Author response: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation. https://doi.org/10.7554/eLife.18299.001 eLife digest 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. https://doi.org/10.7554/eLife.18299.002 Introduction 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. Results LTM can be induced by truncated sensitization training following amnesia produced by posttraining PSI Animals were given training that induced long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR); this training (hereafter 5X training) consisted of five bouts of tail shocks spaced 20 min apart (Cai et al., 2011, 2012; Chen et al., 2014) (Figure 1). Control animals received no training. Then, ~15 min (range = 10–20 min) after training, trained animals received an intrahemocoelic injection of anisomycin, a protein synthesis inhibitor, or vehicle solution. Control animals received an injection of vehicle solution at the equivalent experimental time. At 24 h after training the duration of the SWR was tested in all the animals (24-h posttest), after which two of the groups—the control group (Control-Veh-3XTrained) and a group that had received the long-term training plus the posttraining injection of anisomycin (5XTrained-Aniso-3XTrained) were given additional sensitization training, which consisted of three bouts of tail shocks spaced 20 min apart. Previously, we found that this training (hereafter 3X training), which is insufficient to induce LTM in naïve animals, can successfully reinstate LTM following its disruption by inhibition of PKM Apl III—the Aplysia homolog of PKMζ (Bougie et al., 2012, 2009)—or memory reconsolidation blockade (Chen et al., 2014). All the groups, including the two that received the 5X training but not the 3X training (5XTrained-Veh and 5XTrained-Aniso groups), were given another test at 48 h after training or at the equivalent experimental time (48-h posttest). Figure 1 Download asset Open asset LTS can be established by truncated training following its disruption by posttraining PSI. (A) Experimental protocols. The times of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the fifth bout of sensitization training (time = 0). The time of the intrahemocoelic injection of anisomycin or vehicle is indicated by the red arrow. After the 24-h posttest, animals in the Control-Veh-3XTrained and 5XTrained-Aniso-3XTrained groups received truncated sensitization training (3 bouts of tail shocks). (B) The mean duration of the SWR measured at 24 h and 48 h for the Control-Veh-3XTrained (n = 7), 5XTrained-Veh (n = 6), 5XTrained-Aniso (n = 5), and 5XTrained-Aniso-3XTrained (n = 6) groups. A repeated-measures ANOVA indicated that there was a significant group x time interaction (F[6,40] = 210.9, p < 0.0001). Subsequent planned comparisons indicated that the overall differences among the four groups were highly significant on all of the posttests (24 h, F[3,20] = 456.7, p < 0.0001; and 48 h, F[3,20] = 250.6, p < 0.0001). SNK posthoc tests revealed that the initial sensitization training produced significant LTS, as indicated by the increased mean duration of the SWR, in the 5XTrained-Veh group (56.7 ± 2.2 s) at 24 h compared with that in the Control-Veh-3XTrained (1.9 ± 0.9 s, p < 0.001). The mean duration of the SWR in the 5XTrained-Veh group at 24 h was also significantly longer than that in the 5XTrained-Aniso (1.4 ± 0.4 s, p < 0.001), and 5XTrained-Aniso-3XTrained groups (1.7 ± 0.7 s, p < 0.001). The differences among the Control-Veh-3XTrained, 5XTrained-Aniso and 5XTrained-Aniso-3XTrained groups were not significant at 24 h. The mean duration of the SWR in the 5XTrained-Veh group (58.2 ± 1.8 s) was still protracted at 48 h, and was significantly longer than that in the Control-Veh-3XTrained group (1.1 ± 0.2 s), as well as that in the 5XTrained-Aniso group (1.2 ± 0.2 s, p < 0.001 for both comparisons). LTS was induced in the 5XTrained-Aniso-3XTrained group by the three additional tail shocks applied after the 24-h posttest. The mean duration of the SWR in this group at 48 h was 49.7 ± 3.4 s, which was significantly longer than that for the Control-Veh-3XTrained group. In addition, the mean duration of the reflex in the 5XTrained-Aniso-3XTrained group was longer than that in 5XTrained-Aniso group, but still significantly