Cells called neurons allow information to travel quickly around the body so that we can rapidly respond to any changes that we sense in our environment. This includes non-conscious reactions, such as the knee-jerk reflex in humans.

Reflexes and other behaviors can be influenced by long-term memory, and it is thought that long-term memory is stored by changes in the synapses that connect neurons to each other. The reflexes of a sea slug known as Aplysia are often used to study memory because it has a simple nervous system in which individual sensory neurons (which detect changes) only form synapses with single motor neurons (which control muscles).

Chen et al. have now studied whether long-term memory is actually stored in these synapses. Sensory neurons and motor neurons removed from Aplysia were grown together in Petri dishes and allowed to form synapses. Next, the cells were treated with the hormone serotonin, which promotes long-term memory by, in part, causing the neurons to grow more synapses.

Afterwards, the cells were given treatments that disrupted long-term memory and also reversed the synaptic growth caused by serotonin. However, it was not only new synapses that retracted: some synapses that had existed before the serotonin treatment were also lost. This apparently random loss of synapses suggests that the memory was not stored in specific synapses. Moreover, long-term memory could be restored after these treatments, which supports that idea that memory does not depend on synapses between the neurons being maintained.

This work offers hope that it might be possible to develop treatments that help to restore long-term memory in people suffering from Alzheimer's disease and other conditions that affect long-term memory.

There is significant empirical support for the idea, proposed by Ramón Y Cajal more than a century ago (Cajal, 1894), that long-term memories are expressed in the brain, in part, by changes in synaptic connectivity. A corollary of this idea, accepted by many, if not most, modern neuroscientists, is that memories are maintained by persistent molecular and cellular alterations in synaptic structures themselves (Bailey and Kandel, 2008; Kandel et al., 2014). Here, we have tested the idea that long-term memory (LTM) is stored at synapses using the marine mollusk Aplysia californica. The relatively simple behavior and nervous system of this model invertebrate organism offer several major advantages for understanding memory at the level of modifications of individual synapses (Kandel, 2001). A form of learning in Aplysia whose cellular and molecular substrates are particularly well understood is sensitization of the gill- and siphon-withdrawal reflex (Carew et al., 1971; Brunelli et al., 1976; Antonov et al., 1999; Kandel, 2001; Glanzman, 2010). Sensitization of the withdrawal reflex exhibits a long-term (≥24 hr) form (Pinsker et al., 1973) due, in part, to long-term facilitation (LTF) of the monosynaptic connection between the sensory and motor neurons that mediate the reflex (Frost et al., 1985). Importantly, the monosynaptic sensorimotor connection can be reconstituted in dissociated cell culture, and LTF of the in vitro synapse can be induced by training with pulses of serotonin (5HT), the monoaminergic neurotransmitter that mediates sensitization in Aplysia (Brunelli et al., 1976; Glanzman et al., 1989; Marinesco and Carew, 2002). Cellular and molecular analyses of this form of long-term synaptic plasticity have provided major mechanistic insights into long-term memory in Aplysia (Goelet et al., 1986; Dash et al., 1990; Bartsch et al., 1995; Martin et al., 1997), insights that have generalized to learning and memory in other organisms, including mammals (Yin et al., 1994, 1995; Frey and Morris, 1997; Kogan et al., 1997; Josselyn et al., 2001). Accordingly, we used the in vitro sensorimotor synapse in initial experiments to determine whether LTM is stored at synapses.

Current evidence supports the idea that LTM can be modified or even eliminated under certain circumstances. One of these goes under the rubric of reconsolidation blockade. Here, a stimulus is delivered to an animal that serves to reactivate the LTM for a previous learning experience. If, immediately after delivery of this reminder, the animal is treated with an inhibitor of protein synthesis (Nader et al., 2000), or subjected to electroconvulsive shock (Misanin et al., 1968), the LTM will be apparently eliminated. Based on this evidence, it has been proposed that the reminder stimulus returns the LTM to a labile state in which new protein synthesis is required to reconsolidate the memory; and that inhibition of protein synthesis during this period of reconsolidation can erase the original memory (Nader and Hardt, 2009) (but see Lattal and Abel, 2004). Another manipulation that can apparently erase LTM permanently is inhibition of the constitutively active catalytic fragment of the atypical protein kinase Cζ; the ongoing activity of this catalytic fragment, named PKMζ, appears to be required for the maintenance of several forms of LTM in mammals (Sacktor, 2011); inhibition of PKMζ, in the absence of a reminder, has been reported to abolish consolidated memory (but see Lee et al., 2013; Volk et al., 2013).

We, and others, have shown that the synaptic memory for LTF of in vitro sensorimotor connections can be apparently eliminated when sensorimotor cocultures are treated with a protein synthesis inhibitor following reminder training (Cai et al., 2012; Lee et al., 2012; Hu and Schacher, 2014). In addition, inhibiting the activity of PKM Apl III, the constitutively active fragment of the Aplysia atypical protein kinase C (PKC Apl III) (Villareal et al., 2009; Cai et al., 2011; Bougie et al., 2012), also erases consolidated LTF (Cai et al., 2011). LTF is mediated partly by the growth of new sensory neuron varicosities (Glanzman et al., 1990; Bailey and Kandel, 2008). Here we investigated whether blocking the reconsolidation of the memory for LTF, or inhibiting PKM Apl III, altered this long-term change in presynaptic structure. We found that the synaptic growth induced by LTF training was reversed by these two memory-disrupting manipulations; however, although the overall number of presynaptic varicosities reverted to the original, pretraining level, the resultant morphological pattern of sensorimotor synapses differed significantly from the original one. These results imply that the persistence of memory does not require the stability of particular synaptic connections.

