Figure 5. Inhibition of CaMKII prevents the effect of NT3. A peptide inhibitor for CaMKII (CaMKII-pep), together with rhodamine- dextran, was injected into Xenopus embryo at the two-cell stage, and the nerve–muscle co-cultures were prepared from the injected em- bryos. (A) Super-imposed phase and fluorescence micrographs of the 1-d-old nerve–muscle co-cultures showing cells loaded with ( 1) or without (2) CaMKII-pep. N, spinal neurons; M, myocytes. (B) A pair of recordings showing that loading of CaMKII peptide into presynaptic neurons N(1) prevented the NT3-induced increase in SSC frequency, whereas NT3 still enhanced transmitter release in a N(2) neuron in the same culture. (C) Summary of the effect of CaMKII-pep loaded into either presynaptic spinal neurons or postsyn- aptic myocytes. The number of synapses recorded are indicated above each pair of columns. In all N2 conditions, application of NT3 elicited a significant increase in SSC frequency (P , 0.01, t test). pressed the SSC frequency. Quantitative analysis indi- cated that KN62 virtually reversed the NT3 effect (Fig. 6 B). Thus, continuous activity of CaMKII appears to be necessary for NT3 modulation of transmitter release at the developing neuromuscular synapses. Discussion Previous work has shown that neurotrophins rapidly po- tentiate synaptic transmission through presynaptic mecha- nisms. The acute potentiation of transmitter release by BDNF is accompanied by a rise in [Ca21]i in both the NMJ (Stoop and Poo, 1995) and at the CNS synapses (Bern- inger and Garcia, 1993; Marsh and Palfrey, 1996; Sakai et al., 1997; Li et al., 1998). However, it is unclear whether and how the increase in [Ca21]i mediates the neurotro- phin-induced synaptic potentiation and which downstream signaling events are involved. In this paper we report a surprising finding that the acute potentiation of transmit- ter release by NT3 at the neuromuscular synapses is inde- pendent of Ca21 influx from extracellular sources. Instead, this potentiation is mediated by Ca21 released from intra- cellular stores through IP3 and ryanodine receptors. Thus, the mechanisms by which BDNF and NT3 modulate trans- mitter release could be quite different. Furthermore, we demonstrated that Ca21 released from intracellular stores is capable of activating CaMKII, and the continuous acti- vation of CaMKII is required for the effect of NT3. Taken together, this study provides, to our knowledge, the first evidence for a link between neurotrophins and CaMKII. These findings may provide new insights into the general mechanisms of neurotransmitter release and exocytosis, The Journal of Cell Biology, Volume 149, 2000 788