这项工作是由美国国立卫生研究院资助DA15169和DA035224到下摆

所有的动物处理和组织收获是按照美国马萨诸塞大学医学院实验动物管理使用委员会大学（IACUC）的指引进行，继批准的协议＃A1506（Melikian，PI）。 所需的解决方案 人工脑脊液（学联） -使每日新鲜 125 mM氯化钠，2.5 mM的氯化钾，1.2毫米的NaH 2 PO 4，1.2毫摩尔MgCl 2，2.4 mM的氯化钙 ，26毫米的NaHCO 3，和11毫摩尔葡萄糖 注：准备学联为10倍原液，不含碳酸氢钠和葡萄糖。每天做从10倍股票1X工作溶液，用新鲜的碳酸氢钠和葡萄糖补充。 蔗糖补充学联（SACSF） -使每日新鲜 250毫米SUC升，2.5毫米氯化钾，1.2毫米的NaH 2 PO 4，1.2毫摩尔MgCl 2，2.4 mM的氯化钙 ，26毫米的NaHCO 3，和11毫摩尔葡萄糖 注：准备SACSF为10倍的贮备液，不含碳酸氢钠和葡萄糖。每天做从10倍股票1X工作溶液，用新鲜的碳酸氢钠和葡萄糖补充。 磺基-N-羟基琥珀酰基-SS-生物素 （磺基-NHS-SS-生物素，Pierce化学公司） 储备溶液应为200 mg / ml的DMSO中，并能抵抗多种冷冻/解冻循环。等分试样贮存于-20℃。琥珀酰酯水解迅速在水溶液中，使工作液应立即应用到片前做好准备。 片淬火解决方案 ACSF补充有100mM甘氨酸 RIPA裂解BuffeŘ 的10mM Tris，pH值7.4，150 mM氯化钠，1.0毫摩尔EDTA，1％的Triton-X-100，0.1％SDS，1％脱氧胆酸盐钠 RIPA与蛋白酶抑制剂（RIPA / PI） -使每日新鲜 RIPA补充了1μM的亮肽素，1μM的胃蛋白酶抑制剂，1μM的抑肽酶和1mM苯甲基磺酰氟。 1。准备脑片<ol><li>让新鲜的1X SACSF和1x学联</li><li>在一个小烧杯寒意SACSF冰上。这将被用于保持新鲜收获小鼠脑。 </li><li>通过用95％/ 5％的O 2 / CO 2，20分钟，在冰上鼓泡饱和的ACSF和SACSF与氧气。 </li><li> P30-38的小鼠应使用最佳的组织的生存能力。牺牲动物通过颈椎脱位和断头，迅速取出大脑进入预冷，含氧SACSF。 </li><li>用振动切片机，使感兴趣区域300μm的脑切片。 </li><li>如果需要的话，可以将它们进一步回收前解剖以富集特定脑区域或单独的左右半球分别作为对照组和实验的切片使用。 </li><li>用火抛光的巴斯德吸液管，转移切片网眼底套入24孔板腔。 </li><li>允许切片恢复为40分钟，31℃，含氧学联，具有持续，温和起泡。 </li></ol> 2。药物治疗（如适用）和生物素片<ol><li>以下恢复，洗净切片3倍于预热（37℃），含氧学联冒泡不断与95％/ 5％O 2 / CO 2。 </li><li>添加测试化合物，并培育具有连续充氧。 <ol><li>为方便起见，加10倍浓缩的药物一个1/10量，并通过轻轻颠倒混匀。 </li><li>摇板轻轻地在水浴中在所需的温度。 </li></ol></li><li>继药物治疗，迅速寒意片在冰冷学联洗3次。 </li></ol> 3。表面生物素化的蛋白质<ol><li>准备1.0毫克/毫升的前夕标签硫代NHS-SS-生物素在冰冷的学联。 </li><li>添加0.75毫升硫代NHS-SS-生物素片和切片孵育在冰上，45分钟。 </li><li>用冰冷学联快速洗片三次，然后孵育在冰冰冷学联10分钟。 </li><li>用冰冷的切片淬灭缓冲液冲洗切片3次，并培育与0.75毫升片骤冷缓冲液2次，25分钟，在冰上骤冷自由磺基-NHS-SS-生物素。 </li></ol> 4。准备组织裂解液<ol><li>在冰冷学联洗净切片三次，用火抛光的巴斯德吸管每片转移到离心管中。 </li><li>轻轻离心200×g离心1分钟，小心吸出剩余学联沉淀切片。 </li><li>加入400μl冰冷的RIPA / PI和移液器向上打破了组织，下一次通过P200的点子ETTE。 </li><li>转移解离片/ RIPA到一个新的管和孵育30分钟，4℃，旋转，完成溶解。 </li><li>通过离心沉淀细胞碎片，18,000×g离心，15分钟，4°C。 </li><li>确定裂解物中的蛋白浓度使用BCA蛋白测定法，以牛血清白蛋白（BSA）作为标准。 </li></ol> 5。