Figure 6. Reversal of NT3 effects by addition of KN62 after NT3 application. (A) Examples showing the time courses that KN62 reverses the NT3 effect. NT3 was first applied (filled arrow) to the culture medium to elicit an increase in the frequency of SSCs. After the SSC frequency reached the peak, KN62 (open arrow) was either applied (open circles) or not applied (filled circles), and synaptic currents were continuously monitored. (B) Sum- mary of the reversal effect of KN62. SSC frequencies are calcu- lated in the same way as Fig. 3 C. n 5 5. Asterisk indicates P , 0.01, t test. and how neurotrophic factors may regulate these pro- cesses. In addition to extracellular Ca21, the release of Ca21 from intracellular stores could either modulate or contrib- ute directly to transmitter release (Berridge, 1998). Signals that result in the opening of either IP3 receptors or ryano- dine receptors can generate local increases in [Ca21]i, which in turn participates in the exocytotic process. Al- though still a fairly new concept, transmitter release trig- gered or modulated by the release of Ca21 from intracellu- lar stores has been shown in a number of systems such as the cholinergic synapse in Aplysia, reticulospinal synapse in lamprey, and sympathetic nerve terminals (Smith and Cunnane, 1996; Cochilla and Alford, 1998; Mothet et al., 1998). An important question then is whether neurotro- phins, which are capable of eliciting an IP3 signal through the activation of phospholipase-g pathway (Segal and Greenberg, 1996), can serve as endogenous neuromodula- tors to regulate synaptic transmission under physiological conditions. In this study, we have provided strong evi- dence that NT3 potentiates transmitter release by stimu- lating Ca21 release from intracellular stores. We have shown that NT3 increased transmitter release in Ca21-free or Cd21-containing medium, and that pretreatment with thapsigargin prevented the NT3 effect. Moreover, inhibi- tion of IP3 receptors blocked the NT3 effect. Thus, NT3 induces the release of Ca21 through IP3 receptors at the terminals of developing spinal neurons, leading to an in- crease in spontaneous transmitter secretion. It is conceiv- able that similar mechanisms are used for NT3 to enhance evoked synaptic transmission, although we could not test this possibility because most of our experiments have to be done in Ca21-free medium. Consistent with our results, neurotrophins have been shown to induce an increase in [Ca21]i in hippocampal neurons (Berninger and Garcia, 1993; Marsh and Palfrey, 1996), possibly by enhancing the release of Ca21 from intracellular stores (Sakai et al., 1997; Li et al., 1998). The release of Ca21 from IP3 receptors could further trigger Ca21-induced Ca21 release from ry- anodine receptors (Berridge, 1998). We found that the acute modulation of transmitter release by NT3 was blocked by the ryanodine receptor antagonist TMB-8 or a high concentration of ryanodine (100 mM). However, acti- vation of the ryanodine receptor alone by low concentra- tions of ryanodine (2.5–5 mM) was not sufficient to en- hance transmitter release, and application of NT3 on top of that still increased SSC frequency. Thus, both IP3 re- ceptors and ryanodine receptors are involved in the acute effect of NT3, but the primary effect of NT3 is probably on the IP3 receptors. Although the electrophysiological anal- ysis clearly indicates that NT3 potentiates neurotransmit- ter release through presynaptic mechanisms, our pharma- cological experiments can not formally establish that NT3 acts on presynaptic terminals directly. We can not rule out the possibility that NT3 initially acts on postsynaptic mus- cle cells to trigger the release of Ca21 from intracellular stores, leading to the secretion of some retrograde signal to activate presynaptic CaMKII. Although our results suggest a role of Ca21 release from internal stores in NT3-induced synaptic potentiation, a previous study has shown that the acute potentiation by BDNF in the same preparation requires external Ca21 (Stoop and Poo, 1996). BDNF binds and interacts almost exclusively with the TrkB receptor, whereas NT3 binds primarily to the TrkC receptor (Kaplan and Stephens, 1994). It is possible that the activation of TrkB triggers Ca21 influx, whereas that of TrkC is coupled to internal Ca21 stores in the developing spinal neurons. Indeed, we found that inhibition of Ca21 release from internal stores can not block the BDNF-induced synaptic potentiation. Similarly, both BDNF and NT3 attract growth cone turn- ing in these developing spinal neurons, but the intracellu- lar mechanisms that mediate the turning responses to the two factors are completely different (Song et al., 1998). The BDNF effect requires Ca21 influx into the terminals and elevation of [cAMP]i, whereas the NT3 effect is inde- pendent of extracellular Ca21. In this study, we show that the activation of IP3 receptors is required for the synaptic effect of NT3, but not for that of BDNF. Thus, although He et al. Mechanisms of NT3-induced Synaptic Potentiation 789