这项工作是由美国NINDS和美国心脏协会，以S.Ge.这种方法最初是笔者在红军宋博士在美国约翰霍普金斯大学的实验室建立和发展。我们要感谢他的指导和支持红军宋博士。

1。病毒注射高滴度工程逆转录病毒（1 × 109单位/毫升）是由逆转录病毒载体共转染HEK293T细胞，成由病毒上清离心VSVG。来源和生产方法，看到优秀的Jove示范（3）。 注：年轻成人（4-6周龄）雌性C57BL / 6小鼠（查尔斯河）是在标准条件下安置。所有的程序都遵循美国国家研究理事会的大学石溪分校IACUC批准了一项协议下的实验室动物护理和使用指南。 <ol><li>每头牲畜被冻结的逆转录病毒在冰上解冻2ul。 </li><li>麻醉（100微克氯胺酮+每克体重10微克甲苯噻嗪）和安装在立体定位框架（Steolting）的动物。删除在头上的头发和皮肤用70％乙醇擦拭。 </li><li>暴露的颅骨钻4个浅孔使用一个牙钻（0.6毫米钻头）在下面的坐标： <ul><li> anterioposterior = -2毫米;从囟外侧= ± 1.6毫米;腹= 2.5毫米; anterioposterior =囟-3毫米;横向= ± 2.6毫米;腹= 3.2毫米。 </li><li> anterioposterior = -2毫米;从囟外侧= ± 1.6毫米;腹= 2.5毫米; anterioposterior =囟-3毫米;横向= ± 2.6毫米;腹= 3.2毫米。 </li></ul></li><li>登上一个加入1μl汉密尔顿，平头注射器注入到齿状回率在0.25μL/ min的0.5％μL逆转录病毒网站。 （见上述腹侧深入的坐标。）暂停2分钟，每次注射后，才慢慢退出注射器，以防止流体倒流。注射之间，简单地冲洗外表面用无菌PBS的注射器针尖和撒施去除血迹。 </li><li>关闭伤口用无菌手术缝合材料（型号P - 1型;尺寸6-0），并返回所需的时间阶段，直到注射后动物标准的住房条件。 </li></ol> 2。切片的制备为了获得高品质的成年小鼠急性片，我们使用切片准备修改后的切割解决方案（见下文）。 <ol><li>前寒意裁剪解决方案，以0-4 °上冰和95％的泡沫的C = O 2 -5％ 的 CO 2为至少30分钟。 </li><li>麻醉和transcardially灌注冰冷的氧饱和切削液的动物。 </li><li>滤纸与寒冷，含氧的切削液预湿，迅速取出培养皿盘的大脑。切断小脑和片安装表面至少1毫米前（可见光）注射坐标。胶的大脑上一个vibrotome台阶，并安装到它切割室充满了寒冷和含氧切割解决方案。 </li><li>准备冠状或横向切片（300 -350μm）和大口径的吸管转移到孵化室含室温学联95％O 2 / 5％的CO 2气泡。 </li><li>片，不断冒泡，在32 ° C孵育30-60分钟。返回会议厅，录音时间室温。 </li></ol> 3。电生理实验<ol><li>片被转移到录音室，这是连续灌注与氧饱和学联在32 ° C - 34 ° C。 </li><li>双极型钨刺激电极放置在使用低倍率的显微镜物镜（10X）的齿状回分子层。 </li><li>在新生儿DGCs分粒状区/颗粒细胞层表达GFP或dTomato确定。在高放大倍率（40X）下的新生神经元进行全细胞patchclamp记录。 </li><li>射击细胞的属性得到了一系列的电流注入电流钳模式下。 </li><li>电刺激刺激电极通过录音软件控制的刺激隔离的交付，并记录在新生儿DGCs引起突触后电流。 </li></ol> 4。数据分析成人出生的神经元的不同发育阶段的分析，引起突触后反应的振幅。 5。代表性的成果使用上述协议，是常见的绿色荧光蛋白的表达在新生儿的齿状回神经元与图 1类似。请注意，新生的神经元易于可视化和强劲标记树突和轴突。在薄的DGC轴突和小刺的表达依赖于质量和滴度的病毒，促进剂，在体内表达的长度。在我们的手中，新生细胞和亚细胞器通常可见注射后几天内，脊柱的表达，可以追溯到从发展的早期阶段。 W孔在不同的时间阶段的新生神经元细胞的录音让新生细胞的独特属性，如动作电位（ 图2a），以及突触融合的过程中成熟（图2b）期间的研究到现有的神经回路。 <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="图1" /> 图1 2 -光子共聚焦图像，新生神经元在一个水平的成年小鼠齿状回部分。绿色细胞21dpi（天注射后）绿色荧光蛋白表达与pUX - GFP逆转录病毒标记的新生DGCs。 <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="图2" /> 图2）动作电位射击一个新生DGC的属性在21 DPI，B）代表诱发兴奋性突触后电流（EPSCs）记录在一个新生儿在21 dpi的DGC。

