感谢Ricardo Dolmetsch博士（斯坦福大学医学院;现任隶属机构:诺华生物医学研究所），协助开展此项分析，马修·伍德博士，宝马爱原，以及董秀金博士对手稿的批判性阅读。

所有动物手术均经筑波大学动物护理与使用委员会（IACUC）批准进行。 解剖<ol><li>为了准备胚胎13-14日ICR或C57BL / 6胎儿，通过颈椎脱位安乐死怀孕的小鼠。取出子宫，并用汉克平衡盐溶液（HBSS）将其放入培养皿中。冲洗干净，以清除血液。 </li><li>使用镊子，将子宫转移到具有70％乙醇的新培养皿中以消毒。 </li><li>将子宫转移到带有HBSS的新培养皿中。使用消毒的解剖镊子和手术剪刀从子宫去除胎儿。 </li><li>使用外科剪刀切断胎儿。将它们放入带有HBSS的新培养皿中。 </li><li>取出皮肤和头骨，取出大脑使用灭菌解剖钳。将它们放入带有HBSS的培养皿中。 注意:从这一步开始，最好在干净的情况下进行该程序替补。 </li><li>在立体显微镜下使用灭菌解剖镊子取出脑膜。使用镊子切除基底神经节，海马和小脑。使用弯尖钳，将皮质转移到带有HBSS的新培养皿中。 </li><li>使用一次性滴管或25 mL移液管收集所有皮质。将其放入15 mL管中，然后用移液器取出HBSS。 </li><li>将3mL脑皮质酶溶液加入到含有皮质的管中。将该管放入水浴中，37℃下孵育5分钟。 注意:用0.25％胰蛋白酶和1mM EDTA制备由磷酸盐缓冲盐水（PBS）组成的脑皮质培养物。用0.22μm过滤器灭菌。 </li><li>在含有皮质的管中再加入3 mL脑皮质酶溶液，并在37°C的水浴中孵育另外5 min。 </li><li>使用5 mL移液管取出脑皮质酶溶液，加入5 mL Dulbe含有10％胎牛血清（FBS）的cco&#39;s Modified Eagle Medium（DMEM）。 </li><li>通过使用无菌P1000提示的5 mL移液管轻轻上下移动皮质分离皮质。再次使用带有P200尖端的5 mL移液管移液。 </li><li>将40μm尼龙细胞过滤器放在50mL管上，过滤悬浮液以除去碎屑。 </li><li>在室温下以420×g离心5分钟，然后除去DMEM培养基。 </li><li>加入5 mL脑皮层培养液，轻轻重悬细胞沉淀。 注意:准备一个脑皮层培养基，由补充有1×B-27补充剂，2mM L-谷氨酰胺和100U / mL青霉素链霉素的神经元基础培养基组成。 </li><li>使用血细胞计数器计数细胞，并使用脑皮层培养基调节细胞浓度（根据需要）。 </li><li>平板3.0×10 6皮质神经元，每个涂覆的35毫米玻璃底盘2.0毫升。 注意:电镀前，涂上具有0.1mg / mL聚-D-赖氨酸氢溴酸盐（PDL）或0.01％聚-L-鸟氨酸（PLO）的35mmm玻璃底盘。在37℃下孵育3小时-O / N，并用PBS洗涤至少3次。 </li></ol>成像囊泡运动<ol><li>准备标记每种类型囊泡的质粒。例如，使用EGFP-Rab5用于早期内体，EGFP-Rab7用于晚期内含体，LAMP-EGFP用于溶酶体，EGFP-LC3用于自噬体8,13。 注意:这些质粒可根据tsuruta.fuminori.fn@u.tsukuba.ac.jp的要求提供。通常使用转染试剂可以用这些质粒转染3〜5天体外 （DIV）神经元。 </li><li>通过移液将200μg无血清培养基混合4.0μg质粒。在单独的管中，在200μL无血清培养基中稀释8μL转染试剂（参见表格 ）。在室温下孵育5分钟。 </li><li>通过移液将步骤2.2中的质粒溶液和转染剂溶液混合。