We next examined whether depleting ApNRX also blocks the 5-HT-induced synaptic growth that accompanies LTF. Again, we found a significant decrease in the number of new presynaptic varicosities when presynaptic ApNRX was downregulated by the injection of antisense

oligonucleotides (Figures 6C and 6D; % increase in varicosity numbers: no injection –10.4 ± 4.8, n = 8; antisense alone –12.2 ± 3.8, n = 5; sense alone −6.8 ± 6.3, n = 4; 5-HT 35.2 ± 8.2, n = 13; 5-HT+antisense 0.6 ± 5.0, n = 17, p < 0.01 versus 5-HT; 5-HT+sense 36.8 ± 7.8, n = 9). We have hypothesized that the cytoplasmic tail of ApNRX is necessary to recruit new molecular components important for the learning-related SB203580 cell line assembly of the presynaptic active zone and its consequent 5-HT-induced remodeling and growth associated with LTF at the Aplysia sensory-to-motor

neuron synapse. To test this idea, we generated a C-terminal deletion construct of ApNRX (ApNRXΔC) that lacks the cytoplasmic tail. We expressed this construct in the sensory neurons and found that there was no obvious Entinostat research buy difference in the expression of ApNRXΔC-GFP compared to ApNRX-GFP (data not shown). To bring the expression level to a steady state, we waited two days after injecting ApNRX-GFP or ApNRXΔC-GFP into presynaptic sensory neurons and treated sensory-to-motor neuron cocultures with five pulses of 5 min of 5-HT (10 μM) and measured EPSPs before and 24 hr after 5-HT treatment. We found that the overexpression of ApNRXΔC in the presynaptic sensory neuron making functional connections with the postsynaptic motor neuron in culture leads to a significant reduction of LTF at 24 hr, but that overexpression of wild-type ApNRX did not enhance LTF ( Figure 6E;

% initial EPSP amplitude: 5-HT 70.4 ± 9.4, n = 51; 5-HT + ApNRXΔC overexpression 3.9 ± 5.6, n = 14, p < 0.001 versus 5-HT; 5-HT + ApNRX overexpression 71.8 ± 13.7, n = 10). Overexpression of wild-type ApNRX and or ApNRXΔC had no effect on basal transmission (% initial EPSP amplitude: no expression −12.2 ± 4.2, n = 28; ApNRXΔC overexpression alone –12.4 ± 11.6, n = 5; ApNRX overexpression alone −5.5 ± 13.2, n = 6). Unlike its effect on LTF, ApNRXΔC overexpression had no effect on STF induced by one pulse of 5-HT (10 μM) ( Figure 6F; % initial EPSP amplitude: no injection –8.5 ± 4.0, n = 19; ApNRXΔC overexpression alone 1.5 ± 5.7, n = 4; ApNRX overexpression alone 7.3 ± 12.8, n = 6; 5-HT 61.0 ± 7.3, n = 26; 5-HT + ApNRXΔC overexpression 77.0 ± 12.0, n = 9; 5-HT + ApNRX overexpression 78.9 ± 16.4, n = 11). These results with ApNRXΔC provide additional support for the notion that ApNRX is an important regulatory component of long-term memory storage in Aplysia perhaps via intracellular signaling cascades mediated by the cytoplasmic domain.