The effect of brain derived neurotrophic factor on presynaptic function

项目摘要 脑源性神经营养因子（BDNF）是一种重要的神经营养因子，调节突触生理 以及中枢神经系统的可塑性。多种证据表明它与精神疾病有关 抑郁症和精神分裂症等健康疾病，以及抗抑郁药的生物学基础 效果然而，尽管它已被证明与神经精神疾病有关，但很少有研究调查 BDNF如何影响突触前末梢，尽管它是神经传递的基本组成部分。 这部分是由于研究前突触的困难和缺乏可用的技术，直到 更近一些先前发表的工作使用海马切片中的病毒操纵证明BDNF 作用于突触前的高亲和力TrkB受体，以增加释放概率，或 囊泡将响应于动作电位而释放。由此产生的假设是BDNF信号 通过TrkB信号通路特异性靶向突触前功能，该提案旨在研究 BDNF在突触前作用的分子基础。这将通过以下方式实现：首先，检查 BDNF对兴奋性神经传递的作用（子目的1.1）。子目标1.1的初步数据显示， 分离的海马培养物，兴奋性突触的释放概率增加，以响应急性 BDNF治疗依赖于TrkB激活。Subaim 1.2将剖析潜在的机制 通过使用药理学方法筛选潜在的下游途径来解释这种效应 与这种效应有关。由于BDNF活动的时空背景在其影响中至关重要，我将 我还使用cTrkB KO海马培养物来检查自发和诱发的释放事件， 内源性BDNF操纵（子目的1.3）。突触处的局部钙瞬变与 神经递质释放，尤其是诱发释放。因此，我将研究突触前钙瞬变 受外源性BDNF影响（子目标2.1），使用cTrkB培养物的内源性BDNF影响（子目标2.2），和 BDNF是否利用钙调节神经传递（子目标2.3）。拟议的研究将涉及 用荧光探针转染或感染的体外神经元培养物的光学成像以研究前同步酶， 以及药理学和遗传操作工具，以提供对发现的进一步机制见解。 这项研究将进一步了解BDNF在突触中的基本作用， 用于改进当前的治疗方法，例如用于抗抑郁药。所提出的研究将完成 通过精心制定的培训计划，包括进一步提高批判性思维技能的机会， 技术专长和科学交流。此外，MSTP提供的研究环境， 范德比尔特脑科学研究所和精神病学系提出的研究和培训计划是理想的， 协作和支持的环境，并专注于学员的发展。

PROJECT SUMMARY Brain derived neurotrophic factor (BDNF) is a prevalent neurotrophin that modulates synaptic physiology and plasticity in the central nervous system. Multiple lines of evidence point towards its involvement in mental health disorders such as depression and schizophrenia, as well as the biological basis for an antidepressant effect. Yet despite its demonstrated involvement in neuropsychiatric diseases, very few studies have investigated how BDNF affects the presynaptic terminal, despite it being a fundamental component of neurotransmission. This is in part due to the difficulty of studying the presynapse and the lack of available technology to do so until more recently. Previously published work using viral manipulation in hippocampal slices demonstrates that BDNF acts upon the high affinity TrkB receptor at the presynapse to increase release probability, or the likelihood that a vesicle will be release in response to an action potential. With the resulting hypothesis that BDNF signaling specifically targets presynaptic function via TrkB signaling pathways, this proposal aims to investigate the molecular basis of BDNF’s effect at the presynapse. This will be done through the following: first, examining the effect of BDNF on excitatory neurotransmission (subaim 1.1). Preliminary data from subaim 1.1 show that in dissociated hippocampal cultures, release probability at excitatory synapses is increased in response to acute BDNF treatment in a manner dependent on TrkB activation. Subaim 1.2 will dissect the potential mechanisms underlying this effect through using pharmacological approaches to screen for potential downstream pathways that are involved in this effect. Because the spatiotemporal context of BDNF activity is critical in its effect, I will also use cTrkB KO hippocampal cultures to examine the spontaneous and evoked release events as a result of endogenous BDNF manipulation (subaim 1.3). Local calcium transients at synapses are strongly linked to neurotransmitter release, especially evoked release. I will thus investigate how presynaptic calcium transients are affected by exogenous BDNF (subaim 2.1), endogenous BDNF using cTrkB cultures (subaim 2.2), and whether BDNF utilizes calcium to modulate neurotransmission (subaim 2.3). The proposed studies will involve optical imaging of in vitro neuron cultures transfected or infected with fluorescent probes to study the presynase, as well as pharmacological and genetic manipulation tools to provide further mechanistic insight into findings. This research will further current understanding of the fundamental role of BDNF at the synapse, with implications for improving current therapeutics such as for antidepressants. The proposed research will be accomplished through a thoughtfully developed training plan that involves opportunities to further critical thinking skills, technical expertise, and scientific communication. Additionally, the research environment provided by the MSTP, Vanderbilt Brain Institute, and Department of Psychiatry is ideal for the proposed research and training plan, a collaborative and supportive environment, and focus on trainee development.