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。

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			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
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			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">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 &amp;#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

Research

JoVE Journal

Neuroscience

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

15.7K 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 &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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 ...

科研

神经科学

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

使用近红外光谱MazeSuite和功能研究学习空间导航

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

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 ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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

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

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

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...