Name: real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons
Uploaded: 2014-09-15T15:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

我们要感谢岳（保罗）胡锦涛，雷切尔SINIT为他们提供技术援助。这项研究是由美国国立卫生研究院资助（PN2 EY016525），并从唐氏综合症的研究与治疗基金会和拉里·L.希尔布鲁姆基金会的资助支持。

手术和动物的程序进行了严格按照美国国立卫生研究院指南的护理和使用实验动物。所有涉及使用动物实验是由加州大学圣地亚哥分校机构动物照顾及使用委员会批准。 1质粒的克隆，单生物素BDNF的表达和纯化（mBtBDNF） 注：建设前期proBDNFavi和BirA的cDNA插入在HEK293FT细胞10 pcDNA3.1载体和共表达。使用Ni-NTA珠根据生产成熟的和生物活性monobiotinylated神经...

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在这项研究中，我们报道了一种新技术的发展，以产生具有生物活性，monobiotinylated BDNF（mBtBDNF），可以用来跟踪BDNF的轴突运输。通过缀合的蛋白质，以量子点的链霉亲和使用的微流体腔室，该方法允许一个检测BDNF在与单个分子的灵敏度，在实时的和具有空间分辨率和时间分辨率原代神经元的轴突运输。这里所使用的工具提供了由研究介导的BDNF / TrkB的信号内吞体的健康和疾病的神经元轴突运输?...

神经元是高度分化的细胞，其长，通常高度阐述的过程是基本建立和保持神经回路的结构和功能。轴突起着运载的货物和从突触至关重要的作用。蛋白质和细胞器的细胞胞体合成需要通过轴突运输到达突触前末梢，支持神经元功能。相应地，接收的信号在远端轴突需要被转并输送到体细胞。这些方法可用于神经元的存活，分化和维持所必需的。在某些神经元的轴突运输必须通过的距离超过1000倍的细胞体的直径进行，有可能容易地预想到，即使是很小的缺陷可显着影响神经元和电路功能。 脑源性神经营养因子（BDNF），生长因子的神经营养因子家族中的一员，是目前在马纽约州的大脑区域，包括海马，大脑皮层和基底前脑。 BDNF起着认知和记忆的形成所配套的存活，分化，并参与认知神经回路的功能至关重要的作用。 BDNF结合到其受体的酪氨酸激酶受体TrkB，在轴突终端那里它激活的TrkB介导的信号传导途径，包括有丝分裂原激活的蛋白激酶/细胞外信号调节蛋白激酶（MAPK / ERK），磷脂酰肌醇3激酶（PI3K）和磷脂酶C-γ（PLCγ）。在参与这些信号传导途径的蛋白被包装到内吞囊泡结构，以形成BDNF / TrkB的信令内体1-6被再逆行转运到神经元胞体。 微流体培养室是研究在正常条件下，以及在损伤和疾病7,8的设定轴突生物学一个非常有用的平台。通过隔离轴突从细胞体，该装置允许一个具体研究运输中的轴突8-10。在与在该研究中使用450微米的微沟槽屏障基于PDMS的微流体平台进行商业购买（参见材料表）。为了研究BDNF的运输，我们开发了一种新的技术，生产monobiotinylated BDNF（mBtBDNF）。我们把生物素受体肽，美联社（又称重组蛋白质）的优势。它是一个包含一个赖氨酸残基，可以由大肠杆菌酶的生物素连接酶，BirA的具体连接到生物素15的氨基酸序列。我们融合的重组蛋白质的小鼠预先proBDNF基因的C末端，通过PCR（ 图1A）。该构建体被克隆到哺乳动物表达载体pcDNA3.1 myc基因-His载体。我们还克隆的细菌BirA的DNA导入的pcDNA3.1 myc基因-His载体。两个质粒瞬时共转染到HEK293FT细胞表达两种蛋白。 BirA的催化毕结扎在具体的赖氨酸驻留在重组蛋白质中的BDNF的在1的C末端：1的比例以产生monobiotinylated BDNF单体。生物素化的，成熟的BDNF与〜18kDa的分子质量，回收和使用的Ni-树脂（ 图1C）的介质纯化。 BDNF的生物素化是完全的，作为判断无法通过免疫印迹法（ 图1D），以检测未修饰BDNF。链霉亲和共轭量子点，量子点655，分别用来标明mBtBDNF使QD-BDNF。的重组蛋白质的存在下不与BDNF的活性干扰作为mBtBDNF能够激活磷酸化的TrkB（ 图1E）和刺激神经 ​​突向外生长（ 图1F），以重组人BDNF（rhBDNF）的程度。免疫组化显示，QD-BDNF共定位与TrkB的海马神经轴突，表明QD-BDNF的生物活性（ 图1G）。研究了BDNF的运输，QD-BDNF加入到远端轴突隔室含E18大鼠海马神经元（ 图2A）微流控文化。在轴突QD-BDNF逆行运输被抓获的红色荧光标记（ 支持视频S1，S2）的实时现场图像。通过分析所产生的kymograph，QD-BDNF的观察可以在约1.06微米/秒（ 图3A）的移动速度逆向转移。 GFP或mCherry-标记的BDNF已被用于跟踪BDNF的神经轴突的运动。主要的缺点是，他们不是为单个分子的研究不够明亮。另外，无论是顺行性与逆行BDNF运动的存在使得难以评估的逆向转移BDNF是否是一种神经营养蛋白/受体复合体。 在这段视频中，我们展示了用于检查QD-BDNF在利用原代神经元的微流控室现场贩卖的技术。该ultrabrightness和量子点麦出色的耐光性ES能够进行BDNF传输的长期跟踪。这些技术可被利用来提高AD中，HD和其他神经变性疾病的轴突功能的研究。

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF在神经元存活，分化和功能的若干方面具有重要作用。结构和功能的缺陷在轴突日益被看作神经变性疾病，包括阿尔茨海默氏病（AD）和亨廷顿病（HD）的早期特征。尚不清楚是由轴索损伤诱导机制（S）。我们报道了一种新技术的发展，以产生具有生物活性，monobiotinylated BDNF（mBtBDNF），可以用来跟踪BDNF的轴突运输。量子点标记的BDNF（QD-BDNF）是由共轭量子点655到mBtBDNF制备。一种微流体装置被用于分离从神经元胞体的轴突。除了QD-BDNF的到轴突舱允许的BDNF运输轴突的实时成像。我们证明了QD-BDNF的移动基本上是完全逆行，除了极少数停顿，约为1.06微米/秒的移动速度。此系统可用于研究我的破坏轴突功能在AD或HD，以及其他退行性疾病chanisms。

生产具有生物活性的单 - 生物素化的BDNF的纯化 BDNF的稠合有一个重组蛋白质序列（GGGLNDIFEAQKIEWHE）的表达载体，根据以前发表的协议10创建。全长融合蛋白的分子质量预测为〜32kDa的（ <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ）Monobiotinylated成熟的BDNF为18 kDa的（ 图1A）的预测分子量制作在HEK293FT细胞和纯化的条件培?...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF，神经营养因子，对轴突运输是几个神经元群体的生存和功能是至关重要的。一些退行性疾病的标志是轴索结构和功能的破坏。我们展示了用于检查QD-BDNF在使用初级神经元微流控室现场贩卖的技术。

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Research

JoVE Journal

Neuroscience

Mechanical Manipulation of Neurons to Control Axonal Development

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...