The formation of long-term memory depends on new proteins being made in the brain. These new proteins are used partly to build the new connections among neurons that essentially store the memory, and must be made within a critical period of time. Experiments on animals have found that new proteins must be made during or shortly after training to form a stable memory; if protein synthesis is blocked during this period, the memory will not be stabilized (a process also known as memory consolidation).

Changes that alter the activity of genes in neurons also play essential roles in memory consolidation. One such change involves the attachment of a methyl group – a molecule that contains one carbon atom surrounded by three hydrogen atoms – to the DNA of a gene. This process, called DNA methylation, typically inhibits the activity of the gene.

Pearce et al. looked at how completely preventing protein synthesis and DNA methylation disrupted memory consolidation in a type of marine snail called Aplysia. Previously, researchers have exploited this animal’s simple nervous system and behavior to discover basic biological mechanisms of memory that are common to all animals.

The snails were given training that increased the likelihood that they would reflexively withdraw part of their body (called the siphon) in response to touch. When Pearce et al. inhibited protein synthesis soon after training, the snails did not remember the training when tested 24 hours later, as expected. Further analysis showed, however, that a trace of the memory, referred to as the “priming trace”, remained. Snails that had this priming trace could form a long-term memory after partial training, whereas untrained snails did not form memories after such partial training.

Inhibiting the synthesis of proteins during the original training blocked the priming trace, as did inhibiting DNA methylation during or after training. Moreover, inhibiting DNA methylation erased a previously established memory and prevented it from being reinstated by partial training.

Overall, the findings of Pearce et al. show that proteins produced in the brain by learning have multiple roles. In addition, both the consolidation and maintenance of long-term memory depend on one or more genes that otherwise suppress memory being inhibited via DNA methylation. Future work will now aim to identify the priming trace and the genes that suppress memory. Knowledge of the priming trace could lead to new treatments for memory-related disorders such as Alzheimer’s disease. Furthermore, identifying genes that can suppress memory might allow us to reduce some of the harmful effects of traumatic experience.

Since the pioneering work of the Flexners (Flexner et al., 1963), Agranoff (Agranoff and Klinger, 1964) and their colleagues, it has been widely accepted that memory consolidation—the process by which a labile, short-term memory trace is transformed into a stable, long-term trace (Lechner et al., 1999; Müller and Pilzecker, 1900)—requires protein synthesis (Davis and Squire, 1984; Goelet et al., 1986; Hernandez and Abel, 2008). Moreover, the protein synthesis underlying memory consolidation has been observed to exhibit a temporal gradient: inhibition of protein synthesis during or shortly after training appears to be maximally disruptive of memory consolidation; significantly less amnesia is caused by protein synthesis inhibition (PSI) that commences approximately an hour or more after training (Davis and Squire, 1984).

In general, there has been little evidence for a functional distinction with respect to memory consolidation between protein synthesis that occurs during training—hereafter ‘early’ protein synthesis—and protein synthesis that occurs within the first hour or so after the end of training—hereafter ‘late’ protein synthesis. Thus, studies have classically observed significant disruptive effects on the consolidation of long-term memory (LTM) whether a protein synthesis inhibitor is applied either immediately prior to, or immediately after, training (e.g., Agranoff et al., 1966; Barondes and Cohen, 1968; Flexner and Flexner, 1968). Consequently, both early and late protein synthesis are commonly regarded as participating in a more-or-less unitary consolidative process. In particular, protein synthesis is believed to mediate critical late events in memory consolidation, including late gene transcription via the synthesis of transcription factors, such as the CCAAT/enhancer-binding protein (C/EBP), and the consequent synthesis of proteins involved in the construction of new synaptic connections (Bailey et al., 2015; Kandel et al., 2014).

