Figure 2. NT3-induced synaptic potentiation does not require Ca21 influx. (A) Time courses of NT3 effect on spontaneous syn- aptic activity in zero external Ca21 (open circles). The culture medium was replaced by Ca21-free solution. The SSC frequency was monitored before and after NT3 application. Each point rep- resents averaged SSC frequency in 3 min of recording. An exam- ple of recordings in normal medium (filled circles) is included for comparison. (B) The effect of NT3 on SSC frequency in Ca21- free medium (n 5 12) or in medium containing the Ca21 channel blocker, Cd21 (CdCl2, 0.4 mM, n 5 13). For each synapse, a time course of SSC frequency was first constructed on a minute-to- minute basis. SSC frequencies are averaged from a 10-min re- cording right before NT3 application for controls, and a 10-min period starting from the highest number after NT3 application for NT3-treated groups. Error bars in this and all other figures are SEM. NT3 induced significant increases in SSC frequency in both conditions (P , 0.005, t test). Similar methods were used to calculate averaged SSC frequencies in all other figures, unless in- dicated otherwise. C). Thus, the synaptic action of NT3 is primarily mediated by the Ca21 release from IP3 receptors, which further trig- gers Ca21 release from the ryanodine receptors. We then tested whether BDNF, which requires Ca21 in- flux to enhance transmitter release (Stoop and Poo, 1996), also depends on Ca21 release from intracellular stores. Application of BDNF (50 ng/ml) elicited a fivefold in- crease in SSC frequency in normal Ca21-containing me- dium (Fig. 3 D). In cultures pretreated with XeC (1 mM) Figure 3. Enhancement of transmitter release by NT3, but not BDNF, requires Ca21 release from intracellular Ca21 stores. All experiments were carried out in Ca21-free conditions, except that shown in D. The cultures were incubated with various drugs for 15–60 min, and SSCs were recorded continuously to monitor the effects of the drug and NT3 application. Thapsigargin was used to deplete Ca21 from intracellular stores. Asterisk indicates data that are significantly different from the rest (P , 0.01, ANOVA test followed by post hoc comparison). (A) An example showing that thapsigargin (2 mM) prevents the NT3 effect. Application of thapsigargin (open arrow) elicited a transient but marked in- crease in SSC frequency due to the Ca21 release from intracellu- lar stores. Application of NT3 after SSC frequency returned to normal could no longer increase the SSC frequency. (B) Sum- mary of the drug effects. Final concentration of the drugs in the culture media: thapsigargin, 2 mM; XeC, 1 mM; TMB-8, 30 mM; and ryanodine as an antagonist, 100 mM. The number of synapses recorded are indicated in the control bar of each pair. Asterisk indicates P , 0.005, t test. SSC frequencies are calculated in the same way as Fig. 2. (C) Summary of the effects of ryanodine as an agonist (2.5–5 mM). The SSC frequencies from a single synapse are counted for a 10-min period in control and a 10-min period after ryanodine application, and then a 10-min period after NT3 application. The data are then averaged and normalized to con- trols (n 5 9). Note that ryanodine at the low concentration could not block the NT3 effect. (D) BDNF-induced synaptic potentia- tion is independent of Ca21 release from intracellular stores through IP3 receptors. BDNF was applied to culture dishes in the presence (right, n 5 5) or absence (left, n 5 5) of the IP3 receptor inhibitor XeC (1 mM). The Journal of Cell Biology, Volume 149, 2000 786