这项工作得到了国家耳聋与其他交流障碍研究所（NIDCD）校内计划。

1。神经元转<ol><li>文化胚胎第18天（E18）大鼠海马神经元- D -聚赖氨酸包MatTek 35毫米的玻璃底菜 1 。 （DIV），在16-18天的体外转染的神经元，使用Clontech公司CalPhos哺乳动物转染试剂盒。首先，用1.5毫升培养基代替<a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/Classical_Media/dmem.html">贝科改良Eagle培养基</a> （DMEM），每35毫米盘0.5小时之前转。 （步骤1.6）使用，保存在一个无菌的15毫升管的原培养液。 </li><li>混合使用无菌H 2 O（Clontech公司）和12.4μL2米钙的解决方案（Clontech公司）的总体积100μL的10μg的pEGFP - N1质粒DNA。 </li><li>从步骤1.2）中添加的混合物100μL2 ×哈佛商学院滴加而涡旋2 ×哈佛商学院在中等速度。 </li><li>让坐在混合物在室温下放置20分钟，然后添加步骤1.3）DMEM培养液培养的神经元最终混合物。 </li><li>为1-1.5小时，放入37℃培养箱中培养的神经元。 </li><li>删除磷酸钙介质中，然后用DMEM三次洗细胞。在返回培养皿中的孵化器，交流与原培养液DMEM培养基。 </li></ol> 2。在脊柱的FRAP实验<ol><li>用于转染后二至四天的FRAP实验的神经元。 </li><li>从35毫米的玻璃底菜更换培养液，立即加入预热Tyrode液，其中包含（毫米），HEPES 10 5 145氯化钠，氯化钾，葡萄糖10，甘氨酸0.005，2.6氯化钙和氯化镁2 1.3（用NaOH调整pH至7.4）。 </li><li>蔡司LSM 710共聚焦显微镜是用于FRAP实验。蔡司TempModule系统是用来控制温度（37℃），湿度和工作系统的CO 2（5％）。确保连接和一瓶水，这是用来平衡湿度，是装满了水，CO 2坦克。 </li><li>查找100 ×目标（αplan的复消色差透镜100 × / 1.46油）的转染成熟树突。如果在盘转染细胞稀疏，寻找所需的细胞与40 ×的目标（计划NEOFLUAR 40 × / 1.3油），然后切换到100 ×目标捕捉图像。 </li><li>使用5 ×光学变焦和256 × 256像素分辨率的图像一小段几个刺的枝蔓。要拍摄图像，使用额定转速（像素停留时间为3.15微秒）需要0.5秒完成扫描。针孔设置为2μm的获得较强的荧光。到图像时，尝试使用激光传输低，例如1-5％，以避免漂白的整个形象。 </li><li>选择感兴趣的脊椎。在我们的实验中，我们选择与他们的脊柱头部直径约1微米的蘑菇刺。 </li><li>为了做一个FRAP实验，漂白前5个控制影像，然后漂白脊柱利益标称100％激光传输的10倍[氩激光功率为30兆瓦，488激光线的50％（15毫瓦），和约3-4毫瓦，达到通过显微镜488激光线]，然后漂白后，立即捕捉了一系列的图像。在这个实验中，图像被捕获漂白后每15秒1秒。调整的时间间隔应根据不同的目标蛋白质和不同的实验设计（ 图1） 。 </li><li>保存图像。 </li></ol> 3。数据分析<ol><li>与ImageJ软件中打开图像。 </li><li>对齐使用对齐工具图像的堆栈 - ImageJ（“插件”→“栈洗牌”→“对齐堆栈片”→“翻译”和/或“刚体”），使脊柱的利益不浮换句话说 - 它仍然在同一位置上的图像。 </li><li>测量相对利益（F S）的脊椎，一个转，但漂白地区（对照组），在时间的推移图像和背景区域（F B）的荧光强度，使用ImageJ工具“力度V时间显示器”（ “插件“→”栈 - 洗牌“→”强度V时间显示器“）。控制区域可以是一个倍受关注的远端树突的一块。背景区是一个非荧光的地区。 </li><li>计算前（F C0）和（F C）漂白后，通过比较控制区的荧光光漂白率（R ）。 R = F C / F C0。 </li><li>目标脊柱的荧光强度（F）的规范如下： F =（F S - F B）/ R。 </li><li>曲线拟合Graphpad Prism软件一阶段的指数方程（ 图2）或官方所用的荧光强度的目标脊柱ř类似软件。 </li><li>计算移动的分数（F M）和不动的一小部分（一 ）由下列公式： F M = F∞/ F 0， 其中，F∞全面复苏后的荧光强度，F 0是前漂白的荧光强度。 F的第I = 1 - F 米。 </li></ol> 4。代表性的成果： 在这项研究中，我们执行一个成熟的海马神经元的FRAP实验。在18-22格，已形成了蘑菇刺。用我们的方法，在一个小区域的荧光强度的动态变化，如脊椎，可以被记录下来。 要分析的EGFP荧光恢复过程中，我们采取控制之前，漂白和5张1图像的每1在15秒漂白后立即第二。图像分辨率是足够的定量分析。标记的荧光蛋白的荧光恢复型材高度重复性。 我们还简要说明如何定义一个荧光蛋白的移动和静止的分数，使用ImageJ和Graphpad Prism软件。我们在这里展示的FRAP方法和分析，可以广泛应用于神经科学，细胞生物学等研究。 <img src="/files/ftp_upload/2568/2568fig1.jpg" alt="图1" /> 图1。FRAP EGFP荧光测量在脊柱从培养的海马神经元。红色箭头指示的漂白时间。照片代表前同一地区（预），0，1，5，10，15秒​​后漂白。字母S，C和B，分别标有脊柱，控制和背景区域。在实验过程中，神经元保持在37 ° C。比例尺，1微米。 <img src="/files/ftp_upload/2568/2568fig2.jpg" alt="图2" /> 图2。FRAP EGFP荧光曲线超过15秒的时间。绿线显示了原始的曲线;红线显示正常化的曲线。曲线上的点显示FRAP每隔1秒。一个阶段的指数方程拟合曲线。漂白前的平均荧光计为100％。在这个实验中，移动的比例（MF）是94％，在不动的分数（IF）为6％。