One mechanism increasingly implicated in the consolidation of LTM is the epigenetic process of DNA methylation (Levenson et al., 2006; Maddox et al., 2014; Miller et al., 2008; Monsey et al., 2011; Oliveira, 2016; Rajasethupathy et al., 2012). However, the relationship between protein synthesis and DNA methylation in memory consolidation is unclear. Mechanistically, is protein synthesis upstream or downstream of DNA methylation during consolidation? DNA methylation is usually associated with gene silencing. If DNA methylation is required for the synthesis of necessary consolidative proteins, this would imply that a prerequisite for this synthesis is the silencing of one or more repressor genes. On the other hand, it is possible that activation of DNA methyltransferase (DNMT), the family of enzymes that catalyze the transfer of a methyl group to DNA, during memory consolidation itself depends on protein synthesis. Of course, these two possibilities are not mutually exclusive.

Here, we have examined the potentially distinctive roles of early and late protein synthesis in the consolidation of the LTM for behavioral sensitization in Aplysia. In addition, we have tested the effect on memory consolidation of both early and late inhibition of DNA methylation. We find that LTM can be induced by partial training, which is insufficient to induce LTM in naïve (untrained) animals, after the disruption of LTM by late, but not early, administration of a protein synthesis inhibitor. By contrast, both early and late inhibition of DNMT block LTM consolidation as indicated by the preclusion of subsequent memory induction by partial training. These results point to a functional distinction between early and late protein synthesis in memory consolidation, and suggest a potential role for early protein synthesis in DNA methylation. Finally, we show that inhibition of DNMT disrupts not only the consolidation, but also the persistence, of LTM; thus, the maintenance of consolidated LTM requires ongoing DNA methylation.

We have shown that protein synthesis during and shortly after sensitization training is essential for the normal consolidation of LTM in Aplysia. Our results therefore confirm previous results obtained in Aplysia by Montarolo et al. (1986) and Castellucci et al. (1989), as well in vertebrates by many groups (reviewed in Davis and Squire, 1984; Hernandez and Abel, 2008). In addition, however, we have significantly extended prior findings regarding protein synthesis and memory consolidation through our demonstration that LTM can be induced by supplemental partial training following its disruption by PSI shortly after the original (full) LTS training, but not following PSI during the original LTS training. Thus, the present results reveal a novel functional distinction between the mnemonic role of protein synthesis during training and that of protein synthesis shortly after training. An early study in Aplysia indicated that bath-applied anisomycin (18 µM) produces rapid (≤15 min), nearly complete (95–99%) inhibition of protein synthesis, as measured by the incorporation of leucine into proteins in identified central neurons (Schwartz et al., 1971). Because the pretraining injections of anisomycin in our study were made 10–20 min prior to the onset of training, and because the duration of the 5X training in our study was ~80 min, the pretraining anisomycin treatment would be expected to produce >90% disruption of protein synthesis in the animals throughout much, if not all, of the training period. The posttraining injections of anisomycin in our study were made 10–20 min after the end of 5X training; if one assumes a maximum post-injection time of 15 min for the onset of significant PSI within the central nervous system (CNS) of the animals (Schwartz et al., 1971)—drugs injected into the hemocoel of Aplysia have ready access to the CNS due to the open circulatory system and lack of a blood-brain barrier in gastropod mollusks (Abbott et al., 1986)—then the posttraining injections of anisomycin should have begun to inhibit protein synthesis by >90% within 30 min after the end of 5X training. Our results indicate that proteins synthesized during training (early protein synthesis) play a special role in the consolidation of LTM. Specifically, early protein synthesis causes the generation of a priming component that allows LTM to be later established by partial training if it is disrupted by posttraining PSI (Figures 1 and 2). Barzilai et al. (1989) reported that a 1.5-h treatment with 5HT produces the rapid onset (<30 min) of the synthesis of several (unidentified) proteins; in addition, the synthesis of some of these proteins depends on gene transcription, suggesting that they represent immediate-early proteins. Thus, the priming component induced by early protein synthesis in our study may be the product of immediate-early gene transcription. In support of this idea, Rajasethupathy et al. (2012) found that exposure to five spaced pulses of 5HT down regulated the expression of the transcriptional repressor CREB2 in Aplysia sensory neurons; moreover, this downregulation depended on methylation of the CREB2 gene, because it was blocked by the DNMT inhibitor RG108. CREB2 is regarded as a memory suppressor in Aplysia (Abel et al., 1998) (although see Hu et al., 2015); blockade of CREB2 activity (by means of a function-blocking antiserum) in Aplysia sensory neurons has been shown to facilitate the induction of LTF (Bartsch et al., 1995). The memory suppressive effect of CREB2 is due, at least in part, to the repression of CREB1 activation and the downstream expression of immediate-early genes, including the C/EBP gene (Alberini et al., 1994; Alberini and Kandel, 2014). Thus, the expression of CREB1-dependent immediate-early genes, enabled through 5HT-induced DNA methylation of CREB2, could form the priming memory component revealed by the present experiments. According to this idea, DNA methylation would be upstream from the early protein synthesis required for memory consolidation in Aplysia.