shorter than that in the 5XTrained-Veh group at 48 h (p < 0.01). Asterisks, comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained group, the 5XTrained-Aniso group, and 5XTrained-Aniso-3XTrained group at 24 h; and comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained group and the 5XTrained-Aniso group at 48 h. Pound signs, comparison of the 5XTrained-Veh with the 5XTrained-Aniso-3XTrained group at 48 h. Plus signs, comparisons of the 5XTrained-Aniso-3XTrained group with the Control-Veh-3XTrained and 5XTrained-Aniso groups at 48 h. Here and in subsequent figures one symbol indicates p < 0.05; two symbols, p < 0.01; and three symbols, p < 0.001. Error bars in this and subsequent figures represent ± SEM. https://doi.org/10.7554/eLife.18299.003 The group given 5X training followed by vehicle injection (5XTrained-Veh group) exhibited significant sensitization at 24 h after training compared with the two groups that received the 5X training followed by anisomycin injection (5XTrained-Aniso and 5XTrained-Aniso-3XTrained groups), and with the control group (Control-Veh-3XTrained). The subsequent 3X training produced LTS in the 5XTrained-Aniso-3XTrained group, as shown by the results for the 48-h posttest. (Notice that the 3X training did not induce LTS in animals that did not receive prior 5X training.) Thus, although posttraining PSI produced complete amnesia for sensitization at 24 h, it did not preclude the subsequent establishment of LTS by partial training. Previous work (Montarolo et al., 1986) examined the effect of PSI at various times after training on serotonin (5HT)-dependent, long-term facilitation (LTF) of the sensorimotor synapse in dissociated cell culture, an in vitro homolog of LTS in Aplysia (Kandel, 2001). This work demonstrated that LTF, tested at 24 h after training, was disrupted by anisomycin applied during a 3-h period that extended from 1 h before the onset of spaced 5HT training through 0.5 h after training (Montarolo et al., 1986). (The 5HT training lasted 1.5 h.) By contrast, a 3-h period of anisomycin treatment beginning at either 0.5 h or 4 h after the end of 5HT training did not block LTF. Considering our behavioral results (above), we wished to determine whether exposure to a protein synthesis inhibitor immediately after 5HT training would disrupt LTF at 24 h and, if so, whether LTF could subsequently be induced by partial training. Accordingly, some sensorimotor cocultures were treated with anisomycin (10 µM, 2 h) immediately after training with five 5-min pulses of 5HT (100 µM; 5X5HT training), spaced at 15-min intervals (Cai et al., 2008; Chen et al., 2014) (Figure 2A). To test whether LTF could be induced by partial training following its potential disruption by posttraining anisomycin treatment, three spaced pulses of 5HT (3X5HT training) were used. The experiment included a group of cocultures (5X5HT group) that received the full 5HT training, but not the posttraining anisomycin, as well as a group (Control) that received neither the 5HT nor the posttraining anisomycin. Finally, there was a group (3X5HT) that received the three pulses of 5HT at 24 h, but not the initial 5X5HT training. (Cocultures not treated with a drug at a particular point in the experiment were treated with standard perfusion medium instead.) The application of anisomycin immediately after the 5X5HT training blocked LTF at 48 h (5X5HT-Aniso group; Figure 2B,C). However, three pulses of 5HT applied 24 h after the original 5HT training and subsequent PSI induced LTF at 48 h (5X5HT-Aniso-3X5HT group). Notice that the mean EPSP in the 5X5HT-Aniso-3X5HT group was not significantly different from that in the 5X5HT group, which indicates that the supplemental partial training induced normal LTF. These cellular results accord with our behavioral finding that, although immediate posttraining PSI disrupts the consolidation of LTM, later abbreviated training can result in full LTM. Montarolo et al. (1986) observed significant LTF when cocultures were treated with anisomycin for 3 h, and even for 22 h, beginning 0.5 h after the end of 5X5HT training; the present results, taken together with those of Montarolo et al., indicate that protein synthesis during a period of 30 min or so immediately following the 5X5HT training is