胶原基质试验已制定和完善了许多不同的研究小组在过​​去的二，三十年的工作。多巴胺和纹状体植这里描述的方法，从这些研究中大大受益。此外，要感谢她的帮助下设立纹状体植文化Asheeta普拉萨德。在实验室工作是由人类前沿科学计划组织（职业发展奖），荷兰健康研究与发展组织（ZonMW VIDI和弦ZonMW），Europanian联盟（mdDA NeuroDev，FP7/2007-2011的格兰特222999）（RJP），荷兰科学研究组织（TopTalent; ERES）时。

1。鼠尾胶原的制备<ol><li>收集6-10成年鼠尾（这是可能的尾巴，直到使用储存在-20°C）。 </li><li>隔夜浸泡在95％乙醇的尾巴在室温（RT）。 </li></ol> 解剖老鼠尾巴（组织培养罩）： （保持在70％乙醇的工具时，不使用他们，并确保在整个过程中使用的所有解决方案，工具和玻璃器皿是无菌） <ol start="3"><li>收集从尾巴肌腱，切断尾巴尖。同一个钳子的大尾巴结束。用另一个钳子举行，弯曲和折断尾部接近其他镊子。拉开和肌腱会被留下（ 图1A，B）的。 </li><li>收集在无菌的H 2 O的肌腱和收集所有筋后用无菌的H 2 O移动到一个新的petridish </li><li>服用2-3筋，切丝用2对为CEPS在一个无菌的H 2 O的新petridish删除非肌腱组织，如静脉（肌腱组织是有光泽和反射图1C）。拆除所有非肌腱组织后，每尾产生约100-150毫克的肌腱。 </li><li>肌腱转移300毫升无菌的3％醋酸和隔夜慢慢搅拌4°C。 </li><li>添加额外的200毫升无菌的3％醋酸，搅拌慢慢一夜之间在4°C。 </li><li>离心机的醋酸溶液中，包含120分钟，4〜2700 XG筋°C间沉淀不溶解的肌腱和非肌腱组织。 </li><li>准备在离心透析管： <ul><li>切件40厘米</li><li> 5分钟烧开500μMEDTA </li><li>无菌Ĥ2 O的凉爽</li></ul></li><li>在透析管的一端绑一个结，并填写用上清液离心肌腱解决方案，在组织文化引擎盖冰的油管使用自动pipettE和25毫升吸管。避免产生气泡。结无菌铝箔冰商店的油管和油管的另一端，直到所有管件填补（ 图1D）。 </li><li>准备在组织文化引擎盖10无菌0.1X MEM，pH值4.0升。浮子管件和添加这些预冷0.1X的MEM溶液10升烧杯。透析在4°C，而在一夜之间慢慢搅拌。此透析除去多余的酸性物质，同时保持低的pH值。 </li><li>更换新的预冷10升0.1X的MEM搅拌一夜之间在4°C。 </li><li>更换新的预冷10升0.1X的MEM，重复上一步搅拌一夜之间在4°C。 </li><li>分装到无菌的50毫升管的胶原溶液。在4°C店等分保持6-12个月，仅处理在组织文化引擎盖的胶原股票。 </li><li>重要的是要测试是否需要稀释纯化的胶原质（0.1X的MEM）胶原MATR获得最佳效果第九检测。因此，产生胶原蛋白系列稀释（稀释的胶原蛋白，1:1，1:2和1:5稀释），并执行如下所述，以确定最佳的胶原蛋白稀释胶原基质检测。 </li></ol> 2。多巴胺中脑的解剖7,8 <ol><li>从母亲的子宫解剖E14.5小鼠胚胎和L15培养基上冰，直到需要。 </li></ol> 所有后续的解剖步骤进行在L15介质上冰。 <ol start="2"><li>剖析了大脑。 </li><li>取出切割沿内侧部分每端脑泡（为纹状体植使用的端脑泡）的前脑。 </li><li>取出脑膜护套。 </li><li>使背腹切只是脑弯曲的喙。 </li><li>再拍背腹切只是脑曲尾。 </li><li>使用1 microdissectio使沿背中线rostrocaudal切列印刀，露出底层的腹侧中脑组织。小心打腹侧脑组织，因为它包含的多巴胺能神经元（ 图2）。 </li><li>使两个rostrocaudal削减横向和腹侧中线平行，消除背部的脑组织。 </li><li>其余腹侧脑组织分为植用显微切割刀。 </li><li>存储在L15冰，直到使用含有5％胎牛血清培养基外植体。 </li></ol> 3。纹状体的剖析 所有的解剖步骤进行在L15介质上冰。 <ol><li>使用端脑泡在脑解剖解剖。 </li><li>取下丘脑，在丘脑和纹状体使用镊子之间的切割。 </li><li> 1正中侧面切喙纹状体删除，如嗅球延髓结构。位于尾端纹状体组织重复此步骤。 </li><li>位置其余切片获得的纹状体冠状（ 图2）。 </li><li>纹状体可以确认，作为一个组织的密度较小（更加透明）的一部分。到外植体，用显微切割刀解剖纹状体和切割。避免中线稍深组织紧密，其中包含神经细胞迁移的外侧和内侧的神经节隆​​起。 </li><li>存储在L15冰，直到使用含有5％胎牛血清培养基外植体。 </li></ol> 4。