cell, voltage-clamp recording techniques. The SSCs are in- duced by spontaneous secretion of individual ACh-con- taining synaptic vesicles from motor nerve terminals inde- pendent of action potentials, since they are not affected in the presence of tetrodotoxin (Song et al., 1997). As shown before (Lohof et al., 1993; Stoop and Poo, 1995, 1996), acute application of NT3 (50 ng/ml) to the synapses in the presence of extracellular Ca21 dramatically enhanced spon- taneous transmitter release, as reflected by a rapid increase in the frequency of SSCs (Fig. 1 A). A previous report showed that in the same type of cultures, BDNF enhances synaptic transmission by facilitating Ca21 influx into the presynaptic terminals (Stoop and Poo, 1996). We thus tested whether the acute NT3 effect uses a similar mecha- nism. The culture medium was substituted with Ca21-free extracellular solution after several washes with the same solution. Surprisingly, application of NT3 still elicited an increase in the frequency of SSCs under the zero external Ca21 condition (Fig. 1 B and Fig. 2). The time courses of the NT3-induced increase in SSC frequency in both Ca21- free and Ca21-containing media were very similar, although the basal level of SSC frequency in Ca21-free media before NT3 application was slightly lower (Fig. 2, A and B). Quantitative analysis indicated that NT3 only increased the frequency, without affecting the amplitude or decay time of SSCs (data not shown), suggesting that NT3 facili- tates presynaptic transmitter release in the absence of ex- tracellular Ca21. To further examine the role of membrane Ca21 channels, we blocked Ca21 influx by the general Ca21 channel blocker Cd21 (0.4 mM). NT3 was still capable of elevating SSC frequency in the presence of Cd21 (Fig. 2 B). Thus, NT3-induced potentiation of transmitter release does not depend on Ca21 influx from extracellular sources. Our further analyses were thus performed mostly in Ca21- free medium. Role of Intracellular Ca21 Stores We next determined whether the NT3 effect is mediated by an increase in Ca21 release from intracellular Ca21 stores. Thapsigargin inhibits Ca21-ATPase activity and therefore has frequently been used to deplete all intracel- lular Ca21 stores (Thastrup et al., 1990). Application of thapsigargin (2 mM) in both normal (n 5 5, data not shown) and Ca21-free media (Fig. 3 A) elicited a transient increase in SSC frequency, which returned to control lev- els within 20–60 min. NT3 no longer elicited any changes in SSC frequency in thapsigargin-treated synapses (Fig. 3, A and B). When intracellular Ca21 stores were depleted by thapsigargin, application of hypertonic solution (sucrose, 500 mM) to the synapses in Ca21-free medium still elicited a transient but marked increase in transmitter release, sug- gesting that there are still synaptic vesicles in the nerve terminals (data not shown). Thus, the lack of NT3 effect in thapsigargin-treated synapses was not due to vesicle de- pletion. These results suggest that an increase in [Ca21]i due to Ca21 release from intracellular stores may con- tribute to the facilitation of transmitter release by in- duced NT3. There are two major pathways for the release of Ca21 from intracellular stores: the inositol 1, 4, 5-trisphosphate (IP3) receptor and the ryanodine receptor (Berridge, 1998). Application of the IP3 receptor inhibitor xestospon- gin C (XeC; 1 mM) (Gafni et al., 1997) prevented the in- crease of SSC frequency elicited by NT3 (Fig. 3 B). The re- lease of Ca21 from IP3 receptors could further trigger Ca21-induced Ca21 release from ryanodine receptors (Ber- ridge, 1998). Low concentration of ryanodine (2.5–5 mM) may be used as a ryanodine receptor agonist, whereas high concentration (100 mM) may serve as an antagonist. In Ca21-free medium, application of ryanodine at high con- centration (100 mM) had no effect on basal SSCs, but pre- vented the increase in SSC frequency induced by NT3 (Fig. 3 B) (P . 0.5, ANOVA). Pretreatment of the cul- tures with another ryanodine receptor antagonist 8-(deth- ylamino)octyl 3, 4, 5-trimethoxybenzoate (TMB-8) (30 mM) (Hunt et al., 1990) also blocked the NT3 effects (Fig. 3B). Unlike thapsigargin, however, ryanodine at lower concentration (2.5–5 mM) was not sufficient to elicit a con- sistent increase in SSC frequency (Fig. 3 C). Furthermore, application of NT3 in the presence of low concentration of ryanodine still elicited an increase in SSC frequency (Fig. 3 Figure 1. Acute potentiation of transmitter release at NMJ by NT3 is independent of ex- tracellular Ca21. (A) A sam- ple recording showing that application of NT3 (50 ng/ml) rapidly increases the fre- quency of SSCs in the normal culture medium. (B) Appli- cation of NT3 also rapidly in- creases the frequency of SSCs in the Ca21-free me- dium. He et al. Mechanisms of NT3-induced Synaptic Potentiation 785