly different selection pressures use common molecular cascades in the service of memory formation. From this review, three kinases, PKA, MAPK and PKC, have emerged as crucial regulators of transcriptionally independent memory formation. Current Opinion in Neurobiology 2006, 16:672–678
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The cAMP signaling cascade is required for memory formation in Aplysia, Apis and Drosophila. In Aplysia, PKA activity is required for STF and RT-ITM. In Drosophila, the cAMP signaling mutants dunce and rutabaga are defective in STM and amnesiac is unable to form MTM. In Apis, a NO–cGMP–PKA pathway mediates E-LTM. In both Aplysia and Drosophila, MAPK signaling is crucial for memory formation. For instance, in Aplysia, two forms of ITM, RT-ITM and SS-ITM, require MAPK activity. In Drosophila, the importance of MAPK signaling appears especially pervasive because several mutants disrupt all memory phases. Finally, all three species have a PKC-dependent phase of memory. Perhaps even more striking is the fact that they all require some form of PKM during the intermediate phase of memory. Both Aplysia and Apis generate PKM from the cleavage of PKC by calpain. In Drosophila, however, it appears that ARM requires the activity of a unique PKM gene. The relationship between these three kinases and translation is especially interesting. In all instances, phases of memory that rely on PKA and MAPK cascades also require translation. By contrast, those that require PKC activity are independent of new protein synthesis. These observations are consistent with the known roles of these kinases in downstream signaling. Both PKA and MAPK have been extensively linked to the control of translational machinery [25,26,72] (for reviews see [73,74]). However, PKC typically phosphorylates downstream effectors, such as release machinery or ion channels, that directly regulate synaptic plasticity [30,31] (for a review see [75]). The fact that these cascades, that have very different temporal profiles, are often triggered by the same training procedures supports the view that multiple memory mechanisms are required for the continuity of memory expression over diverse temporal domains.
Acknowledgements This work was supported by National Institute of Mental Health Grant R01 M414-10183 and National Science Foundation Grant IBN-0049013 (to TJ Carew)
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Ghirardi M, Montarolo PG, Kandel ER: A novel intermediate stage in the transition between short- and long-term facilitation in the sensory to motor neuron synapse of Aplysia. Neuron 1995, 14:413-420. This important study was the first demonstration of an intermediate phase of plasticity (ITF), which is mechanistically distinct from previously studied short-term and long-term forms. The authors show that ITF induced by repeated applications of the neuromodulator, 5HT, requires translation but not transcription
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2.
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