大会胶原基质的检测 胶原蛋白的制备（上冰的所有步骤，使用冷却枪头） <ol><li> 100μL的MEM 10倍和40微升1M碳酸氢钠（ 碳酸氢钠 ）混合860μL稀释后的胶原蛋白和保持在冰上。如果在这一点上的胶原蛋白升温，这将巩固。 </li><li>在4以及NUNC菜以及准备胶原（直径约5毫米）下降了20μL添加到盖玻片并让它在二氧化碳培养箱站在37°C和5％的CO 2为30分钟（环境氧浓度就足够了）。在此孵化的胶原蛋白会涂胶。 </li></ol> 设置共培养在胶原蛋白凝胶 <ol start="3"><li>胶原蛋白已糊化后，用200μl的尖端吸管传输多巴胺或纹状体植的胶原蛋白。 </li><li>移动在接近使用针彼此的外植体。让他们除了在距离约直径一植（200-300微米）（ 图2）。 </li><li>去除多余的介质，并加入20微升外植体上制备的胶原。这往往会导致植走动。定位用针如4.4中所述的外植体。 </li><li>让胶原在室温下15分钟固化，30分钟之后，在37°C和5％CO 2。 </li><li>胶原成立后，加入400μL电子xplant中期和增长为2-3天，在二氧化碳培养箱37℃，5％CO 2。 </li></ol> 5。通过免疫组化和定量分析 免疫组化 <ol><li>要解决植，轻轻在PBS中添加8％多聚甲醛400μL（PFA）培养基400μL（从而稀释至4％的煤灰）和静置在室温下为1小时。 </li><li>在室温下洗3倍的PBS 15分钟。 </li><li>阻止缓冲区（BB孵化; PBS + 1％FBS + 0.1％的Triton X-100）2小时。 </li><li>在4°C孵育过夜BB抗体对于多巴胺神经轴突使用的抗酪氨酸羟化酶抗体（1:1000）7，反DARPP32抗体（1:500）9纹状体轴突和可视化所有轴突反βIII微管蛋白（1:3000）7。 </li><li>在室温下在PBS洗5倍1小时。 </li><li> 4 BB（1:500）在适当的荧光标记的第二抗体，孵育过夜°C。从这个步骤，尽量减少外植体暴露在光线（例如覆盖铝箔）。 </li><li>在PBS洗一夜之间在4°C间在第二天洗。 </li><li>安装在显微镜植幻灯片使用延长抗淬灭试剂安装介质。添加玻璃显微镜幻灯片〜10微升下降。盖玻片，轻轻地放在植朝下安装介质下降。避免捕获的盖玻片下的空气非常轻轻地降低它的安装介质下降。 </li></ol> 量化计算的P / D比值 <ol start="9"><li>使用萤光显微镜的外植体（ 图3）获得数字图像。 </li><li>使用这些图像，每个象限分为外植体产生近端象限（即最接近相邻植植）和远端象限（最远从相邻expla远离外植体的一部分NT），4（图2）。 </li><li>测量在近端和远端象限植20最长的新兴突起的长度。 </li><li>使用20最长突起的平均长度来计算的P / D比值为每个个别的外植体。美联社/ D比值&gt; 1表明轴突的吸引力，而比&lt;1表示轴突的排斥。 </li><li>在某些情况下，密集的突起生长，防止个别突起长度的评估。在这种情况下，量化可以通过测量外植体的优势和领先的前轴突生长在近端和远端象限之间的距离。 </li></ol> 6。代表性的图像多巴胺和纹状体植培养成功后的轴突大量使用明场显微镜可见或可视作为反βIII的微管蛋白的免疫细胞化学（未显示）。这些轴突的一个子集，将多巴胺或S<html>作为可视化用免疫细胞化学triatal轴突（ 图3）。由外植体划分成象限描述和确定的P / D比值，假定轴突的吸引或排斥效果可量化的（ 图3E）。 <img alt="图1" src="/files/ftp_upload/3691/3691fig1.jpg" /> 图1。照片说明从鼠尾肌腱胶原蛋白的制备。拉除鼠尾）公开筋。乙）筋绳状束可见。 c）筋容易从静脉区分他们闪亮的白色外观。 d）溶解在乙酸筋后，将溶液转移至透析管。要保持胶原蛋白溶解冷，管放置在冰上的无菌铝箔。 E 2“SRC =”/ files/ftp_upload/3691/3691fig2.jpg“/&gt; 图2显示该程序的不同步骤。示意图。适当的大脑区域的解剖和削减产生植。一个单一的脑和纹状体植在胶原基质放置在靠近，离开增长为48-72小时，在37°C轴突荧光免疫组化，可视化和计算的P / D比值量化chemotropic反应。 <img alt="图3" src="/files/ftp_upload/3691/3691fig3.jpg" /> 图3。代表结果显示轴突生长在胶原基质的文化。一）中脑外植体染色与抗酪氨酸羟化酶抗体揭示轴突生长。虚线表示相邻的外植体。乙类）放大显示个人轴突和生长锥（箭头）。三）纹状体植沾满反DARPP32的。 d）C的放大显示个别突起。 é）为例，的P / D比值量化。外植体分为相等象限。近端象限正面临着相邻的外植体，而远端象限正面临着远离它。规模横道100微米。

