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How the brain maintains long-term memories is one of the major outstanding questions in modern neuroscience. Evidence from mammalian studies indicates that activity of a protein kinase C isoform, PKMζ, plays a critical role in the maintenance of long-term memory. But the range of memories whose persistence depends on PKMζ, and the mechanisms that underlie PKMζ's effect on long-term memory, remain obscure. Recently, a PKM isoform, known as PKM Apl III, was cloned from the nervous system of Aplysia. Here, we tested whether PKM Apl III plays a critical role in long-term memory maintenance in Aplysia. Intrahemocoel injections of the pseudosubstrate inhibitory peptide ZIP or the PKC inhibitor chelerythrine, erased the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) as late as 7 d after training. In addition, both PKM inhibitors disrupted the maintenance of long-term (≥ 24 hr) facilitation (LTF) of the sensorimotor synapse, a form of synaptic plasticity previously shown to mediate LTS of the SWR. Together with prior results (Bougie et al., 2009), our results support the idea that long-term memory in Aplysia is maintained via a positive-feedback loop involving PKM Apl III-dependent protein phosphorylation. The present data extend the known role of PKM in memory maintenance to a simple and well-studied type of long-term learning. Furthermore, the demonstration that PKM activity underlies the persistence of LTF of the Aplysia sensorimotor synapse, a form of synaptic plasticity amenable to rigorous cellular and molecular analyses, should facilitate efforts to understand how PKM activity maintains memory.
Traditionally, memories have been believed to undergo a single, time-dependent process of consolidation during the transition from short-term to long-term (Müller and Pilzecker, 1900; McGaugh, 2000). Early in this process, memories are labile and subject to disruption by such treatments as application of an electroconvulsive shock (McGaugh, 1966) and inhibition of protein synthesis (Agranoff and Klinger, 1964); once consolidated, however, memories are thought to be relatively permanent and resistant to disruption by amnestic treatments. However, the standard model of memory consolidation has been challenged by evidence indicating that the persistence of memory depends on some active, ongoing process, and that even well established memories can become rapidly degraded if this process is interrupted, (Drier et al., 2002; Ling et al., 2002; Pastalkova et al., 2006; Shema et al., 2007).
In Aplysia the induction of serotonin (5-HT)-induced LTF of sensorimotor synapses, the form of synaptic plasticity that underlies LTS (Frost et al., 1985), requires cyclic AMP response element-binding protein (CREB) (Dash et al., 1990; Bartsch et al., 1995). Activation of CREB has been hypothesized to initiate the processes of transcription and translation that mediate long-term memory in Aplysia (Goelet et al., 1986). Recently, it has been proposed that early maintenance, at least, of long-term memory in Aplysia is mediated by ongoing activity of cytoplasmic polyadenylation element binding protein (ApCPEB) (Si et al., 2003b; Si et al., 2003a). ApCPEB can undergo a change in its state from an inactive monomer, to an active multimer; in the latter state the protein is self-perpetuating (Si et al., 2010), a capacity that endows it, potentially, with the ability to subserve the persistence of memory. Although most of the work implicating CPEB in memory maintenance has been done in Aplysia, there is evidence that homologs of ApCPEB have a similar function in Drosophila (Keleman et al., 2007) and, possibly, in mammals (Alarcon et al., 2004).
An alternate molecular mechanism for memory maintenance has emerged from the work of Sacktor and colleagues. They have shown that a constitutively active fragment of the mammalian atypical protein kinase Cζ (PKCζ), PKMζ, plays a critical role in the persistence of long-term potentiation (LTP) in the mammalian hippocampus (Ling et al., 2002; Pastalkova et al., 2006), as well as several forms of mammalian memory (Pastalkova et al., 2006; Shema et al., 2007; Serrano et al., 2008). Until now, most of the studies examining the role of PKM-type isoforms in memory have been performed on rodents (but see Drier et al., 2002).
An atypical PKC has been cloned from the nervous system of Aplysia (Bougie et al., 2009). This Aplysia PKC, PKC Apl III, can undergo proteolytic cleavage by calpain, thereby yielding a PKM fragment, PKM Apl III. Furthermore, 5-HT appears to activate PKM Apl III in motor neurons of Aplysia (Villareal et al., 2009). The discovery and initial characterization of PKM Apl III have set the stage for the present examination of the role of PKM in memory retention in Aplysia.
Adult Aplysia californica (80–120 g) were obtained from a local supplier (Alacrity Marine Biological, Redondo Beach, CA, USA). (Note that Aplysia are hermaphroditic organisms.) Animals were housed in a 190-liter aquarium filled with cooled (12–14° C), aerated seawater (Catalina Water Company, Long Beach, CA, USA). The behavioral training and testing methods were similar to those previously described (Fulton et al., 2008). Three pretests were performed at once per 10 min, beginning 25 min before the start of training. During each pretest, as well as in the post-training tests, the siphon was lightly stimulated with a broom bristle, and the duration of the resulting SWR was timed. Sensitization training consisted of five bouts of electrical shocks delivered to the tail at 20-min intervals. During each bout, the animal received three trains of shocks spaced 2 s apart. Each train was 1 s in duration; the shocks (10-ms pulse duration, 40 Hz, 120 V) were delivered via a Grass stimulator (S88, Astro-Med, West Warwick, RI) connected to platinum wires implanted in the tail. After training the animals were given posttests as indicated in the figures.
