作者承认的想法执行后，小鼠胚胎5片文化的原始来源。我们要感谢我们gofer在拍摄约阿希姆基尔希的慷慨科学支持和安娜德劲。这项工作是由德国研究基金会（德意志研究联合会:Sonderforschungsbereich 488 Teilprojekt B7/B9）和海德堡大学（卓越集群无线通信网络）。

第1部分:准备切片和培养。 <ol><li>切片培养基（DMEM培养液，25％1X HBSS中，25％胎牛血清，0.5％葡萄糖，1毫米谷氨酰胺​​，2.5毫米的HEPES，pH值7.3）和3厘米Millicell小CM 0.4微米的文化准备10厘米的组织培养板膜插入和孵化器保持在37 ° C和5％的CO 2。 </li><li>在微波炉中加热4％，熔点低，在PBS点琼脂糖，并继续加热板，使其保持在约37 ° C。 </li><li>填补10厘米的细菌培养皿中，用PBS（140毫米氯化钠，氯化钾2.7毫米，10毫米的Na 2 HPO 4，1.8MM KH 2 PO 4），置于冰上。 </li><li>设置切片机的冷却装置，或确保缓冲区托盘和冷却元件在预冷，冷冻储存。 </li></ol>第2部分:嵌入胚胎。 <ol><li>解剖胚胎从子宫和审查倒置荧光显微镜检查GFP的表达。 </li><li>在一个倒置的10厘米的培养皿和东方他们胚胎，使用的Whatman纸条，以除去过多的PBS。 </li><li>应用胚胎琼脂糖固定在这个位置上。让琼脂糖巩固。 </li><li>限制的区域，周围用刀片切割胚胎。 </li><li>旋转到另一边嵌入式胚胎。 </li><li>应用胚胎组织的额外琼脂糖，以确保胚胎完全嵌入。 </li><li>准备好干净的边缘和vibratome夹头使用类似"Krazy胶"一个特殊的胶水乐泰406，装载琼脂糖块。 </li></ol>第3部分:切片过程。 <ol><li>预冷的缓冲盘和冷却元件。 </li><li>插入粘组织夹头和添加1X的HBSS（ 钙+镁+免费的HBSS，10毫米HEPES缓冲液pH值7.3，500 U / ml青霉素/链霉素），直到覆盖。 </li><li>插入和修复precleaned（70％乙醇）切片机刀片。 </li><li>准备350 - 450微米的薄片，他们转移到组织培养板缩短玻璃巴氏吸管不停地在冰。 </li><li>使用一对镊子，小心地从每个切片和转移到Millicell小文化膜琼脂糖。一个充满了6毫升培养液在10厘米的组织培养板膜，可培养约4片。 </li><li>在37片° C和5％的CO 2（培养基:DMEM培养液，25％1X的HBSS，25％胎牛血清，0.5％葡萄糖，1mm的谷氨酰胺，2.5毫米的HEPES，pH值7.3）。如果一个执行时间推移成像系列，应保持介质的恒定量。在很短的时间内定时成像系列的情况下，它是足够的离开的媒介，因为它是。对于较长的文化时期的变化后12-20小时，建议。 </li></ol>第4部分:在显微镜成像脊髓神经生长。 <ol><li>图像放置一个10厘米的组织培养板中，含有一个堂堂正正的荧光显微镜下切片，Millicell小文化膜脊神经。 </li><li>标签的显微镜舞台上的Millicell小文化膜的方向，正确的位置，在未来的成像时间点。 </li><li>图像的脊神经生长使用的4倍（数值孔径NA] 0.1），10X（NA 0.3），或20X（NA 0.5）的目标。 </li></ol>图1显示了成像系列在20小时的文化与4倍和10倍的目标描绘的脊髓神经生长。 <img src="/files/ftp_upload/2309/2309fig1.jpg" alt="图1" /> 图1成像系列在横片tauGPF一个纯合子胚脊髓神经生长。 * =电机的腹侧脊髓，箭头=背根神经节神经元。背片（D）腹（五）轴表示。 Scalebars:200微米。

