Actions of BDNF on Ca2+ Signals in Hippocampal Neurons

描述（由申请人提供）：BDNF已成为活动依赖性突触发育和可塑性的有效调节剂。转运和释放的功能障碍，以及通过其受体TrkB信号传导的功能障碍，与许多发育性和神经退行性脑疾病的病因有关。普遍的观点是，Ca2+从ip3敏感存储释放是BDNF调节Ca2+稳态的唯一机制。然而，直接证据确定特定的信号和来源的Ca2+离子介导这些行动是有限的和矛盾的。此外，尽管有大量证据表明外源性BDNF的作用，但在神经元活动期间释放的天然BDNF的作用尚不清楚。该项目的长期目标是确定TrkB激活对海马神经元和突触产生广泛影响的BDNF的机制。在这一竞争性更新中，我们将重点关注我们的观察，即BDNF引发与膜电流相关的缓慢和持续的Ca2+信号，分别让人想起由TRPC通道介导的容性Ca2+进入和非选择性阳离子电流。具体的假设是，BDNF触发trkb依赖性PLCgamma激活，随后CA1锥体神经元中ip3敏感储存的Ca2+被动员，从而激活容性Ca2+进入和由TRPC通道介导的持续内向电流。前两个目标将确定外源性BDNF对膜电流和细胞内Ca2+水平的基本作用，而第三个目标将利用这一知识来确定传入刺激过程中释放的天然BDNF所引起的类似反应。同时Ca2+成像和电生理记录，结合药理学抑制剂，功能阻断抗体和sirna介导的敲低将用于识别信号通路的组成部分。BDNF清除剂可以确定传入活动释放的BDNF是否引起类似的Ca2+信号和内向电流。我们希望提出的研究能够提供迄今为止最全面的理解BDNF对膜电流和细胞内Ca2+稳态的直接作用，从而导致突触功能、结构和可塑性的持久变化。

DESCRIPTION (provided by applicant): BDNF has emerged as a potent modulator of activity-dependent synaptic development and plasticity. Dysfunctions in its trafficking and release, as well as in signaling through its receptor TrkB, have been implicated in the etiology of numerous developmental and neurodegenerative brain disorders. The prevailing notion is that Ca2+ release from IP3-sensitive stores is the only mechanism for BDNF to modulate Ca2+ homeostasis. However, direct evidence identifying the specific signaling and the sources of Ca2+ ions mediating those actions is limited and contradictory. In addition, nothing is known about the actions of native BDNF released during neuronal activity, despite the extensive evidence of the effects of exogenously applied BDNF. The long-term goal of this project is to identify the mechanisms by which TrkB activation sets in motion the wide range of BDNF effects on hippocampal neurons and synapses. In this competing renewal we will focus on our observation that BDNF elicits slow and sustained Ca2+ signals associated with membrane currents, reminiscent of capacitative Ca2+ entry and non-selective cationic currents mediated by TRPC channels, respectively. The specific hypothesis is that BDNF triggers TrkB-dependent PLCgamma activation followed by Ca2+ mobilization from IP3-sensitive stores in CA1 pyramidal neurons, leading to the activation of capacitative Ca2+ entry and a sustained inward current mediated by TRPC channels. The first two Aims will identify the elementary actions of exogenously applied BDNF on membrane currents and intracellular Ca2+ levels, while the third Aim will use this knowledge to identify similar responses evoked by native BDNF released during afferent stimulation. Simultaneous Ca2+ imaging and electrophysiological recording, combined with pharmacological inhibitors, function-blocking antibodies, and siRNA-mediated knockdown will be used to identify the components of the signaling pathway. BDNF scavengers will allow determining whether BDNF released by afferent activity evokes similar Ca2+ signals and inward currents. We expect the proposed studies to provide the most comprehensive understating to date of the immediate actions of BDNF on membrane currents and intracellular Ca2+ homeostasis, leading to enduring changes in synaptic function, structure, and plasticity.