Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ
Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ
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DOI：
10.1523/jneurosci.20-19-07417.2000
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发表时间：
2000-10-01
影响因子：
5.3
通讯作者：
Katz, DM
中科院分区：
文献类型：
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作者：
Balkowiec, A;Katz, DM
为了确定内源性脑源性神经营养因子（BDNF）的活性依赖性释放，我们开发了一种体外模型，使用初级感觉神经元和改良的ELISA，称为ELISA原位。新生大鼠的结节岩神经节细胞的分离培养物在预涂有抗BDNF抗体的威尔斯孔中生长，以捕获释放的BDNF，随后使用常规ELISA检测。传统的ELISA单独不能检测到任何增加BDNF浓度高于对照值后，慢性去极化与40 mM KCl为72 hr. However，ELISA原位显示了一个非常显着的增加BDNF的释放，从65 pg/ml的控制到228 pg/ml的KCl处理的文化。原位测定的功效似乎主要与快速捕获释放的BDNF有关，其防止BDNF与培养的细胞结合。因此，我们使用这种方法来比较BDNF释放从暴露30分钟的培养物，以连续去极化与升高的KCl或图案化的电场刺激（50个双相矩形脉冲的25毫秒，在20 Hz，每5秒）。短期KCl去极化在引起任何可检测的BDNF释放方面完全无效，而模式化电刺激使细胞外BDNF水平增加20倍。此外，BDNF释放的幅度取决于刺激模式，高频爆发最有效。这些数据表明，BDNF释放的最佳刺激曲线类似于其他神经活性肽。此外，我们的研究结果表明，BDNF的释放可以编码突触前神经元活动的时间特征。
To define activity-dependent release of endogenous brain-derived neurotrophic factor (BDNF), we developed an in vitro model using primary sensory neurons and a modified ELISA, termed ELISA in situ. Dissociate cultures of nodose-petrosal ganglion cells from newborn rats were grown in wells precoated with anti-BDNF antibody to capture released BDNF, which was subsequently detected using conventional ELISA. Conventional ELISA alone was unable to detect any increase in BDNF concentration above control values following chronic depolarization with 40 mM KCl for 72 hr. However, ELISA in situ demonstrated a highly significant increase in BDNF release, from 65 pg/ml in control to 228 pg/ml in KCl-treated cultures. The efficacy of the in situ assay appears to be related primarily to rapid capture of released BDNF that prevents BDNF binding to the cultured cells. We therefore used this approach to compare BDNF release from cultures exposed for 30 min to either continuous depolarization with elevated KCl or patterned electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec). Short-term KCl depolarization was completely ineffective at evoking any detectable release of BDNF, whereas patterned electrical stimulation increased extracellular BDNF levels by 20-fold. In addition, the magnitude of BDNF release was dependent on stimulus pattern, with high-frequency bursts being most effective. These data indicate that the optimal stimulus profile for BDNF release resembles that of other neuroactive peptides. Moreover, our findings demonstrate that BDNF release can encode temporal features of presynaptic neuronal activity.