We provide additional support for this idea with data from behavioral experiments in which we show that LTM can be reinstated in intact Aplysia following its apparent disappearance due to reconsolidation blockade or PKM inhibition. Because these two antimnemonic manipulations not only disrupt the behavioral expression of LTM, but also eliminate the synaptic changes—both electrophysiological (Cai et al., 2011, 2012; Lee et al., 2012) and morphological (present data)—closely associated with LTM in Aplysia (Bailey and Chen, 1983, 1988; Frost et al., 1985; Glanzman et al., 1990), our results challenge the idea that the synapse is a cellular site for long-term memory storage in Aplysia. In other behavioral experiments we show that both the disruption of LTM through inhibition of PKM Apl III and LTM induction require epigenetic changes. These results point to the nucleus of neurons as the potential locus of the engram in Aplysia.

The present morphological results, together with those of our previous behavioral and electrophysiological investigations (Cai et al., 2011, 2012), suggest that the persistence of sensitization-related LTM in Aplysia does not require the persistence of the synaptic connections generated during learning. Rather, LTM appears to be regulated by a homeostatic mechanism that specifies the net synaptic strength according to experience. Following 5X5HT stimulation, we observed that the number of presynaptic varicosities in the sensorimotor cocultures increased to a new overall value, and that this value was maintained despite the appearance and disappearance of individual varicosities. Furthermore, blockade of memory reconsolidation, or inhibition of PKM, reset the number of varicosities to the original, non-facilitated value. Remarkably, this resetting did not result from the straightforward retraction of the varicosities that appeared by 24 hr after 5X5HT stimulation, varicosities whose growth coincides with LTF (Glanzman et al., 1990); rather, the resetting was produced by a complex reorganization that involved retraction of both 5HT-induced and original varicosities, as well as growth of new, additional varicosities. Thus, for any given sensorimotor connection, the homeostatic regulatory mechanism appeared indifferent to the identities of the particular presynaptic varicosities and, presumably, active zones (Schacher et al., 1990), that mediated baseline synaptic transmission and synaptic facilitation. Our data argue that synapses are not ‘tagged’ with respect to memory storage, at least for a connection involving a single SN and a single MN; the data may therefore reflect a fundamental distinction between the cellular and molecular processes of LTM storage and those of LTM induction in Aplysia, in which synaptic tagging plays a critical role (Martin et al., 1997). The data also suggest that synaptic change is an expression mechanism, rather than a storage mechanism, for LTM in Aplysia (although see below).

Importantly, neither reconsolidation blockade nor inhibition of PKM caused the varicosity number to drop below the starting value. (Notice that the same is true for the amplitude of the EPSP in our earlier synaptic physiological studies, which used the same training regimens [Cai et al., 2011, 2012].) This result provides additional evidence for the operation of a homeostatic regulatory mechanism, and suggests that the homeostatic mechanism toggles between two all-or-none states, a facilitated, sensitization-related state and a nonfacilitated one. (There must also be a third state, synaptic depression, in which the strength of the sensorimotor connection is reduced below the nonfacilitated level [Montarolo et al., 1988].) Our results are reminiscent of those from studies in the Drosophila neuromuscular junction and the mammalian CNS, which also indicate that synaptic plasticity is regulated by homeostatic mechanisms (Turrigiano, 2007).

The present conclusions appear to conflict with those from morphological investigations of LTM in mice in which dendrites have been chronically imaged in the intact, living brain. Several such studies have reported stability of some dendritic spines for periods of weeks-to-months after training (Xu et al., 2009; Yang et al., 2009; Liston et al., 2013). These results, as well as evidence that spines in the adult mammalian cortex exhibit little turnover (Grutzendler et al., 2002), have led some to argue that stable spines provide a physical basis for the lifelong maintenance of memories (Bhatt et al., 2009). But other studies of the mouse brain using very similar in vivo imaging techniques have reported a high degree of spine instability in the adult cerebral cortex (Trachtenberg et al., 2002; Holtmaat et al., 2005); the contradiction between these two sets of opposed findings remains unresolved. Furthermore, even in those studies where spines of adult cortical neurons were reported to be highly stable, there was significant instability of both pre-existing and new spines within the first 2–4 days after a training protocol (Xu et al., 2009; Yang et al., 2009), and we monitored SN varicosities for only 48 hr after training with 5HT. Therefore, the present results are not necessarily inconsistent with those from in vivo imaging studies in the mouse brain.

We found that LTS could be fully reinstated following its disruption by the same antimnemonic treatments previously shown to eliminate LTF (Cai et al., 2011, 2012; Lee et al., 2012; Hu and Schacher, 2014) (Figures 7 and 8). This finding is consistent with two possible explanations. First, the LTM for sensitization may be intact in animals following reconsolidation blockade and inhibition of PKM, despite the elimination of both behavioral enhancement and synaptic growth. According to this scheme, synapses serve merely to express LTM, they are not sites of LTM storage. The reinstatement of LTM expression, then, involves restoring the appropriate number of synapses between the SNs and MNs, as determined by an experience-registering homeostatic mechanism. The second possible explanation incorporates the notion of an as-yet-unidentified priming mechanism; here, reconsolidation blockade and inhibition of PKM, can erase the stored sensitization memory through the reversal of pre- and postsynaptic structures induced by the long-term training, but the antimnemonic treatments do not eliminate the primer. The primer does not constitute LTM, but is required for its reconstitution via new synaptic growth. The priming signal might interact with fresh facilitatory input to the withdrawal circuit, due to the additional (3×) tail shocks, to upregulate the number of synaptic contacts; the appropriate number of contacts could be determined by the homeostatic process, and involve signals from existing and new synapses.

In a previous study we attempted to reinstate LTS following its disruption by inhibition of PKM Apl III but failed (Cai et al., 2011) (see Figure 3B of the earlier study). Interestingly, we used only a single bout of tail shocks in that attempt. In light of the present results (Figures 7 and 8), our previous failure indicates that one bout of tails shocks is insufficient to fully recruit the signaling pathways that reinstate LTS (Abel et al., 1998; Sossin, 2008; Mayford et al., 2012; Zhang et al., 2012). In future work we will seek to discover how the molecular signals evoked by three bouts of tail shocks differ from those evoked by a single bout.

