这项研究是由部分启动资金，从得克萨斯A＆M大学

前夹层的制备 手术工具：由高压灭菌消毒清扫剪刀和镊子 盖玻片清洁： <ol><li>在1L草烧杯中33％的盐酸中浸泡至少24小时的所有盖玻片（直径为24孔板15毫米） </li><li>偶尔晃动的水10分钟，清除所有残留的盐酸洗</li><li>用微孔水冲洗</li><li>排水关闭</li><li>在95-98％的乙醇浸泡盖玻片</li><li>用干净的纸巾将盖玻片上平板和空气干燥的盖玻片</li><li>传输盖玻片一个玻璃烧杯中，盖上铝箔和高压灭菌器 注：盖玻片可以存储在这个阶段</li><li>个人盖玻片放入培养皿。涂层准备就绪</li></ol> 盖玻片涂层： <ol><li>稀聚- D -赖氨酸100 x的PBS和过滤，消毒（0.22微米）的股票（PDL，10 mg / ml的0.5％的牛血清白蛋白的PBS存储，在-20 ° C等分） </li><li> 1个客运专线解决方案（〜0.15毫升/厘米2），2小时在37℃的孵化器中的C大衣盖玻片</li><li>删除PDL的解决方案，完全洗3次，用无菌DDH 2 O和干燥的盖玻片</li></ol> 注：大衣足够的盖玻片/每个夹层板。 PDL涂层的盖玻片上可以存储在几个星期在4 ° C。 媒体和解决方案 夹层介质（DM）：使用无菌冰冷汉克的平衡盐溶液（W / O型的Ca 2 +，Mg 2 +的）（HBSS中，Invitrogen公司14175），辅以10毫米的HEPES（Invitrogen公司15630 ）。 总结文化传媒：DMEM/NBB27介质包含DMEM / Neurobasal（1:1卷：第一卷），2％B27，1 ×佐藤0.5毫米丙酮酸钠，0.75毫米GlutaMAX，60μg/mlN -乙酰半胱氨酸，5微克/毫升胰岛素，10NM D -生物素和1％青霉素/链霉素。为了使100毫升的总的培养液，混合50毫升的DMEM（W / O型丙酮酸/谷氨酰胺，Invitrogen公司的11960），50毫升Neurobasal培养基（Invitrogen公司211034），375μLGlutaMax（100 × 35050 Invitrogen公司），5.5毫克丙酮酸钠（Sigma公司P2256），2毫升B27（Invitrogen公司17504），6.3毫克N -乙酰半胱氨酸（Sigma公司A8199），1毫升佐藤股票（100 ×股票，见下文），25μLD -生物素的股票（股票，4000 × 40在PBS微米存储为分装于-20 ° C.六西格玛B4639），100μL胰岛素（1000 ×股票，在5毫克/毫升0.01N盐酸作为分装保存于-20 ° C，西格玛16634），和1 ml青霉素/链霉素（100 × 15140）股票，Invitrogen公司，过滤消毒和储存在4 ° C。 佐藤100 x股票的解决方案：为了使股票的40毫升：400毫克的apo -转（Sigma公司T2252），400毫克的BSA（Sigma公司A9647），存储为黄体酮的10μL（25毫克/毫升乙醇，混合40毫升Neurobasal分装于-20 ° C. Sigma公司P8783），64毫克腐胺（Sigma公司P7505），亚硒酸钠（30微米PBS中，作为分装保存于-20 ° C，西格玛S5261）和40μL。过滤消毒佐藤股市的解决方案，并储存为-20 ° C等分 神经元电镀液（下午）：Neurobasal中等，2％B27，2MM谷氨酰胺（100倍的股票，在-20℃，Invitrogen公司25030存储等分），25μm的谷氨酸（100种股票，分装储存在-20 ° C）和1％青霉素/链霉素。 神经元培养基（CM）：Neurobasal中等，2％B27，2MM谷氨酰胺（100倍的股票，分装储存在-20 ° C）和1％青霉素/链霉素。 5 FDU股票：100倍的股票，1mm的5 -氟-2&#39; -脱氧尿苷（Sigma公司F0503）and1mM尿苷Neurobasal培养基（Sigma公司U3003）。过滤消毒，并作为分装储存在-20 ° C 木瓜蛋白酶消化解决方案（做出新的，以清扫前）： <ol><li>溶解在4毫升DM 3.2毫克L -半胱氨酸（SIGMA的C - 7352） </li><li> 1N氢氧化钠（pH试纸测试），在水浴地方调整pH值至7.4左右，在37 ° C 注：组织消化前执行下一个步骤（见下文）。 </li><li>添加木瓜蛋白酶的终浓度为20单位/毫升</li><li>过滤消毒（0.22毫米），并于37℃水浴中放置° C </li></ol> 胰蛋白酶抑制剂的解决方案（做出新的，以清扫前）： <ol><li>溶解0.2克胰蛋白酶抑制剂在20毫升马克（西格玛T7295） </li><li>检查pH值，用1N氢氧化钠调整pH值至7.4〜 </li><li>过滤消毒，并在37℃水浴中放置° C </li></ol>脑解剖 安装： <ul><li>消毒的镊子和剪刀</li><li> 70％的乙醇</li><li>清洁纸巾和diap二垫，在实验台上</li><li>用冰冷的替补的子宫和胎儿的DM10厘米培养皿</li><li>在解剖镜下ICE平台</li><li> 2-36厘米菜与DM上的冰</li><li>暖日下午在37℃水浴</li></ul><ol><li>安乐死怀孕根据您的机构的动物保健批准的程序只Sprague - Dawley大鼠（E16）和我们委员会（IACUC） </li><li>为上腹部地区的尿布垫和绝育酒精的老鼠</li><li>使用镊子举行的腹部皮肤和一把剪刀，一个V形切口，切割只有皮肤</li><li>分开皮肤和使用另一消毒的镊子和剪刀穿过肌肉层</li><li>胚囊中提取的链，并转移到一个10厘米含有冰冷的DM菜</li><li>采用显微切割剪刀，小心地取出每一个胚胎，从每个胚胎囊和大脑，并释放到清洁10厘米菜与DM冰平台上的大脑</li><li>在层流罩下解剖镜下，脑/后脑部分删除，并用细钳去除脑膜，冰袋转移到一个新的6厘米菜与DM的清理皮层/海马 注：在这个阶段，组织准备内切酶消化。 </li><li>将木瓜蛋白酶和胰蛋白酶抑制剂的解决方案和过滤消毒</li><li>删除多余的DM从皮质/海马</li><li>添加4毫升准备木瓜蛋白酶消化解决方案</li><li>所有内容转移至50ml管，并在37℃水浴中放置猎鹰管℃，整整5分钟</li><li>用吸管取出木瓜蛋白酶溶液</li><li>添加为2-3分钟5毫升胰蛋白酶抑制剂的解决方案，旋流管，水浴中放置在37 ° C </li><li>删除胰蛋白酶抑制剂的解决方案</li><li>重复步骤13和14的三倍</li><li>加20ml的温暖！ </li><li>磨碎已经消失，直到所有的团块（20-30倍） </li><li>在100xg离心为7分钟</li><li>悬浮于10ml PM的沉淀和离心洗两次</li><li>重悬沉淀于10ml下午进行定期的神经元文化或聚合文化NBB27/DMEM </li><li>穿过一个70μm的细胞筛细胞</li><li>活细胞计数</li><li>下文所述或钢板定期神经元培养的细胞聚合文化 注：在这一步，神经高度丰富的文化可以通过电镀到PDL的涂层板游离细胞密度在200-640细胞/ 毫米 2根据实验的目的。细胞附着后（1〜2 4小时），用温水下午取代的媒介。如果时间过长，在第4天，培养，取代以热烈的CM培养基的一半。 5 FDU（终浓度为10微米），对于高度纯化的神经元的文化，有丝分裂抑制剂，可以添加在DIV2抑制（即在DIV2 - 3，）非神经元细胞的增殖。 CM 2天的复苏后，细胞被脉冲治疗与5 - FDU再次为一个完整的中期变化的另外2天（6-7格）。此后，更换新鲜，每3-4天暖厘米中期的一半。长达4周的神经元是可行的。 </li></ol>聚合制备和电镀<ol><li>暂停NBB27/DMEM介质中分离的细胞中含有1X佐藤在10ng/ml，CNTF和10μMforskolin的2 × 10 6细胞/ ml密度</li><li>细胞悬液转移到每一个无涂层的6孔板以及2毫升</li><li>文化3晚。每一天，轻轻悬浮细胞，一旦使用P1000的Pipetman 注：细胞开始形成聚集体（图1）。 </li><li>总电镀的前一天，涂层板/与基质胶的盖玻片： <ul><li>稀股票基质胶（生长因子减少，BD公司354230）用冷水冰的DMEM 1:20 注：基质胶分装，应按照制造商协议准备。所有的提示，Eppendorf管和股票等分的解决方案应冷，以避免基质胶聚合。基质胶分装保存于-80 ° C，并在4 ° C解冻</li><li>外套以前PDL涂层的盖玻片用300μl/well稀释后的基质胶溶液在37℃培养箱中过夜</li><li>用温水PBS洗净，然后用温暖无菌DDH 2 O板和离开孵化器板。 </li></ul></li><li>聚合形成后3天，轻轻重悬细胞，再通过成50毫升猎鹰管200μm的丝网筛暂停。允许聚合解决试管底部的比重（1〜3分钟） </li><li>小心取出上清液</li><li>添加介质，轻轻悬浮聚合，并让他们重新定居。此过程重复几次以去除死细胞，单个细胞和碎片。用显微镜检查是否上清液中还含有非聚合的细胞和debri小号</li><li>轻轻重悬在同一NBB27/DMEM介质中的聚集（图1）和计数聚合</li><li>调整的聚集密度约25-30 aggreggats/50ul </li><li>传输等分总悬浮液（500 -1000μl）至2ml的Eppendorf管电镀 注：由于聚合倾向于安定下来的管底部，关键是使总悬浮的多个管镀。轻轻重悬之前往往播种到涂盖玻片或培养板，以确保每个盖玻片上镀聚合同样数量的聚集。 </li><li>从孵化器中含有基质胶的涂布盖玻片取出培养板。从每口井的解决方案，用温暖的PBS和 DDH 2 O盖玻片，然后干燥简略。他们现在已经准备好为总播种</li><li>反转一个试管中总悬浮液，让他们可以均匀分布播种前。加载到每个盖玻片中心总悬浮液50μL，轻轻把板背面没有任何进一步的干扰，让骨料均匀地附着盖玻片孵化器 注：集料的密度是至关重要的。如果聚合种子太接近对方，或如果播种密度高，他们往往合并到一起，因为他们成长。 </li><li>添加额外的DMEM/NBB27medium（500μL），每孔4-6 h后或第二天一大早</li><li>要保持总的文化，一半改变中期每3-4天。当改变培养基，小心，不要破坏聚合，这是轻轻沿井侧墙加入中等。 注：可以观察几个小时后，板块聚集，聚集的突起生长。轴突快速增长中的头两个星期，并形成丰富的聚合体（图2）之间的连接。神经胶质祖细胞移植放射状聚集，并随着时间的推移，分化成星形胶质细胞和成熟的少突胶质细胞。 </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="图1" /> 图1，洗涤后在不同的日子和聚合形成聚集体细胞的相衬图像。 <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="图2" /> 图2。期后在基质胶涂盖玻片的种子2和12天的总文化的对比度的图像。

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

早期的研究报告突触介导的转自由浮动总文化的成熟髓鞘的形成。这里描述的中枢神经系统的总文化系统在三个维聚集，与传统的2D文化便利，以促进细胞的发育，迁移和体外分化的分析相结合的无血清多能祖细胞的生长，可以修改系统和神经前体细胞神经胶质细胞相互作用的调查研究和。例如，如少突胶质祖细胞的转基因细胞5月2日被添加到总文化。如小胶质细胞和巨噬细胞或神?...

preparation of rat brain aggregate cultures for neuron and glia development studies

在体外系统概括了发展和祖细胞分化为成熟的神经元和神经胶质细胞在中枢神经系统（CNS）的，将提供一个强大的神经学家平台，调查轴突胶质细胞相互作用，性能和多能祖细胞的分化，少突胶质细胞的进展在细胞和分子水平的细胞系细胞。我们在这里介绍从胚胎大鼠forebrains，在无血清培养基可维持3-4周，并在我们的实验室用来作为模型来研究神经元的神经胶质细胞的相互作用和中枢神经系统的髓鞘的中枢神经系统的总的培养体系。本短片将展示如何隔离和成长E16大鼠脑组织中的中枢神经系统的总文化。此外，从相同的大脑解剖，经常游离神经文化高度浓缩，可随时获得和使用各种研究，对中枢神经系统神经元或用于与其他细胞共培养。

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

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

胚胎大鼠脑组织总文化系统的一个协议描述。在聚集的多能祖细胞可以发展和分化成神经元，星形胶质细胞和少突胶质细胞。

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) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K 次观看.  Texas A&M University (TAMU). 胚胎大鼠脑组织总文化系统的一个协议描述。在聚集的多能祖细胞可以发展和分化成神经元，星形胶质细胞和少突胶质细胞。

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

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 ...

科研

生物学

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 ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS - 运动神经元疾病：机制和新疗法的发展

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

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 ...

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 of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

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 ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

制备鼠尾肌腱的生物力学和Mechanobiological研究

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...