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Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.
Figure 1. Acute potentiation of transmitter release at NMJ by NT3 is independent of extracellular Ca2+. (A) A sample recording showing that application of NT3 (50 ng/ml) rapidly increases the frequency of SSCs in the normal culture medium. (B) Application of NT3 also rapidly increases the frequency of SSCs in the Ca2+-free medium.
Figure 2. NT3-induced synaptic potentiation does not require Ca2+ influx. (A) Time courses of NT3 effect on spontaneous synaptic activity in zero external Ca2+ (open circles). The culture medium was replaced by Ca2+-free solution. The SSC frequency was monitored before and after NT3 application. Each point represents averaged SSC frequency in 3 min of recording. An example of recordings in normal medium (filled circles) is included for comparison. (B) The effect of NT3 on SSC frequency in Ca2+-free medium (n = 12) or in medium containing the Ca2+ channel blocker, Cd2+ (CdCl2, 0.4 mM, n = 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 recording 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 indicated otherwise.
Figure 3. Enhancement of transmitter release by NT3, but not BDNF, requires Ca2+ release from intracellular Ca2+ stores. All experiments were carried out in Ca2+-free conditions, except that shown in D. The cultures were incubated with various drugs for 15â60 min, and SSCs were recorded continuously to monitor the effects of the drug and NT3 application. Thapsigargin was used to deplete Ca2+ 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 μM) prevents the NT3 effect. Application of thapsigargin (open arrow) elicited a transient but marked increase in SSC frequency due to the Ca2+ release from intracellular stores. Application of NT3 after SSC frequency returned to normal could no longer increase the SSC frequency. (B) Summary of the drug effects. Final concentration of the drugs in the culture media: thapsigargin, 2 μM; XeC, 1 μM; TMB-8, 30 μM; and ryanodine as an antagonist, 100 μM. 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 (2.5â5 μM). 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 controls (n = 9). Note that ryanodine at the low concentration could not block the NT3 effect. (D) BDNF-induced synaptic potentiation is independent of Ca2+ release from intracellular stores through IP3 receptors. BDNF was applied to culture dishes in the presence (right, n = 5) or absence (left, n = 5) of the IP3 receptor inhibitor XeC (1 μM).
Figure 4. Effect of KN62 on NT3-induced synaptic potentiation. (A) A sample recording showing that pretreatment of the neuromuscular synapse with KN62 prevents the NT3-induced facilitation of transmitter release. (B) Blockade of NT3-induced synaptic potentiation of SSC frequency by KN62. In KN62 groups, the nerveâmuscle cultures were pretreated with KN62 for 10â20 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 before NT3 application. Asterisk indicates P < 0.01, t test.
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 nerveâmuscle co-cultures were prepared from the injected embryos. (A) Super-imposed phase and fluorescence micrographs of the 1-d-old nerveâmuscle co-cultures showing cells loaded with (+) or without (â) CaMKII-pep. N, spinal neurons; M, myocytes. (B) A pair of recordings showing that loading of CaMKII peptide into presynaptic neurons N(+) prevented the NT3-induced increase in SSC frequency, whereas NT3 still enhanced transmitter release in a N(â) neuron in the same culture. (C) Summary of the effect of CaMKII-pep loaded into either presynaptic spinal neurons or postsynaptic myocytes. The number of synapses recorded are indicated above each pair of columns. In all Nâ conditions, application of NT3 elicited a significant increase in SSC frequency (P < 0.01, t test).
Figure 6. Reversal of NT3 effects by addition of KN62 after NT3 application. (A) Examples showing the time courses that KN62 reverses the NT3 effect. NT3 was first applied (filled arrow) to the culture medium to elicit an increase in the frequency of SSCs. After the SSC frequency reached the peak, KN62 (open arrow) was either applied (open circles) or not applied (filled circles), and synaptic currents were continuously monitored. (B) Summary of the reversal effect of KN62. SSC frequencies are calculated in the same way as Fig. 3 C. n = 5. Asterisk indicates P < 0.01, t test.
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