The data from our behavioral experiments involving the use of an HDAC inhibitor (Figure 9) implicate epigenetic modifications (Levenson and Sweatt 2005; Rahn et al., 2013; Graff and Tsai 2013a)—either in the SN or MN or, most likely, both—in the storage mechanism for LTM in Aplysia. Data from an in vitro study of LTF have also implicated histone acetylation in LTM in Aplysia (Guan et al., 2002). How might HDAC inhibition block chelerythrine's apparent disruption of LTM? Possibly, TSA, by inhibiting histone deacetylase, activates the transcription of genes that promote LTM; the activation of these genes, in turn, may compensate for a disruptive action of chelerythrine on LTM. Findings by others are consistent with this idea. Thus, treatment with an HDAC inhibitor rescues cognitive deficits and learning-related synaptic plasticity in mice that are heterozygous for a mutant form of CREB binding protein (CBP) (Alarcon et al., 2004). In addition, it has been found that phosphorylation of CBP by the atypical PKCζ is required for the histone acetylation that promotes the differentiation of developing neural precursors into neurons, astrocytes, and oligodendrocytes; mutant mice haploinsufficient for CBP exhibit abnormal development of the cerebral cortex (Wang et al., 2010). Furthermore, chelerythrine, by inhibiting the phosphorylation of CBP by PKCζ, blocks the differentiation of cultured precursor cells into astrocytes, and oligodendrocytes (Wang et al., 2010). A recent study in Aplysia has found that reducing the activity of CBP in SNs through intracellular injection of CBP small interfering RNA (siRNA) impaired the induction of LTF in sensorimotor cocultures (Liu et al., 2013). Interestingly, this deficit in LTF induction could be rescued through the use of a 5HT training protocol designed to maximize the phosphorylation and synthesis of CCAAT/enhancer-binding protein (C/EBP). Taken together, these studies suggest that chelerythrine can disrupt epigenetic processes, possibly involving histone acetylation stimulated by CBP, that promote the induction and maintenance of LTM. Moreover, because HDAC inhibition can block chelerythrine's deleterious effects on memory, as well as promote the induction of LTM, it is intriguing to speculate that these effects might be due to enhancement of a CBP-dependent pathway.

But the above explanation for why TSA blocks chelerythrine's effect on consolidated LTM raises a critical question. Our data suggest that consolidated LTM persists after chelerythrine treatment (as well as reconsolidation blockade), possibly as nuclear changes. If so, then the molecular basis for the persistence of LTM is unlikely to be histone acetylation, because an apparent action of chelerythrine is to reverse histone acetylation. Another prominent epigenetic mechanism known to play a role in LTM is DNA methylation (Day and Sweatt, 2010). Although early evidence indicated that learning-induced DNA methylation in the hippocampus was transient and readily reversible (Miller and Sweatt, 2007), a more recent study has reported that contextual fear conditioning in rats induces DNA methylation of the gene for calcineurin in cortical neurons that persists for at least a month (Miller et al., 2010). Furthermore, Sweatt and colleagues have shown in a rat model of childhood maltreatment that early trauma can produce changes in the DNA methylation of the gene for brain-derived neurotrophic factor (BDNF) in the cortex, changes that persist into adulthood (Roth et al., 2009). Thus, DNA methylation may constitute an epigenetic mechanism for the lifelong storage of memory (Day and Sweatt, 2010). Interestingly, 5HT has recently been reported to induce DNA methylation of the promoter of the transcriptional repressor of memory CREB2, thereby facilitating the induction of LTF (Rajasethupathy et al., 2012). Finally, another potential candidate for a nuclear mechanism of LTM storage is suggested by a recent study by Suberbielle et al. (2013) that found, remarkably, that learning causes DNA double-strand breaks (DSBs) in neurons in the brains of control mice; thus, chromatin remodeling subsequent to DNA DSBs may also encode LTM.

Could a nonsynaptic storage mechanism based on nuclear changes mediate the maintenance of associative memories, particularly those induced in complex neural circuits in the mammalian brain, where a given neuron may have 1000s or 10s of 1000s of synaptic partners? An obvious difficulty confronting any hypothetical nuclear storage mechanism in the mammalian brain is how the appropriate number of connections can be maintained in a synapse-specific manner after learning has occurred. For example, if a synaptic contact that has undergone Hebbian long-term potentiation (Bliss and Collingridge, 1993) as a consequence of associative learning retracts, how could a nuclear storage mechanism restrict the growth of a replacement contact to the correct pair of pre- and postsynaptic neurons? Possibly, there are nonsynaptic ways for neurons to communicate that ensure specificity of associative synaptic plasticity in the face of the significant lability of synaptic structure documented here.

There are, of course, somal, non-nuclear, mechanisms of memory storage or priming that could account for the apparent persistence of memory following synaptic erasure. One such mechanism would be the persistent activity of a kinase (Hegde et al., 1993; Bougie et al., 2012), or persistent inhibition of a phosphatase (Sharma et al., 2003), in the cell body of the SN or MN or in both cell bodies. Another somal mechanism consistent with our data is ubiquitination of one or more somal proteins (Hegde et al., 1997; Chain et al., 1999); up-regulation of a ubiquitin-proteasome pathway could degrade proteins that inhibit the storage of LTM, or that block the priming of LTM.

