Treatment with either ZIP or chelerythrine disrupts established LTS of the withdrawal reflex
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 (). 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.
Figure 1 Biotin-labeled ZIP penetrates the connective tissue and enters inside neurons. Biotin-labeled peptide was conjugated to HRP-avidin, and then visualized by DAB reaction (see the Materials and Methods). A, Low-power micrograph of a cryostat section of the (more ...)
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 (). 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.
Figure 2 Inhibiting PKM Apl III disrupts established LTS in Aplysia. A, Experimental protocol. The timing of the pretests, training, posttests, and drug/vehicle injections is shown relative to the end of the last training session. The time of the intrahemocoel (more ...)
The disruption of LTS by inhibition of PKM is distinct from memory reconsolidation
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 (). As in our earlier experiment (), 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 [below], because we previously found that the chelerythrine injection had no effect on the SWR .) 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.
Figure 3 Disruption of established LTS is not a reconsolidation-related phenomenon, and once disrupted, LTS exhibits neither spontaneous recovery nor reinstatement. A1 and B1, Experimental protocols (see ). In the experiments in A1, the animals did not receive (more ...)
After its disruption, the memory for LTS does not spontaneously recover, and cannot be reinstated by brief sensitization training
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 (). (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 ). 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.
Treatment with chelerythrine or ZIP disrupts LTS even one week after training
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 (). 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.
Figure 4 The effect of chelerythrine or ZIP treatment at 7 d after training. A1 and B1, Experimental protocols, as in . A2, Chelerythrine injection at Day 7 blocked the subsequent expression of LTS. There was significant LTS at Day 7 prior to vehicle/chelerythrine (more ...)
We also tested the effect of an injection of the peptide inhibitor ZIP on one week-old memory for sensitization (). The design of this experiment was similar to that in the test of chelerythrine above (), 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 .) 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.
Inhibition of protein synthesis one week after training does not disrupt LTS
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 (). 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.
Figure 5 Anisomycin disrupts the induction, but not the maintenance, of LTS. A1 and B1, Experimental protocols, as in . A2, Anisomycin injection prior to training blocked the induction of LTS. The mean SWR in the Trained-Veh group was significantly sensitized (more ...)
Next we tested whether the anisomycin treatment could disrupt well-established LTS. Two groups of animals received LTS training (). 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.
Treating sensorimotor synapses with either ZIP or chelerythrine 24 hr after 5-HT training disrupts LTF
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]) (). 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 (). 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.
Figure 6 Maintenance of LTF in Aplysia depends PKM Apl III. A1, Sensorimotor coculture. Scale bar, 20 βm. A2, Sample electrophysiological records from a pretest of a synapse. Scale bars, 20 mV and 200 ms. B1, Experimental protocol for the demonstration (more ...)
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 (). The differences among the groups at 48 hr in this experiment (2) 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 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 ().
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 (). 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 ().
Figure 7 LTF does not exhibit reinstatement after disruption by chelerythrine. A1, Experimental protocol. 24 hr after 5-HT treatment some cocultures were treated with 510 βM chelerythrine for 1 hr. Afterwards, the drug was washed out with culture medium. (more ...)
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 (). 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 for the neuronal input resistances and presynaptic spike thresholds for this experiment.)