The blockade of protein synthesis shortly after (i.e., starting ~30 min after) training, albeit disruptive of LTM, as indicated by the absence of LTM at 24 h following posttraining PSI, nonetheless does not preclude the subsequent induction of LTM by partial training. (Note that our results appear to partly contradict those of Montarolo et al. (1986) who observed that anisomycin applied to sensorimotor cocultures starting at 30 min after 5X5HT training did not block LTF; however, the onset of the anisomycin application in our experiments was ~15 min earlier than in the Montarolo et al. study, which may explain the apparent discrepancy in results.) In support of the present findings, Shobe et al. (2016) have recently reported that consolidation of the LTM for sensitization is disrupted by posttraining application of the protein synthesis inhibitor emetine to a reduced preparation of Aplysia. Therefore, PSI after training does not disrupt the memory primer, which is induced by early protein synthesis (Figure 3). It seems likely, moreover, that it is the persistence of this primer that underlies the ability of truncated training to reinstate LTM following its disruption by reconsolidation blockade or inhibition of PKM (Chen et al., 2014).

How can truncated training establish LTM following impairment of memory consolidation by posttraining PSI, and also reinstate consolidated LTM after its disruption by reconsolidation blockade or inhibition of PKM (Chen et al., 2014)? One possibility is that protein synthesis—in addition to signaling by one or more growth-related factors (Hu et al., 2004; Kopec et al., 2015; Zhang et al., 1997)—during long-term training results in persistent activation of mitogen-activated protein kinase (MAPK) (Martin et al., 1997; Sharma et al., 2003b), and it is the sustained MAPK activity that enables reinstatement of LTM by partial training. In support of this idea, sensitization training that is sufficient to induce LTM has recently been found to cause prolonged (1 h) posttraining activation of MAPK (Shobe et al., 2016). But posttraining PSI disrupts this persistent MAPK activity (Shobe et al., 2016), and we have shown that the memory priming signal is maintained despite posttraining blockade of translation (Figures 1 and 2). Although the priming signal cannot therefore be MAPK itself, it could be a signal downstream from activated MAPK. Martin et al. (1997) showed that during 5HT-induced LTF MAPK translocates to the nucleus of sensory neurons; this nuclear translocation is required for LTF. Possibly, the priming signal is a nuclear change downstream of MAPK. Epigenetic modifications are attractive candidates for the priming signal. Indeed, MAPK-dependent increases in the phosphorylation of histone H3 have been implicated in LTM in rats (Chwang et al., 2006) and snails (Danilova et al., 2010). Furthermore, we previously showed that inhibition of histone deacetylase (HDAC) permitted 3X sensitization training to induce LTM in naïve, untrained animals (Chen et al., 2014). Thus, persistent histone modifications induced by 5X training may provide the scaffolding necessary for later reconstruction of LTM by partial training during memory reinstatement. Besides MAPK-dependent epigenetic modifications, other candidates for the priming signal include small, non-coding RNAs, the expression of which is induced by 5HT training during LTF (Rajasethupathy et al., 2012). In future research we will seek to identify the memory priming signal.