The present data necessitate a reappraisal of the mnemonic consequences of blockade of memory reconsolidation and inhibition of PKM. It has been previously argued that these manipulations can erase consolidated memory (Nader and Hardt, 2009; Sacktor, 2011; Glanzman, 2013) (although see Lattal and Abel, 2004). But our results indicate that the effect of reconsolidation blockade and PKM inhibition is not to delete LTM but, rather, to impair its expression. In other words, the antimnemonic actions of these manipulations may result from their ability to at least partially reverse the cellular and molecular changes, including synaptic growth, that mediate the expression, rather than the storage, of LTM. Possibly, neither reconsolidation blockade nor PKM inhibition can reverse the nuclear remodeling—involving epigenetic modifications, particularly DNA methylation (Day and Sweatt, 2010; Graff et al., 2011; Rahn et al., 2013), as well as, perhaps, chromatin remodeling subsequent to DNA DSBs (Suberbielle et al., 2013)—that represents the stored memory trace. Alternatively, these antimnemonic manipulations might indeed disrupt the stored memory trace, but leave intact some memory-priming signal. Likely candidates for a residual priming signal include the persistent repression of transcription of the CREB repressor, CREB2 (Bartsch et al., 1995; Upadhya et al., 2004; Liu et al., 2011), and persistent degradation of the regulatory subunit of protein kinase A (PKA) (Chain et al., 1999).

Because chelerythrine's disruption of LTM in Aplysia is blocked by TSA, chelerythrine's antimnemonic actions must involve histone deacetylation. Previous speculation regarding how chelerythrine and the zeta inhibitory peptide (ZIP) disrupt memory maintenance in both Aplysia and rats has focused on the ability of these pharmacological agents to inhibit the phosphorylation of proteins by atypical PKM (Cai et al., 2011; Sacktor, 2011) (Box 1). The present results imply that chelerythrine and ZIP may interfere with epigenetic processes induced by atypical PKM, as well as inhibit protein phosphorylation by atypical PKM. A prior study reported that, following inhibition of PKMζ activity in the insular cortex of rats by the peptide inhibitor ZIP, conditioned taste aversion could not be reinstated by an application of the unconditioned stimulus (UCS, intraperitoneal injection of lithium chloride) (Shema et al., 2007). Our experience (Cai et al., 2011, and the present results) suggests that the failure to reinstate conditioned taste aversion in this study was not because LTM had been extinguished, but because a single application of the UCS was too weak to reverse the disruptive effects of ZIP on mechanisms of LTM expression, some of which may involve chromatin remodeling (Figure 7A,B).

In summary, we have found that LTM, or a primer for LTM, can persist following reconsolidation blockade and inhibition of PKM. Furthermore, the residual memory/primer must be independent of synaptic plasticity, because it persists following elimination of the synaptic changes induced during learning. Our results indicate that consolidated memories may be far more refractory to modification or elimination than generally supposed. If confirmed and extended to mammals, the present results would have important implications for treating disorders of LTM, such as posttraumatic stress disorder (PTSD).

1. 1. Abel T
   2. Martin KC
   3. Bartsch D
   4. Kandel ER

   (1998) Memory suppressor genes: inhibitory constraints on the storage of long-term memory

   Science 279:338–341.

   https://doi.org/10.1126/science.279.5349.338

   • Google Scholar
2. 1. Alarcón JM
   2. Malleret G
   3. Touzani K
   4. Vronskaya S
   5. Ishii S
   6. Kandel ER
   7. Barco A

   (2004) Chromatin acetylation, memory, and LTP are impaired in CBP+/- mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration

   Neuron 42:947–959.

   https://doi.org/10.1016/j.neuron.2004.05.021

   • Google Scholar
3. 1. Antonov I
   2. Kandel ER
   3. Hawkins RD

   (1999)

   The contribution of facilitation of monosynaptic PSPs to dishabituation and sensitization of the Aplysia siphon withdrawal reflex

   The Journal of Neuroscience 19:10438–10450.

   • Google Scholar
4. 1. Bailey CH
   2. Chen M

   (1988) Long-term memory in Aplysia modulates the total number of varicosities of single identified sensory neurons

   Proceedings of the National Academy of Sciences of USA 85:2373–2377.

   https://doi.org/10.1073/pnas.85.7.2373

   • Google Scholar
5. 1. Bailey CH
   2. Chen M

   (1983) Morphological basis of long–term habituation and sensitization in Aplysia

   Science 220:91–93.

   https://doi.org/10.1126/science.6828885

   • Google Scholar
6. Book

   1. Bailey CH
   2. Kandel ER

   (2008)

   Synaptic remodeling, synaptic growth and the storage of long-term memory in Aplysi

   In: Sossin WS, Lacaille JC, Castellucci VF, Sylvie B, editors. Essence of memory. Amsterdam: Elsevier. pp. 179–198.

   • Google Scholar
7. 1. Bartsch D
   2. Ghirardi M
   3. Skehel PA
   4. Karl KA
   5. Herder SP
   6. Chen M
   7. Bailey CH
   8. Kandel ER

   (1995) Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change

   Cell 83:979–992.

   https://doi.org/10.1016/0092-8674(95)90213-9

   • Google Scholar
8. 1. Bhatt DH
   2. Zhang S
   3. Gan WB

   (2009) Dendritic spine dynamics

   Annual Review of Physiology 71:261–282.

   https://doi.org/10.1146/annurev.physiol.010908.163140

   • Google Scholar
9. 1. Bliss TV
   2. Collingridge GL

   (1993) A synaptic model of memory: long-term potentiation in the hippocampus

   Nature 361:31–39.

   https://doi.org/10.1038/361031a0

   • Google Scholar
10. 1. Bougie JK
    2. Cai D
    3. Hastings M
    4. Farah CA
    5. Chen S
    6. Fan X
    7. McCamphill PK
    8. Glanzman DL
    9. Sossin WS

    (2012) Serotonin-induced cleavage of the atypical protein kinase C Apl III in Aplysia

    The Journal of Neuroscience 32:14630–14640.