在室温下孵育20分钟。 </li><li>将无血清培养基和400μL来自步骤2.3的质粒溶液加入到每个涂覆的玻璃底盘中。在37℃下在5％CO 2培养箱中孵育30分钟。用2 mL大脑皮质培养基代替培养基。在37℃下在5％CO 2培养箱中孵育神经元1-2天。 </li><li>转染1 - 2 d后，选择转染的细胞进行图像采集。 注意:小于7 DIV的早产神经元具有呈现动态囊泡运动的趋势。选择适度表达荧光探针的神经元，因为当选择高度表达的神经元时，无法获得清晰的图像。此外，选择具有长轴突和复杂树突的典型神经元形状，例如锥体神经元，以便于鉴定轴突和树突（参见图1A）。 </li><li>使用延时成像系统进行图像采集: 对于成像，请使用配备有40X Plan Apo 0.95NA或100X Plan Apo VC NA1.4物镜的电荷耦合器件（CCD）相机的荧光显微镜。使用孵育系统控制温度在37°C。在图像采集软件控制的100s周期内以1帧/ 5s获取神经元图像。或者，以更短的间隔和更长的时间段采集图像，例如在300s的时间段内每2秒一帧。 注意:各种应用程序可用于拍摄顺序图像，其中许多应用将适用于此方法。因此，没有特别的应用推荐;相反，每个研究者都应该使用任何可用的应用程序。 </li></ol>图像分析<ol><li>使用手动跟踪14在ImageJ软件中打开所有顺序图像。 注意:到目前为止，手动跟踪一直是wi用于跟踪实验15,16,17,18,19。手动跟踪由FabriceCordelières博士研发。详细的书面说明可在线20 。为了分析，利用足够数量的图像来提高成像数据的质量。建议使用20张以上的图像。 </li><li>要组合顺序图像，请选择&lt;图像&gt;→&lt;堆叠&gt;→&lt;图像到堆叠&gt;。单击&lt;确定&gt;。 </li><li>选择&lt;插件&gt;→&lt;手动跟踪&gt;;将弹出跟踪窗口。 </li><li>单击&lt;显示参数？&gt;复选框，并在参数部分中定义跟踪参数。 注意:可以设置几个参数，包括时间间隔，x / y校准，z校准，搜索中心的正方形尺寸点数，点大小，行宽和字体大小。在这个简化的测定中，定义了时间间隔和x / y校准。在其他实验情况下，也可能需要定义其他参数。这里使用的参数如下:&lt;时间间隔&gt;为5秒，对于40X Plan Apo 0.95NA物镜，&lt;x / y校准&gt;为0.26642μm，对于100X Plan Apo VC NA1.4为0.10657μm物镜。 </li><li>定义参数后，单击&lt;添加轨迹&gt;开始新的轨道。 </li><li>在确定感兴趣的囊泡（ 例如， 图1A中的蓝色和红色箭头所示的囊泡（右图））后，点击顺序图像中信号的中心记录xy坐标。记录的xy坐标，距离和速度的结果显示在新窗口中。 注意:在溶酶体的情况下，大的明亮的荧光信号（ 例如，ure 1A（右图），橙色箭头）经常在神经元中观察到。由于这些信号偶尔表现出积聚的囊泡或病理性静脉曲张，因此应避免这些信号以量化运动性。 </li><li>重复步骤3.6从所有顺序图像收集囊泡的xy坐标;在选择参考点后，每个图像自动前进到连续图像。 </li><li>将这些数据导出到新表（ 例如电子表格）。使用适当的软件创建一个合适的图表（参见表格材料 ）。 注意:要分析数据，选择&lt;距离&gt;列，总结该列的所有值，并将运动距离显示为条形图， 如图1B所示 。此外，重复此步骤，计算总运动距离的平均值，并显示为条形图， 如图1C所示 。创建条形图和波形数据的软件如图所示材料表。 </li></ol>

