that the signaling mechanisms for BDNF and NT3 may be quite different. Due to the complexity of CNS synapses, the mecha- nisms underlying the synaptic actions of neurotrophins are difficult to study. The neuromuscular junction (NMJ) of- fers a simple and easily accessible model to study the role and the mechanisms of neurotrophins in synaptic devel- opment and function in great detail. Two modes of neurotrophic regulation have been identified using the Xenopus nerve–muscle co-cultures: acute potentiation of neurotransmitter release and long-term regulation of syn- apse maturation. In the long-term mode, the spontaneous synaptic currents (SSCs) and impulse–evoked synaptic currents exhibit more mature properties after a prolonged treatment with NT3, and to a lesser extent, with BDNF (Wang et al., 1995; Liou and Fu, 1997; Liou et al., 1997). The neurotrophins induce an increase in the expression of synaptic vesicle proteins, and in the number of synaptic varicosities in the presynaptic site (Wang et al., 1995), as well as changes in the acetylcholine (ACh) receptors in the postsynaptic site (Wang and Poo, 1997; Gonzalez et al., 1999). In the acute mode, application of BDNF or NT3 rapidly enhances synaptic transmission at the NMJ (Lohof et al., 1993). The acute effect of neurotrophins is due strictly to an enhancement of transmitter release probabil- ity in the presynaptic site (Lohof et al., 1993; Stoop and Poo, 1995). The SSC frequency is markedly increased, whereas the quantal sizes are not affected. The expression of NT3, but not BDNF or NT4, in the postsynaptic muscle cells is activity-dependent (Xie et al., 1997). Further, the secretion of NT4 in muscle cells seems to be induced by re- petitive stimulation of presynaptic neurons (Wang and Poo, 1997). These results suggest that neurotrophins may serve as target-derived, retrograde messengers that acutely modulate transmitter release at the developing neuromus- cular synapses (Xie et al., 1997). A critical and yet unresolved question is: what are the intracellular signaling mechanisms that mediate such rapid synaptic effects of neurotrophins? In the hippocampus, BDNF-induced enhancement of high frequency transmis- sion at CA1 synapses appears to be mediated through the activation of mitogen-associated protein kinase and phos- phatidylinositol-3 kinase pathways, but not phospholipase C-g pathway (Gottschalk et al., 1999). The acute modula- tion of synaptic transmission by BDNF at NMJ appears to require Ca21 influx into the presynaptic terminals, but sig- naling events downstream of Ca21 influx are not known (Stoop and Poo, 1996). Do neurotrophins share similar mechanisms in modulating synapses in the CNS and at the NMJ? Do BDNF and NT3 use the same signaling pathway to potentiate the neuromuscular synapses? In this report, we address the role of the nerve terminal Ca21 in the acute regulation of neurotransmitter release at the NMJ by NT3. Specifically, we focus on the intracellular Ca21 stores and the presynaptic Ca21/calmodulin-dependent kinase II (CaMKII). A number of recent studies have suggested the involvement of intracellular Ca21 stores in synaptic trans- mission (for review see Berridge, 1998). Although exten- sive studies have revealed diverse effects of CaMKII in postsynaptic functions (Chapman et al., 1995), the only clearly defined presynaptic effects of CaMKII is to regu- late the availability of readily releasable synaptic vesicles at the nerve terminals (Llinas et al., 1985; Greengard et al., 1993). We have now provided evidence that the acute po- tentiation of transmitter release by NT3 depends on a rise of Ca21 concentrations ([Ca21]i) in the presynaptic termi- nals. Surprisingly, the increase in [Ca21]i was due to Ca21 released from intracellular stores, but not to Ca21 influx from extracellular sources. Furthermore, the continuous activation of CaMKII, which is triggered by the increase in [Ca21]i, appears to be required for the effect of NT3. These results may help understand how neurotrophins acutely modulate neurotransmitter release. Materials and Methods Embryo Injection Specific peptide inhibitor for CaMKII (Ishida et al., 1995) (500 mM; Cal- biochem) was mixed with rhodamine-dextran (10 mg/ml, mol wt 10,000) at 1:1 ratio. Approximately 6–12 nl of the solution was injected into one blas- tomere of embryos at the 2- to 4-cell stage by a Picospritzer. The final con- centration of the peptide within an injected blastomere was z12.5 mM. 1 d after injection, the neural tube and the associated myotomal tissues were dissected and used to prepare nerve–muscle cultures. Cells containing CaMKII-pep were identified by rhodamine fluorescence. Culture Preparation Xenopus nerve–muscle cultures were prepared according to the proce- dure described previously (Lu et al., 1992). In brief, the neural tube and the associated myotomal tissue of Xenopus embryos at stage 20 to 22 were dissociated in Ca21-Mg21–free saline supplemented with EDTA (58.2 mM NaCl, 0.7 mM KCl, 0.3 mM EDTA, pH 7.4) for 15–20 min. The cells were grown on glass coverslips for 24 h at room temperature (20–228C). The culture medium consisted (vol/vol) of 50% Leibovitz L-15 medium (Sigma), 1% FCS (Life Technologies), and 49% Ringer’s solution (115 mM NaCl, 2 mM CaCl2, 2.5 mM KCl, 10 mM Hepes, pH 7.6). NT3 (2–5 3 1029 M; kindly provided by Regeneron Pharmaceuticals, Inc.) and various inhibitors were applied directly to the culture media at the time of recording. Electrophysiology Synaptic currents were recorded at room temperature in culture medium from myocytes innervated by spinal motoneurons using whole cell, volt- age-clamp recording techniques (Lu et al., 1992). The solution inside the recording pipette contained: 150 mM KCl, 1 mM NaCl, 1 mM MgCl2, and 10 mM Hepes buffer, pH 7.2. Membrane currents in all recordings were monitored by a patch clamp amplifier (EPC-7), with a current signal filter at 3 kHz. The membrane potentials of the muscle cells were generally in the range of 255–275 mV and were voltage clamped at 270 mV after measuring the membrane potentials. For experiments performed in the absence of external Ca21, the culture medium was replaced with a Ca21- free extracellular solution containing 115 mM NaCl, 2 mM MgCl2, 10 mM Hepes, 3 mM EGTA, and 0.1% BSA. All data were stored on a videotape recorder for later playback on a storage oscilloscope (Tektronix TDS 420) and a chart recorder (Gould EasyGraf 240), or analysis using the SCAN program. To quantitatively measure the changes in neurotransmitter re- lease, a time course of SSC frequency was first constructed on a minute- to-minute basis. The SSC frequencies in a 10-min period right before drug application were averaged as control. The changes in SSC frequency were measured by averaging a 10-min period recording starting from the high- est number after drug application. Results Ca21 Influx Is Not Required for NT3-induced Synaptic Potentiation We recorded synaptic activities at the neuromuscular syn- apses in 1-d-old Xenopus nerve–muscle cultures using whole The Journal of Cell Biology, Volume 149, 2000 784