Myristoylated pseudosubstrate inhibitor ZIP (myr-SIYRRGARRWRKL-OH) (Invitrogen, Carlsbad, CA) was dissolved in dH2O (vehicle) to a concentration of 5 mM. A scrambled peptide (myr-RLYRKRIWRSAGR-OH; ScrZIP) (Tocris, Ellisville, MO), also dissolved in dH2O to a concentration of 5 mM, or the vehicle alone, was used in Control experiments. Chelerythrine (EMD Bioscience, San Diego, CA) was dissolved in dH2O to a concentration of 10 mM. Injections of 200 μl per 100 g of body weight of ZIP, ScrZIP or chelerythrine were made into the animal's neck. Anisomycin was first dissolved in dimethyl sulfoxide (DMSO) to a concentration of 40 mM, and then diluted in artificial seawater (ASW) to a concentration of 8 mM (20% DMSO). 500 μl per 100 g of body weight of anisomycin was injected into the animals. Injections of the same amount of vehicle solution (DMSO in ASW) were made in Control experiments. The final concentrations of ZIP/ScrZIP, chelerythrine and anisomycin in the animal were approximately 10 μM, 20 μM and 40 μM, respectively. The final concentration of DMSO in the hemocoel was ~ 0.1%. The specific times at which the intrahemocoel injections were made are indicated in the relevant figures.
To confirm that myristoylated ZIP can penetrate the connective tissue sheath surrounding the ganglia of the Aplysia CNS, and enter inside neurons, biotinylated myristoylated ZIP (von Kraus et al., 2010) was injected into animals at the same concentration (200 μl per 100 g body weight) as was used for myristoylated ZIP in the behavioral experiments. Five hours after the intrahemocoel injection of biotin-labeled ZIP, the pleural-pedal ganglia were removed from the animal and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) with 30% sucrose overnight at 4 °C. The ganglia were then rinsed with fresh PBS/30% sucrose and transferred to plastic molds, where they were embedded in Optimal Cutting Temperature compound (Sakura Finetek, Torrance, CA) and frozen on dry ice. The frozen tissue block was cut into 20 μm-thick sections with a cryotome. The tissue sections were placed onto glass slide, rinsed three times (5 min per rinse) with PBS, and incubated with PBS containing 0.3% Triton for 10 min. Slices were incubated with the VECTASTAIN Elite ABC reagent (Vector Laboratories, Burlingame, CA) for 1 hr and rinsed in PBS (pH 7.4). To visualize the avidin-biotinylated horse radish peroxidase (HRP) complex, the slices were incubated in diaminobenzidine (DAB) for 5 min. The DAB reaction was stopped by washing with H2O, and the sections were then mounted with VectaMount (Vector). Differential interference contrast images of the sections were made using a Zeiss LSM 5 Pascal Laser Scanning microscope (Zeiss, Germany) equipped with 10× and 20× objective lenses.
The synaptic experiments used sensorimotor cocultures, each consisting of one pleural sensory neuron and one small siphon (LFS-type) motor neuron; the neurons were individually dissociated from central ganglia of Aplysia (60–100 g), and placed into cell culture together (Lin and Glanzman, 1994). The culture medium contained 50% Aplysia hemolymph and 50% Leibowitz-15 (L-15, Sigma, St Louis, MO, USA). The cultures were maintained at 18° C for 3–4 d before the start of the experiments to allow them to form robust monosynaptic connections. The mean size of the sensorimotor excitatory postsynaptic potentials (EPSPs) evoked on the Day 1 pretest in the cocultures included in the study was 24.5 ± 0.9 mV. One-way analyses of variance (ANOVAs, see below) performed on the pretest EPSPs for each of the synaptic experiments indicated that the group differences were not significant (P > 0.6 for each experiment).
The electrophysiological methods have been previously described (Lin and Glanzman, 1994; Cai et al., 2008). Briefly, during electrophysiological recording cocultures were perfused with 50% sterile ASW and 50% L-15 (perfusion medium). All experiments were performed at room temperature. Synaptic strength was determined on Day1 by eliciting a single EPSP in the motor neuron using intracellular activation of the sensory neuron (pretest). After this initial synaptic assessment the microelectrodes were removed from the neurons. 5-HT was prepared fresh daily as a 10 mM stock solution in ASW, and then diluted to the final concentration of 100 μM in the perfusion medium immediately before the first application. To induce long-term facilitation (LTF), cocultures were treated with repeated, spaced applications of 5-HT (five 5-min applications of 5-HT, 20-min interval between applications). After each 5-min application the 5-HT was rapidly washed out with normal perfusion medium for 15 min. The Control cocultures were treated with the perfusion solution alone. Following 5-HT or control treatment, the perfusion medium was replaced with culture medium, and the cocultures were returned to the 18° C incubator. 48 hr later the neurons were reimpaled with microelectrodes, and the synaptic strength was reassessed (posttest).