critical for the normal consolidation of LTF in Aplysia. Figure 2 Download asset Open asset Partial training induces LTF following its disruption by PSI immediately after long-term training. (A) Experimental protocols. The initial training consisted of five 5-min pulses of 100 µM 5HT (5X5HT) spaced at 15-min intervals. Cocultures in the 5X5HT-Aniso and 5X5HT-Aniso-3X5HT groups were treated with anisomycin (10 µM, red bar) for 2 h immediately after the 5X5HT training. Three 5-min pulses of 5HT (100 µM; 3X5HT training) were given to cocultures in the 5X5HT-Aniso-3X5HT group at 24 h after 5X5HT training, as well as to cocultures in the 3X5HT group at the equivalent experimental time. (B) Sample EPSPs. Each pair of traces shows EPSPs recorded from the same coculture on the pretest and posttest. Scale bars: 10 mV, 100 ms. (C) Graph presenting the mean normalized EPSPs, measured at 48 h, for the five experimental groups: Control (n = 13), 3X5HT (n = 12), 5X5HT (n = 16), 5X5HT-Aniso (n = 12), and 5X5HT-Aniso-3X5HT (n = 7). A one-way ANOVA indicated that the overall differences among the five groups were highly significant (F[4,55] = 7.9, p < 0.0001). SNK posthoc tests showed that the mean normalized EPSP in the 5X5HT group (216.6% ± 30.6%) at 48 h was significantly larger than that in the Control (113.1% ± 11.1%, p < 0.01), 3X5HT (90.8% ± 17.9%, p < 0.001), and 5X5HT-Aniso (76.1% ± 16.4%, p < 0.001) groups. The mean normalized EPSP in the 5X5HT-Aniso-3X5HT group (196.5% ± 26.8%) was also significantly larger than that in the Control (p < 0.05), 3X5HT (p < 0.05), and 5X5HT-Aniso (p < 0.05) groups. None of the other differences among the groups was significant. https://doi.org/10.7554/eLife.18299.004 Amnesia produced by PSI during training cannot be subsequently reversed by partial training Castellucci et al. (1989) found that PSI during the original period of sensitization training produced amnesia. We wished to determine whether LTM could be induced by partial training following its disruption by PSI during sensitization training. Accordingly, we performed an experiment using the identical protocol as that shown in Figure 1, except that the animals received an injection of either anisomycin or vehicle ~15 min before the onset of the 5X training (Figure 3). The 5X training induced LTM at the 24-h posttest in animals given the vehicle (Veh-5XTrained group), but not in animals given anisomycin (Aniso-5XTrained and Aniso-5XTrained-3XTrained groups) prior to training. Furthermore, 3X training did not produce LTM in animals that received the protein synthesis inhibitor prior to 5X training, as indicated by the lack of sensitization in the Aniso-5XTrained-3XTrained group at 48 h. In contrast to posttraining PSI, therefore, PSI during training prevented induction of LTM by the supplemental truncated training. Figure 3 Download asset Open asset LTS cannot be induced by partial training when PSI occurs during the original (5X) sensitization training. (A) Experimental protocols. The times at which the pretests, training, posttests, and drug/vehicle injections occurred are shown relative to the end of the last training session. The red arrow indicates when either anisomycin or vehicle was injected into the hemocoel. (B) The mean duration of the SWR measured at 24 h and 48 h for the Veh-Control-3XTrained (n = 5), Veh-5XTrained (n = 8), Aniso-5XTrained (n = 6), and Aniso-5XTrained-3XTrained (n = 6) groups. A repeated-measures ANOVA showed a significant group x time interaction (F[6,42] = 40.9, p < 0.0001). Planned comparisons indicated that the group differences were highly significant for both 24-h (F[3,21] = 43.9, p < 0.0001) and 48-h (F[3,21] = 45.4, p < 0.0001) posttests. SNK posthoc tests revealed that the 5X training produced sensitization of the SWR in the Veh-5XTrained group (mean duration = 50.1 ± 5.6 s) at 24 h compared with the results for the Veh-Control-3XTrained group (mean duration of the SWR = 1.4 ± 0.4 s, p < 0.001). In addition, the SWR in the Veh-5XTrained group was significantly longer than that in the Aniso-5XTrained group (2.2 ± 1.2 s, p < 0.001) and the Aniso-5XTrained-3XTrained group (4.0 ± 2.3 s, p < 0.001). The differences among the Veh-Control-3XTrained, Aniso-5XTrained, and Aniso-5XTrained-3XTrained groups were not significant