分离蛋白质生物素<ol><li>优化珠/总蛋白的比例。 注：个别蛋白表达水平可以有很大的不同在不同的脑区。除非所有的组织裂解液的一定量的生物素化的蛋白质的被捕获，这是不可能精确地检测在表面上表达的任何潜在的变化。因此，就必须根据经验确定特定蛋白质/大脑区域产生一个新的切片生物素化研究着手之前最佳珠/总蛋白比率。 <ol><li>孵育25μL链亲和素琼脂糖珠的增加量组织裂解液（大致范围25-200微克），然后第继续进行如下所述的结合，洗涤和洗脱步骤。 </li><li>量化所产生的免疫反应带，并选择绑定，这将允许增加或减少蛋白质表面表达准确定量的线性范围珠/裂解液的比例。 </li></ol></li><li>准备链霉亲和琼脂糖珠<ol><li>确定所需的所有样品总珠体积。准备了充足的珠子体积的金额加上一个额外的（ 即 4个样品在25μL/样本= 100μL珠+一个额外= 125μL珠。 </li><li>旋涡链霉亲珠股票和吸管出所需的音量。如果使用的是P200的移液器，切断尖底，以防止堵塞或损坏珠。 </li><li>在0.5-1.0毫升RIPA / PI珠洗3次离心18,000×g离心1分钟以去除防腐剂，每RIPA加入后涡旋和清洗间收集珠子，在室温脾气ATURE。 </li><li>吸过使用连接到一个保温瓶玻璃巴斯德吸管清洗之间的缓冲区。吸气时，附着在玻璃巴斯德吸管的末端的塑料P200尖提供更精细的控制。它没有必要完全除去所有的缓冲器的第2次洗涤，如它的风险珠吸进移液管。最后一次洗涤后，去除尽可能多的多余的缓冲尽量不吸珠。 </li><li>加入RIPA / PI带来珠回到他们原来的音量，上下吹打重悬。避免涡流在这一点上，作为珠会坚持到管壁。 </li><li>分装成小珠用P200与尖端的离心管切断。吸管向上和向下采样之间数次，以确保珠保持均匀悬浮分散管中。 </li></ol></li><li>结合生物素蛋白链亲和素磁珠<ol><li>分发细胞裂解物，以含有的琼脂糖珠的管中。对于实验磨片<html>再多个样品进行比较，确保使用的蛋白质相同数量的每个样品进行精确的比较。 </li><li>添加额外的RIPA / PI带给样品200μl的最小体积。这保证了样品将在孵化过程中充分混合，也统一了整个样品的蛋白浓度。 </li><li>放置在管的管肩和搅拌过夜，在4℃下</li><li>在单独的管中，分配用于每个样品的总裂解物体积的等效。另外，如果样品体积都高，总裂解物体积的一小部分可以被保留，而不是，以适应在SDS-PAGE凝胶上的最大负载量。这些将被用于归一化的表面表达的总蛋白量。 </li><li>加2倍的任一个等于或体积的1/5体积的6倍SDS-PAGE样品缓冲液并任孵育在4℃下，在平行珠的样品，或储存在-20℃直至样品进行了分析。 </li></ol></li></ol>ove_title“&gt; 6。洗脱和样品分析<ol><li>通过离心18,000×g离心2分钟，在室温下沉淀的珠子。 </li><li>吸上清，洗珠三次0.75毫升RIPA。为了尽量减少损失珠，留下缓冲的一个小头体积上面珠洗之间。最后一次洗涤后，除去尽可能多的RIPA尽可能不破坏珠粒料。 </li><li>通过减少二硫键洗脱生物素蛋白链菌素的磁珠。加25μl的2×SDS-PAGE还原样品缓冲液，涡旋以及与旋转样品30分钟，室温。 注意：许多膜蛋白具有较高的聚集倾向时，在SDS-PAGE样品缓冲液煮沸，严重影响其电泳迁移率。如果这是正在研究的蛋白质的情况下，避免加热样品，相反，洗脱在室温下缓慢。如果煮沸是绝对必要的（ 例如 ，如果在平行评估的另一种蛋白，需要煮沸），SAMPLë缓冲器可以用2M尿素（终浓度）进行补充，以减少聚集。而有效地减少聚集在某些情况下，尿素往往损害带的外观。 </li><li>通过免疫印迹分析样品。 </li><li>解冻总裂解物样品，并旋转的同时，胎圈的样品，30分钟，室温。 </li><li>分离的蛋白质在SDS-PAGE凝胶。 </li><li>通过免疫印迹鉴定目的蛋白（S）。 <ol><li>可以肯定的是，乐队在检测的正确量化的线性范围检测。 </li><li>以确保该生物素化试剂并没有通过受损/受损细胞得以进入细胞内的蛋白质。这最好是通过免疫印迹法在平行于特定于被研究的细胞类型的细胞内蛋白质来实现的。 </li><li>量化带的密度，并计算相对蛋白质表面密度为总蛋白的表达水平的百分比。 </li></ol></li></ol>