<ol>
	<li>Zheng, C. Y., Petralia, R. S., Wang, Y. X., Kachar, B., Wenthold, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAP102+is+a+highly+mobile+MAGUK+in+spines.">SAP102 is a highly mobile MAGUK in spines.</a> J Neurosci. 30, 4757-4766 (2010).</li><li>Grati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+turnover+of+stereocilia+membrane+proteins:+evidence+from+the+trafficking+and+mobility+of+plasma+membrane+Ca(2+)-ATPase+2.">Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.</a> J Neurosci. 26, 6386-6395 (2006).</li><li>Liu, L. N., Aartsma, T. J., Thomas, J. C., Zhou, B. C., Zhang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+on+Red+Alga+Reveals+the+Fluorescence+Recovery+Is+Ascribed+to+Intrinsic+Photoprocesses+of+Phycobilisomes+than+Large-Scale+Diffusion.">FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion.</a> PLoS ONE. 4, e5295-e5295 (2009).</li><li>Wiedenmann, J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+with+EosFP+and+other+photoactivatable+marker+proteins+of+the+GFP+family.">Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.</a> Expert Rev Proteomics. 3, 361-374 (2006).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Sprague, B. L., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+binding:+proper+and+fitting.">FRAP analysis of binding: proper and fitting.</a> Trends Cell Biol. 15, 84-91 (2005).</li></ol>

