我们感谢克里斯·罗杰斯博士（美国塔夫茨大学）用于编程和手稿建议。我们感谢亚历克斯朱利亚诺和德博拉·李支持视频制作。 提供的资金由美国国家科学基金会研究生研究奖学金在合成生物学和ONR穆里。

1。构建机器人模型<ol><li>选择一种模式生物来研究神经行为学文献中得到很好的体现。无脊椎动物通常做出很好的候选人，由于其相对简单的神经系统得到很好的研究，主要是由先天的反射。我们将证明这种方法使用美国龙虾， 龙虾美洲 。 </li><li>选择研究自反行为建模的目的。我们选择了龙虾的反应，触角弯曲到爪凹凸检测作为动物本能反应触角弯曲，引起rheotaxis（水流方向），而爪接触调解避障。非常适合这种类型的研究1简单的反射，依靠从两侧对称传感器decussating神经连接。 </li><li>建立或选择相应的传感器的机器人平台，为选定的行为，为了运行的神经网络仿真。虽然可以构造一个专用的机器人如RoboLobster 12或蔓延（蟑螂）机器人家族13，这里我们使用市售的试剂盒，以加快实验过程。 LEGO MINDSTORMS NXT 2.0套件，提供模板，模块化组件和建立的传感器，可以快速的物理结构的机器人。 </li><li>选择或建立传感器相匹配的步骤1.2中选择的行为方式。预制传感器可用于LEGO MINDSTORMS套件或自制的传感器可以设计14,15。我们已经选择了包括LEGO触摸传感器和定制的触角弯曲传感器构成的FlexPoint弯曲传感器拼接NXT连接。要建立一个自定义的电阻连接，切断NXT连接线和焊接黑色和白色的电缆连接到传感器的引线。 </li><li>使用先前公布的神经行为学研究，以寻找或开发一个假设的神经网络为建模purposes。文献提出了各种神经网络，以解释各种模式生物的行为，从龙虾16鳗17。功能神经单位应查明他们的突触连接理论。新型神经行为学的实验，也可以开发和实施，如果实验室的情况下允许。 </li></ol> 2。神经系统编程<ol><li> LEGO MINDSTORMS NXT的LabVIEW模块一起使用LabVIEW软件来创建虚拟仪器技术（VIS），运行方程的数学的神经元和突触模型。虽然任何发布的模型可以使用，我们建议基于离散时间地图（DTM）模型[18]。这使得计算效率的实时操作，同时保持输出神经元放电的各种制度。两个控制参数，α和适马 ，确定动力学模拟神经元，产生多样的神经输出，包括托尼Ç扣球，乱射击，爆破和内在的沉默。 LEGO MINDSTORMS的LabVIEW VI这个神经元模型可以在网上找到这里： <a href="http://myfiles.neu.edu/blustein.d/neurobots" target="_blank">http://myfiles.neu.edu/blustein.d/neurobots</a> </li><li>地方到一个While循环，使网络将更新迭代的VI。 </li><li>突触后神经元通过突触连接突触前神经元秒杀输出。 </li><li>通过迭代更新参数通过移位寄存器到下一个计算周期。 DTM模型更新快（x）和慢（Y）的动力被传递到下一次迭代计算的变量。 </li><li>将突触的参数来定义网络的特征。对于每个突触的突触的强度（gsyn），松弛率（γ），和反转电位（XRP）是系数在DTM突触行为的模型方程，确定。的gsyn的值是一个多钳决定突触电流的影响从一个尖峰事件的幅度，范围从0.0（没有效果）1.0（100％的效果）。 伽玛值是一个乘数无论决定的突触电流衰变率的秒杀活动，从0.0（完全衰减）到1.0（无衰减）。 XRP值的高低决定了激励或抑制突触，通常值范围从-3.0至3.0代表激励和抑制负值代表正面的价值观。值可以调整为特定的网络特性。在这里，我们使用以下设置：γ= 0.95，gsyn = 0.60，XRP = 2.2（兴奋性突触）-1.8（抑制性突触）。 </li><li>调整字母和sigma控制参数修改基线神经元活动。多样的控制参数的模型的动态别处18详细描述，但通常，α </em&gt; &lt;4 西格玛值低或强直扣球高西格玛值的神经活动产生内在的沉默。 “低”和“高” 西格玛值之间的过渡，通常会发生的 Alpha值介于-1.0和1.0之间。爆破神经元活动产生阿尔法 &gt; 4时西格玛值接近零。在这里，我们使用α= 4.05，σ= -3.10。 </li><li>使用LabVIEW VI的输入传感器的信息进入你的网络。所提供的传感器，这些VI已经MINDSTORMS的LabVIEW模块内。对于定制电阻传感器，使用光传感器VI作为一种编程框架。有关创建自制传感器的信息可以在别处找到14。 </li><li>将LabVIEW程序可视化神经元的活动面板上的图表。 </li><li>暂时取代以手动操作传感器传感器输入，前面板控制箱将神经网络的信息。 </li><li>运行神经网络和手动调节前面板控制，呈现多样的感官输入。确认，该网络的功能定性的预测对已知的条件。 </li><li>根据需要通过改变神经元和突触参数调整网络。第一次尝试调整网络应该做的，通过改变突触强度的各种突触（gsyn）。可能需要进行调整，以及其他参数。 </li><li>一旦网络的功能与定性准确，更换前面板控制盒与传感器输入的VI。 </li></ol> 3。神经系统仿真测试<ol><li>设置受控制的环境中，在类似的条件下，观察动物和机器人。我们使用了一个含有岩障碍（ 图1）的矩形槽。可以清空的容器，在相同的空间以容纳LEGO机器人。 </li><li>安装AVIDEO相机的开销记录的动物/机器人行为的。确保主体和背景之间的对比度高，允许自动跟踪。我们画测试箱白色的底部。 </li><li>将在受控环境中的动物和视频记录的行为。一定限制的龙虾，以配合机器人的感官能力。在这里，我们用铝箔面膜遮住眼睛和淡水19 deafferenting化学感受器限制龙虾的感官能力。 </li><li>调整，以适应环境的LEGO机器人（除去水分，如果必要的话），并运行相同的控制环境中的机器人。 </li><li>使用MATLAB脚本来自动跟踪标本上的标记。我们使用20 MouseLabTracker。 </li><li>创建和比较的动物和机器人运动矢量图。 </li><li>修改的中枢神经系统，并重复步骤3.3-3.6观察不同方面的电子神经系统的影响机器人的行为。在这里，我们调整爪的凹凸传感器神经元和运动神经系统和监控机器人的性能相比，龙虾之间的突触强度。或者，感官测试领域的条件下可以被修改，机器人和动物中观察到的效应。例如，这里所描述的舞台上可能会受到不同的水流速。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

