支持从匹兹堡大学医学院的药理学及化学生物学系提供启动基金。确认雅各布实验室成员谁促成了视频提交:尼古拉斯·格拉夫。

下面描述的所有协议都是按照IACUC和匹兹堡大学医学院的内部评级机构审查委员会。 1。海马神经细胞在组织培养罩的准备注意:使用无菌技术和试剂整个协议1。 <ol><li>制备聚-D-赖氨酸（0.1 mg / ml的H 2 O）涂覆的玻璃盖玻片上作为底物的神经元的培养。 <ol><li>将每个3.5厘米组织培养皿内4-5圆形玻璃盖玻片。 </li><li>点70微升聚-D-赖氨酸到每个12毫米盖玻片。注:对于实时成像，玻璃底3.5厘米组织培养皿可以使用（现货200微升聚-D-赖氨酸上的嵌入式14毫米玻璃盖玻片）。 </li><li>离开烹制的菜肴在组织培养罩的O / N。为了尽量减少聚-D-赖氨酸蒸发，并保持表面消毒的组织文化引擎盖，关闭窗扇和关闭日ê鼓风机的O / N。注:O / N孵化期间不使用紫外线灯。 </li><li>第二天早上，洗碗3倍用2毫升H 2 O </li><li>删除最后一个H 2 O的洗涤后，加入2毫升媒体到每个3.5厘米菜，菜品留在孵化器，直到准备板的神经元在步骤1.4。 </li></ol></li><li>海马神经元从胚胎18-19天（E18-19）大鼠（从22戈斯林修改）编制。 </li><li>转染新鲜分离的神经元在培养的一天1-4微克maxiprepped结构的DNA。 注意:通常为3的DNA微克转染到1-2万神经元，用50％的生存力和60％的转染效率（ 例如，开始与2000000的神经元:（（2×10 6个神经元×0.5）0.6）提供了约100万神经元;。故障排除潜在的问题，当其中600,000转染1不同量的结构的DNA可转过度表达，其次是结构定位和功能的相应特性。 </li><li>镀上以每3.5厘米菜约200,000神经元的最终密度的聚-D-赖氨酸包被的盖玻片的转染的神经元。注:为玻璃底培养皿，板40,000-50,000神经元上14毫米的玻璃区域。 </li><li>更换培养基制备神经元培养后4-24小时，然后让神经元发展的孵化器，直到在体外 （DIV）或所需的阶段14-17天。 </li></ol> 2。内吞作用分析 注意:注意对α-银环蛇毒素和废物筒箭毒碱和处理: <ol><li>处理所有肽神经毒素的建议的生物安全2级（BL-2）研究方案的指导方针。所有适当的个人防护装备必须佩戴。冻干粉剂毒素应根据制造商的说明进行配制。毒素废物的寿LD在遵守监管机构的环境，健康与安全准则处置。 </li><li>以去除可能在储存期间形成的潜在的肽聚集体，α-银环蛇毒素的等分试样应简要使用前离心，并将上清液应该用于实验。 </li><li> α-银环蛇毒素（BGT）股票是在-20℃下再悬浮至1毫克/毫升的浓度在无菌H 2 O和存储于10μl等分试样荧光再加BGT股票是用在3微克/毫升。筒箭毒碱的用量为150μM的最终浓度。 </li></ol><ol><li>设置台式加热块到16℃，在寒冷的房间，薄铝或不锈钢板之上。或者，如果有的话，一个台式冷却/加热设备可用于每个步骤需要16℃。 </li><li>含有以mM凉爽外的Hepes缓冲盐溶液（HBS:135氯化钠，氯化钾4.7，10 HEPES，11葡萄糖，1.2的MgCl 2，和2.5氯化钙 ，pH值7.4），以16℃的水浴中。 </li><li>传递神经元菜铝板在16℃，冷却5分钟。 </li><li>移除媒体（ 保留条件培养基和储备孵化器的步骤2.8，测定细胞内吞作用的时间点 ）和替换用1毫升16°C HBS +筒箭毒碱（150微米）2分钟。 </li><li>删除HBS +筒箭毒碱，以标签的废弃物容器，并更换1毫升16°C HBS +筒箭毒碱（150微米），+α-银环蛇毒素的Alexa 594 [3微克/毫升]，孵育在16°C下15分钟。 </li><li>删除HBS +筒箭毒碱+α-银环蛇毒素的Alexa 594，以标签的废弃物容器和洗碗3倍用2毫升16°C哈佛商学院（洗涤液可以通过在抽吸含有50ml 50％的漂白粉的保温瓶收集）。 </li><li>对于实时成像时间序列实验用玻璃下面受体的内吞作用见底3.5厘米培养皿中，执行步骤2.1-2.6，然后转移到菜加热阶段（珀耳帖元件）上的显微镜，并开始成像共聚焦或全内反射荧光显微镜的设置。 </li><li>转让T = 0的时间点盖玻片到RT 4％多聚甲醛/ 4％蔗糖20分钟的菜，与其他盖玻片继续步骤2.9。 </li><li>对于其他的时间点，与哈佛商学院的更换平衡调节37℃介质（保留在步骤2.4），并在37°C 注回到孵化器内吞作用:建议对T = 15额外的时间点，30，和60。 </li><li>在时间点需要，采取菜肴从培养箱中，用2毫升RT的DPBS洗涤两次快速（DPBS无钙，镁），然后在4％低聚甲醛/ 4％蔗糖20分钟固定。 </li><li> 20分钟后固定，除去4％多聚甲醛/ 4％蔗糖浪费容器，用2ml RT DPBS洗两次菜。 </li><li>孵育盖玻片在RT中免疫块溶液10分钟（块溶液= 5％马血清，0.5％BSA的DPBS），含0.2％的Triton X-100对渗透率IZE的神经元，使细胞内的受体池和/或其他感兴趣的蛋白标记的抗GFP免疫染色。 </li><li>删除块+ 0.2％的Triton X-100和孵化的免疫块解决方案无需盖玻片的Triton X-100为20-30分钟。 </li><li>在4℃下进行块盖玻片一抗孵育在室温或O / N数小时注:与盖玻片初选在4℃的CO / N孵化常规生产改善免疫荧光结果。 </li><li>洗盖玻片3X用DPBS（5分钟，每次洗涤步骤）。 </li><li>孵育盖玻片，在1小时在室温块溶液的第二抗体。 </li><li>洗盖玻片3X用DPBS（5分钟，每次洗涤步骤）。 </li><li>摩盖玻片，单独搬运每个盖玻片:从盖玻片与实验室组织的去除背部多余的液体，然后倒置盖玻片到4微升玻片上安装介质。 </li><li>允许片干燥在室温覆盖30分钟，然后ST在4℃的矿石，直到准备进行显微镜检查。 </li><li>图像采集和实验，共聚焦显微镜（盲目的实验条件）进行分析。 60X油NA以下激光器1.49浸泡的目的:氩气488纳米，561二极管，640二极管。 <ol><li>使用相同的图像获取的设置，取得与该神经元的细胞体和在焦点几个树枝状突起单个Z截面图像。 </li><li>沿20μm的每神经元3-4近端树突量化α-银环蛇毒素的Alexa荧光信号和GFP免疫染色，使用相同的阈值对所有的内吞作用的数据进行分析。 </li><li>对于每个时间点分析从10-12神经元数据时，重复该实验与几个独立的神经元培养物。 </li></ol></li></ol>

