to block IP3 receptors, BDNF elicited the same magnitude of synaptic potentiation (Fig. 3 D), suggesting that the Ca21 release from intracellular stores is not required for BDNF-induced synaptic potentiation. Thus, BDNF and NT3, two proteins from the same neurotrophin family, can both potentiate neurotransmitter release, but they use to- tally different intracellular mechanisms. The Effect of NT3 Requires Continuous Activation of CaMKII The release of Ca21 from intracellular Ca21 stores induced by NT3 may trigger the activation of the presynaptic CaMKII. CaMKII has been shown to enhance transmitter release in adult squid giant synapses and mammalian brain synaptosomes, presumably due to an increase in the avail- ability of readily releasable synaptic vesicles at the nerve terminals (Llinas et al., 1985; Nichols et al., 1990). We first tested whether CaMKII is involved in modulating trans- mitter release at the developing NMJ in the Xenopus cul- ture system using KN62, a frequently used inhibitor for CaMKII (Tokumitsu et al., 1990). We found that bath ap- plication of KN62 (3 mM) rapidly and reversibly reduced the amplitude of evoked synaptic currents. The average evoked synaptic current amplitudes before and 10 min af- ter KN62 application were 1.77 1 0.36 nA and 1.01 1 0.24 nA, respectively (n 5 8, P , 0.01, t test). In contrast, appli- cation of KN62 had little effect on the spontaneous release of neurotransmitters (Fig. 4, A and B). These results are consistent with the idea that CaMKII may regulate trans- mitter release when the terminal [Ca21]i is elevated, but may not be very effective at the quiescent level of [Ca21]i. Since NT3 acts presynaptically at the NMJ and CaMKII is capable of regulating transmitter release, we determined whether CaMKII is involved in the synaptic action of NT3 at the developing NMJ. Pretreatment of the nerve–muscle cultures with KN62 completely prevented the increase of SSC frequency elicited by NT3 (Fig. 4). The average SSC frequencies before and 10 min after KN62 remained un- changed (before KN62; 5.9 6 0.6 events/min; after KN62, 5.6 6 0.6 events/min; n 5 8, P . 0.1). These results suggest that CaMKII is necessary for the NT3 regulation of neu- rotransmitter release. KN62 may also inhibit other Ca21/calmodulin-depen- dent protein kinases (Enslen et al., 1994). To ensure that the NT3 effect is indeed mediated by CaMKII, we loaded a specific peptide inhibitor (CaMKII-pep) (Ishida et al., 1995) into the presynaptic neurons using embryo injection techniques (Alder et al., 1992; Lu et al., 1992). CaMKII- pep was injected together with rhodamine-dextran into one of the blastomeres of Xenopus embryos at the two-cell stage. The embryos were allowed to develop until stage 22 before being used to prepare the nerve–muscle co-cul- tures. Introduction of the CaMKII-pep did not appear to affect the development and morphology of the Xenopus embryos at the time of cell culture (not shown). The spinal neurons and the myocytes 1 d after cultures also exhibited normal morphology (Fig. 5 A). It has been shown that the rhodamine fluorescence faithfully reflects the cells con- taining the coinjected exogenous proteins (Alder et al., 1992; Lu et al., 1992). In cultures derived from injected embryos, substantial numbers of neurons and myocytes Figure 4. Effect of KN62 on NT3-induced synaptic potentiation. (A) A sample recording showing that pretreatment of the neuro- muscular synapse with KN62 prevents the NT3-induced facilita- tion of transmitter release. (B) Blockade of NT3-induced synap- tic potentiation of SSC frequency by KN62. In KN62 groups, the nerve–muscle cultures were pretreated with KN62 for 10–20 min before application of NT3. The number of synapses recorded are indicated above each pair of columns. In each experiment, SSC frequency was calculated from a 10-min recording period, and then the number after NT3 treatment was normalized to that be- fore NT3 application. Asterisk indicates P , 0.01, t test. were CaMKII-pep–positive, as indicated by the rhoda- mine fluorescence (Fig. 5 A, N1 and M1). Fig. 5 B shows SSCs recorded from a pair of synapses in which the presynaptic neurons were loaded with or with- out CaMKII-pep into normal Ca21 medium, respectively. At the CaMKII-pep (2) synapse, application of NT3 still resulted in a marked increase in SSC frequency (Fig. 5, B and C). In contrast, loading of CaMKII-pep into the pre- synaptic neurons completely prevented the effects of NT3 (Fig. 5, B and C). Similar to KN62, loading of CaMKII- pep to the presynaptic neurons did not affect basal sponta- neous transmitter release, but blocked the effect of NT3 (Fig. 5 C). Similar results were obtained in Ca21-free me- dium (Fig. 5 C). Furthermore, when CaMKII-pep was loaded into the postsynaptic myocytes (M1), NT3 was still capable of eliciting a significant increase in SSC frequency (Fig. 5 C). Taken together, these results strongly suggest that potentiation of transmitter release at the developing neuromuscular synapses by NT3 is achieved through the activation of CaMKII in the presynaptic neurons, but not the postsynaptic muscle cells. Does the NT3-induced potentiation require a continu- ous activation of CaMKII? To address this question, we applied KN62 after synaptic transmission was potentiated by NT3. Fig. 6 A shows that within z20–30 min after NT3 application, the increase in SSC frequency reached the peak. Application of KN62 at the peak gradually sup- He et al. Mechanisms of NT3-induced Synaptic Potentiation 787