这项工作是由神经生物有限公司资助的。我们想感谢 Ferrati 博士和塞尔希奥·比埃拉·德梅洛博士圣乔瓦尼罗 (神经生物学) 对手稿的评论和建议。

所有动物研究都是根据批准的议定书进行的。
注意: 在本节中, 提供了在实验过程中执行的主要阶段的顺序和建议的时间间隔 (图 1)。此外, 对该议定书的一步一步的描述由一个说明性的小组补充, 显示了在潜伏期后从脑切除到组织均匀化的关键行动 (图 2)。前面介绍了用于构建该设备的材料和说明的详细信息以及对世行分析的后续阶段的细节22。
1. 外科工具、Vibratome 设置和治疗准备
注意: 在开始实验过程之前, 请执行以下步骤:
<ol><li>准备两种不同的人工脑脊液 (aCSFs), 一个用于脑切片, 另一个用于孵化步骤, 如前所述的29。简单地说, 两个 aCSFs 的工作浓度 (毫摩尔) 为 "切片" aCSF: 120 氯化钠, 5 氯化钾, 20 NaHCO 3, 2.4 CaCl 2, 2 MgSO 4, 1.2. 2 PO 4 和10葡萄糖;6.7 HEPES 盐和 3.3 HEPES 酸;pH 值: 7.1。而为 "录音" aCSF: 124 氯化钠, 3.7 氯化钾, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1.3 2 PO 4 和10葡萄糖;pH 值: 7.1。</li>
	<li>混合解决方案, 然后放在冰和氧切片 aCSF, 以便让它冷, 而删除大脑和后续切片。保持室温 (RT), 并向录音 aCSF 提供氧气。</li>
	<li>将器械置于解剖罩下, 用于斩首和脑切除,即、断头台、手术解剖包 (剪刀、镊子、刀片) 和刮刀。</li>
	<li>通过选择切片厚度 (300 µm) 来设置 vibratome 参数, 频率为 7, 速度为6。将 vibratome 腔插入适当的空间, 并将其与冰环绕。</li>
	<li>打开卡波金 (95% O2, 5% CO2) 阀, 氧在脑切片过程中的 "切片" (aCSF)。选择最小流量, 大约2毫升/分钟。</li>
	<li>用两个15毫升的管子来准备测试的条件。添加一个管10毫升的 "记录" aCSF 单独 (对照组) 和其他, 10 毫升的 "录音" aCSF 丰富的 T30 集中2µM (治疗组)。涡流两管, 并保持他们在冰上, 直到他们使用在安装准备阶段 (3 节)。</li>
</ol>2. 麻醉、脑提取和切片
注: 本研究采用9种野生 P14 雄性大鼠。脑提取和后续切片构成了该议定书的一个关键部分, 因为它们应迅速执行 (10 分钟以内), 以防止或至少减慢组织退化过程。
<ol><li>在棉层添加1.5 毫升 100% w/瓦的异氟醚到感应腔内, 将动物放在里面, 然后关闭盖子。等待, 直到动物被深深麻醉, 确认没有踏板反射, 然后斩首动物使用断头台。</li>
	<li>把动物的头放在装有冰冷切片 aCSF 的罐子里, 在脑移除步骤中 (图 2A -c)。</li>
	<li>用手术刀片切开头骨上的皮肤, 然后用剪刀在中线后切开头骨, 从后部开始前方直到到达 OBs 上方的骨骼 (图 2A)。</li>
	<li>用一对镊子将头骨两侧侧拉, 以便能够进入大脑 (图 2B)。插入一个铲腹部到大脑, 轻轻地舀出来, 并保持它在冰冷的 "切片" aCSF 水合约5分钟 (图 2C)。</li>
	<li>使用刀片 (图 2D) 将大脑放在滤纸上, 并切断小脑。将大脑垂直粘附在 vibratome 盘上, 并将其插入切片腔内, 用冰冷 "切片" aCSF 填充, 并提供氧气 (图 2E)。</li>
	<li>调整切割间隔, 然后进行脑切片 (图 2F)。收集包含感兴趣区域的部分, 如前面描述的22所述的内侧隔膜 (MS)、DBB 的对角线带和 innominata (SI)。从上到下, 三切片包含 BF (图 2G) 的延髓 (R)、中间 (I) 和尾部 (C) 部分。</li>
	<li>沿中线除以小剪刀, 获得两个镜面 hemislices (图 2H), 分别用作同一解剖平面上的控制和处理条件。</li>
	<li>将 hemisections 在 vibratome 室中保持 5-10 分钟, 然后用玻璃吸管将其转移到含有记录 aCSF 的鼓泡锅中。保持在 RT 约30分钟恢复。</li>
	<li>同时填写三个玻璃瓶, 3 毫升记录 aCSF (以前在步骤1.6 中编写) 为对照组和三瓶与3毫升的 aCSF 丰富的 T30 (以前在步骤1.6 中编写) 为治疗组。</li>
</ol>3. 安装准备
<ol><li>将 hemisections 转移到相应的玻璃小瓶 (图 2I) 中, 以便为控制组 (绿色框架、图 2J) 连续三个 hemislices (包括延髓、中间和尾部高炉区域) 及其匹配处理组的对应项 (红色框架,图 2J)。</li>
	<li>用两个不同的刷子转移脑组织, 一个用于控制 hemislices, 另一种用于治疗的对应物, 以防止条件之间的任何污染。</li>
	<li>用相应的盖子封住每个玻璃瓶, 呈现氧气针 (21G) (图 2J)。将设备的主管连接到卡波金源 (图 2K)。在整个潜伏期提供氧气到 hemislices 的最低流速为2毫升/分钟, 以避免脑组织的任何移动和可能的机械损伤在实验期间。开始5小时的孵化。在5分钟内执行步骤 3.1-3.3。 注: 经常检查 (每 15-20 分钟) 是否将氧气供应给所有的瓶子, 其流量不会从底部移动组织。</li>
</ol>4. 均匀化样品
<ol><li>停止孵化后的氧气流, 并从瓶子里取出所有盖子。使用适当的画笔将 hemislices 转移到含有250µL 溶解缓冲器的1.5 毫升管中, 在冰上保持管 (图 2L)。为了使裂解缓冲, 混合 PBS 1x 与磷酸酶和蛋白酶抑制剂鸡尾酒都稀释 1:100, 并融汇组织与不同的杵, 以防止污染之间的样本。在 10-15 分钟内完成这些操作。</li>
	<li>离心机的脑裂解在 1000 x g 5 分钟4摄氏度。将上清液转移到新管中, 将样品保持在-80 摄氏度。</li>
</ol>5. 蛋白质浓度和印迹分析的阅读
<ol><li>确定每个样品的蛋白质浓度, 并随后为西方印迹分析准备整除数, 如前所述的 22.</li>
	<li>进行统计学分析, 评价治疗对蛋白质表达的影响。</li>
</ol>