    https://doi.org/10.1523/JNEUROSCI.3026-11.2012

    • Google Scholar
11. 1. Bougie JK
    2. Lim T
    3. Farah CA
    4. Manjunath V
    5. Nagakura I
    6. Ferraro GB
    7. Sossin WS

    (2009) The atypical protein kinase C in Aplysia can form a protein kinase M by cleavage

    Journal of Neurochemistry 109:1129–1143.

    https://doi.org/10.1111/j.1471-4159.2009.06045.x

    • Google Scholar
12. 1. Brunelli M
    2. Castellucci V
    3. Kandel ER

    (1976) Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP

    Science 194:1178–1181.

    https://doi.org/10.1126/science.186870

    • Google Scholar
13. 1. Cai D
    2. Pearce K
    3. Chen S
    4. Glanzman DL

    (2011) Protein kinase M maintains long-term sensitization and long-term facilitation in Aplysia

    The Journal of Neuroscience 31:6421–6431.

    https://doi.org/10.1523/JNEUROSCI.4744-10.2011

    • Google Scholar
14. 1. Cai D
    2. Pearce K
    3. Chen S
    4. Glanzman DL

    (2012) Reconsolidation of long-term memory in Aplysia

    Current Biology 22:1783–1788.

    https://doi.org/10.1016/j.cub.2012.07.038

    • Google Scholar
15. 1. Cajal SR

    (1894) The croonian lecture: La fine structure des centres nerveux

    Proceedings of the Royal Society of London 55:444–467.

    https://doi.org/10.1098/rspl.1894.0063

    • Google Scholar
16. 1. Carew TJ
    2. Castellucci VF
    3. Kandel ER

    (1971) An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aplysia

    The International Journal of Neuroscience 2:79–98.

    https://doi.org/10.3109/00207457109146995

    • Google Scholar
17. 1. Chain DG
    2. Casadio A
    3. Schacher S
    4. Hegde AN
    5. Valbrun M
    6. Yamamoto N
    7. Goldberg AL
    8. Bartsch D
    9. Kandel ER
    10. Schwartz JH

    (1999) Mechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia

    Neuron 22:147–156.

    https://doi.org/10.1016/S0896-6273(00)80686-8

    • Google Scholar
18. 1. Dash PK
    2. Hochner B
    3. Kandel ER

    (1990) Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation

    Nature 345:718–721.

    https://doi.org/10.1038/345718a0

    • Google Scholar
19. 1. Day JJ
    2. Sweatt JD

    (2010) DNA methylation and memory formation

    Nature Neuroscience 13:1319–1323.

    https://doi.org/10.1038/nn.2666

    • Google Scholar
20. 1. Frey U
    2. Morris RG

    (1997) Synaptic tagging and long-term potentiation

    Nature 385:533–536.

    https://doi.org/10.1038/385533a0

    • Google Scholar
21. 1. Frost WN
    2. Castellucci VF
    3. Hawkins RD
    4. Kandel ER

    (1985) Monosynaptic connections made by the sensory neurons of the gill- and siphon-withdrawal reflex in Aplysia participate in the storage of long-term memory for sensitization

    Proceedings of the National Academy of Sciences of USA 82:8266–8269.

    https://doi.org/10.1073/pnas.82.23.8266

    • Google Scholar
22. 1. Fulton DJ
    2. Condro MC
    3. Pearce K
    4. Glanzman DL

    (2008) The potential role of postsynaptic phospholipase C activity in synaptic facilitation and behavioral sensitization in Aplysia

    Journal of Neurophysiology 100:108–116.

    https://doi.org/10.1152/jn.90389.2008

    • Google Scholar
23. 1. Glanzman DL

    (2010) Common mechanisms of synaptic plasticity in vertebrates and invertebrates

    Current Biology 20:R31–R36.

    https://doi.org/10.1016/j.cub.2009.10.023

    • Google Scholar
24. 1. Glanzman DL

    (2013) PKM and the maintenance of memory

    F1000 Biology Reports 5:4.

    https://doi.org/10.3410/B5-4

    • Google Scholar
25. 1. Glanzman DL
    2. Kandel ER
    3. Schacher S

    (1990) Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons

    Science 249:799–802.

    https://doi.org/10.1126/science.2389145

    • Google Scholar
26. 1. Glanzman DL
    2. Mackey SL
    3. Hawkins RD
    4. Dyke AM
    5. Lloyd PE
    6. Kandel ER

    (1989)

    Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

    The Journal of Neuroscience 9:4200–4213.

    • Google Scholar
27. 1. Goelet P
    2. Castellucci VF
    3. Schacher S
    4. Kandel ER

    (1986) The long and the short of long-term memory—a molecular framework

    Nature 322:419–422.

    https://doi.org/10.1038/322419a0

    • Google Scholar
28. 1. Gräff J
    2. Kim D
    3. Dobbin MM
    4. Tsai LH

    (2011) Epigenetic regulation of gene expression in physiological and pathological brain processes

    Physiological Reviews 91:603–649.

    https://doi.org/10.1152/physrev.00012.2010

    • Google Scholar
29. 1. Gräff J
    2. Tsai LH

    (2013a) Histone acetylation: molecular mnemonics on the chromatin

    Nature Reviews. Neuroscience 14:97–111.

    https://doi.org/10.1038/nrn3427

    • Google Scholar
30. 1. Gräff J
    2. Tsai LH

    (2013b) The potential of HDAC inhibitors as cognitive enhancers

    Annual Review of Pharmacology and Toxicology 53:311–330.

    https://doi.org/10.1146/annurev-pharmtox-011112-140216

    • Google Scholar
31. 1. Grutzendler J
    2. Kasthuri N
    3. Gan WB

    (2002) Long-term dendritic spine stability in the adult cortex

    Nature 420:812–816.