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作者什么都没有透露。

此处所描述的信息为基础的固定和活的技术可以被用来跟踪受体或其它细胞质膜蛋白质运输中的细胞系，神经元和其它的原代细胞。此法已成功地用于研究膜的插入和移除的配体门控离子通道和G蛋白偶联受体，并评估变化贩卖由于受体激动剂和调节剂的存在下进行。主要方面包括标签的适当定位到细胞外的位置，并进行控制，以确保增加的BBS（作为标准的其他记者，如GFP）在功能上是沉默的。对...

利用荧光再加α-银环蛇毒素研究受体定 ​​位和动态是率先在烟碱乙酰胆碱受体13-15，毒素的内源靶的研究。随后，最小BGT结合肽（BBS）的掺入已被用于研究贩卖既兴奋性和抑制性配体门控离子通道和G蛋白偶联受体2,10,16-21的。本次论坛为基础的技术提供的优势，用于贩运研究，如表面生物素的方法，活细胞的抗体标记的抗体与细胞外抗原表位，和光漂白（FRAP）后荧光恢复其他方法。在细胞表面生物素游离胺的共价修饰，以影响细胞活性的潜力。基于抗体的研究常常受到阻碍表面抗原簇或封盖，它可以改变拐卖事件。由于对FRAP S中的漂白步骤tudies，一个重要的问题是损害底层的细胞结构。一个额外的优点是，信息标记的构建体也可用于生化方法学与生物素偶联银环蛇毒素对驴受体贩卖。这种技术是很容易适用于细胞系和原代细胞。对于在细胞表达烟碱乙酰胆碱（胆碱）受体的使用，胆碱受体拮抗剂筒箭毒碱，必须使用整个协议所指示的。对未转染的细胞在缺乏筒箭毒碱的执行一个简单的表面BGT标签（等同于内吞协议T = 0的时间点），将提供内源性胆碱受体的证据。 使用这种技术的一个重要的考虑因素是BBS的适当的插入，使得它存在于当目的蛋白被递送到质膜的细胞外的位置。例如，GABAAR亚基的N-末端结构域T​​RAF期间驻留在囊泡腔ficking并成为细胞外质膜受体插入后，使细胞表面的受体和其从细胞表面通过内吞事件去除的评估特异性标记。之前我们已经表明，除了GFP，myc和或BBS的表位GABAAR亚基这个领域在功能上保持沉默。标准控件应该执行，以确保标签的蛋白的表达水平相似，以不加标记的构建物，它适当地本地化的，并且它不影响受体的功能。转结构的这一特点也将有助于在故障排除过度担忧。

<table><tbody><tr><td>P3 Primary Cell 4D kit&amp;nbsp;</td><td>Lonza</td><td>V4XP-3024</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 647 conjugate</td><td>Molecular Probes</td><td>B35450</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 488 conjugate</td><td>Molecular Probes</td><td>B13422</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 594 conjugate</td><td>Molecular Probes</td><td>B13423</td><td></td></tr><tr><td>(+)-Tubocurarine chloride</td><td>Tocris</td><td>2820</td><td></td></tr><tr><td>Mounting medium&amp;nbsp;</td><td>DAKO</td><td>cS703</td><td></td></tr><tr><td>DPBS no calcium, magnesium</td><td>Invitrogen</td><td>14190-136</td><td></td></tr><tr><td>Poly-D-lysine&amp;nbsp;</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>Gephyrin antibody</td><td>Synaptic Systems</td><td>147 011</td><td>1:300</td></tr><tr><td>VIAAT/VGAT antibody</td><td>Synaptic Systems</td><td>131 002</td><td>1:1,000</td></tr><tr><td>anti GFP</td><td>Life Technologies</td><td>A6455</td><td>1:2,000</td></tr><tr><td>Glass bottom tissue culture dish</td><td>MatTek Corporation</td><td>P35G-1.5-14-C - Mattek Dishes</td><td></td></tr><tr><td>Coverslips (round cover glass), #1 thickness, 12 mm</td><td>Warner Instruments</td><td>640-0702</td><td></td></tr></tbody></table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