at 24 h. The SWR in the Veh-5XTrained group (mean duration = 49.8 ± 5.8 s) remained sensitized at 48 h, as indicated by the comparison with the reflex in the Veh-Control-3XTrained group (mean duration = 1.2 ± 0.2 s). The SWR was also significantly prolonged in the Veh-5XTrained group compared with that in the Aniso-5XTrained group (mean duration = 2.0 ± 1.0 s, p < 0.001 for both comparisons). The three tail shocks applied after the 24-h posttest did not establish LTS in the Aniso-5XTrained-3XTrained group. The mean duration of the SWR in this group at 48 h was 2.2 ± 1.2 s, which was not significantly different from that in the Veh-Control-3XTrained and Aniso-5XTrained groups. The SWR of the Veh-5XTrained group at 48 h was significantly longer than that in the Aniso-5XTrained-3XTrained group (p < 0.001). Asterisks, comparisons of the Veh-5XTrained group with the Veh-Control-3XTrained group, the Aniso-5XTrained group, and the Aniso-5XTrained-3XTrained group. https://doi.org/10.7554/eLife.18299.005 Inhibition of DNA methyltransferase, whether during or shortly after training, causes irreversible amnesia Why should PSI during training be so devastating for the consolidation of LTM? An intriguing possibility is that PSI during training obstructs DNA methylation required for memory consolidation. To test this possibility, we performed experiments in which the DNA methyltransferase (DNMT) inhibitor RG108 was injected into animals just before the onset of 5X training (Figure 4). DNMT inhibition during 5X training resulted in amnesia at 24 h and 48 h posttraining (comparison of the Veh-5XTrained group with the RG-5XTrained group); furthermore, subsequent 3X training did not induce LTM, as shown by the 48-h data (comparisons of the RG-5XTrained-3XTrained group with the Veh-5XTrained and Veh-Control-3XTrained groups). Figure 4 Download asset Open asset DNMT inhibition during the original (5X) sensitization training precludes the ability of subsequent partial training to induce LTS. (A) Experimental protocol. The times of occurrence of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. Either RG108 or vehicle was injected into the hemocoel at the time indicated by the red arrow. (B) The mean duration of the SWR measured at 24 h and 48 h for the Veh-Control-3XTrained (n = 7), Veh-5XTrained (n = 7), RG-5XTrained (n = 8), and RG-5XTrained-3XTrained (n = 7) groups. A repeated-measures ANOVA indicated that there was a significant group x time interaction (F[6,50] = 73.6, p < 0.0001). Subsequent planned comparisons showed that the overall differences among the four groups for the 24-h and 48-h posttests were highly significant (24 h, F[3,25] = 197.9, p < 0.0001; and 48 h, F[3,25] = 82.8, p < 0.0001). As revealed by SNK posthoc tests, the SWR exhibited sensitization at 24 h in the Veh-5XTrained group (mean duration = 54.0 ± 3.4 s) compared with that in the Veh-Control-3XTrained group (mean duration = 1.3 ± 0.3 s, p < 0.001). The differences in duration of the SWR at 24 h among the Veh-Control-3XTrained, RG-5XTrained (3.6 ± 1.3 s), and RG-5XTrained-3XTrained (1.9 ± 0.6 s) groups were not significant. Sensitization of the SWR was maintained in the Veh-5XTrained group (mean duration of the reflex = 55.3 ± 3.6 s) at 48 h, as shown by the comparison with the Veh-Control-3XTrained group (mean duration of the reflex = 1.6 ± 0.6 s, p < 0.001). There were no significant differences among the Veh-Control-3XTrained, RG-5XTrained (mean duration of the SWR = 3.1 ± 1.2 s), and RG-5XTrained-3XTrained (mean duration of the SWR = 5.7 ± 4.4 s) groups at 48 h, indicating that the three additional bouts of tail shocks given to the latter group after the 24-h posttest failed to induce LTS. Asterisks, comparisons of the Veh-5XTrained group with the Veh-Control-3XTrained group, the RG-5XTrained group, and the RG-5XTrained-3XTrained group. https://doi.org/10.7554/eLife.18299.006 Next we examined whether DNMT inhibition that commenced after long-term training caused amnesia and, if so, whether subsequent abbreviated training resulted in LTM. Accordingly, we performed an experiment like the previous one, except that RG108 was injected into some animals 10–20 min after, rather than before, 5X training (Figure 5). Posttraining DNMT inhibition, like pretraining DNMT