这项工作得到了国家卫生研究院 (R01-NS084941) 和亚利桑那生物医学研究委员会 (ADHS16-162406) 提供给 PTR 的赠款的支持。T32-HL007249 已经获得了从博士后任命到国家卫生培训补助金研究所的支持。

下面概述的所有程序都已由一个机构动物护理和使用委员会 (IACUC) 批准, 并符合国家卫生研究院和动物研究: 报告体内实验 (到达) 指南。协议的过程流程在图 1中描述。
1. 程序的设置
<ol><li>准备脑微血管缓冲 (BMB)。从称重54.66 克 d-甘露醇, 1.90 克 EGTA, 1.46 克 2-氨基-2-(羟甲基)-13-丙二醇 (即, 三) 基为清洁烧杯开始。加1.0 升去离子水。使用磁力搅拌器混合 BMB 缓冲器的元件。一旦溶液彻底混合, 将 pH 值调整为 7.4, 使用1.0 米 HCl。</li>
	<li>在麻醉动物之前, 准备26% 葡聚糖 (兆瓦 7.5万) 溶液 (w/v)。按照以下步骤中所述, 准备葡聚糖溶液。 注意: 提前准备这个解决方案是至关重要的, 因为葡聚糖可以大约 1.5-2 小时的时间溶解在水中的缓冲液中。<ol>
<li>将粉葡聚糖 (26 克/100 毫升缓冲) 称量成干净的烧杯。</li>
		<li>测量出适当的 BMB 体积, 并分配到一个单独的, 干净的烧杯。</li>
		<li>慢慢地将 BMB 倒入含粉状葡聚糖的烧杯中。用玻璃搅拌棒在 BMB 的浇注过程中, 手工混合粉葡聚糖。</li>
		<li>使用前立即将 pH 值调整为7.4 和1.0 米 HCl。 注: 典型的从12个大大鼠 (男性或女性) 脑微血管的分离; 3 月的年龄; 200-250 克每一个) 需要200毫升葡聚糖溶液 (即52毫升的 BMB)。</li>
	</ol>
</li>
</ol>2. 大大鼠脑组织的提取
<ol><li>经过所需的实验治疗, 麻醉大大鼠使用氯胺酮 (50 毫克/千克; 2.5 毫升/千克 ip) 和甲苯噻嗪 (10 毫克/毫升; 2.5 毫升/千克 i, p.)。稀释氯胺酮和甲苯噻嗪在0.9% 盐水到最后浓度的20毫克/毫升和4.0 毫克/毫升分别。弄死动物通过斩首使用削尖的断头台根据 IACUC 指南。对雄性和雌性大大鼠使用相同的手术方法。</li>
	<li>用手术剪刀做一个横切口切除大鼠颅骨的皮肤。</li>
	<li>使用 rongeurs, 小心地取出颅骨板并暴露大脑。</li>
	<li>用刮刀取出大脑。将大脑分离, 将孤立的脑组织放置在含有5毫升 BMB 的50毫升圆锥管中。添加 1.0 ul 的蛋白酶抑制剂鸡尾酒每1.0 毫升的 BMB 缓冲区立即使用前。</li>
</ol>3. 脑处理
<ol><li>将脑组织从50毫升锥形管转移到干净的培养皿中。</li>
	<li>使用镊子, 轻轻滚动12.5 厘米直径滤纸的大脑, 以消除外层脑膜, 这是松散地坚持到大脑皮层。轻轻地按下脑组织对滤纸和卷的组织再次。在此步骤中经常打开过滤器纸张。</li>
	<li>用镊子将脉络丛从大脑半球分离出来。 注: 脉络丛出现于脑室表面的透明膜组织。</li>
	<li>用镊子轻轻地压扁脑组织, 取出残余的脑膜和嗅觉灯泡。</li>
	<li>将皮质脑组织放入冷冻玻璃砂浆中。加入5.0 毫升的 BMB 含有蛋白酶抑制剂鸡尾酒的砂浆。</li>
	<li>使用架空功率均质机, 融汇脑组织使用15上下中风在 3700 rpm。使用10毫升砂浆和杵组织磨床进行均匀化。在每个样品的均匀化之间, 用70% 乙醇清洗杵。 注意: 均匀性笔画应在速度和幅度上保持一致。</li>
	<li>将匀浆倒入标签离心管。</li>
</ol>4. 离心步骤
<ol><li>添加8.0 毫升26% 葡聚糖溶液, 每个标签离心管包含脑匀浆。</li>
	<li>反转两次管, 然后彻底涡流样品。使用多个角度进行涡流, 以确保大脑匀浆液与26% 葡聚糖溶液彻底混合。</li>
	<li>离心机样品在 5000 x g为15分钟在4°c。 注意: 对于某些分析, 将脑微血管与脑实质进行比较是有益的。如果需要对脑实质中蛋白质表达的评估, 从这个离心步骤 (即脑实质分数) 中的上清可以收集和储存在-80 摄氏度, 以供将来使用。</li>
	<li>使用真空吸尘器和玻璃巴斯德吸管去除上清液。 注意: 必须注意不要干扰颗粒。否则, 将减少所采集的脑微血管数量, 这将显著降低该过程中蛋白质的产量。</li>
