To test whether ApNRX and ApNLG are

required for the stable maintenance of LTF, we injected antisense oligonucleotides directed against ApNRX into sensory neurons at 24 hr after repeated pulses of 5-HT and measured EPSPs at 48 hr and 72 hr. Basal synaptic transmission was not affected by the oligonucleotide injections ( Figure 7A; % initial EPSP amplitude: no injection at 24 hr 8.6 ± 8.2, at 48 hr –4.1 ± 8.0, at 72 hr –4.2 ± 9.3, n = 14; antisense alone Z VAD FMK at 24 hr 3.5 ± 7.1, at 48 hr –17.5 ± 5.7, at 72 hr –13.7 ± 7.8, n = 11; sense alone at 24 hr –0.7 ± 5.5, at 48 hr –12.3 ± 5.6, at 72 hr –8.6 ± 10.0, n = 11). However, we find that injection of antisense oligonucleotides leads to a significant reduction of LTF measured at 48 and 72 hr ( Figure 7A; % initial EPSP amplitude: 5-HT at 24 hr 99.2 ± 15.2, at 48 hr 75.1 ± 9.7, at 72 hr 52.2 ± 10.8, n = 16; 5-HT + antisense at 24 hr 89.2 ± 15.3, at 48 hr 24.6 ± 10.5, at 72 hr 10.6 ± 8.9, n = 19, p <

0.001 versus 5-HT at 48 and 72 hr; 5-HT + sense at 24 hr 103.7 ± 22.7, at 48 hr 69.0 ± 16.1, Vorinostat at 72 hr 56.8 ± 15.8, n = 14). We also injected antisense oligonucleotides directed against ApNLG into the motor neuron at 24 hr after repeated pulses of 5-HT and measured EPSPs at 48 hr and 72 hr. We again found that injection of antisense oligonucleotides leads to a significant reduction of LTF measured at 48 and 72 hr (Figure 7B; Ketanserin % initial EPSP amplitude: 5-HT at 24 hr 115.2 ± 20.6, at 48 hr 81.5 ± 16.5, at 72 hr 67.2 ± 10.3, n = 11; 5-HT + antisense at 24 hr

89.8 ± 11.3, at 48 hr 18.6 ± 6.1, at 72 hr 1.8 ± 5.4, n = 18, p < 0.001 versus 5-HT at 48 and 72 hr; 5-HT + sense at 24 hr 103.7 ± 20.5, at 48 hr 103.0 ± 22.0, at 72 hr 78.0 ± 5.5, n = 11). Basal synaptic transmission was not affected by the oligonucleotide injections (% initial EPSP amplitude: no injection at 24 hr 4.1 ± 4.0, at 48 hr –13.6 ± 5.9, at 72 hr –14.4 ± 5.2, n = 7; antisense alone at 24 hr 8.4 ± 6.6, at 48 hr 5.0 ± 8.9, at 72 hr –1.9 ± 8.1, n = 7; sense alone at 24 hr 15.1 ± 10.7, at 48 hr 7.8 ± 8.3, at 72 hr 7.8 ± 10.6, n = 9). These results indicate that ApNRX and ApNLG play a critical role not only in the initiation of LTF and the associated presynaptic structural changes but also in the stabilization and persistence of these learning-induced synaptic changes. The human NLG-3 R451C point mutation has been linked to ASD (Jamain et al., 2003). Since there has not been any study investigating the mutant’s effect in synaptic plasticity, we made an arginine (R) to cysteine (C) point mutation in ApNLG at the homologous position, overexpressed this mutant in the motor neurons, and investigated its effect on 5-HT-induced changes in the strength of the sensory-to-motor neuron synapse.

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