Volume 27, Issue 13, Pages 1888-1899.e4 (July 2017) Selective Erasure of Distinct Forms of Long-Term Synaptic Plasticity Underlying Different Forms of Memory in the Same Postsynaptic Neuron  Jiangyuan Hu, Larissa Ferguson, Kerry Adler, Carole A. Farah, Margaret H. Hastings, Wayne S. Sossin, Samuel Schacher  Current Biology  Volume 27, Issue 13, Pages 1888-1899.e4 (July 2017) DOI: 10.1016/j.cub.2017.05.081 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Two Pairings at One Input Produced Persistent Plasticity at Both Sensory Neuron Inputs (A) Experimental protocol. See the Results and STAR Methods. (B) Two sensory neurons plated with a single motor neuron L7. The scale bar represents 100 μm. (C) Summary of the changes in synaptic strengths produced by treatment. A two-way ANOVA indicated a significant effect of group × repeated measures (F9, 72 = 100.086, p < 0.0001 at S1; F9, 72 = 144.302, p < 0.0001 at S2). Pairwise comparisons at each time point (Bonferroni) indicated that two pairings of activity plus 5-HT (stimulated alone) evoked significant facilitation at each input compared to Cont (∗∗p < 0.01) and the other stimulated groups (#p < 0.05 and ##p < 0.01) on days 3, 5, and 7. Error bars represent SEM. See also Figures S1 and S2. Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Reactivation-Dependent Synaptic Reconsolidation Blockade at Each Input after Rapamycin (A) Experimental protocol. See the Results and STAR Methods. (B) Summary of the changes in synaptic strengths ±5-HT application on day 3 followed by ±rapamycin. A two-way ANOVA indicated a significant effect of group × repeated measures for both S1 (F18, 120 = 237.19, p < 0.0001) and S2 (F18, 120 = 119.814, p < 0.0001). Pairwise comparisons at each time point indicated significant facilitation in S1 for all stimulated groups compared to Cont + 5-HT on days 3, 4, and 6 (∗∗p < 0.01). In S2, pairwise comparisons indicated significant facilitation for all stimulated groups compared to Cont + 5-HT on days 3, 4, and 6 (∗∗p < 0.01), except for 5-HT reactivation plus rapamycin, which was significantly different than stimulated alone on days 4 and 6 (##p < 0.01). Error bars represent SEM. (C) Summary of the changes in synaptic strength ±HS followed by ±rapamycin. A two-way ANOVA indicated a significant effect of group × repeated measures for both S1 (F18, 120 = 72.987, p < 0.0001) and S2 (F18, 120 = 74.701, p < 0.0001). Pairwise comparisons indicated significant facilitation in S1 for all stimulated groups compared to Cont + HSS1 on days 3, 4, and 6 (∗∗p < 0.01), except for HSS1 reactivation plus rapamycin, which was significantly different than stimulated alone on day 4 and 6 (##p < 0.01). In S2, pairwise comparisons indicated significant facilitation for all stimulated groups compared to Cont + HSS1 (∗∗p < 0.01) on days 3, 4, and 6. Error bars represent SEM. Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Overexpression of Specific dn-PKM Constructs in L7 Reversed Distinct Forms of Persistent LTF at Each Input (A) Experimental protocol. See the Results and STAR Methods. (B) Phase-contrast views of sensorimotor cultures and epiflourescent views of construct expression in the same view area 24 hr after injection on day 3. Scale bar represents 100 μm. (C) Summary of the changes in synaptic strengths after overexpression of control or dominant-negative (dn) PKM constructs. A two-way ANOVA indicated a significant effect of group × repeated measures for both S1 (F21, 132 = 74.8, p < 0.0001) and S2 (F21, 132 = 35.802, p < 0.0001). Pairwise comparisons at each time point indicated significant facilitation in S1 for all stimulated groups compared to Cont on days 3, 4, and 6 (∗∗p < 0.01), except after overexpression of dn-PKM Apl III, which was significantly different than stimulated alone on days 4 and 6 (#p < 0.05 and ##p < 0.01). In S2, pairwise comparisons indicated significant facilitation for all stimulated groups compared to Cont on days 3, 4, and 6 (∗∗p < 0.01), except after overexpression of dn-PKM Apl I, which was significantly different than stimulated alone on days 4 and 6 (##p < 0.01). Error bars represent SEM. See also Figure S3. Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 KIBRA Stabilizes PKMs in Isolated Aplysia Sensory Neurons (A) Description of domains and conservation in Aplysia KIBRA. The homology in the atypical PKC-binding domain and the residues mutated to form KIBRAAAA are shown. (B) Examples of expression of EGFP and mRFP PKM Apl I (top), mRFP PKM Apl II (middle), and mRFP PKM Apl III (bottom), with vector (pNEX3), KIBRA, or KIBRAAA in neurites of isolated Aplysia sensory neurons. (C) Quantification of stabilization. All results are normalized in each experiment to the average mRFP/EGFP ratio of vector alone (pNEX3) (solid horizontal line). ANOVAs were performed separately for PKM Apl I (pNEX, n = 29; KIBRA, n = 43; KIBRAAA, n = 26; F2, 97 = 6.7, p < 001), PKM Apl II (pNEX, n = 18; KIBRA, n = 15; KIBRAAA, n = 22; F2, 54 = 4.04, p < 0.05), and PKM Apl III (pNEX, n = 16; KIBRA, n = 31; KIBRAAA, n = 31; F2, 77 = 12.1, p < 0.0001). ∗p < 0.05 is significant for a post hoc comparison between this group and the control pNEX (Bonferroni post hoc test). All experiments were repeated in at least three separate preparations of sensory neurons (n, number of neurons). Error bars are SEM. (D) After live imaging of mRFP and EGFP, a subset of neurons was fixed and stained with anti-KIBRA antibody. Levels of KIBRA increased in KIBRA-expressing cells (4.2 ± 1.3-fold, n = 15) and KIBRAAAA-expressing cells (4.8 ± 2.1-fold, n = 18) to a similar extent (p > 0.1, two-tailed Student’s t test between the fold increase in KIBRA- and KIBRAAAA-expressing cells). Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Blocking KIBRA Interactions in L7 Selectively Reversed Persistent Associative Plasticity (A) Experimental protocol. See the Results and STAR Methods. (B) Summary of changes in synaptic strength following overexpression of dn-KIBRA in L7 on day 3. A two-way ANOVA indicated a significant effect of group × repeated measures for both S1 (F9, 69 = 154.881, p < 0.0001) and S2 (F6, 69 = 254.243, p < 0.0001). Pairwise comparisons at each time point indicated significant facilitation in S1 and S2 for all stimulated groups compared to Cont on days 3, 4, and 6 (∗∗p < 0.01), except in S1 after overexpression of dn-KIBRA, which was significantly different than stimulated alone on days 4 and 6 (##p < 0.01). Error bars represent SEM. See also Figures S4–S6. Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Expression of Persistent LTF Required Calpain Activity (A) Experimental protocol. See the Results and STAR Methods. (B) Summary of the changes in synaptic strength following overexpression of either dn-SOL or dn-Classical calpain in L7. A two-way ANOVA indicated significant effect of group × repeated measures for both S1 (F15, 99 = 71.482, p < 0.0001) and S2 (F15, 99 = 47.761, p < 0.0001). Pairwise comparisons at each time point indicated significant facilitation in S1 for all stimulated groups compared to Cont on days 2, 3, and 5 (∗∗p < 0.01), except after overexpression of dn-Classical calpain, which was significantly different than stimulated alone on days 2, 3, and 5 (#p < 0.05 and ##p < 0.01). In S2, pairwise comparisons indicated that only stimulation alone produced a significant facilitation compared to Cont on days 2, 3, and 5 (∗∗p < 0.01). After overexpression of either dn-Classical or dn-SOL calpain, these stimulated groups were significantly different than stimulated alone on days 2, 3, and 5 (#p < 0.05 and ##p < 0.01). Error bars represent SEM. (C) Summary of the changes in synaptic strength following overexpression of dn-Classical calpain in the sensory neuron (input S1) receiving two pairings of activity plus 5-HT. A two-way ANOVA indicated a significant effect of group × repeated measures for S1 (F9, 66 = 80.395, p < 0.0001) and S2 (F9, 66 = 76.666, p < 0.0001). Pairwise comparisons at each time point indicated significant facilitation in both S1 and S2 after stimulation alone compared to Cont on days 2, 3, and 5 (∗∗p < 0.01). After overexpression of dn-Classical calpain, there were significant differences in both S1 and S2 compared to stimulated alone on days 2, 3, and 5 (#p < 0.05 and ##p < 0.01). Error bars represent SEM. Current Biology 2017 27, 1888-1899.e4DOI: (10.1016/j.cub.2017.05.081) Copyright © 2017 Elsevier Ltd Terms and Conditions