<ol>
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X., Lidsky, T., Wegiel, J., Grundke-Iqbal, I., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolically+active+rat+brain+slices+as+a+model+to+study+the+regulation+of+protein+phosphorylation+in+mammalian+brain.">Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain.</a> Brain Research Protocols. 6 (3), 134-140 (2001).</li></ol>

作者宣布了相互竞争的金融利益。苏珊. 绿地是一家私营公司的创始人和总裁, 拥有公司的股份。艾玛努艾拉 Brai 和安东内拉 Cogoni 是神经生物有限公司的员工。

该协议的主要方面是基于已建立良好的 "体外" 大脑技术, 允许同步测试两个镜面 hemislices, 从同一解剖平面获得, 监测其在应用特定条件 (控制或治疗);因此, 这提供了一个实验范式, 尽可能严密控制。如在神经退行性事件22中所见, 以时间、剂量和地点特定方式评估与神经元损伤有关的不同 neurochemicals 的可能性是一个基本特征。此方法将体内生理环境的优点与体外</...

AD 是一种慢性病理学特征的逐步神经退行性损伤影响不同的大脑区域, 如嗅皮质 (EC), BF, 海马 (HC), 和嗅觉灯泡 (OB)1,2,3, 4,5。AD 开发的后期阶段导致了逐渐的认知下降, 使这种疾病成为最常见的痴呆症, 大约占所有病例的 70%6。尽管人们广泛尝试了解导致 AD 的最初阶段, 但目前还没有一个明确的实验性指示来阐明它们。此外, 最流行的理论-"淀粉样假说"-越来越受到质疑, 因为它没有提供一个完整的形象解释广告病理, 也没有一个药物的目标已证明有效的7,8 ,9。
另一种被越来越关注的理论表明, 变性期间发生的初始机制与主要易受 AD3、10、11的神经元簇有关.,12,13,14. 这种异构的蜂窝中枢包含在 BF、中脑和脑干中, 项目到多个区域, 如 EC、HC 和 OB15,16。尽管它的神经元形态学和神经递质合成的多样性, 这一核心细胞在表达疼痛的共同特征, 也可以有一个非酶功能17,18。这种非经典的角色作为一个新的信号分子介导钙 (Ca2 +) 流入神经元, 可以接受营养或有毒事件有关 Ca2 +剂量, 可用性和神经元年龄17,18,19。
在变性期间, 观察到的细胞损失可能因此与这个非酶函数17,18,20, 这是归因于30mer 肽 (T30) 从疼痛 C 总站裂了20. 根据以前的结果, 进行细胞培养和光学成像18,21的准备工作, 我们通过一种新的方法, 通过一种基于前体大鼠脑切片的高炉结构, 表明 T30 诱导类似于 AD 的配置文件22。具体地说, 这种新的方法提供了比细胞培养更具生理的情况, 因为它保持了完整组织的许多特征, 从解剖到电路保存, 尽管时间窗口小时。我们应用这个协议来探索在变性的早期阶段发生的事件, 监测 T30 应用时的急性反应。
尽管大量的文献, 使用脑切片来调查隐含在神经损伤或神经发生的分子通路23,24, 此协议首次提供更直接和敏感的读出比较脊髓切片的常用用法。然而, 正如脊髓脑部分的情况一样, 这种急性切片程序也可以用于几个目的, 如评估神经保护或毒性分子, 发现在特定过程中发生的主要分子变化,中枢神经系统相关病理学的免疫组化分析和药理学测定。