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作者没有什么可以披露的。

该协议介绍了囊泡运动量的定量程序。在原代神经元中，内含体和溶酶体往往在年轻神经元（4-6 DIV）中表现出较高的运动能力。鉴于神经元必须将一些成分递送到前端以延长神经过程，因此膜运输应在此阶段动态发生。因此，使用年轻神经元观察神经元树突中的动态运动是重要的。此外，即使在年轻的神经元中，较大的囊泡也不会表现出较高的运动性。囊泡大小的调节可能涉及融合裂变步骤的频...

内源 - 溶酶体运输的精确控制对于调节神经元功能是必不可少的。值得注意的是，这些囊泡的动态运动是神经元形态，发育和生存调节的关键因素。该系统的缺陷引起严重的神经元疾病1,2 。将囊泡运输与神经元疾病相关联的分子机制被认为是复杂的，并且有几个组织试图检查这种相关性。例如，已经报道，扰动的晚期内体运动性与尼曼 - 皮克C病3 （由溶酶体缺陷引起的遗传性神经变性疾病）显着相关。另一个例子是溶酶体Ca 2+通道的突变， trpml1 ，其损害溶酶体运动，导致溶酶体贮积病4,5 ， </sup&gt; 6 。我们的研究小组报道，PtdIns（3,5）P 2转换的失调抑制了神经元中的内体和溶酶体运动，导致应激反应脆弱性增加7,8 。 PtdIns（3,5）P 2的代谢调节，其主要定位于晚期内体和溶酶体上，在多种细胞功能中起重要作用，包括囊泡运输和融合裂变过程9,10 。由于受损的PtdIns（3,5）P 2周转导致严重的神经变性11,12 ，内体 - 溶酶体运动的异常调节可能是了解神经变性发病机理的关键因素。对囊泡运动基础的分子机制的调查可能因此提供有希望的线索，可以加深我们的不便了解几种神经元疾病。 在本文中，我们引入了一种有价值的方法来量化神经元中的囊泡运动，使用自由软件包称为手动跟踪。目的是开发一种快速量化方法来分析囊泡运动。该定量是通过点击延时电影的每个帧中的参考点的标准方法进行的。与其他应用程序不同，使用手动跟踪软件使此方法非常简单，具有广泛的实用性。此外，该方法也适用于其他细胞，例如胶质细胞。虽然这种方法是原始的，但它可以应用于各种分析，包括细胞运动和形态变化。例如，在跨越图像序列定义参考点之后，可以使用数据分析和图像处理软件从连续图像中提取关于参考点的位置和每个位置处的时间的信息洁具。综合起来，这种方法简单而有力，有助于提高基于膜运输的研究的效率的发展，例如检查内体 - 溶酶体功能的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Thermo Fisher</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin Streptmycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsine</td>
			<td>Thermo Fisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine&#160;</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Polyethyleneimine &#34;Max&#34;</td>
			<td>polysciences</td>
			<td>24765</td>
			<td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/ml Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td>
		</tr>
		<tr>
			<td>BIOREVO BZ-9000</td>
			<td>Keyence</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation system INUG2-K13</td>
			<td>Tokay Hit</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 6.0</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel version 15</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ verion 1.47</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes

在大脑中，膜运输系统在调节神经元功能，如神经元形态，突触可塑性，存活和胶质细胞通讯中发挥重要作用。迄今为止，许多研究报道，这些系统中的缺陷引起各种神经元疾病。因此，了解囊泡动力学的机制可能会提供有助于治疗几种神经元疾病的有影响力的线索。在这里，我们使用ImageJ平台的软件插件描述了一种量化囊泡运动的方法，例如运动距离和运动速度。为了获得定量图像，我们用EGFP标记的囊泡标记蛋白标记神经元内体 - 溶酶体结构，并使用延时显微镜观察囊泡的运动。该方法非常有用，简化了神经突，如轴突和树突，以及神经元和神经胶质细胞的神经元测量囊泡运动。 Furthermo这种方法可以应用于其他细胞系，如成纤维细胞和内皮细胞。这种方法可以为我们对膜运输的理解提供有价值的推动。

该测定设计用于测量体外培养中的囊泡动力学。该测定用于确定与神经元形态和存活相关的囊泡运动性。 图1A和B显示了显示神经元溶酶体运动性的代表性数据。皮质神经元用溶酶体标记LAMP-EGFP转染，并使用标准荧光显微镜观察。以前曾报道，即使在静息神经元中，特异性内体溶酶体囊泡也是高度移动的。实际上，这些结果表明在相同的枝?...

Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

膜运输的调查对于了解神经元功能至关重要。在这里，我们介绍一种量化神经元囊泡运动性的方法。这是一种方便的方法，可以适应于神经系统膜运输的量化。

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

quantification-endosome-lysosome-motilities-cultured-neurons-using

Research

JoVE Journal

Neuroscience

8.0K 次观看.  University of Tsukuba. 膜运输的调查对于了解神经元功能至关重要。在这里，我们介绍一种量化神经元囊泡运动性的方法。这是一种方便的方法，可以适应于神经系统膜运输的量化。 

视频: Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...