Our demonstration that LTM can be fully established by abbreviated training after being disrupted by posttraining PSI echoes recent findings for contextual fear memory in mice by Tonegawa and colleagues (Ryan et al., 2015). These investigators used optogenetic stimulation of hippocampal neurons that had been active during fear conditioning to restore LTM after the induction of retrograde amnesia by posttraining treatment with anisomycin. Similar to our finding that LTF was absent at 24 h in sensorimotor cocultures after 5X5HT training followed by exposure to anisomycin (Figure 2), Ryan et al. observed an absence of learning-induced long-term potentiation (LTP) (Herring and Nicoll, 2016) in hippocampal slices from anisomycin-treated animals; this synaptic disruption correlated with retrograde amnesia. Despite the lack of persistent synaptic changes widely regarded as hallmarks of consolidated memory (Bailey et al., 2015; Dudai et al., 2015), some aspect of LTM nonetheless endured in the two studies.

Ryan et al. (2015) did not test whether LTM could be induced by optogenetic stimulation subsequent to PSI during fear conditioning, nor did they propose a specific storage mechanism for LTM. Our data point to DNA methylation as being essential for the consolidation of LTM. Prior work in mammals also supports a critical role for DNA methylation in memory consolidation (Halder et al., 2016; Levenson et al., 2006; Miller et al., 2008; Miller and Sweatt, 2007; Monsey et al., 2011; Oliveira, 2016). More relevantly, Kandel and colleagues reported that RG108 blocks the establishment of LTF in Aplysia sensorimotor cocultures, and that this effect is due, at least in part, to blockade of 5HT-induced methylation of the gene for the transcriptional repressor CREB2 (Rajasethupathy et al., 2012). The present results elaborate upon this idea; besides showing that the consolidation of LTM in Aplysia requires epigenetic suppression of one or more memory repressor processes, our results suggest the intriguing possibility that this suppression may depend on early protein synthesis. Although little is known at present regarding the potential role of protein synthesis in DNA modification, a report that activity-dependent induction of a specific gene, Gadd45b, is essential for DNA demethylation in the mammalian hippocampus (Ma et al., 2009) is consistent with this idea. Furthermore, in their study of the epigenetic regulation of memory consolidation in Aplysia, Rajasethupathy et al. (2012) found that DNA methylation of CREB2 was regulated by a neuronally expressed Piwi protein. Perhaps the early protein synthesis mediating the consolidation of LTM involves the expression of Piwi in Aplysia. In the absence of direct evidence that protein synthesis triggers DNA methylation in Aplysia, however, it is at least as plausible that the process of DNA methylation is upstream, rather than downstream, of early protein synthesis in the consolidation of LTM, as discussed above.

Given, as our results indicate, that the consolidation of LTM in Aplysia depends critically on the silencing of one or more genes whose protein products act to repress memory, what are potential candidates for this memory repressive function? An obvious candidate, of course, is CREB2 (Bartsch et al., 1995; Rajasethupathy et al., 2012), but there are others. For example, phosphatases have been proposed to subserve memory repression in mammals. Miller and Sweatt (2007) found that infusion of a DNMT inhibitor into the hippocampus of rats immediately after contextual fear conditioning blocked the consolidation of fear memory as assessed 24 h later, and that this effect was due, in part, to DNA methylation of the gene for protein phosphatase 1 (PP1). Another phosphatase that may subserve memory repression, and whose gene may become silenced by DNA methylation during learning, is calcineurin (protein phosphatase 2B) (Baumgärtel and Mansuy, 2012). Calcineurin activity suppresses the induction of hippocampal LTP (Winder et al., 1998; Winder and Sweatt, 2001). Moreover, genetically overexpressing calcineurin in the brains of mice disrupts the consolidation of LTM (Mansuy et al., 1998), whereas genetically inhibiting calcineurin enhances hippocampal LTP and LTM in mice (Malleret et al., 2001). In addition, Baumgärtel et al. (2008) reported that calcineurin activity is inhibited in the amygdala during the consolidation of conditioned taste aversion (CTA) in mice, and that the level of calcineurin activity during learning determines the strength of the CTA memory. In Aplysia the effects of genetically inhibiting or overexpressing either PP1 or calcineurin on LTM have yet to be examined. However, both phosphatases modulate the CREB-mediated response to extracellular stimuli in Aplysia signaling pathways (Hawkins et al., 2006). Moreover, pharmacological inhibition of calcineurin has been shown to facilitate the induction of the LTM for sensitization in Aplysia (Sharma et al., 2003a).