    https://doi.org/10.1038/nature01276

    • Google Scholar
32. 1. Guan Z
    2. Giustetto M
    3. Lomvardas S
    4. Kim JH
    5. Miniaci MC
    6. Schwartz JH
    7. Thanos D
    8. Kandel ER

    (2002) Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure

    Cell 111:483–493.

    https://doi.org/10.1016/S0092-8674(02)01074-7

    • Google Scholar
33. 1. Hegde AN
    2. Goldberg AL
    3. Schwartz JH

    (1993) Regulatory subunits of cAMP-dependent protein kinases are degraded after conjugation to ubiquitin: a molecular mechanism underlying long-term synaptic plasticity

    Proceedings of the National Academy of Sciences of USA 90:7436–7440.

    https://doi.org/10.1073/pnas.90.16.7436

    • Google Scholar
34. 1. Hegde AN
    2. Inokuchi K
    3. Pei W
    4. Casadio A
    5. Ghirardi M
    6. Chain DG
    7. Martin KC
    8. Kandel ER
    9. Schwartz JH

    (1997) Ubiquitin C-terminal hydrolase is an immediate-early gene essential for long-term facilitation in Aplysia

    Cell 89:115–126.

    https://doi.org/10.1016/S0092-8674(00)80188-9

    • Google Scholar
35. 1. Hernandez AI
    2. Blace N
    3. Crary JF
    4. Serrano PA
    5. Leitges M
    6. Libien JM
    7. Weinstein G
    8. Tcherapanov A
    9. Sacktor TC

    (2003) Protein kinase Mζ synthesis from a brain mRNA encoding an independent protein kinase Cζ catalytic domain. Implications for the molecular mechanism of memory

    The Journal of Biological Chemistry 278:40305–40316.

    https://doi.org/10.1074/jbc.M307065200

    • Google Scholar
36. 1. Holtmaat AJ
    2. Trachtenberg JT
    3. Wilbrecht L
    4. Shepherd GM
    5. Zhang X
    6. Knott GW
    7. Svoboda K

    (2005) Transient and persistent dendritic spines in the neocortex in vivo

    Neuron 45:279–291.

    https://doi.org/10.1016/j.neuron.2005.01.003

    • Google Scholar
37. 1. Hu JY
    2. Schacher S

    (2014) Persistent long-term facilitation at an identified synapse becomes labile with activation of short-term heterosynaptic plasticity

    The Journal of Neuroscience 34:4776–4785.

    https://doi.org/10.1523/JNEUROSCI.0098-14.2014

    • Google Scholar
38. 1. Josselyn SA
    2. Shi C
    3. Carlezon WA Jr
    4. Neve RL
    5. Nestler EJ
    6. Davis M

    (2001)

    Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala

    The Journal of Neuroscience 21:2404–2412.

    • Google Scholar
39. 1. Kandel ER

    (2001) The molecular biology of memory storage: a dialogue between genes and synapses

    Science 294:1030–1038.

    https://doi.org/10.1126/science.1067020

    • Google Scholar
40. 1. Kandel ER
    2. Dudai Y
    3. Mayford MR

    (2014) The molecular and systems biology of memory

    Cell 157:163–186.

    https://doi.org/10.1016/j.cell.2014.03.001

    • Google Scholar
41. 1. Kim JH
    2. Udo H
    3. Li HL
    4. Youn TY
    5. Chen M
    6. Kandel ER
    7. Bailey CH

    (2003) Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long-term facilitation in Aplysia

    Neuron 40:151–165.

    https://doi.org/10.1016/S0896-6273(03)00595-6

    • Google Scholar
42. 1. Kogan JH
    2. Frankland PW
    3. Blendy JA
    4. Coblentz J
    5. Marowitz Z
    6. Schütz G
    7. Silva AJ

    (1997) Spaced training induces normal long-term memory in CREB mutant mice

    Current Biology 7:1–11.

    https://doi.org/10.1016/S0960-9822(06)00022-4

    • Google Scholar
43. 1. Lattal KM
    2. Abel T

    (2004) Behavioral impairments caused by injections of the protein synthesis inhibitor anisomycin after contextual retrieval reverse with time

    Proceedings of the National Academy of Sciences of USA 101:4667–4672.

    https://doi.org/10.1073/pnas.0306546101

    • Google Scholar
44. 1. Lee AM
    2. Kanter BR
    3. Wang D
    4. Lim JP
    5. Zou ME
    6. Qiu C
    7. McMahon T
    8. Dadgar J
    9. Fischbach-Weiss SC
    10. Messing RO

    (2013) Prkcz null mice show normal learning and memory

    Nature 493:416–419.

    https://doi.org/10.1038/Nature11803

    • Google Scholar
45. 1. Lee SH
    2. Kwak C
    3. Shim J
    4. Kim JE
    5. Choi SL
    6. Kim HF
    7. Jang DJ
    8. Lee JA
    9. Lee K
    10. Lee CH
    11. Lee YD
    12. Miniaci MC
    13. Bailey CH
    14. Kandel ER
    15. Kaang BK

    (2012) A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia

    Proceedings of the National Academy of Sciences of USA 109:14200–14205.

    https://doi.org/10.1073/pnas.1211997109

    • Google Scholar
46. 1. Levenson JM
    2. Sweatt JD

    (2005) Epigenetic mechanisms in memory formation

    Nature Reviews. Neuroscience 6:108–118.

    https://doi.org/10.1038/nrn1604

    • Google Scholar
47. 1. Ling DS
    2. Benardo LS
    3. Serrano PA
    4. Blace N
    5. Kelly MT
    6. Crary JF
    7. Sacktor TC

    (2002) Protein kinase Mζ is necessary and sufficient for LTP maintenance

    Nature Neuroscience 5:295–296.

    https://doi.org/10.1038/nn829

    • Google Scholar
48. 1. Liston C
    2. Cichon JM
    3. Jeanneteau F
    4. Jia Z
    5. Chao MV
    6. Gan WB

