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 injection of either drug or vehicle is indicated by the red arrow in this figure and in Figures 3, 4 and 5 (below). B, ZIP treatment at 24 hr after training blocked LTS. Animals in all three trained groups exhibited significant sensitization at 24 hr, as indicated by SNK post-hoc tests. Thus, the SWR was longer in the Trained-Vehicle (Veh) group (n = 4) than in the Control-Veh group (n = 6) (q = 8.6, p < 0.001); in the Trained-ZIP group (n = 6) than in the Control-ZIP group (n = 4) (q = 7.4, p < 0.001); and in the Trained-ScrZIP group (n = 4) than in the Control-ZIP group (q = 6.6, p < 0.001). The Trained-Veh and Trained-ScrZIP groups also exhibited significant sensitization at 48 hr, as compared to their respective control groups: Trained-Veh vs. Control-Veh, q = 13.8 (p < 0.001); and Trained-ScrZIP vs. Control-ZIP, q = 12.4 (p < 0.001). By contrast, the Trained-ZIP animals did not exhibit sensitization at 48 hr. Thus, the SWR in the Trained-ScrZIP animals was significantly longer than that in the Trained-ZIP animals (q = 13.6, p < 0.001), and there was no significant difference between the Trained-ZIP and Control-ZIP groups. Notice that there were no significant differences between the Trained-Veh and Trained ScrZIP data for either the 24- or 48-hr test. Also, the differences between the Control-Veh and Control-ZIP groups were not significant in the experiments in B, or in any of our other experiments. Asterisks, comparison between Trained-Veh and Control-Veh groups; plus signs, comparison between Trained-ZIP and Control-ZIP groups; pound signs, comparison between Trained-ScrZIP and Control-ZIP groups; and paragraph signs, comparison between the Trained-ScrZIP and Trained-ZIP groups. Here and in subsequent figures, one symbol, P < 0.05; two symbols, P < 0.01; three symbols, P < 0.001. C, Chelerythrine treatment at 24 hr also disrupted established LTS. SNK post-hoc comparisons indicated that the training produced significant LTS in the Trained-Veh and Trained-Chel groups at 24 hr (Trained-Veh vs. Control-Veh, q = 8.6 [p < 0.001], and Trained-Chel vs. Control-Chel, q = 8.4 [p < 0.001]). Sensitization was also evident at 48 hr in the Trained-Veh group (Trained-Veh vs. Control-Veh, q = 8.4 [p < 0.001]), but not in the Trained-Chel group. The difference between the two trained groups on the 48-hr test was highly significant (q = 8.8 [p < 0.001]). There was no significant difference between the Trained-Chel and Control-Chel groups at 48 hr. Also, there were no significant differences between the two control groups at either 24 hr or 48 hr. Asterisks, as in B; plus signs, comparison between Trained-Chel and Control-Veh groups; pound signs, comparison between the Trained-Veh and Trained-Chel groups. Data in this figure, and in Figures 3–5, are the mean duration (in seconds) of the SWR. Error bars in this and subsequent figures represent SEM.