<table>
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S7653</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9333</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5761</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>63138</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5655</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes salt</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H7006</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes acid</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H3375</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>G7528</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>223506</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>T30 peptide</td>
			<td>Genosphere Biotechnologies, France</td>
			<td></td>
			<td>AChE-derived peptide tested</td>
		</tr>
		<tr>
			<td>Surgical dissecting kit</td>
			<td>World Precision Instruments, USA</td>
			<td>Item #: MOUSEKIT</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Swann-Morton, UK</td>
			<td>BS 2982</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific, USA</td>
			<td>11566873</td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td></td>
			<td></td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Germany</td>
			<td>VT1000 S</td>
			<td>Slicing</td>
		</tr>
		<tr>
			<td>Brushes</td>
			<td></td>
			<td></td>
			<td>Tissue handling</td>
		</tr>
		<tr>
			<td>Oxygen canister</td>
			<td></td>
			<td></td>
			<td>Sectioning and incubation phase</td>
		</tr>
		<tr>
			<td>1x Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2438-4</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors</td>
			<td>Fisher Scientific, USA</td>
			<td>1284-1650</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche complete PIC, USA</td>
			<td>4693116001</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Starlab, UK</td>
			<td>I1415-5390</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm Protein Assay</td>
			<td>Thermo Scientific, USA</td>
			<td>22660</td>
			<td>Protein concentration</td>
		</tr>
	</tbody>
</table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

尽管许多研究试图开发可靠的动物模型, 反映了变性的主要过程, 但很少有人被广泛接受。在这里, 我们提出了一个新的程序, 适应于已知的前体脑切片技术, 它提供了一个更接近的在体内类似的情况下, 比的试管准备, 以调查早期事件触发细胞变性, 作为观察到阿尔茨海默病 (AD)。这个变异包括简单和容易地可再生的步骤, 使保存选定的脑区域的解剖细胞构筑和它的地方功能在一个生理环境中。不同的解剖区域可以从同一大脑中获得, 提供了在一个站点、剂量和时间依赖性的方式下进行多项实验的机会。可能影响与此方法有关的结果的潜在限制与组织的保存有关,即,在切片和孵化步骤和剖面厚度期间保持其解剖完整性, 这能影响生化和免疫组化分析。这种方法可以用于不同的目的, 如探索分子机制参与生理或病理条件, 药物筛选, 或剂量反应化验。最后, 该议定书还可以减少在行为研究中使用的动物数量。在这里报告的应用最近首次被描述和测试的体外大鼠脑切片包含基底前脑 (BF), 这是一个主要影响的脑区的 AD. 具体地说, 它已经证明, 从乙酰胆碱酯酶 (疼痛) 的 C 终点产生的毒性肽的管理可能会提示一个 AD 样的轮廓, 触发, 沿 antero-后轴的 BF, 差异表达AD 中的蛋白质改变, 如 alpha7 烟碱受体 (α7 nAChR), 磷酸化头 (p 头) 和淀粉样蛋白β (Aβ)。

这里提出的协议表明, 在含 BF 的部分 (图 3A) 中, 对有毒肽 T30 的管理, 以与站点相关的方式调节α7-nAChR、p 头和 Aβ的表达式。烟碱受体显示, 延髓处理的 hemislice 显著增加, 与它的控制对应 (切片 1, p = 0.0310) (图 3B), 而中间切片不显示任何变化的两个条件 (切片 2, p = 0.1195) (图 3B)。在后段中, T30-exp...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

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

我们提出了一种方法, 它可以进一步深入了解基础变性的早期事件, 并基于建立的前体脑技术, 结合体内和试管内实验的优点。此外, 它是一个独特的机会, 直接比较治疗和未经治疗的小组在同一解剖平面。

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, ...

an-alternative-approach-to-study-primary-events-neurodegeneration

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

6.8K 次观看.  Culham Science Centre. 我们提出了一种方法, 它可以进一步深入了解基础变性的早期事件, 并基于建立的前体脑技术, 结合体内和试管内实验的优点。此外, 它是一个独特的机会, 直接比较治疗和未经治疗的小组在同一解剖平面。

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

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

在大鼠脑片的单通道和补品GABA激活电流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

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 ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

利用体外大鼠脑切片研究变性主要事件的一种替代方法

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在 C 在体内激光干切断线虫

一个快速高分辨率的荧光成像的方法在半厚的脑片

在大鼠脑片的单通道和补品GABA激活电流

神经元形态的研究方法:

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

大脑切片生物素化:一个

鼠标后脑

一个

在脑片恐惧电路体外光遗传学解剖

在基于生物发光钙指标GFP的水母体内脑功能成像方法