<ol>
	<li>Tucker, K. L., Meyer, M., Barde, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+are+required+for+nerve+growth+during+development.">Neurotrophins are required for nerve growth during development.</a> Nat Neurosci. 4, 29-37 (2001).</li><li>Brachmann, I., Jakubick, V. C., Shaked, M., Unsicker, K., Tucker, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+slice+culture+system+for+the+imaging+of+nerve+development+in+embryonic+mouse.">A simple slice culture system for the imaging of nerve development in embryonic mouse.</a> Dev Dyn. 236, 3514-3523 (2007).</li><li>Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+slice+preparation:+a+tribute+to+the+pioneer+Henry+McIlwain.">The brain slice preparation: a tribute to the pioneer Henry McIlwain.</a> Journal of neuroscience methods. 59, 5-9 (1995).</li><li>Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+circuitry+of+identified+projection+neurons+in+cat+visual+cortex+brain+slices.">Local circuitry of identified projection neurons in cat visual cortex brain slices.</a> J Neurosci. 7, 1223-1249 (1987).</li><li>Hotary, K. B., Landmesser, L. T., Tosney, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+slices.">Embryo slices.</a> Methods Cell Biol. 51, 109-124 (1996).</li></ol>

在比较广泛的方法，准备妊娠中期的小鼠胚胎的胚胎切片文化（E10 - E12），我们观察到一个vibratome毫无疑问，尊重文化的整体可行性和重复性生产的最可靠的结果神经生长模式。相比之下，使用斩波器3被证明是完全inviable一个McIlwain组织切片准备。我们原任一个断头台的方法4，在整个胚胎的垃圾可以同时缠一堑长一连续篦生产400 微米的第1钨丝切片准备。虽然这种技术已?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14180</td>
<td>&nbsp;</td>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>FST</td>
<td>various</td>
<td>&nbsp;</td>
<tr>
<td>L.M.P. agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-022</td>
<td>&nbsp;</td>
<tr>
<td>Whatmann paper</td>
<td>&nbsp;</td>
<td>Whatman Int.</td>
<td>3030917</td>
<td>&nbsp;</td>
<tr>
<td>Shortened firepolished pipettes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>41966</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10270-106</td>
<td>&nbsp;</td>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine 100x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Microm International GmbH</td>
<td>HM 650 V</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX61WI</td>
<td>&nbsp;</td>
<tr>
<td>analySIS</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG 50</td>
<td>&nbsp;</td>
<tr>
<td>10 cm culture plates</td>
<td>&nbsp;</td>
<td>Greiner Bio-One</td>
<td>633171</td>
<td>&nbsp;</td>
<tr>
<td>LOCTITE 406</td>
<td>&nbsp;</td>
<td>Henkel</td>
<td>142580</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>none</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9105.2</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>x4 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>PL series</td>
<td>&nbsp;</td>
<tr>
<td>x10 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>UPLFL &#x2013;PH series</td>
<td>&nbsp;</td>
<tr>
<td>Filter</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>U-MNIBA2</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>SIS F-View II</td>
<td>&nbsp;</td>
<tr>
<td>Equipment for heated chamber</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>CTI-Controller 3700 and incubator S #11531171</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth

多种用途，小鼠胚胎体外培养器官切片是可取的。例如，我们采用了转基因小鼠线（tauGFP）在绿色荧光蛋白（EGFP）的增强版本是专门表示，在发展的中枢和外周神经系统1所有神经元的可能性，让这两个电影的支配前肢和操纵这个过程中，药理学和基因工程技术生产的 2 。切片文化的培育成功，最关键的参数，是这片准备的方法。多种方法进行广泛的测试后，我们发现一个vibratome设备切片胚胎等，他们经常在文化，展示了可行性的几天内，最重要的结果是最好的，在这样一个时代的发展特定的方式。对于妊娠中期的胚胎，这包括脊神经从脊髓和背根神经节，他们在外围和适当的骨骼和肌肉组织的决心目标的正常生长。 在这项工作中，我们提出了一种用于加工成300整个胚胎的胚胎一天（五）E10的E12的方法 - 种植400微米片在一个标准的组织文化的孵化器，可研究后切片准备两天。这种方法成功的关键是使用vibratome每个琼脂糖包埋胚胎切片。其次是种植后Millicell小文化膜插入后放在一个中等体积小，接口中的文化技术的切片。凋落物平均7胚胎常规生产，至少有14片（2-3片每胚胎前肢地区），由于胚胎的年龄以及切片厚度略有不同。大约80％的培养切片显示神经生长，它可以测量througout培养期2。使用tauGFP鼠标线的代表结果表明。

Watch this Scientific Journal Video about Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth at JoVE.com

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

我们现在准备妊娠中期的小鼠胚胎的器官切片周围神经生长的培养和时间推移成像的方法。

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

organotypic-slice-culture-gfp-expressing-mouse-embryos-for-real-time

Research

JoVE Journal

Neuroscience

16.1K 次观看.  University of Heidelberg. 我们现在准备妊娠中期的小鼠胚胎的器官切片周围神经生长的培养和时间推移成像的方法。

视频: Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

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