A stock solution of myristoylated 1 mM ZIP/ScrZIP or 10 mM chelerythrine was prepared as in the behavioral experiments. The stock solution of ZIP/ScrZIP/chelerythrine was added to the cocultures at 24 hr after the 5-HT treatment, unless otherwise indicated. The final concentration of ZIP/ScrZIP and chelerythrine in the culture medium was 1 μM and 5–10 μM, respectively; the drug treatment period was 1 hr. Following ZIP or chelerythrine treatment, the drug was washed out of the culture dish with culture medium.
The data from the behavioral experiments are the mean ± SEM duration (in seconds) of the SWR for each test. The significance of the group differences were first assessed with a repeated measures analysis of variance (ANOVAs); given significance of the group differences (p < 0.05), Student-Newman-Keuls (SNK) post-hoc tests were subsequently used for pairwise comparisons. For the synaptic experiments, the peak amplitude of the posttest EPSP was normalized to the amplitude of the pretest EPSP for the same coculture. The membrane input resistances for both neurons (sensory and motor) were measured, and the threshold for evoking an action potential (spike threshold) was determined. The input resistances and the spike threshold obtained in the posttest were normalized to those in the pretest. The normalized data were expressed as means ± SEM. Nonparametric tests were used to assess the significance of the electrophysiological results. A Mann-Whitney U-test was used for a single comparison of two groups. Kruskal-Wallis tests were used to assess the significance of differences among several groups; if the group differences were significant (P < 0.05), Dunn's tests were then used for post-hoc comparisons between pairs of groups. All reported levels of significance are two-tailed values.
To explore the possibility that PKM Apl III maintains long-term memory in Aplysia, we tested whether injecting the zeta inhibitory peptide (ZIP) (Ling et al., 2002) into animals disrupts previously established LTS. ZIP consists of the autoinhibitory pseudosubstrate sequence of the regulatory domain of PKCζ, and this sequence is conserved in PKC Apl III (Bougie et al., 2009). In our experiments the peptide was injected into an animal's hemocoel through its neck. Gastropod mollusks do not possess a blood-brain barrier (Abbott et al., 1986); furthermore, the central nervous system of Aplysia is directly and richly vascularized by a branch of the anterior aorta (Furgal and Brownell, 1987). Therefore, small molecules introduced into the hemolymph would be expected to be rapidly delivered to the abdominal ganglion, as well as the other central ganglia, of Aplysia (Furgal and Brownell, 1987). Nonetheless, to ensure that the inhibitory peptide had free access to neurons of the CNS, we injected biotinylated ZIP (von Kraus et al., 2010) into the hemocoel. The concentration of the biotinylated ZIP in the hemolymph (~10 μM) was the same as the concentration of ZIP used in the behavioral experiments (below). Biotin labeling was clearly evident inside central ganglia; in particular, unambiguous staining was observed inside individual central neurons (Fig. 1). We therefore conclude that the ZIP was able to readily enter central neurons from the hemocoel in living animals, despite the presence of the connective tissue sheath surrounding the ganglia.
To test the ability of ZIP to disrupt long-term memory maintenance, animals were given sensitization training and then tested 24 hr later. Shortly (≤ 15 min) after the 24-hr test three groups of animals that had been subjected to sensitization training received an intrahemocoel injection of myristoylated ZIP (~ 10 μM final concentration in the hemolymph here and in subsequent experiments), the myristoylated, scrambled version of the ZIP peptide (ScrZIP, same concentration as ZIP), or the vehicle (dH2O) (Fig. 2A, B). Two groups of control animals that did not receive sensitization training also received an injection of ZIP or vehicle. The SWR of all animals was tested once more at 48 hr. A one-way ANOVA indicated that the group differences for the 24-hr and 48-hr posttests were highly significant (F[4,19] = 18.2 and 52.1, p < 0.0001 for the results of each ANOVA). Post-hoc tests on the 24-hr data indicated all of the trained groups showed significant sensitization at 24 hr compared to the control groups, and that the ZIP injection at 24 hr blocked the expression of sensitization at 48 hr. This effect was unlikely to have been due to a nonspecific effect of ZIP, because the injection of the scrambled peptide did not affect LTS. Furthermore, there were no significant differences between the Control-Veh and Control-ZIP groups.
The PKC inhibitor chelerythrine is specific for PKM Apl III at low (≤ 20 μM) concentrations in Aplysia (Villareal et al., 2009). Accordingly, we examined the effect of a low concentration (~ 20 μM in the hemolymph here and in subsequent experiments) of chelerythrine on maintenance of LTS (Fig. 2C). The behavioral testing and training methods, and injection method were the same as in the ZIP experiments. There were four groups: Control-Veh (n = 6), Control-Chelerythrine (Chel) (n = 5), Trained-Veh (n = 7) and Trained-Chel (n = 7). The differences among the groups were highly significant for both the 24-hr and 48-hr posttests (one-way ANOVAs, F[3,21] = 24.1 and 18.4, p < 0.0001 for both posttests). The two trained groups showed significant sensitization at 24 hr, as indicated by post-hoc comparisons with their respective control groups. Trained animals that received an injection of vehicle solution were also sensitized at 48 hr, but sensitization was absent in the trained animals treated with chelerythrine. Although, like ZIP, chelerythrine disrupted maintenance of LTS, the drug did not appear to have a deleterious effect on the animals, as indicated by the lack of significant differences between the Control-Veh and Control-Chel groups on any of the post-hoc comparisons.