FRAP分析已被广泛使用在体内和体外1-2研究。这种技术通常利用GFP <a href="http://en.wikipedia.org/wiki/Fusion_protein" title="融合蛋白">融合蛋白</a> ，虽然它也可以用红藻融合蛋白3。这种分析是敏感的，可以用来描述GFP标记的蛋白质的流动性。 要产生有意义的FRAP分析，重要的是要避免不必要的漂白FRAP实验之前和期间。有两种方法来实现这一。首先，搜索和观察实验的神经元的过程要快。特别是，神经元与100X的目标很长一段时间的观察明显漂白的荧光。二，高的激光功率和频繁扫描，往往会增加漂白的可能性。因此，将需要计算在控制区域的漂白率，然后在实验地区的FRAP曲线正常化。正火的方法已被描述的协议（见步骤，细节为3.3-3.5）。 漂白步骤，也是确保获得良好FRAP结果的关键。在这个实验中，我们漂白利息的10倍，100％的激光传输的脊椎。这条件是足够的一个背景在一个固定的编制水平的脊柱荧光漂白的。因此，我们设置漂白后的荧光强度在0秒作为背景相同数量。根据在第一次扫描的速度，可能已经被检测到的荧光恢复大量感兴趣的蛋白质是流动性很强。 已经开发了许多荧光蛋白探针研究蛋白质动力学与FRAP。互补性办法，例如，使用photoactivatable GFP（PA - GFP的），或photoconvertible变种。连同FRAP还原技术，这些工具成为不可或缺的活细胞成像研究4-6。

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<td>35 mm glass-bottom dishes </td>
<td>&nbsp;</td>
<td>MatTek</td>
<td>P35GC-1.0-14-C</td>
<td>&nbsp;</td>
<tr>
<td>CalPhos Mammalian Transfection Kit</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>631312</td>
<td>&nbsp;</td>
<tr>
<td>pEGFP-N1 plasmid</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>6085-1</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss confocal microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>LSM 710</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss TempModule system</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>N.A.</td>
<td>&nbsp;</td>
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<td>Graphpad Prism software</td>
<td>&nbsp;</td>
<td>Graphpad software</td>
<td>N.A.</td>
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fluorescence recovery after photobleaching (frap) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons

FRAP已被用来量化GFP标记的蛋白质的流动性。使用一个强大的激励激光，一个GFP标记蛋白的荧光漂白，在感兴趣的区域。 GFP标记的来自该地区以外的的漂白蛋白扩散到感兴趣的区域时，该区域的荧光恢复。蛋白质的流动性进行了分析，通过测量荧光恢复率。这种技术可以用来描述蛋白质的流动性和周转率。 在这项研究中，我们对培养海马神经元（增强型绿色荧光蛋白）EGFP的载体。我们使用蔡司710共聚焦显微镜，光漂白的在一个单一的脊椎的绿色荧光蛋白的荧光信号，然后取时间的推移图像记录荧光漂白后恢复。最后，我们估计在刺的绿色荧光蛋白的移动和静止的分数的百分比，通过分析成像数据，使用ImageJ和Graphpad软件。 这FRAP协议显示了如何执行基本FRAP实验，以及如何对数据进行分析。

Watch this Scientific Journal Video about Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons at JoVE.com

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

FRAP已被用来量化绿色荧光蛋白（GFP）标记在培养细胞中的蛋白质的流动性。我们研究分析了GFP的荧光漂白后恢复的百分比移动/静止的分数。在这项研究中，FRAP刺海马神经元。

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 ...

fluorescence-recovery-after-photobleaching-frap-fluorescence-tagged

Research

JoVE Journal

Neuroscience

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

54.1K 次观看.  National Institutes of Health, Bethesda. FRAP已被用来量化绿色荧光蛋白（GFP）标记在培养细胞中的蛋白质的流动性。我们研究分析了GFP的荧光漂白后恢复的百分比移动/静止的分数。在这项研究中，FRAP刺海马神经元。

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

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 ...

科研

神经科学

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 ...

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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 ...

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 ...

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

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

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. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.