The effect of chelerythrine or ZIP treatment at 7 d after training. A₁ and B₁, Experimental protocols, as in Figure 2. A₂, Chelerythrine injection at Day 7 blocked the subsequent expression of LTS. There was significant LTS at Day 7 prior to vehicle/chelerythrine injection in both the Trained-Veh group (Trained-Veh vs. Control-Veh, q = 9.1 [p < 0.001]) and Trained-Chel group (Trained-Chel vs. Control-Chel, q = 7.3 [p < 0.001]). The Trained-Veh group also exhibited robust sensitization compared to the Control-Veh group on Days 8 (q = 7.6 [p < 0.001]) and 9 (q = 7.0 [p < 0.01]). But sensitization was not apparent in the Trained-Chel group following the injection of chelerythrine, as indicated by the lack of significance in the Days 8 and 9 comparisons between the two chelerythrine-treated groups. Also, the duration of the SWR was significantly longer in the Trained-Veh group than in the Trained-Chel group on posttraining Days 8 (q = 8.1 [p < 0.001]) and 9 (q = 7.4 [p < 0.001]). Asterisks, comparison between the Trained-Veh and Control-Veh groups; plus signs, comparison between Trained-Chel and Control-Chel groups; and pound signs, comparison between the Trained-Veh and Trained-Chel groups. B₂, ZIP injection at Day 7 also blocked subsequent LTS. The training produced significant LTS in the Trained-Veh group at Days 8 and 9, as indicated by the comparison with the Control-Veh group (Day 8 comparison, q = 9.2 [p < 0.001]; and Day 9 comparison, q = 11.4 [p < 0.001]). However, following the injection of ZIP, sensitization was no longer apparent in the Trained-ZIP, as indicated by the lack of a significant difference between the Trained-ZIP and Control-Veh groups. Moreover, animals in the Trained-Veh group were significantly more sensitized than those in the Trained-ZIP group on both posttraining tests (Day 8 comparison, q = 9.2 [p < 0.001]; and Day 9 comparison, q = 11.4 [p < 0.001]). Notice that there was no posttest preceding the injection of ZIP/vehicle solution at Day 7. Asterisks, comparison between the Trained-Veh and Control-Veh groups; plus signs, comparison between Trained-Veh and Trained-ZIP groups.

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 of 24 hr LTF. B₂, Sample EPSPs. Each pair of traces shows EPSPs recorded from the same sensorimotor synapse on Day 1 (Pre) and ~ 24 hr later (Post). Training with 5-HT (100 βM) produced LTF (lower traces). Scale bars in this and subsequent figures, 10 mV and 80 ms. B₃, 5-HT-trained synapses showed significant LTF at 24 hr. Data in this figure and in Figure 7 are the mean normalized amplitude of the posttest EPSPs. The mean normalized EPSP in the synapses “trained” with 5-HT (5-HT group, n = 10) at 24 hr after training was 147% ± 13%, whereas the mean EPSP in the Control group (n = 10) was 74% ± 10% (U = 6.0 [p < 0.001]). C, Protocol for the synaptic experiments using ZIP/ScrZIP. 24 hr after 5-HT training, some cocultures were treated with 1 βM ZIP/ScrZIP for 1 hr, after which the ZIP/ScrZIP was rapidly washed out with culture medium. D₁, Sample EPSPs. D₂, ZIP treatment at 24 hr blocked the subsequent expression of LTF. Dunn's post-hoc tests indicated that the mean normalized EPSP at the 48 hr posttest in the 5-HT group (153% ± 23%, n = 17) was significantly greater than those in the Control (52% ± 8%, n = 15) and 5-HT-ZIP (50% ± 7%, n = 15) groups (5-HT vs. Control, Q = 22.0 [p < 0.01]; 5-HT vs. 5-HT-ZIP, Q = 24.2 [p < 0.001]). There was no significant difference between the EPSPs in the 5-HT-ZIP and ZIP alone (47% ± 9%, n = 11) groups, nor between the Control and ZIP groups. Asterisks, comparison between the 5-HT and Control groups; plus signs, comparison between the 5-HT and 5-HT-ZIP groups. E₁, Sample EPSPs. E₂, Treatment of the sensorimotor synapses with the scrambled ZIP peptide at 24 hr did not block the expression of LTF. As indicated by Dunn's post-hoc tests, the mean normalized EPSP at 48 hr in the 5-HT group (165% ± 21%, n = 13) was significantly greater than those for the Control group (71% ± 10%, n = 13; Q = 20.2 [p < 0.01]). Moreover, the mean normalized EPSP in the 5-HT-ScrZIP group (161% ± 28%, n = 11) was significantly greater than that in the Control-ScrZIP group (78% ± 12%, n = 10; Q = 16.2 [p < 0.05]). There was no significant difference between the EPSPs in the 5-HT and 5-HT-ScrZIP groups, nor between the Control and ScrZIP groups. Asterisks, comparison between the 5-HT and Control groups; plus signs, comparison between the 5-HT-ScrZIP and ScrZIP alone groups. F₁, There were no significant changes in the mean input resistance of the sensory neurons in the experiments shown in D. F₂, The mean input resistance of the motor neurons in these experiments also did not change (same cocultures as in F₁). F₃, ZIP treatment did not alter the mean spike threshold of the sensory neurons (experiments shown in D).