Because we elicited the SWR just prior to the injection of ZIP/chelerythrine, it could be argued that the lack of LTS in the experimental animals at 48 hr was due to disruption of memory reconsolidation triggered by the 24 hr posttest (see Nader et al., 2000a; Sara, 2000). To evaluate this explanation for our data, we repeated the chelerythrine experiment, omitting the 24 hr posttest (Fig. 3A). As in our earlier experiment (Fig. 2C), some animals (Trained-Chel, n = 4) received an intrahemocoel injection of chelerythrine 24 hr after sensitization training. Another group (Trained-Veh, n = 8) received an injection of the vehicle at 24 hr after training. The third group (Control-Veh, n = 4) also received an injection of the vehicle at 24 hr, but was not trained. (We did not include a control chelerythrine group in this experiment, or in the experiment presented in Fig. 3B [below], because we previously found that the chelerythrine injection had no effect on the SWR [Fig. 2C].) All three groups were given just a single posttest at 48 hr. The differences among the groups on the 48 hr posttest were highly significant (one-way ANOVA, F[2,13] = 34.7, p < 0.0001). The Trained-Veh group, but not the Trained-Chel group, exhibited sensitization at 48 hr. Thus, the apparent elimination of established LTS by chelerythrine did not depend on evoking the SWR immediately preceding the drug injection, and cannot be attributed to disruption of memory reconsolidation.
After their extinction, conditioned reflexes can exhibit spontaneous recovery with the passage of time, or be reinstated if the animal is exposed to the original unconditioned stimulus (Rescorla and Heth, 1975). Sensitization is, of course, a nonassociative form of learning; nonetheless, we wished to know whether LTS would show either spontaneous recovery or reinstatement after its apparent elimination by inhibition of PKM Apl III. In this experiment animals were first retested at 72 hr after training (or at the equivalent time in the control group). Chelerythrine was injected into two groups of animals at 72 hr after LTS training, and then at 96 hr post-training one of the groups (Trained-Chel-Reinstate group) received an additional bout (three 1-s trains) of tail shocks (Fig. 3B). (Notice that this training comprises one fifth of the number of tail shocks used to induce LTS.) The SWR was tested at 72 hr, 96 hr, 120 hr and 144 hr after sensitization training in the trained groups, or at the equivalent times in the control group. There were three groups of trained animals—Trained-Veh (n = 13), Trained-Chel (n = 7), and Trained-Chel-Reinstate (n = 7)—and a single control group (Control-Veh, n = 7). (We did not include a control group treated with chelerythrine alone in this experiment, because we had previously found that the chelerythrine treatment had no effect on the baseline SWR [Fig. 2C]). The differences among the four groups were highly significant for each of the posttests (one-way ANOVAs, F[3,30] for 72 hr = 19.5, p < 0.0001; F[3,30] for 96 hr = 97.4, p < 0.0001; F[3,30] for 120 hr = 50.3, p < 0.0001; and F[3,30] for 144 hr = 52.8, p < 0.0001). All three trained groups exhibited significant LTS at 72 hr after training. But following the drug injection, only the Trained-Veh group subsequently exhibited sensitization; there was no evidence of spontaneous recovery of sensitization for the 72-hr period after chelerythrine injection in either the Trained-Chel or the Trained-Chel-Reinstate groups. Furthermore, the additional bout of sensitization training at 96 hr failed to reinstate LTS in the Trained-Chel-Reinstate group.
The above results provide compelling evidence that an early stage (24–72 hr) of memory maintenance for LTS in Aplysia requires ongoing activity of PKM Apl III. However, LTS of the SWR has been shown to persist for at least 3 weeks (Pinsker et al., 1973). To test whether the maintenance of later stages of the memory for LTS also depends on PKM Apl III, animals were treated with chelerythrine at one week after sensitization training (Fig. 4A). The drug was injected into the animals immediately after the SWR was tested at Day 7 to assess whether the training produced sensitization that lasted at least one week; the animals were then retested on Days 8 and 9. There were two trained groups of animals (Trained-Veh [n = 5] and Trained-Chel [n = 5]), and two control groups (Control-Veh [n = 4] and Control-Chel [n = 3]). One-way ANOVAs indicated that the overall differences among the four groups were highly significant on all of the posttests (Day 7, F[3,13] = 22.7, p < 0.0001; Day 8, (F[3,13] = 15.4, p < 0.001; and Day 9, F[3,13] = 12.9, p < 0.001). Post-hoc tests showed that the Trained-Veh group exhibited significant sensitization 7–9 days after training compared to the Control-Veh group. The SWR in the Trained-Chel group was significantly sensitized compared to the Control-Chel group on the Day 7 posttest, but not on either the Day 8 or Day 9 posttest. Finally, there was no difference between the Control-Veh and Control-Chel groups on any of the posttests, indicating that the chelerythrine did not affect the baseline SWR. These results demonstrate that LTS was present in the Trained-Chel group on Day 7 after training, but was absent on Days 8 and 9. Therefore, the chelerythrine injection disrupted the week-old memory for LTS, and the disrupted memory did not spontaneously recover over the next two days.