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

连续在成年哺乳动物大脑海马神经发生提供了一个独特的实验模型系统来研究神经元的发育和成熟的大脑整合。这里介绍的协议结合逆转录病毒标签和电生理的方法来研究新生儿在成年小鼠的大脑的齿状颗粒神经元的整合。 为了确保您的实验是成功的，我们建议在整个程序的关键步骤以下内容： 为了避免不必要的感染和炎症，所有工具应消毒（高压灭菌，任何类型的消毒柜，或70％的乙醇），术前。使用无菌PBS洗注射前注射器针头的尖端。 病毒注射，动物必须要很好地安装在立体定位装置和不必要的移动源必须在注射过程中最小化。确保头是固定的和坚定的，并计算注射坐标前水平前囟和lambda调整鼠标的鼻子位置。解压缩到注射器迅速，以尽量减少暴露在室温下的病毒 - 逆转录病毒，特别是温度敏感。慢慢注入病毒在推荐率，以尽量减少压力损失。使用推荐的平头的注射器（与常见的斜面TIP）将确保一个均匀分布的齿状回感染面积。 在准备切片，切割解决方案和孵化学联必须起泡至少30分钟，让氧气饱和的解决方案，与95％Ø 2 -5％的CO 2 。动物应进行灌注和组织保持在寒冷，氧饱和片的质量为最佳的解决方案。 录音，逐渐增加刺激强度，直到有一个突触后响应。必要的齿状回内分子层变化的刺激电极的位置。 综上所述，推荐的方法提供了一种探索在早期一体化的成人出生的神经元的不同性质和可能的功能作用，转变为主动，成熟的电路。

<ul><li>切割的解决方案（毫米）：110 1.3 KH 2 PO 4 25.0 0.5 3碳酸氢钠，氯化钙2，7，10 MgCl 2的葡萄糖，1.3抗坏血酸钠，0.6丙酮酸钠，5.0 kynurenic酸2.5氯化钾，氯化胆碱，。 </li><li>人工脑脊髓液（学联，毫米）：125.0氯化钠，碳酸氢钠3 25.0 2.5氯化钾， 氯化钙 ，1.3，1.3 KH 2 PO 4 MgCl 2的抗坏血酸钠，1.3，0.6丙酮酸钠，10葡萄糖。 </li><li>内部解决方案，为全细胞patchclamp（毫米）：120钾葡萄糖酸钙，15氯化钾， 氯化镁2，EGTA 0.1，10.0 HEPES，4 MgATP，0.3 NA 3的GTP，7磷酸（pH值7.4，300 mOsm） 。 </li></ul>

studying the integration of adult-born neurons

分侧脑室脑室区（SVZ），并在小组整个生命的海马齿状回颗粒区（SGZ），在成年哺乳动物大脑的神经再生发生。以前的报告显示，成年海马神经发生是多样的脑部疾病，包括癫痫，精神分裂症，抑郁和焦虑（1）关联。解读成人出生的正常和异常的神经元整合的过程中可能会揭示这些疾病的病因，并告知新疗法的发展。 SGZ成年神经发生反射镜胚胎和产后的神经元的发育，包括命运规范，迁移，突触的整合，和成熟的阶段。然而，全面整合多长时间，6周期间发生。初步GABA能突触输入成年出生SGZ齿状颗粒细胞（DGCs），其次是谷氨酸能在14天（2）输入。这对规范成人出生在齿状回神经元电路形成的特定因素，目前尚不得而知。 我们的实验室利用复制缺陷型逆转录病毒载体莫洛尼鼠白血病病毒的基础上，提供荧光蛋白和调控基因推测这些增殖细胞。该病毒的技术，提供高特异性细胞的出生日期，血统，形态，和突触的分析和解决。 一个典型的实验中往往采用两个或三个病毒含有独特的标签，转基因，启动子和单细胞在体内的发育过程中所需的分析。以下协议描述分析功能的新生神经元的整合，使用单一的绿色（GFP）或（dTomato）红色荧光蛋白逆转录病毒和膜片钳电生理的方法。

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

描述一个在成年动物的新生儿齿状颗粒细胞融合的方法来研究。该技术使用一个精心设计的逆转录病毒标签新生神经元的电生理记录，以确定在体内的功能整合。

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

Research

JoVE Journal

Neuroscience

13.7K 次观看.  State University of New York at Stony Brook. 描述一个在成年动物的新生儿齿状颗粒细胞融合的方法来研究。该技术使用一个精心设计的逆转录病毒标签新生神经元的电生理记录，以确定在体内的功能整合。

视频: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

利用生命病毒介导的人神经元转导研究头塔聚集的体外模型

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由游泳鱼中可移动的特龙对神经元的无线电生理记录

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...