	<li>并用重悬在含有蛋白酶抑制剂鸡尾酒的5.0 毫升 BMB 中的颗粒 (即 1.0 ul 蛋白酶抑制剂鸡尾酒每1.0 毫升的 BMB 缓冲器)。涡流颗粒, 确保彻底搅拌。</li>
	<li>将8.0 毫升26% 葡聚糖添加到每个离心管和涡流中, 如本协议步骤 4.1-4.2 所述。</li>
	<li>离心机样品在 5000 x g为15分钟在4°c。</li>
	<li>使用真空瓶和玻璃吸管, 吸入上清, 并确保含有脑微血管的小球不会被破坏。 注: 附着在管壁上的多余材料不含微血管, 应仔细清洗和清除。</li>
	<li>重复步骤4.5 至 4.8, 另外两次。</li>
	<li>4葡聚糖离心步骤完成后, 在每个颗粒和涡流中加入5.0 毫升的 BMB, 以并用重悬样品。 注意: 在步骤4.10 完成后, 可以收集整个微血管来分析蛋白质的定位。这可以通过采取 50 ul 整除重新悬浮微血管颗粒和涂抹在玻璃显微镜幻灯片上完成。微血管然后热固定在95°c 10 分钟在一个加热块, 然后在冰冷的乙醇固定, 另外10分钟, 然后可以存储在4°c, 直到需要进行成像研究。</li>
</ol>5. 离心制备全脑微血管膜标本
<ol><li>将样品转移到冷冻玻璃均质机上。</li>
	<li>使用架空功率均质机, 融汇脑组织使用8-10 上下中风在 3000 rpm。采用10毫升砂浆和杵组织磨床进行均匀化。在每样样品均匀化后用70% 乙醇清洗杵。</li>
	<li>将样品转移到清洁 ultracentrifuge 管。数和重量每个单独管。平衡和对管之前加载到 ultracentrifuge 转子。 注: 所有 ultracentrifuge 管的称量和配对必须与瓶盖一起进行, 以确保准确测量管的重量。</li>
	<li>离心机样品在 15万 x g为1小时在4°c。</li>
	<li>使用真空瓶和玻璃巴斯德吸管, 吸入上清, 小心不要扰乱毛细血管膜颗粒。</li>
	<li>根据颗粒的大小, 添加适当的存储缓冲量, 这是由去离子水和 BMB 的 1:1 (v/v) 比率组成。通常, 颗粒在 400-500 ul 的存储缓冲区中悬浮。添加蛋白酶抑制剂鸡尾酒到存储缓冲区的比率为 1.0 ul 每1.0 毫升的存储缓冲区。</li>
	<li>在存储缓冲区中并用重悬颗粒的涡流样品。</li>
	<li>使用玻璃巴斯德吸管, 转移毛细管膜样品到一个标记的1.5 毫升离心管。</li>
	<li>通过针头注射器 5x, 以确保样品完全混合, 并没有细胞聚集存在。</li>
	<li>将样品存放在摄氏-80 摄氏度, 直到需要进行分析。 注: 用布拉德福德法测定各微血管膜样品的蛋白质含量。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+maintenance+and+disruption+of+the+blood-brain+barrier.">Development, maintenance and disruption of the blood-brain barrier.</a> Nat Med. 19 (12), 1584-1596 (2013).</li><li>Abdullahi, W., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Expression+of+P-glycoprotein+and+Organic+Anion+Transporting+Polypeptides+at+the+Blood-Brain+Barrier:+Understanding+Transport+Mechanisms+for+Improved+CNS+Drug+Delivery?.">Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?.</a> AAPS J. 19 (4), 931-939 (2017).</li></ol>

作者没有什么可透露的。

本文介绍了一种简单有效的从大鼠脑组织中分离的微血管制备膜蛋白样品的方法。在文献13、20、21、22中, 已报告了几种分离大鼠脑微血管和/或生成来自孤立微孔的膜制剂的方法。,24. 虽然上文所述的微血管隔离协议在原理上是相似的, 但这一方法已经得到了优化, 能够制备出...