We also tested the effect of an injection of the peptide inhibitor ZIP on one week-old memory for sensitization (Fig. 4B). The design of this experiment was similar to that in the test of chelerythrine above (Fig. 4A), but we did not test the SWR prior to the ZIP injection at Day 7 to exclude a contribution from disruption of memory reconsolidation to any resulting positive results. Three groups were included: Trained-Veh (n = 5), Trained-ZIP (n = 5) and Control-Veh (n = 5). (Neither a Control-ZIP group nor a Trained-Scr-ZIP group was included in this experiment, because we did not observe any effect of ZIP on the baseline SWR, nor a disruptive effect of the scrambled peptide on sensitization, in our earlier behavioral experiment [Fig. 2B].) The group differences for the posttests on Days 8 and 9 were highly significant (Day 8 one-way ANOVA, F[2,12] = 28.3, p < 0.0001; Day 9 one-way ANOVA, F[2,12] = 43.5, p < 0.001). Post-hoc tests showed that Trained-Veh group was significantly sensitized on the posttests compared to both the Control-Veh and Trained-ZIP groups. Therefore, the week-old memory for LTS appeared to be eliminated when PKM Apl III activity was inhibited with ZIP.
Recent work has implicated the Aplysia homolog of cytoplasmic polyadenylation element binding protein (ApCPEB) in the maintenance of LTS. According to one model, ApCPEB, when activated by sensitization-related stimulation, becomes constitutively active and drives local protein synthesis; this ApCPEB-dependent ongoing protein synthesis, it is believed, plays a critical role in maintaining LTS (Si et al., 2003b; Si et al., 2010). In support of this idea, inhibition of protein synthesis has been reported to reverse LTF of the sensorimotor synapse 24–48 hr after training (Miniaci et al., 2008). Until now, however, empirical support for a role for ApCPEB in memory maintenance in Aplysia has come exclusively from experiments on isolated synapses in dissociated cell culture (Si et al., 2003b; Si et al., 2003a; Miniaci et al., 2008; Si et al., 2010); the potential role of ApCPEB in maintaining memory following actual learning in Aplysia has not been examined. This information is critical to understanding how the memory for LTS persists in Aplysia, because our evidence (above) suggests that a requirement for PKM Apl III in long-term memory maintenance may temporally overlap, at least partly, with that for ApCPEB (Miniaci et al., 2008). As an initial step toward delineating the relative roles of ApCPEB and PKM Apl III in the persistence of the memory for LTS, therefore, we tested whether temporarily inhibiting protein synthesis can disrupt well-consolidated LTS.
To ascertain the efficacy of our method for inhibiting translation in Aplysia, we first confirmed a previous finding (Castellucci et al., 1989) that induction of LTS requires protein synthesis. Twenty min before the start of an experiment one group of animals (Trained-Aniso group [n = 5]) received an intrahemocoel injection of anisomycin (final concentration in the hemolymph was ~ 40 μM in 0.1% DMSO), while another group (Trained-Veh group [n = 5]) received an injection of the vehicle (DMSO in artificial seawater). There were two untrained control groups, one that received an injection of the vehicle solution (Control-Veh group [n = 4]), and another that received an injection of anisomycin in DMSO (Control-Aniso [n = 4]) at the equivalent time in the experiment as the trained groups. Prior to training the duration of the SWR in response to light touch of the siphon was measured in a series of three pretests spaced 10 min apart (Fig. 5A). Some animals then received the tail-shock sensitization training. Twenty-four hr after the training, or at the equivalent time in control animals, the SWR was retested. There was a single posttest at 24 hr after training (or at the equivalent time for the controls). The overall differences among the groups on the posttest were highly significant (one-way ANOVA, F[3,14] = 393.2, P < 0.0001). The Trained-Veh group was significantly sensitized compared to both the Control-Veh and Trained-Aniso groups. There were no significant differences among the Control-Veh, Control-Aniso and Trained-Aniso groups. Thus, as previously reported (Castellucci et al., 1989), treatment with anisomycin prior to sensitization training blocks the induction of LTS.
Next we tested whether the anisomycin treatment could disrupt well-established LTS. Two groups of animals received LTS training (Fig. 5B). The SWR of both groups was tested one week later. One of the trained groups (Trained-Veh [n = 4]) then received an intrahemocoel injection of the vehicle solution, while the other group (Trained-Aniso [n = 5]) received an injection of the protein synthesis inhibitor. An untrained group (Control-Veh [n = 5]) was given an injection of the vehicle solution on Day 7. (The previous experiment indicated that the anisomycin injection did not affect the baseline SWR, so an anisomycin-injected control group was not included.) The SWR of the animals in each group was then retested on Days 8 and 9. The overall group differences were significant for each of the posttests, as indicated by one-way ANOVAs (Day 7, F[2,9] = 10.2, p < 0.005; Day 8, F[2,9] = 12.1, p < 0.003; and Day 9, F[2,9] = 17.3, p < 0.001). There were no significant differences between the two trained groups on any of the posttests. Therefore, by one week after training, temporary inhibition of protein synthesis does not disrupt the memory for LTS.
