Figure 9. The SOL calpain isoform is required for the expression of persistent nonassociative LTF, while the classical calpain isoform is required for the expression of persistent associative LTF. A, The experimental protocol for examining the effects of overexpressing dominant-negative forms of calpain isoforms on the expression of persistent nonassociative LTF. Cultures were treated with two sets of 5-HT applications on days 0 and 1. Dominant-negative constructs of the different calpain isoforms were injected into L7 or sensory neuron 4–5 h after last application of 5-HT on day 1. EPSP amplitudes were tested before control or experimental treatments on day 0 and were retested on days 2, 3, and 5. B, C, Summary of the change in the EPSP amplitudes for control and stimulated groups (2 5-HT) with and without overexpression of dn-SOL or dn-classical in L7 (B) or sensory neurons (C) on day 1. A two-factor ANOVA indicated a significant effect of treatment over time for L7 (F(15,102) = 70.256; p < 0.001) and for the sensory neurons (F(15,102) = 25.562; p < 0.001). Individual comparisons indicated the following: (1) overexpression of dn-classical or dn-SOL in L7 on day 1 after control treatment had no significant effect on synaptic baseline compared with control (#) on day 2 (F(5,34) = 0.0003 or 0.0001; all p > 0.9) and day 3 (F(5,34) = 0.0004 or 0.0003; all p > 0.9) and day 5 (F(5,34) = 0.004 or 0.002; all p > 0.7); (2) overexpression of dn-SOL or dn-classical in the sensory neurons on day 1 after control treatment had no significant effect on synaptic baseline compared with control (#) on day 2 (F(5,34) = 0.0005 or 0.0001; all p > 0.9) and day 3 (F(5,34) = 0.0001 or 0.002; all p > 0.8) and day 5 (F(5,34) = 0.0002 or 0.003; all p > 0.8); (3) after stimulation to produce persistent nonassociative LTF (2 5-HT) and overexpression of dn-classical either in L7 or sensory neurons, the changes in synaptic strength remained significantly greater than control (*) on days 2, 3, and 5 (F(5,34) = 5.669, 5.738, and 5.332, all p < 0.01 in L7; F(5,34) = 2.615, 2.789, and 2.854, all p < 0.05 in the sensory neuron); (4) after stimulation to produce persistent nonassociative LTF (2 5-HT) and overexpression of dn-SOL either in L7 or sensory neurons, the changes in synaptic strength were no longer significantly different from controls (#) on days 2, 3, and 5 (F(5,34) = 1.162, 0.348, and 0.211, all p > 0.05 in L7; F(5,34) = 0.527, 0.205, and 0.095, all p > 0.1 in the sensory neuron); (5) after stimulation to produce persistent nonassociative LTF (2 5-HT) and overexpression of dn-SOL either in L7 or sensory neuron, the changes in synaptic strength were significantly smaller (o) than the change detected in 2 5-HT alone on days 3 and 5 in L7 (F(5,34) = 3.169 and 2.922; all p < 0.05), and on days 2, 3, and 5 in the sensory neuron (F(5,34) = 2.909, 4.382, and 5.335, all p < 0.05). D, The experimental protocol for examining the effects of overexpressing dominant-negative forms of calpain isoforms on the expression of persistent associative LTF. Cultures were treated with pairings of tetanic stimulation in the sensory neuron plus a brief 5-HT application on days 0 and 1. Dominant-negative constructs of the different calpain isoforms were injected into L7 or sensory neuron 4–5 h after the last application of 5-HT on day 1. EPSP amplitudes were tested before control or experimental treatments on day 0 and retested on days 2, 3, and 5. E, F, Summary of the change in the EPSP amplitudes for control and stimulated groups [2 (Tet + 5-HT)] followed by overexpression of dn-classical or dn-SOL in L7 (E) or sensory neurons (F) on day 1. A two-factor ANOVA indicated a significant effect of treatment over time for L7 (F(9,72) = 23.888; p < 0.001) and for the sensory neurons (F(9,72) = 61.12; p < 0.001). Individual comparisons indicated the following: (1) after stimulation to produce persistent associative LTF [2 (Tet + 5-HT)] and overexpression of dn-classical either in L7 or sensory neurons, the changes in synaptic strength were no longer significantly different from control (#) on days 2, 3, and 5 (F(3,24) = 0.829, 0.072, and 0.063, all p > 0.1 in L7; F(3,24) = 1.08, 0.261, and 0.063, all p > 0.05 in the sensory neuron), which were significantly smaller (o) than the changes detected in 2 (Tet + 5-HT) alone (F(3,24) = 2.277, 6.968, and 6.736, all p < 0.05 in L7; F(3,24) = 5.19, 6.857, and 7.229, all p < 0.01 in the sensory neuron); (2) after stimulation to produce persistent associative LTF [2 (Tet + 5-HT)] and overexpression of dn-SOL either in L7 or sensory neurons, the changes in synaptic strength remained significantly greater than those for control (*) on days 2, 3, and 5 (F(3,24) = 3.727, 3.456, and 3.923, all p < 0.01 in L7; F(3,24) = 10.337, 9.726, and 8.652, all p < 0.01 in the sensory neuron), which were not significantly different from the changes detected in 2 (Tet + 5-HT) alone (F(3,24) = 0.239, 1.101, and 0.749, all p > 0.05 in L7; F(3,24) = 0.01, 0.001, and 0.0001, all p > 0.1 in the sensory neuron).