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).

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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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