LTF of the monosynaptic connection between the siphon sensory and motor neurons mediates, at least partly, behavioral sensitization of the SWR (Frost et al., 1985). Accordingly, we wished to know whether PKM Apl III activity maintains LTF, as well as LTS. Long-term (≥ 24 hr) facilitation can be induced in synapses between sensory and motor neurons in dissociated cell culture by repeated treatment with 5-HT (Montarolo et al., 1986). We therefore tested whether inhibition of PKM Apl III disrupts maintenance of LTF of the in vitro sensorimotor connection. As previously reported (Cai et al., 2008), five spaced 5-min bouts of 5-HT (100 μM) produced significant LTF of the excitatory postsynaptic potential (EPSP) at the synapse between a single pleural sensory neuron and a single small siphon (LFS) motor neuron in dissociated cell culture (Mann-Whitney test, U = 6.0 [p < 0.001]) (Fig. 6A, B). However, when synapses were treated with myristoylated ZIP (1 μM, 1 hr) ~ 24 hr after the 5-HT “training”, facilitation was absent 24 hr later (~ 48 hr after 5-HT training); by contrast, synapses trained with 5-HT, but not treated with ZIP at 24 hr were significantly facilitated at 48 hr (Fig. 6D). A nonparametric ANOVA performed on the group data for the 48 hr posttest showed that the differences among the groups were highly significant (Kruskal-Wallis test, H = 23.7, p < 0.0001). Post-hoc tests indicated that there was significant facilitation at 48 hr in the 5-HT trained group, but not in the group treated with ZIP 24 hr after 5-HT training. The EPSPs in synapses treated with PKM inhibitor alone did not differ from those in Control synapses. Therefore, the myristoylated ZIP had no apparent effect on baseline synaptic transmission.
To control for the potential nonspecific effects of the peptide treatment on synaptic facilitation, we performed additional experiments in which sensorimotor cocultures were treated with the scrambled ZIP peptide at 24 hr after 5-HT training; other cocultures were treated with ScrZIP alone at the equivalent time point. Treatment of the cocultures with ScrZIP 24 hr after 5-HT training did not disrupt the expression of LTF at 48 hr (Fig. 6E). The differences among the groups at 48 hr in this experiment (Fig. 6E2) were highly significant (Kruskal-Wallis test, H = 21.4, p < 0.0001). Post-hoc tests showed that both the 5-HT and 5-HT-ScrZIP groups were significantly facilitated at 48 hr compared to the Control and ScrZIP alone groups, respectively.
Notice that in none of the experiments presented in Fig. 6D, E were the synapses tested at 24 hr, so our results could not be due to a reconsolidation-related phenomenon. Furthermore, we observed no significant differences among the groups with respect to the input resistances of the sensory and motor neurons, or in the spike thresholds of the sensory neurons (Fig. 6F).
We also tested the effect of chelerythrine treatment on established LTF. As was true for ZIP, applying chelerythrine (5–10 μM, 1 hr) to sensorimotor cocultures 24 hr after 5-HT training blocked the expression of LTF at 48 hr after training (Fig. 7A). A nonparametric ANOVA indicated that the group differences for the 48 hr posttest were significant (Kruskal-Wallis test, H = 20.4, p < 0.001). Furthermore, post-hoc tests indicated that the 5-HT treated group exhibited significantly more facilitation at 48 hr than either the vehicle-treated Controls or the co-cultures treated with chelerythrine after 5-HT (5-HT-Chel group). Chelerythrine treatment by itself did not affect the sensorimotor EPSP, as indicated by the lack of a significant difference between the EPSPs in cocultures treated with chelerythrine alone and those in the vehicle-treated Control group. Finally, the disruptive effect of chelerythrine on the maintenance of LTF could not be accounted for by effects on neuronal input resistance or presynaptic spike threshold (Fig. 7C).
We attempted to reinstate LTF following chelerythrine treatment using brief 5-HT stimulation. There were four experimental groups used in the attempt. Three of the groups—Control (n = 11), 5-HT (n = 12), Chel (n = 10)—were treated identically to their counterparts in the previous experiment, except that the 1-hr exposure to chelerythrine/vehicle solution started at 18 hr after training with the 5-HT/vehicle solution, rather than at 24 hr (Fig. 7B). Synapses in the fourth group (5-HT-Chel-Reinstate, n = 14) were given the standard 5-HT training, followed by chelerythrine treatment at 18 hr; in addition, at 24 hr this group was given a single, 5-min pulse of 5-HT (100 μM), which, by itself, produces short-term, but not long-term, facilitation (Bartsch et al., 1995). The overall group differences for the 48 hr posttest were significant (Kruskal-Wallis test, H = 15.1, p < 0.002). The LTF produced by 5-HT treatment was disrupted by chelerythrine treatment at 18 hr. Furthermore, brief treatment with 5-HT at 24 hr failed to reinstate the LTF in the chelerythrine-treated group. (See Fig. 7D for the neuronal input resistances and presynaptic spike thresholds for this experiment.)
