Fig. 3. NT3-induced potentiation of SSCs at NMJ requires activation of PI3 kinase and PLC-γ, but not MAP kinase. (a) A sample recording of SSCs from an innervated myocytes before and after NT3 application (final concentration, 2 nM) in 1-day-old cultures. SSCs of varying amplitudes are observed as downward currents. (b) Blockade of NT3-induced synaptic potentiation by WT or Ly. Each data set (two circles connected by a line) represents the frequency of SSCs (averaged from 10 min of recording) from a single synapse before and after NT3 application. In the second and third groups, cultures were pre-treated with WT (0.5 µM) or Ly (5 µM), respectively, for 30 min before NT3 application (2 nM). (c) Prevention of NT3-induced synaptic potentiation by the PI3 kinase inhibitor WT. The SSC frequency was monitored before and after NT3 application (2 nM) in cultures treated with (open symbols) or without (closed symbols) WT (0.5 µM). Each point represents averaged SSC frequency in 3 min of recording. (d) Requirement of activation of PI3 kinase, PLC-γ and IP3 receptors, but not MAP kinase pathways, in NT3-induced synaptic potentiation. The cultures were treated with various inhibitors for 15–30 min before NT3 (2 nM) application: PD (10 µM) for MAP kinase pathway, WT (0.1 µM) and Ly (5 µM) for PI3 kinase, U (5 µM) for PLC-γ, or Xestospongin C for IP3 receptors (XeC, 1 µM). For each synapse, a time course of SSC frequency was first constructed on a minute-to-minute basis. SSC frequencies were averaged from a 10-min recording right before NT3 application, and from a 10-min period starting from the highest number after NT3 application for NT3-treated groups. The SSC frequencies after NT3 application were then normalized to those before NT3 application.