Besides demonstrating a role for DNA methylation in memory consolidation, the present study shows that ongoing DNA methylation plays a crucial role in memory maintenance. This finding resonates with those from recent work in mammals (Halder et al., 2016; Miller et al., 2010; Mizuno et al., 2012), as well as in invertebrates (Biergans et al., 2015; Lukowiak et al., 2014). We do not know the identity of the gene or genes whose persistent methylation underlies memory maintenance in Aplysia. The gene for CREB2 is one possibility; but a recent study using sensorimotor cocultures found that a long-lasting increase in postsynaptic CREB2 expression mediated the maintenance of LTF beginning 48 h after the induction of synaptic plasticity (Hu et al., 2015), which is inconsistent with a role for the continuous silencing of the CREB2 gene in the maintenance of sensitization memory. Interestingly, Miller et al. (2010) observed persistent DNA methylation of the gene for calcineurin in the anterior cingulate cortex of rats trained contextual fear conditioning. The notion that ongoing suppression of calcineurin activity via gene silencing mediates LTM maintenance in Aplysia is attractive in light of previous data implicating calcineurin activity in the inhibition of LTM (Sharma et al., 2003a); nonetheless, to date no direct evidence supports a role for calcineurin in the maintenance of memory in Aplysia.

A unique and fascinating aspect of the present results is that they enable a direct comparison of the effect on the persistence of a single, specific form of memory—LTS in Aplysia—of inhibiting DNA methylation with those for two other manipulations that have been purported to eliminate consolidated LTM, inhibition of PKMζ (Sacktor, 2011) and reconsolidation blockade (Nader, 2015). Previously, we showed that although inhibition of PKM Apl III, the Aplysia homolog of PKMζ (Bougie et al., 2012, 2009), and reconsolidation blockade both disrupt the consolidated LTM for behavioral sensitization in Aplysia (Cai et al., 2011, 2012), the memory can nonetheless be fully reinstated using truncated sensitization training (Chen et al., 2014). By contrast, as shown in the present study, the LTM for sensitization cannot be reinstated after its disruption by inhibition of DNMT (Figures 6–9). This finding indicates that the ongoing DNA methylation of one or more genes is a precondition for memory maintenance, and, moreover, that some essential priming component of LTM, one whose persistence enables LTM reinstatement, must be impervious to the disruptive effects of PKM inhibition and of reconsolidation blockade, but eliminable by inhibition of DNMT.

In summary, we have found that two functionally distinct periods of protein synthesis regulate the consolidation of LTM in Aplysia. The earlier period occurs during training (and, possibly, extends into the immediate posttraining period as well); it involves the production of a memory priming element. The later period starts within 30 min after training; proteins synthesized during this posttraining period are required for the normal expression of LTM. Nonetheless, inhibition of the synthesis of these late proteins, albeit disruptive of LTM, does not impair the priming element, whose occult presence permits LTM to be established by supplemental partial training following its disruption by posttraining PSI. Finally, we have shown that both the consolidation and maintenance of LTM in Aplysia depend, in part, on gene silencing via DNA methylation. Future work will be required to determine the identity of the gene/s whose DNA methylation is required for the induction and persistence of LTM, and whether this methylation is triggered by early protein synthesis.

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Author details

1. Kaycey Pearce

   Department of Integrative Biology and Physiology, UCLA, Los Angeles, United States

   Contribution

   KP, Conceptualization, Data curation, Investigation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

2. Diancai Cai

   Department of Integrative Biology and Physiology, UCLA, Los Angeles, United States

   Contribution

   DC, Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Adam C Roberts

   Department of Integrative Biology and Physiology, UCLA, Los Angeles, United States

   Contribution

   ACR, Software, Formal analysis, Methodology

   Competing interests

   The authors declare that no competing interests exist.

4. David L Glanzman

   1. Department of Integrative Biology and Physiology, UCLA, 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, UCLA, Los Angeles, United States

   Contribution

   DLG, Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   glanzman@ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-5479-0245

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