这是越来越明显的神经递质受体，包括离子型GABA A受体（GABAAR），表现出高度动态的贩运和细胞表面的流动性1-7。为了研究受体细胞表面的定位和内吞作用，这里所描述的技术结合使用荧光​​α-银环蛇毒素与细胞表达含有α-银环蛇毒素（BGT）结合位点（BBS）的结构。在BBS（WRYYESSLEPYPD）是基于肌肉烟碱乙酰胆碱受体，其结合BGT以高亲和力8,9的α亚基。注册成立的BBS站点允许表面定位和受体插入或移除与应用外源性荧光BGT的测量，如以前在GABAA和GABAB代谢的跟踪受体2,10描述。除了 ​​信息的网站，我们氨基酸4与5的成熟GABAAR亚单位通过标准米之间插入一个pH敏感的绿色荧光蛋白（pHGFP 11）olecular生物学和PCR克隆策略（参见图1）12。 BBS的是3&#39;的pH值敏感的GFP报告，由13个氨基酸丙氨酸/脯氨酸接头隔开。对贩运这一出版物是基于固定样本中描述的研究中，pHGFP作为总的记者标记GABAAR亚单位蛋白水平，使BGT正常化标记受体的人口占总人口的受体。这最大限度地减少细胞与细胞的标记GABAAR亚基的较高或较低的基线表达所得BGT染色信号的变异性。此外，pHGFP标签可方便识别构建表达细胞的活的或固定成像实验。

一个信息标记构建体的表征，包括重要的控制，如确定所表达的蛋白质正确装配（特别是与受体的多个亚基组成），贩卖到细胞表面，并适当地局部化。抑制性突触是由γ-氨基丁酸的共定位该具有抑制支架蛋白gephyrin并结合在突触前的抑制端子，确定囊泡抑制性氨基酸转运加载GABA和甘氨酸进入突触小泡（VIAAT / VGAT）A受体表面的集群。 如图2顷 α2pHGFP+ BBS表达神经元与表面GABAAR标记BGT，...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

在这里，我们展示了使用荧光染料的Alexa耦合到α-银环蛇毒素测定GABA A受体表面的定位和内吞作用的海马神经元。通过采用轴承结构结合α-银环蛇毒素，可以实现质膜蛋白的内吞贩运分析短外标签。

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.8K 次观看.  University of Pittsburgh School of Medicine. 在这里，我们展示了使用荧光染料的Alexa耦合到α-银环蛇毒素测定GABA A受体表面的定位和内吞作用的海马神经元。通过采用轴承结构结合α-银环蛇毒素，可以实现质膜蛋白的内吞贩运分析短外标签。 

视频: Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA<sub&gt;一个</sub&gt;受体

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

科研

神经科学

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

生物学

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the animal kingdom and can be recognized as either adaptive or maladaptive responses to a wide range of stress modalities. Rodents provide a proven experimental model for translational studies addressing the integrative mechanisms of anxiety at the molecular, cellular, and circuit levels. In particular, the chronic psychosocial stress paradigm elicits maladaptive responses mimicking anxiety-/depressive-like behavioral phenotypes that are analogous between humans and rodents. While previous studies show significant effects of chronic stress on neurotransmitter contents in the brain, the effect of stress on neurotransmitter receptor levels is understudied. In this article, we present an experimental method to quantitate the neuronal surface levels of neurotransmitter receptors in mice under chronic stress, especially focusing on gamma-aminobutyric acid (GABA) receptors, which are implicated in the regulation of emotion and cognition. Using the membrane-impermeable irreversible chemical crosslinker, bissulfosuccinimidyl suberate (BS3), we show that chronic stress significantly downregulates the surface availability of GABAA receptors in the prefrontal cortex. The neuronal surface levels of GABAA receptors are the rate-limiting process for GABA neurotransmission and could, therefore, be used as a molecular marker or a proxy of the degree of anxiety-/depressive-like phenotypes in experimental animal models. This crosslinking approach is applicable to a variety of receptor systems for neurotransmitters or neuromodulators expressed in any brain region and is expected to contribute to a deeper understanding of the mechanisms underlying emotion and cognition.

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

BS3 化学交联测定:评估慢性应激对啮齿动物大脑中细胞表面 GABAA 受体呈递的影响

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

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在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA