Name: preparation of aplysia sensory-motor neuronal cell cultures
Uploaded: 2009-06-08T00:00:00
Description: Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

在涉及的海兔神经细胞培养实验室工作的经费由美国国立卫生研究院的清单R01和美国国立卫生研究院R21MH077921。

准备（参见解决方案在协议结束部分解决方案的组成） <ol><li>准备培养皿。涂层玻璃Mattek玻璃底培养皿与聚- L -赖氨酸（四硼酸钠）。加入足够的完全覆盖的玻璃和离开&gt; 1小时（可以过夜）。彻底清除聚- L -赖氨酸，在人工海水冲洗（ASW）的4-5倍。删除最后一次冲洗后，加2毫升50％L15（补充盐和含L -谷氨酰胺在终浓度为2mm）/ 50％血淋巴。这种培养基中必​​须至少一个小时，在盘前电镀细胞，...

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	<li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Rayport, S. G., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+vitro:+cell+culture+of+identified+Aplysia+neurons+mediating+short-term+habituation+and+sensitization.">Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization.</a> J Neurosci. 6 (3), 759-763 (1986).</li><li>Goldberg, D. J., Schacher, S., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 213-236 (1998).</li><li>Glanzman, D. L., Kandel, E. R., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+target+motor+neuron+regulates+neurite+outgrowth+and+synapse+formation+of+aplysia+sensory+neurons+in+vitro.">Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro.</a> Neuron. 3 (4), 441-450 (1989).</li><li>Lyles, V., Zhao, Y., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+and+mRNA+localization+in+cultured+Aplysia+neurons.">Synapse formation and mRNA localization in cultured Aplysia neurons.</a> Neuron. 49 (3), 349-356 (2006).</li><li>Martin, K. C., Casadio, A., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-specific,+long-term+facilitation+of+aplysia+sensory+to+motor+synapses:+a+function+for+local+protein+synthesis+in+memory+storage.">Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage.</a> Cell. 91 (7), 927-938 (1997).</li><li>Guan, Z., Giustetto, M., Lomvardas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+long-term-memory-related+synaptic+plasticity+involves+bidirectional+regulation+of+gene+expression+and+chromatin+structure.">Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure.</a> Cell. 111 (4), 483-493 (2002).</li></ol>

海兔感觉运动文化的成功做好准备，有一个有点慢的学习曲线，因为它涉及到显微切割和操纵通过一个立体显微镜观察单个神经元的精细动作技能的发展。根据我们的经验，它需要大约1-3周的实践，获得健康的文化孤立的感觉神经元和一个额外的1-3周，以了解如何对感觉神经与运动神经元。我们经常准备在Labconco超净工作台文化，虽然有可能准备任何实验室的工作台或办公桌上，只要显微?...

为文化需要的解决方案<ol><li> 0.35米氯化镁2，在室温下储存，使用麻醉。 </li><li>聚- L -赖氨酸的解决方案，西格玛P - 1524兆瓦&gt; 300,000，0.1M硼酸钠pH8.2至0.5mg/ml的解决方案。涡和过滤通过0.22微米的过滤消毒，存放在4C °。不要冻融。 </li><li> L - 15的中等粉（莱博维茨）（Sigma公司：L4386）与盐的补充，使1公升 <table border="1" cellspacing="0" cellpadding="0"><tr><td> L - 15粉</td><td> 13.8克</td></tr><tr><td>氯化钠</td><td> 15.4摹</td></tr><tr><td> D -葡萄糖</td><td> 6.24摹</td></tr><tr><td>硫酸镁4 7H 2 0 </td><td> 6.45克</td></tr><tr><td>氯化钾</td><td> 350毫克</td></tr><tr><td>碳酸氢钠3 </td><td> 170毫克</td></tr><tr><td>氯化镁2 · 6H 2 O </td><td> 5.49摹</td></tr><tr><td>氯化钙2 · 2H 2 O </td><td> 1.43克</td></tr><tr><td> HEPES </td><td> 3.53克</td></tr></table>添加ddH20到1升。 pH值7.4-7.5左右，应添加100X笔/链球菌解决方案10毫升，通过一个0.22米过滤器和过滤器消毒。在4C0商店不超过1个月。 </li><li>蛋白酶消化解决方案：1％蛋白酶IX（1unit/mg）L15（辅以如上盐）或反潜立即使用前，通过一个0.22米的微孔过滤灭菌。 5mls应该足够了两种动物的神经节（确保神经节是完全沉浸在蛋白酶溶液）。适马报告说，他们将停止销售蛋白酶第九。替代蛋白酶是：Dispase II（罗氏应用科学部目录＃04942078001）。 </li><li>培养基。前准备文化，解冻10毫升的血淋巴等份和混合10 L15的MLS（辅以如上盐），使20mls培养基。加入200μL200毫米L -谷氨酰胺，拌匀，用于准备文化。这种介质应准备新鲜每次文化。 </li><li>人工海水可从即时海洋（水族馆系统，导师，OH）如下：450毫米氯化钠，氯化钾10毫米，30毫米2（H 2 O）氯化镁， 硫酸镁 20毫米，10毫米氯化钙2（2H 2 O），10毫米的HEPES，pH值，调整至7.4。 </li></ol>

preparation of aplysia sensory-motor neuronal cell cultures

海洋软体动物海兔californica的神经系统是相对简单的，约20,000神经元组成。神经元大（直径可达1毫米）和识别，具有不同的大小，形状，位置和色素沉着，胞体外部暴露在五个配对神经节分布于整个动物的身体。这些属性允许划定的基本电路 1，在动物的特定行为的调查。感觉和运动神经元之间的突触连接，是一个在动物的鳃撤退反射的核心组成部分，其中动物撤回其刺中虹吸触觉刺激的一个简单的防御反射。这种反射的非关联和关联学习经历的形式，包括敏，习惯性和经典性条件反射，。感官电机突触突触可塑性的研究特别的好处，可以在文化重组，特点刺激引起的可塑性在动物的 2,3行为有直接关联的形式。具体来说，羟色胺的应用程序产生一个突触的加强，取决于应用协议，持续几分钟（短期便利），小时（中期的便利）或天（长期便利）。相比之下，肽发射机FMRFamide的应用产生了突触减弱或抑郁症，取决于应用协议，去年从天分钟（长期抑郁症）。的神经元的大尺寸允许反复突触强度的急剧电极记录天期间注射表达载体，siRNA和其他化合物，针对特定的信号级联和分子，从而确定分子和细胞生物学的步骤背后的变化在突触效能。 海兔文化体系的一个额外的好处，从事实的神经元突触特异性展示文化4,5。因此，感觉神经元不与自己（autapses）或与其他感觉神经元形成突触，也没有形成与非目标确定在文化运动神经元突触。静脉曲张，感觉和运动神经元之间的突触联系的网站，都足够大（直径在2-7微米），允许在光镜水平研究目标的运动神经元突触的形成与（以及突触形态的变化）。 在这段视频中，我们展示了准备的感觉，运动神经元的文化，包括anesthetizing成人和青少年海兔的每一步，剖析他们的神经节，神经节蛋白酶消化，去除结缔组织显微切割，感觉和运动神经元的识别和清除每个显微切割细胞类型，运动神经元，感觉神经元和感觉神经轴突的操纵此外电镀形成与培养运动神经元的联系。

Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

小学文化<em&gt;海兔</em&gt;感觉运动神经元突触的形成和突触可塑性研究提供一个模型的制备<em&gt;在体外</em&gt;。这个视频演示的感觉和运动神经元的鉴定和显微切割<em&gt;海兔</em&gt;神经节以及建立和保持文化的感觉运动神经元的方法。

制备海兔感觉运动神经元细胞培养

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

preparation-of-aplysia-sensory-motor-neuronal-cell-cultures

Research

JoVE Journal

Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

科研

生物学

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

制备大鼠脑组织神经元和神经胶质发展研究总结文化

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...