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我们什么都没有透露。

这里描述的胶原基质试验已在过去的几十年里许多不同的实验室使用和改进探讨轴突导向分子和神经系统的各种（例如，见参考文献5-8）。这些研究表明，这种试验是研究轴突导向分子不同（中级）靶组织分泌的影响和调控的有力工具。 然而，应当指出，胶原基质是外环境（ECM）的替代品，但显然缺乏通常在ECM目前许多蛋白质​​。 ECM的组件被称为影响轴突导向分子10</s...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胎牛血清</td><td> BioWhittaker </td><td> 14-801F </td><td></td></tr><tr><td>谷氨酰胺（200MM） </td><td>临时机场管理局</td><td> M11-004 </td><td></td></tr><tr><td> HEPES </td><td> VWR国际</td><td> 441476L </td><td></td></tr><tr><td> β-巯基乙醇</td><td>默克公司</td><td> 444203 </td><td></td></tr><tr><td>最低（MEM）的主要媒介</td><td> Gibco公司</td><td> 61100-087 </td><td></td></tr><tr><td>的Neurobasal </td><td> Gibco公司</td><td> 21103-049 </td><td></td></tr><tr><td> B27的</td><td> Gibco公司</td><td> 17504-044 </td><td></td></tr><tr><td>莱博维茨的L-15中</td><td> Gibco公司</td><td&gt; 11415-049 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司</td><td> 15070-063 </td><td></td></tr><tr><td>延长黄金抗淬灭试剂</td><td> Invitrogen公司</td><td> P36930 </td><td></td></tr><tr><td>透析管</td><td>光谱实验室</td><td> 132660 </td><td></td></tr><tr><td>兔抗酪氨酸羟化酶</td><td> PEL-FREEZ </td><td> P40101-0 </td><td></td></tr><tr><td>兔抗Darpp32（H 62） </td><td>圣 - 克鲁兹</td><td> SC-11365 </td><td></td></tr><tr><td>鼠抗βIII微管蛋白</td><td>西格玛</td><td> T8660 </td><td></td></tr><tr><td>的Alexa Fluor标记的二抗</td><td> Invitrogen公司</td><td></td><td></td></tr></tbody></table>

dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

中脑多巴胺（mdDA）神经元的项目，通过对几个领域，在端脑，包括纹状体内侧前脑束。同样地，中等刺在纹状体的神经元，引起纹状体黑（直接）通路支配黑质2。的这些轴突大片的发展是依赖过多的轴突生长和指导线索，包括由突起或释放（中间）目标地区3,4分子组合行动。这些可溶性因子可以在体外研究的培养mdDA和/或在胶原基质，它提供了一个立体的模仿外环境的轴突基板纹状体植。此外，胶原基质允许放置在附近（例如，见参考文献5和6）其他植或细胞蛋白形成相对稳定的梯度。在这里，我们描述的PUR的方法ification的鼠尾胶原蛋白，多巴胺和纹状体外植体的显微切割，他们的文化，在胶原蛋白凝胶和随后的免疫组化和定量分析。首先，E14.5小鼠胚胎的大脑是孤立和多巴胺和显微纹状体植。这些植（CO）盖玻片上培养48至72小时在体外在胶原凝胶。随后，轴突的预测是使用可视化的神经标记（如酪氨酸羟化酶，DARPP32，或βIII微管蛋白）和轴突生长和吸引力或排斥力的轴突反应进行量化。这神经的编制是一项有用的工具， 在的mesostriatal与纹状体黑轴突生长和发育过程中的指导体外培养的细胞和分子机制研究。采用此法，它也有可能评估其他（中级）多巴胺和纹状体轴突的目标，或测试特定的分子线索。

Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

中脑多巴胺系统及纹状体植为胶原基质检测<em&gt;在体外</em&gt;分析的mesostriatal和纹状体黑通路发展。轴突生长和指导，在此法可以操纵和量化。它也可以被修改为评估其他地区或分子线索。

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

dissection-culture-mouse-dopaminergic-striatal-explants-three

Research

JoVE Journal

Neuroscience

25.6K 次观看.  University Medical Center Utrecht. 中脑多巴胺系统及纹状体植为胶原基质检测在体外分析的mesostriatal和纹状体黑通路发展。轴突生长和指导，在此法可以操纵和量化。它也可以被修改为评估其他地区或分子线索。

视频: Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

补充电机区的光纤连接重新审视：光纤解剖，DTI和三维文档的方法

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...