血脑屏障 (BBB) 存在于中枢神经系统 (CNS) 与系统循环之间的界面, 在维持脑稳态中起着至关重要的作用。具体地说, 脑屏障功能精确控制大脑胞外液中的溶质浓度, 并有效地提供脑组织所需的养分, 以满足中枢神经系统1的相当大的新陈代谢需求。这些作用意味着脑屏障, 主要存在于微血管内皮细胞的水平, 必须拥有离散机制, 使某些物质能够进入大脑实质, 同时确保潜在的有害外来化合物不能积累。事实上, 脑微血管内皮细胞不是 fenestrated 和表现有限的吞饮, 这确保缺乏非选择性通透性 2.此外, 脑微血管内皮细胞表达紧密连接和 adherens 结蛋白, 在相邻的内皮细胞之间形成物理 "海豹", 并极大地限制血液传播物质在脑中的旁细胞扩散。实质。事实上, 内源和外源物质的选择性渗透性需要吸收和流出转运体的功能表达式3。总的来说, 紧密的连接, adherens 连接, 和运输工作, 以保持独特的屏障性能的血屏障。
脑屏障是一个动态屏障, 反应生理, 病理生理学和药理刺激。例如, 缺氧/复氧应力已被证明可以调节临界紧致结蛋白 (即occludin、zonulae occluden-1 (ZO-1)) 的表达, 这与增加旁细胞通透性的血管标记有关, 如作为蔗糖4,5,6。在创伤性脑损伤的设置方面也有类似的观察:7和外围炎症疼痛8,9。这些同样的疾病也可以调节在 BBB10,11,12,13,14的传输机制。事实上, 缺氧/复氧损伤增强了有机阴离子转运多肽 1a4 (Oatp1a4) 在脑屏障中的功能表达, 这可能导致特定 Oatp 运输基质的血液到大脑传输显著增加, 如胆酸和阿托伐他汀13。脑屏障的特性也可以通过药物治疗本身来改变, 这种机制可以为大脑和药物相互作用的药物有效性的深刻变化奠定基础。例如, 扑热息痛靶向脑微血管内皮细胞核受体信号机制, 增加临界外排转运蛋白 p-糖蛋白 (p gp) 的功能表达, 并修改时间依赖性镇痛由吗啡、阿片类镇痛药和建立的 P gp 运输基质15授予。彻底了解血脑屏障的变化, 这可能是由疾病或药物引起的, 也需要识别和鉴定特定的调控机制, 控制这些修改。事实上, 在控制紧结蛋白的分子表达的脑微血管内皮细胞中发现了离散信号通路16,17和运输商15, 18,19。这些观察结果表明, 复杂的分子通路参与了对脑屏障紧密连接和转运体的健康和疾病的调控。
在研究脑屏障的一个重大挑战是一个简单有效的方法, 从实验动物的大脑组织中分离微血管和后续制备膜样品的绝对要求。这些样品必须准备好, 使它们既丰富的脑微血管内皮细胞和有限的存在其他细胞类型。在过去的几年中, 科学家在科学文献13、20、21、22中报告了多种从啮齿动物脑中分离微血管的方法。本文介绍了一种简单、健壮、可重现的方法, 用于分离大鼠脑中的微血管, 并制备出一种能用于蛋白质表达分析的内皮膜富集样品。这种微血管隔离协议的优点是能够获得高质量的样品制备, 并能从单个实验动物那里得到足够的蛋白质产量。这样就可以考虑蛋白质表达中动物间的变异。此项协议的进展大大提高了脑屏障研究的健壮性, 因为现在可以避免对脑屏障蛋白变化的真实程度进行过度估计 (或估计不足)。另外, 加入葡聚糖的多重离心步骤, 可以改善实验样品中微血管的富集, 同时有助于去除不必要的细胞成分, 如神经元。