We found that inhibition of PKM Apl III, using either ZIP or a low concentration of chelerythrine erases both LTS and LTF in Aplysia. Following treatment with ZIP or chelerythrine we did not observe spontaneous recovery of the long-term behavioral and synaptic changes; furthermore, the long-term changes could not be reinstated by stimulation that normally produces short-term sensitization/facilitation. One might question the specificity of our inhibitory treatments for PKM Apl III. However, ZIP mimics the pseudosubstrate of the regulatory domain of PKCζ, and, as pointed out earlier, this pseudosubstrate sequence is conserved in PKC Apl III (Bougie et al., 2009). Furthermore, we found no inhibitory effect of the scrambled ZIP peptide in control behavioral and synaptic experiments. Finally, we have previously shown in biochemical assays that a low concentration of chelerythrine, like that used in the present experiments, is specific for PKM Apl III. In particular, at concentrations < 20 μM chelerythrine does not inhibit classical and novel PKCs in Aplysia (Villareal et al., 2009). Therefore, we do not believe that our results can be attributed to nonspecific actions of the inhibitors.
One caveat concerning our results is that we do not know the extent to which the behavioral effects of the PKM inhibitors were due to direct actions on siphon sensorimotor synapses in the intact animal. Because the drugs were introduced via intrahemocoel injections, both ZIP and chelerythrine had access to the entire Aplysia CNS. Therefore, although facilitation of sensorimotor synapses in the abdominal ganglion has been demonstrated to mediate sensitization of the SWR (Antonov et al., 1999), and although we have shown here that inhibition of PKM Apl III disrupts maintenance of both LTS in vivo and LTF of the sensorimotor synapse in vitro, we cannot rule out the possibility that our behavioral results were due, at least in part, to actions of ZIP and chelerythrine on central sites other than the siphon sensorimotor synapse.
The present results add to the accumulating evidence that PKMs play crucial roles in the persistence of long-term memory and long-term synaptic plasticity in both vertebrates and invertebrates (Drier et al., 2002; Ling et al., 2002; Pastalkova et al., 2006; Shema et al., 2007; Serrano et al., 2008). Whether the maintenance of other forms of long-term memory in Aplysia, such as long-term habituation (Carew et al., 1972; Ezzeddine and Glanzman, 2003) and classical conditioning (Carew et al., 1981), also depends on PKM Apl III activity remains to be determined. Notice, however, that not all forms of vertebrate memory require PKMζ activity for their maintenance (Shema et al., 2007).
Previous studies using the in vitro sensorimotor synapse have provided support for the idea that ongoing local protein synthesis, regulated by a prion-like protein, ApCPEB, mediates an early phase (≤ 48 hr) of the maintenance of long-term memory in Aplysia (Si et al., 2003b; Si et al., 2003a; Miniaci et al., 2008; Si et al., 2010). Our finding that temporary disruption of protein synthesis with anisomycin did not disrupt the 7-d-old memory for LTS in intact animals is consistent with this idea. Moreover, there are theoretical reasons for supposing that ApCPEB's role in maintaining LTS is limited to an early stage. Vertebrate studies have consistently found that, without a reminder stimulus to trigger memory reconsolidation (Nader et al., 2000b), temporary inhibition of protein synthesis does not disrupt well-consolidated memories (Davis and Squire, 1984).
We found that treatment with either ZIP or chelerythrine at 24 hr after 5-HT training disrupts the expression of LTF of the sensorimotor synapse 24 hr later. Interestingly, the same result is obtained when the action of ApCPEB is inhibited at 24 hr, whether by treatment with an inhibitor of protein synthesis, an ApCPEB antisense oligonucleotide, or an antibody that preferentially binds the multimeric form of ApCPEB (Si et al., 2003b; Miniaci et al., 2008; Si et al., 2010). Thus, these two memory-maintaining processes may overlap temporally. If so, it will be interesting to determine whether these processes interact and, if they do interact, how.
Although our anisomycin results are consistent with the idea that ApCPEB does not mediate late-stage memory maintenance in Aplysia, the results must nonetheless be regarded as with some caution. This is because it is possible that ApCPEB has actions at synaptic sites that support long-term memory that are distinct from the regulation of local protein synthesis. Furthermore, recent computational models bring into question the results of classical tests of the role of protein synthesis in memory consolidation. These models indicate that, in those cases where the underlying mechanism of consolidation involves a molecular positive-feedback loop—as is likely to be the case with PKM (see below)— beyond 40 min after training, protein synthesis must be inhibited by > 95% to actually disrupt memory stabilization (Zhang et al., 2010). A recent estimate of anisomycin's efficacy in the molluscan CNS indicates that it blocks protein synthesis by ≥ 81% for 3 hr, and is completely ineffective by 12 hr after treatment (Fulton et al., 2005). Thus, if ApCPEB is also part of a positive-feedback loop that maintains synaptic facilitation in Aplysia, it is possible that our anisomycin injections, which were made 7 d after training, were ineffective in disrupting the actions of ApCPEB.