    (2013) Circadian glucocorticoid oscillations promote learning-dependent synapse formation and maintenance

    Nature Neuroscience 16:698–705.

    https://doi.org/10.1038/nn.3387

    • Google Scholar
49. 1. Liu RY
    2. Shah S
    3. Cleary LJ
    4. Byrne JH

    (2011) Serotonin- and training-induced dynamic regulation of CREB2 in Aplysia

    Learning & Memory 18:245–249.

    https://doi.org/10.1101/lm.2112111

    • Google Scholar
50. 1. Liu RY
    2. Zhang Y
    3. Baxter DA
    4. Smolen P
    5. Cleary LJ
    6. Byrne JH

    (2013) Deficit in long-term synaptic plasticity is rescued by a computationally predicted stimulus protocol

    The Journal of Neuroscience 33:6944–6949.

    https://doi.org/10.1523/JNEUROSCI.0643-13.2013

    • Google Scholar
51. 1. Marinesco S
    2. Carew TJ

    (2002)

    Serotonin release evoked by tail nerve stimulation in the CNS of Aplysia: characterization and relationship to heterosynaptic plasticity

    The Journal of Neuroscience 22:2299–2312.

    • Google Scholar
52. 1. Martin KC
    2. Casadio A
    3. Zhu H
    4. Yaping E
    5. Rose JC
    6. Chen M
    7. Bailey CH
    8. Kandel ER

    (1997) Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage

    Cell 91:927–938.

    https://doi.org/10.1016/S0092-8674(00)80484-5

    • Google Scholar
53. 1. Mayford M
    2. Siegelbaum SA
    3. Kandel ER

    (2012) Synapses and memory storage

    Cold Spring Harbor Perspectives in Biology 4:a005751.

    https://doi.org/10.1101/cshperspect.a005751

    • Google Scholar
54. 1. Miller CA
    2. Gavin CF
    3. White JA
    4. Parrish RR
    5. Honasoge A
    6. Yancey CR
    7. Rivera IM
    8. Rubio MD
    9. Rumbaugh G
    10. Sweatt JD

    (2010) Cortical DNA methylation maintains remote memory

    Nature Neuroscience 13:664–666.

    https://doi.org/10.1038/nn.2560

    • Google Scholar
55. 1. Miller CA
    2. Sweatt JD

    (2007) Covalent modification of DNA regulates memory formation

    Neuron 53:857–869.

    https://doi.org/10.1016/j.neuron.2007.02.022

    • Google Scholar
56. 1. Misanin JR
    2. Miller RR
    3. Lewis DJ

    (1968) Retrograde amnesia produced by electroconvulsive shock after reactivation of a consolidated memory trace

    Science 160:554–555.

    https://doi.org/10.1126/science.160.3827.554

    • Google Scholar
57. 1. Montarolo PG
    2. Kandel ER
    3. Schacher S

    (1988) Long-term heterosynaptic inhibition in Aplysia

    Nature 333:171–174.

    https://doi.org/10.1038/333171a0

    • Google Scholar
58. 1. Nader K
    2. Hardt O

    (2009) A single standard for memory: the case for reconsolidation

    Nature Reviews Neuroscience 10:224–234.

    https://doi.org/10.1038/nrn2590

    • Google Scholar
59. 1. Nader K
    2. Schafe GE
    3. Le Doux JE

    (2000) Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval

    Nature 406:722–726.

    https://doi.org/10.1038/35021052

    • Google Scholar
60. 1. Pastalkova E
    2. Serrano P
    3. Pinkhasova D
    4. Wallace E
    5. Fenton AA
    6. Sacktor TC

    (2006) Storage of spatial information by the maintenance mechanism of LTP

    Science 313:1141–1144.

    https://doi.org/10.1126/science.1128657

    • Google Scholar
61. 1. Pinsker HM
    2. Hening WA
    3. Carew TJ
    4. Kandel ER

    (1973) Long-term sensitization of a defensive withdrawal reflex in Aplysia

    Science 182:1039–1042.

    https://doi.org/10.1126/science.182.4116.1039

    • Google Scholar
62. 1. Rahn EJ
    2. Guzman-Karlsson MC
    3. Sweatt JD

    (2013) Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: Implications for pain and memory

    Neurobiology of Learning and Memory 105:133–150.

    https://doi.org/10.1016/J.Nlm.2013.06.008

    • Google Scholar
63. 1. Rajasethupathy P
    2. Antonov I
    3. Sheridan R
    4. Frey S
    5. Sander C
    6. Tuschl T
    7. Kandel ER

    (2012) A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity

    Cell 149:693–707.

    https://doi.org/10.1016/j.cell.2012.02.057

    • Google Scholar
64. 1. Ren SQ
    2. Yan JZ
    3. Zhang XY
    4. Bu YF
    5. Pan WW
    6. Yao W
    7. Tian T
    8. Lu W

    (2013) PKClambda is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP

    The EMBO Journal 32:1365–1380.

    https://doi.org/10.1038/emboj.2013.60

    • Google Scholar
65. 1. Roth TL
    2. Lubin FD
    3. Funk AJ
    4. Sweatt JD

    (2009) Lasting epigenetic influence of early-life adversity on the BDNF gene

    Biological Psychiatry 65:760–769.

    https://doi.org/10.1016/j.biopsych.2008.11.028

    • Google Scholar
66. 1. Sacktor TC

    (2011) How does PKMzeta maintain long-term memory?