<table>
	<tbody>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>#M4125</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>#E3889</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>#T1503</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Dextran (MW 75,000)</td>
			<td>Spectrum Chemical Mftg Corp</td>
			<td>#DE125</td>
			<td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td>
		</tr>
		<tr>
			<td>Zetamine</td>
			<td>MWI Animal Health</td>
			<td>#501072</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Western Medical Supply</td>
			<td>#5530</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Western Medical Supply</td>
			<td>N/A</td>
			<td>General anesthetic diluent</td>
		</tr>
		<tr>
			<td>Filter Paper (12.5 cm diameter)</td>
			<td>VWR</td>
			<td>#28320-100</td>
			<td>Used for removal of meninges from brain tissue</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td>#60.540.386</td>
			<td>Disposable tubes used for dextran centrifugation steps</td>
		</tr>
		<tr>
			<td>Pierce&#8482; Coomassie Plus (Bradford) Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>#23236</td>
			<td>Measurement of protein concentration in membrane preparations</td>
		</tr>
		<tr>
			<td>Wheaton Overhead Power Homogenizer</td>
			<td>DWK Life Sciences</td>
			<td>#903475</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>10.0ml glass mortar and pestle tissue grinder</td>
			<td>DWK Life Sciences</td>
			<td>#358039</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>#H1758</td>
			<td>Required for pH adjustment of buffers</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>#23210</td>
			<td>Protein standard for Bradford Assay</td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>#91100-12</td>
			<td>Used for dissection of brain tissue</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>#16020-14</td>
			<td>Used for opening skull to isolate brain</td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>#352070</td>
			<td>Used for collection of brain tissue following isolation</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>ThermoFisher Scientific</td>
			<td>#13-678-20C</td>
			<td>Used for aspiration of cellular debris following dextran spins</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>#459836</td>
			<td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>#41121703</td>
			<td>Used for ultracentrifugation of samples</td>
		</tr>
	</tbody>
</table>

a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

血脑屏障 (BBB) 是一种动态屏障组织, 对各种病理生理和药理刺激反应。这些刺激导致的这种变化可以极大地调节药物向大脑的传递, 并通过扩展, 在治疗中枢神经系统疾病方面造成相当大的挑战。许多影响药物治疗的脑屏障变化涉及蛋白质的局部化和表达在内皮细胞的水平。事实上, 这种关于健康和疾病的脑屏障生理学的知识引起了对这些膜蛋白研究的极大兴趣。从基础科学研究的角度来看, 这意味着需要一个简单但健壮和可重复的方法来分离从实验动物身上采集的脑组织中的微血管。为了从新鲜分离的微血管中制备膜样品, 必须在内皮细胞中丰富样品制剂, 但在神经血管单元的其他细胞类型 (如星形胶质细胞、小胶质、神经细胞、毛细血管)。一个额外的好处是能够从单个动物身上制备样本, 以便在实验人群中捕捉蛋白质表达的真实变异性。本文详细介绍了一种用于大鼠脑微血管分离和制备膜样品的方法。通过使用四离心步骤, 在样品缓冲中加入葡聚糖, 可获得从样品中提取的微血管。该协议可以很容易地由其他实验室为自己的具体应用而调整。从该协议生成的样本已经证明, 从蛋白质分析实验中获得健壮的实验数据, 可以极大地帮助理解对生理、病理生理学和药理刺激的脑屏障反应。

实验流程为分离的大鼠脑微血管和为制备血管膜样品显示在图 1中。使用此处介绍的过程, 成功地从大鼠脑中分离出完整的微血管 (图 2A)。这些船只是在完成离心与葡聚糖, 并立即开始离心准备膜样品 (即完成步骤 4.10) 后获得。在这个图像中, 微血管被染色使用抗体对 Oatp1a4, 一个运输者已经证明是良好的表达在血浆膜的脑微血管?...

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

本文介绍了一种分离大鼠脑微血管及制备膜样品的方法。该协议具有明显的优势, 生产丰富的微血管样品与可接受的蛋白质产量从个别动物。样品可用于大脑微血管内皮的健壮蛋白质分析。

一种简便、可再生的新分离大鼠脑微血管膜样品制备方法

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

a-simple-reproducible-method-to-prepare-membrane-samples-from-freshly

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.0K 次观看.  University of Arizona, Tucson. 本文介绍了一种分离大鼠脑微血管及制备膜样品的方法。该协议具有明显的优势, 生产丰富的微血管样品与可接受的蛋白质产量从个别动物。样品可用于大脑微血管内皮的健壮蛋白质分析。

视频: A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

粉条和Capellini处理测试：在大鼠和小鼠的灵巧的前爪功能简单的定量措施

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

科研

神经科学

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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 ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

一种简便、可再生的新分离大鼠脑微血管膜样品制备方法

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