当启动仿生机器人神经系统仿真实验，有几个重要遵循的准则。选择正确的模式生物是至关重要的：挑一个有机体，是很容易获得和维持。无脊椎动物是理想的，因为他们通常不要求实验机构批准，其畜牧业的需求往往比脊椎动物的要求不高。从科学的角度来看，它是有利于选一种动物，有一个既定的神经行为学文献记载，从中可以得出的假设进行测试21。 选择一个?...

神经生理学研究过去100年里已经极大地拓宽我们的知识，神经系统的结构和功能。然而，大多数的神经系统的研究迄今一直依赖于孤立的制剂或内敛科目使用。虽然已经有许多成功的努力，记录神经活动，自由活动动物2-5，仿生机器人的方法提供了一个宝贵的工具，让神经系统操作，以测试系统级神经科学假说6。模拟操作机器人的神经系统可以通过实验操作，并允许软件建模到物理世界的延伸。这种方法已在学术界7,8实施，但假设检验的过程中，建立一个仿生机器人可以是昂贵和费时。我们提出一个方法来执行的仿生机器人的方法，使用市售的机器人K表（LEGO MINDSTORMS NXT 2.0）。这种方法的目的是，机器人9或生物杂交10体现了神经网络模拟测试系统级神经科学假说提供了快速和有效的方式。加快从假设到实验的过程，提高研究效率。简单的LEGO MINDSTORMS平台提供我们演示如何使用美国龙虾（ 美洲龙虾 ）作为模式生物的仿生传感器和神经网络的试验床。该方法还提供了强大的教育工具，在课堂上，学生可以设计和操纵神经系统，为自己的机器人11。

designing and implementing nervous system simulations on lego robots

我们提出一个方法，使用市售的LEGO MINDSTORMS NXT机器人平台测试系统级神经科学假设。第一步的方法是开发一个合适的模型有机体的具体自反行为神经系统模拟;这里我们使用美国龙虾。外感受性反射通过decussating（渡）神经连接介导可以解释动物的出租车朝着或远离刺激作为描述由Braitenberg和调查采用NXT平台特别适合。1的神经系统仿真LEGO使用LabVIEW软件编程头脑风暴的平台。一旦神经系统的正确调谐，运行在机器人上，在相同的环境条件下，对​​动物行为的实验。通过控制试样所经历的感觉周围环境，可以观察到不同的行为输出。这些差异可能指向特定deficienc指神经系统中的模型和服务告知迭代模型下的特定行为研究。这种方法允许实验操作的电子神经系统，并作为一种方式来探索神经科学假说，特别是关于神经生理学基础简单的先天自反行为。 LEGO MINDSTORMS NXT套件提供负担得起的和高效率的平台上测试初步仿生机器人控制计划的。该方法也适用于高中教室作为基础的动手探究为基础的生物机器人课程。

从龙虾的爪子输入到它的神经系统调解障碍，谈判在一种新的环境。 图1显示了一个乐高机器人（ 图1A）和测试领域的一个龙虾（ 图1B）的行为进行分析的视频截图。除的的被掏空水从水箱的机器人的试验，未改性的动物和机器人测试之间的测试领域。 视频跟踪结果显示在图2。在MATLAB中自动跟踪使用MouseLabTracker 20，?...

Watch this Scientific Journal Video about Designing and Implementing Nervous System Simulations on LEGO Robots at JoVE.com

Designing and Implementing Nervous System Simulations on LEGO Robots

LEGO MINDSTORMS机器人平台上的神经网络建模方法。该方法提供了一个模拟工具，无脊椎动物神经科学的研究在研究实验室和课堂。这项技术使调查仿生机器人控制原则。

LEGO机器人神经系统仿真设计和实现

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

designing-and-implementing-nervous-system-simulations-on-lego-robots

Research

JoVE Journal

Neuroscience

14.9K 次观看.  Northeastern University. LEGO MINDSTORMS机器人平台上的神经网络建模方法。该方法提供了一个模拟工具，无脊椎动物神经科学的研究在研究实验室和课堂。这项技术使调查仿生机器人控制原则。

视频: Designing and Implementing Nervous System Simulations on LEGO Robots

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

在自主机器人以昆虫触角电位传感器的嗅觉搜索

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

在五个脊椎动物群中可靠隔离中枢神经系统微血管

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...