    Nature Reviews. Neuroscience 12:9–15.

    https://doi.org/10.1038/nrn2949

    • Google Scholar
67. 1. Schacher S
    2. Glanzman D
    3. Barzilai A
    4. Dash P
    5. Grant SG
    6. Keller F
    7. Mayford M
    8. Kandel ER

    (1990) Long-term facilitation in Aplysia: persistent phosphorylation and structural changes

    Cold Spring Harbor Symposia on Quantitative Biology 55:187–202.

    https://doi.org/10.1101/SQB.1990.055.01.021

    • Google Scholar
68. 1. Sharma SK
    2. Bagnall MW
    3. Sutton MA
    4. Carew TJ

    (2003) Inhibition of calcineurin facilitates the induction of memory for sensitization in Aplysia: Requirement of mitogen-activated protein kinase

    Proceedings of the National Academy of Sciences of USA 100:4861–4866.

    https://doi.org/10.1073/pnas.0830994100

    • Google Scholar
69. 1. Shema R
    2. Sacktor TC
    3. Dudai Y

    (2007) Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKMζ

    Science 317:951–953.

    https://doi.org/10.1126/science.1144334

    • Google Scholar
70. 1. Sossin WS

    (2008) Defining memories by their distinct molecular traces

    Trends in Neurosciences 31:170–175.

    https://doi.org/10.1016/j.tins.2008.01.001

    • Google Scholar
71. 1. Suberbielle E
    2. Sanchez PE
    3. Kravitz AV
    4. Wang X
    5. Ho K
    6. Eilertson K
    7. Devidze N
    8. Kreitzer AC
    9. Mucke L

    (2013) Physiologic brain activity causes DNA double-strand breaks in neurons, with exacerbation by amyloid-beta

    Nature Neuroscience 16:613–621.

    https://doi.org/10.1038/nn.3356

    • Google Scholar
72. 1. Trachtenberg JT
    2. Chen BE
    3. Knott GW
    4. Feng G
    5. Sanes JR
    6. Welker E
    7. Svoboda K

    (2002) Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex

    Nature 420:788–794.

    https://doi.org/10.1038/nature01273

    • Google Scholar
73. 1. Turrigiano G

    (2007) Homeostatic signaling: the positive side of negative feedback

    Current Opinion in Neurobiology 17:318–324.

    https://doi.org/10.1016/j.conb.2007.04.004

    • Google Scholar
74. 1. Upadhya SC
    2. Smith TK
    3. Hegde AN

    (2004) Ubiquitin-proteasome-mediated CREB repressor degradation during induction of long-term facilitation

    Journal of Neurochemistry 91:210–219.

    https://doi.org/10.1111/j.1471-4159.2004.02707.x

    • Google Scholar
75. 1. Villareal G
    2. Li Q
    3. Cai D
    4. Fink AE
    5. Lim T
    6. Bougie JK
    7. Sossin WS
    8. Glanzman DL

    (2009) Role of protein kinase C in the induction and maintenance of serotonin-dependent enhancement of the glutamate response in isolated siphon motor neurons of Aplysia californica

    The Journal of Neuroscience 29:5100–5107.

    https://doi.org/10.1523/jneurosci.4149-08.2009

    • Google Scholar
76. 1. Volk LJ
    2. Bachman JL
    3. Johnson R
    4. Yu Y
    5. Huganir RL

    (2013) PKM-zeta is not required for hippocampal synaptic plasticity, learning and memory

    Nature 493:420–423.

    https://doi.org/10.1038/nature11802

    • Google Scholar
77. 1. Wang J
    2. Weaver IC
    3. Gauthier-Fisher A
    4. Wang H
    5. He L
    6. Yeomans J
    7. Wondisford F
    8. Kaplan DR
    9. Miller FD

    (2010) CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein-Taybi Syndrome brain

    Developmental Cell 18:114–125.

    https://doi.org/10.1016/j.devcel.2009.10.023

    • Google Scholar
78. 1. Xu T
    2. Yu X
    3. Perlik AJ
    4. Tobin WF
    5. Zweig JA
    6. Tennant K
    7. Jones T
    8. Zuo Y

    (2009) Rapid formation and selective stabilization of synapses for enduring motor memories

    Nature 462:915–919.

    https://doi.org/10.1038/nature08389

    • Google Scholar
79. 1. Yang G
    2. Pan F
    3. Gan WB

    (2009) Stably maintained dendritic spines are associated with lifelong memories

    Nature 462:920–924.

    https://doi.org/10.1038/nature08577

    • Google Scholar
80. 1. Yin JC
    2. Del Vecchio M
    3. Zhou H
    4. Tully T

    (1995) CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila

    Cell 81:107–115.

    https://doi.org/10.1016/0092-8674(95)90375-5

    • Google Scholar
81. 1. Yin JC
    2. Wallach JS
    3. Del Vecchio M
    4. Wilder EL
    5. Zhou H
    6. Quinn WG
    7. Tully T

    (1994) Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila

    Cell 79:49–58.

    https://doi.org/10.1016/0092-8674(94)90399-9

    • Google Scholar
82. 1. Zhang Y
    2. Liu RY
    3. Heberton GA
    4. Smolen P
    5. Baxter DA
    6. Cleary LJ
    7. Byrne JH

    (2012) Computational design of enhanced learning protocols

    Nature Neuroscience 15:294–297.

    https://doi.org/10.1038/nn.2990

    • Google Scholar

Author details

1. Shanping Chen

   Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States

   Contribution

   SC, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Diancai Cai

   Competing interests

   The authors declare that no competing interests exist.

2. Diancai Cai

   Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States

   Contribution

   DC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Shanping Chen

   Competing interests

   The authors declare that no competing interests exist.

3. Kaycey Pearce

   Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States

   Contribution

   KP, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Philip Y-W Sun

   Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States

   Present address

   Mayo Medical School, Rochester, United States

   Contribution

   PY-WS, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Adam C Roberts

   Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States

   Contribution

   ACR, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. David L Glanzman

   1. Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, United States
   2. Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, United States
   3. Integrative Center for Learning and Memory, Brain Research Institute, University of California, Los Angeles, Los Angeles, United States

   Contribution

   DLG, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   dglanzman@physci.ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

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