inhibition, blocked the consolidation of LTM, as shown by the absence of LTS at 24 h and 48 h (comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained and 5XTrained-RG groups). In addition, supplemental 3X training following posttraining DNMT inhibition did not induce LTM (comparisons of the 5XTrained-RG-3XTrained group with the 5XTrained-Veh and Control-Veh-3XTrained groups). Figure 5 Download asset Open asset Posttraining inhibition of DNMT precludes later induction of LTS by partial training. (A) Experimental protocol. The times at which the pretests, training, posttests, and drug/vehicle injections occurred are shown relative to the end of the last training session. The time of the intrahemocoelic injection of either RG108 or vehicle is indicated by the red arrow. After the 24-h posttest, animals in the Control-Veh-3XTrained and 5XTrained-RG-3XTrained groups received 3X sensitization training. (B) The mean duration of the SWR measured at 24 h and 48 h for the Control-Veh-3XTrained (n = 8), 5XTrained-Veh (n = 8), 5XTrained-RG (n = 7), and 5XTrained-RG-3XTrained (n = 6) groups. A repeated-measures ANOVA showed that the group x time interaction was significant (F[6,50] = 64.7, p < 0.0001). The overall differences among the four groups for the 24-h and 48-h posttests were highly significant, as indicated by a one-way ANOVA (24 h, F[3,25] = 82.6, p < 0.0001; and 48 h, F[3,25] = 69.2, p < 0.0001). SNK posthoc tests revealed significantly greater sensitization in the 5XTrained-Veh group at 24 h (mean duration of the SWR = 53.1 ± 5.1 s) than in the Control-Veh-3XTrained group (mean duration of the SWR = 2.1 ± 0.9 s, p < 0.001). The differences among the 5XTrained-RG (mean duration of the SWR = 1.7 ± 0.7 s), 5XTrained-RG-3XTrained (mean duration of the SWR = 2.2 ± 0.7 s), and Control-Veh-3XTrained groups at 24 h were not significant. Sensitization persisted in the 5XTrained-Veh group at 48 h (mean duration of the SWR = 48.3 ± 5.0 s) compared with the Control-Veh-3XTrained group (mean duration of the SWR = 1.6 ± 0.3 s, p < 0.001). The failure of the 3X training to induce sensitization in the 5XTrained-RG-3XTrained group was shown by the lack of significant differences between this group (mean duration of the SWR = 2.8 ± 1.2 s) and the Control-Veh-3XTrained group at 48 h. There was also no significant difference between the mean duration of the reflex in the 5XTrained-RG-3XTrained group and that in the 5XTrained-RG (2.3 ± 1.0 s) group at 48 h. Asterisks, comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained group, the 5XTrained-RG group, and the 5XTrained-RG-3XTrained group. https://doi.org/10.7554/eLife.18299.007 Inhibition of DNA methyltransferase eliminates consolidated LTM The failure of partial training to induce LTM following posttraining RG108 treatment, coupled with its ability to establish LTM following posttraining PSI, suggests that DNA methylation is a prerequisite for the establishment of the occult priming trace accessed by partial training after posttraining PSI. To examine this possibility, we gave animals long-term behavioral sensitization training and then administered the DNMT inhibitor 24 h after training (Figure 6). All animals that received the 5X training exhibited LTS at 24 h (5XTrained-Veh, 5XTrained-RG and 5XTrained-RG-3XTrained groups), as indicated by the comparison with the control group (Control-Veh-3XTrained). LTS was also present at 48 h in animals that received the original long-term training and an injection of the vehicle after the 24-h test (5XTrained-Veh group), but not in animals that received the 5X training and an injection of RG108 at 24 h (5XTrained-RG and 5XTrained-RG-3XTrained groups). Moreover, truncated training did not restore LTM in the RG108-treated animals (comparison between the Control-Veh-3XTrained and 5XTrained-RG-3XTrained groups). An additional experiment using a different DNMT inhibitor—5-azacytidine (5-aza)—yielded identical results (Figure 6—figure supplement 1). Figure 6 with 1 supplement see all Download asset Open asset Inhibition of DNMT with RG108 eliminates established LTS in Aplysia. (A) Experimental protocol. The occurrences of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. Either RG108 or vehicle was injected into the animals at the time indicated by the red ar