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 pleural ganglion from an animal given a vehicle injection. Note the absence of DAB staining following biotin-detection processing. B₁, Low-power micrograph of a cryostat section of the pleural ganglion from an animal given an injection of biotin-labeled myristoylated ZIP. B₂, Higher power micrograph of the area indicated by the red box in B₁. There is strong immunocytochemical staining in the connective tissue sheath surrounding the ganglion, as well as inside neurons. Scale bars, 100 μm in A and B₁; 50 μm in B₂.

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 Fig. 2). In the experiments in A₁, the animals did not receive a posttest 24 hr after training. In the experiments in B₁, some animals received one additional bout of sensitization training (black bar) immediately after the 96 hr posttest. A₂, Chelerythrine disrupted established LTS despite the absence of a posttest at 24 hr. Post-hoc tests revealed that the mean SWR in the Trained-Veh group (n = 8) was significantly sensitized compared to that in the Control-Veh group (n = 4, q = 9.6 [p < 0.001]), and the Trained-Chel group (n = 4, q = 9.6 [p < 0.001]). The Control-Veh and Trained-Chel groups did not differ significantly. Asterisks, comparison between Trained-Veh and Control-Veh groups; and plus signs, comparison between the Trained-Veh and Trained-Chel groups. B₂, Absence of spontaneous recovery or reinstatement of LTS following the chelerythrine injection. Animals in the Trained-Chel-Reinstate group were given one additional bout of sensitization training immediately after the 96 hr posttest (black bar in B₁). Sensitization was robust in all three trained groups at 72 hr post-training, as indicated by post-hoc tests (Trained-Veh vs. Control-Veh [q = 10.1, p < 0.001], Trained-Chel vs. Control-Veh [q = 8.4, p < 0.001], and Trained-Chel-Reinstate vs. Control-Veh [q = 8.3, p < 0.001]). However, although the Trained-Veh group exhibited sensitization on all of the subsequent posttests (for the comparison between the Trained-Veh and Control-Veh groups, q = 18.3 [p < 0.001] at 96 hr; q = 13.1 [p < 0.001] at 120 hr; and q = 13.5 [p < 0.001] at 144 hr), sensitization was absent in both groups of chelerythrine-treated animals after 72 hr. Post-hoc tests revealed no significant differences for any of the comparisons between the Control-Veh group and the Trained-Chel and Trained-Chel-Reinstate groups on any of the post-72-hr tests. Asterisks, comparison between the Trained-Veh and Control-Veh groups; plus signs, comparison between Trained-Chel and Control-Veh groups; and pound signs, comparison between Trained-Chel-Reinstate and Control-Veh groups.

Anisomycin disrupts the induction, but not the maintenance, of LTS. A₁ and B₁, Experimental protocols, as in Figure 2. A₂, Anisomycin injection prior to training blocked the induction of LTS. The mean SWR in the Trained-Veh group was significantly sensitized compared to that in the Control-Veh group (SNK test, q = 38.4 [p < 0.001]) and the Trained-Aniso group (SNK test, q = 40.0 [p < 0.001]). The differences among the Control-Veh, Control-Aniso and Trained-Aniso groups were not significant. Asterisks, comparison between the Trained-Veh and Control-Veh groups; plus signs, comparison between the Trained-Veh and Trained-Aniso groups. B₂, Anisomycin injection on Day 7 fails to disrupt LTS expression on Days 8 and 9. Post-hoc tests revealed that the Trained-Veh group was significantly sensitized compared to the Control-Veh groups on each of the three posttests (Day 7, q = 5.5 [p < 0.01]; Day 8, q = 5.8 [p < 0.01]; and Day 9, q = 5.9 [p < 0.01]). The comparison between the Trained-Aniso and Control-Veh groups was also significant on Days 7–9 (Day 7, q = 5.9, p < 0.01; Day 8, q = 6.6, p < 0.01; and Day 9, q = 8.2, p < 0.001). (A Control-Aniso group was not included in this experiment, because we previously found [see A₂] that anisomycin treatment alone had no effect on the SWR.) The differences between the Trained-Veh and Trained-Aniso groups were not significant on any of the posttests. Asterisks, comparison between Trained-Veh and Control-Veh groups, plus signs, comparison between Trained-Aniso and Control-Veh groups.