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作者没有财务利益，这项工作冲突。

尽管长期以来知识内吞贩卖大脑危重影响突触信号，它已被证明具有挑战性的定量测量在成年神经元中的变化的蛋白质的表面表达。在这项工作中，我们提出一个可靠的方法来治疗急性脑片表面标记蛋白体外 。脑片制剂有实用的电生理记录的悠久历史，因为他们保持突触连接和细胞存活率高达小时的准备后。另外，切片的策略可以进行优化，以保持各种感兴趣的大脑区域之间的特定突触连...

内吞贩运是微调的各种整合膜蛋白的细胞膜呈现一个无处不在的细胞机制。内吞作用提供重要的营养物质向细胞内环境1和麻痹受体信号响应受体激活2。内吞再循环回至质膜可以通过增加蛋白质的表达水平在细胞表面3还增强细胞信号传导。此外，膜运输扰动有牵连的许多疾病和病理条件下4,5，强调需要调查支配蛋白质内吞贩运分子机制。而许多蛋白质​​利用经典的网格蛋白依赖的内化机制，越来越多的证据，在过去数年证明了多个网格蛋白独立的内吞机制支配的增加数组的内吞潜在蛋白质6,7。因此，有必要探讨促进生理系统有关拐卖的内吞机制已有很大的增长。 在大脑中，受体，离子通道和神经递质转运蛋白的内吞贩运在建立突触可塑性8-11和应对滥用药物12-15，最终影响神经细胞的兴奋性和突触反应的基础性作用。两者都不迄今为止，大多数神经元拐卖的研究都依赖于异源表达系统或原代培养的神经元，可以可靠地反映在作怪成年神经元的机制。在这里，我们报告说，使用表面生物素定量测量表面蛋白水平在成年啮齿类动物源性急性脑切片的方法。使用这种方法，我们提出了证明小鼠纹状体多巴胺转运蛋白迅速内化在数据重响应对佛波酯介导的蛋白激酶C（PKC）的活化。

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

调节内吞贩运是中央机制，促进多种神经调节活动，通过动态控制在分钟时间尺度受体，离子通道和转运细胞表面呈现。还有就是控制个别蛋白质的内吞机制拐卖了广泛的多样性。研究调查贩卖人口的分子基础主要依赖于表面生物素定量测量响应外源性刺激和基因操作改变膜蛋白的表面表达。然而，这种方法已被主要限于培养的细胞，这可能不是忠实地反映在游戏中成年神经元的生理学相关的机制。此外，培养细胞的方法可能低估贩运机制特定地区的差异。在这里，我们描述了一个扩展的细胞表面生物素的急性脑片制备的方法。我们表明，该方法提供了高精确度的方法来测量在膜表面蛋白水平的快速变化在成年神经元。这种方法可能在神经元内吞贩运领域广泛的实用性。

神经元的多巴胺转运蛋白被内响应于PKC活化在细胞系16-20。尽管许多报告表明在各种细胞系和表达系统的PKC诱导的DAT表面的损失，已经具有挑战性证实这一发现在培养的多巴胺能神经元21-23。我们用小鼠纹状体切片直接测试DAT是否内化响应于PKC活化在成年多巴胺能神经元。以下切片制备，切片沿着从相同平面上的中线和切片切成对半收治±1μM佛波醇肉豆蔻酸酯-13乙酸酯（PMA）处理30...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

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

神经元膜贩运动态控制细胞膜蛋白的可用性和显著影响神经传递。迄今为止，它已经挑战来衡量成人的神经元的神经元内吞贩运。在这里，我们描述了一个非常有效的，量化的方法来衡量在急性脑切片的表面蛋白表达体外快速变化。

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 ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

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

12.8K 次观看.  University of Massachusetts Medical School. 神经元膜贩运动态控制细胞膜蛋白的可用性和显著影响神经传递。迄今为止，它已经挑战来衡量成人的神经元的神经元内吞贩运。在这里，我们描述了一个非常有效的，量化的方法来衡量在急性脑切片的表面蛋白表达体外快速变化。 

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

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

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 ...

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

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

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

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

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 ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门：成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...