An important question to be addressed by future studies is how PKM Apl III maintains LTS and LTF in Aplysia. Note that, at present, the mechanism whereby PKMζ maintains long-term memory in mammals is poorly understood. In particular, it is unclear why the temporary disruption of PKMζ activity by ZIP can erase memories in rodents (Pastalkova et al., 2006; Shema et al., 2007). One might expect that after the peptide is no longer present—due either to diffusion or to degradation—the disrupted memories would reappear. Sacktor (Sacktor, 2011) has hypothesized that PKMζ activity at potentiated synapses is maintained by a positive-feedback loop involving the trafficking of GluR2 subunit-containing AMPA receptors to potentiated synapses. [It is the delivery of additional AMPA receptors to the postsynaptic membrane that represents the molecular engram in most current models of LTP (Blair et al., 2001; Morris et al., 2003; Roberts and Glanzman, 2003).] According to Sacktor's model, PKMζ-dependent phosphorylation of GluR2, or of an accessory protein, causes the AMPA receptor subunit to be trafficked to the postsynaptic membrane, where it forms a “synaptic tag”; the presence of PKMζ at the synapse, in turn, is maintained by its association with GluR2. ZIP, by inhibiting PKMζ, breaks this positive feedback loop by blocking the ongoing phosphorylation of GluR2 (or the accessory protein), which results in the endocytosis of the AMPA receptor subunit; in the absence of the synaptic tag, PKMζ is removed from the synaptic region, and cannot restart the transport of GluR2 subunits to the formerly potentiated synapse once ZIP is eliminated. Consequently, the synapses are reset to their naïve state.
It is unclear whether such a scheme can account for memory maintenance and erasure in Aplysia. Sacktor's model is consistent with our finding (Fig. 5B) that the temporary inhibition of protein synthesis by anisomycin does not disrupt established long-term memory in Aplysia (but see above). It should be pointed out, however, that there is at least one major difference between the Aplysia and mammalian PKM isoforms. PKM Apl III is not formed, as PKMζ is, by transcription from an alternative start site within the atypical PKC, PKC Apl III, gene; rather, as stated earlier, PKM Apl III is produced by calpain-dependent cleavage of PKC Apl III (Bougie et al., 2009). Despite this difference, long-term synaptic plasticity and memory in Aplysia may well be maintained by a positive-feedback loop involving continual PKM Apl III-dependent protein phosphorylation, as Sacktor believes to be true for mammalian LTP and long-term memory.
What are the downstream cellular consequences of continued PKM Apl III activity that serve to maintain stable synaptic facilitation in Aplysia? One possibility is that ongoing PKM Apl III activity maintains learning-induced changes in neuronal structure (see Liu et al., 2009). Both LTS and LTF are accompanied by the growth of new synapses, and this growth involves both presynaptic and postsynaptic structural changes (Bailey and Chen, 1983, 1988b; Bailey and Chen, 1988a; Glanzman et al., 1990; Wainwright et al., 2002). If maintenance of these learning-related, long-term structural changes depends on PKM Apl III, treatment with ZIP or chelerythrine would be expected to reverse them. Another way in which PKM Apl III activity may mediate the persistence of LTS in Aplysia is through enhancement of the trafficking of glutamate receptors at facilitated synapses. Migues et al. (2010) have provided evidence that PKMζ activity in the rat amygdala maintains the memory for conditioned fear by regulating the trafficking of postsynaptic GluR-2 AMPA receptor subunits (above). Modulation of AMPA-type receptor trafficking appears to play a critical role in synaptic plasticity and learning in Aplysia (Zhu et al., 1997; Chitwood et al., 2001; Li et al., 2005; Li et al., 2009; Glanzman, 2010), as it does in mammals (Kessels and Malinow, 2009). Possibly, PKM Apl III activity maintains an increased number of AMPA-type receptors at postsynaptic sites following training that induces LTS. In support of the idea that PKM Apl III is involved in AMPA receptor trafficking in Aplysia, we have recently found that chelerythrine treatment reverses the increased expression of glutamate receptors in the Aplysia CNS that characterizes LTS (Chen et al., 2008, and S.C., K.P., D.C. and D.L.G., unpublished).
We have demonstrated that inhibiting an isoform of PKM eliminates the long-term memory for a form of nonassociative learning, as well as the specific form of long-term synaptic plasticity that underlies the learning, in the well-studied invertebrate Aplysia. The demonstration of memory erasure in this relatively simple model system should greatly facilitate a reductionistic analysis of how PKM's activity supports the persistence of memory. Moreover, given the evidence that ApCPEB also plays a role in the persistence of long-term memory in Aplysia (Si et al., 2003b; Miniaci et al., 2008; Si et al., 2010), we are now in a position to study, potentially, how these two mechanisms interact to maintain long-term memory.
We thank R. Barakat, D. Li, L. Nguyen and T. Phan for assistance with the behavioral training. We also thank F. Krasne, T. O'Dell, T. Sacktor, and W. Sossin for their helpful comments on an earlier version of the manuscript. This study was supported by National Institutes of Health grant R37 NS029563 (D.L.G.).