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. A₂, Sample EPSPs. A₃, Effect of chelerythrine treatment at 24 hr on the expression of LTF at 48 hr. Four groups of cocultures were included in this experiment: one trained with 100 βM 5-HT and then treated with the vehicle solution for 1 hr at 24 hr after 5-HT training (5-HT group, n = 14); one that received only the vehicle solution during training and during the 1 hr treatment 24 hr later (Control group, n = 14); one trained with 5-HT and then treated with chelerythrine (5–10 βM, 1 hr) 24 hr after training (5-HT-Chel group, n = 9); and, finally, a group trained with the vehicle solution and then treated with chelerythrine 24 hr later (Chel, n = 10). Note that the synapses were not tested at 24 hr. SNK post-hoc tests revealed that synapses that received the vehicle treatment following 5-HT training were significantly facilitated at 48 hr (5-HT group, normalized mean EPSP = 147% ± 17%), compared to synapses in the Control group (mean normalized EPSP = 56% ± 9%; Q = 19.5 [p < 0.01]), and to synapses in the 5-HT-Chel group (mean normalized EPSP = 51% ± 8%; Q = 19.6 [p < 0.01]). The EPSPs in the 5-HT-Chel group did not differ significantly from those in the Chel group (mean normalized EPSP = 54% ± 12%). Also, the EPSPs in the Control and Chel groups did not differ significantly, indicating that chelerythrine treatment alone did not affect the synaptic responses in cocultures not trained with 5-HT. Asterisks, comparison between the 5-HT and Control data; plus signs, comparison between the 5-HT and 5-HT-Chel data. B₁, Experimental protocol used to attempt to reinstate LTF. One hour 5–10 βM chelerythrine treatment was given to some cocultures at 18 hr after 5-HT training. At 24 hr one extra 5-min pulse of 5-HT (black bar) was applied to cocultures in the 5-HT-Chel-Reinstate group. B₂, Sample EPSPs. B₃, Retraining with one 5-min pulse of 5-HT, which produces short-term synaptic facilitation, did not reinstate LTF after its disruption by chelerythrine. The mean normalized EPSP in the 5-HT group (161% ± 25%) was significantly facilitated compared to that in the Control group (62% ± 11%, Q = 15.4 [p < 0.05]) and the 5-HT-Chel-Reinstate group (53% ± 8%, Q = 18.6 [p < 0.01]). The difference between the mean normalized EPSPs in the Chel (56% ± 11%) and the 5-HT-Chel-Reinstate groups were not significant, nor was that between the Control and Chel groups. Asterisks, comparison between the 5-HT and Control data; plus signs, comparison between the 5-HT and 5-HT-Chel-Reinstate data. C₁, The mean input resistance of the sensory neurons did not change significantly in the experiments shown in A. C₂, There was also no significant change in the mean input resistance of the motor neurons in these experiments (same cocultures as in C₁). C₃, Chelerythrine treatment did not significantly affect the mean spike threshold of the sensory neurons (experiments shown in A). D₁, No significant changes were observed in the mean input resistance of the sensory neurons in the experiments of B. D₂, Neither did any significant changes occur in the mean input resistance of the motor neurons in these experiments (same cocultures as in D₁). D₃, The sensory neuron spike threshold increased significantly in the chelerythrine-treated cocultures in the experiments of B. However, the synaptic change observed in this experiment cannot be attributed to the effect of this drug on presynaptic spike threshold, because the increase occurred in both the untrained and 5-HT-trained cocultures exposed to chelerythrine. Asterisks, comparison between the 5-HT and Chel groups; plus signs, comparison between the 5-HT and 5-HT-Chel-Reinstate groups.