Name: analysis of dendritic spine morphology in cultured cns neurons
Uploaded: 2011-07-13T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Dendritic Spine Morphology in Cultured CNS Neurons at JoVE.com

我们感谢细心的编辑凯利 - 琼斯。这项工作是支持由美国国立卫生研究院授予R01MH 071316阿尔茨海默氏症协会，全国的精神分裂症和抑郁症（NARSAD）研究联盟和全国自闭症研究联盟（NAAR）（星期三），美国心脏协会博士后奖学金（DPS）;美国心脏协会Predoctoral奖学金（KMW）。

这里所描述的协议，可以用来检查任何原代培养体系的树突棘形态和动态。 1。初级皮层神经元培养的制备<ol><li>准备从高密度的皮层神经元培养SD大鼠E18胚胎和胶质空调无血清培养基 1-2文化。 </li><li>安乐死之一孕鼠（E18）根据ACUC程序，迅速取出子宫中的胎儿，在100毫米的Petri冰盘地方。 </li><li>剖开子宫和羊膜，胎儿颈部举行（脐带完好）用一个镊子，用另一个镊?...

<ol>
	<li>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=Developments+in+neuronal+cell+culture.">Developments in neuronal cell culture.</a> Nature. 336, 185-186 (1988).</li><li>Xie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kalirin-7+controls+activity-dependent+structural+and+functional+plasticity+of+dendritic+spines.">Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines.</a> Neuron. 56, 640-656 (2007).</li><li>Spear, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adolescent+development+and+alcohol+use+in+animals.">Modeling adolescent development and alcohol use in animals.</a> Alcohol Res Health. 24, 115-123 (2000).</li><li>Srivastava, D. P., Woolfrey, K. M., Liu, F., Brandon, N. J., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+ss+activity+modulates+synaptic+signaling+and+structure.">Estrogen receptor ss activity modulates synaptic signaling and structure.</a> J Neurosci. 30, 13454-13460 (2010).</li><li>Srivastava, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enhancement+of+two-step+wiring+plasticity+by+estrogen+and+NMDA+receptor+activity.">Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity.</a> Proc Natl Acad Sci USA. 105, 14650-14655 (2008).</li><li>Woolfrey, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epac2+induces+synapse+remodeling+and+depression+and+its+disease-associated+forms+alter+spines.">Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines.</a> Nat Neurosci. 12, 1275-1284 (2009).</li><li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons:+differential+attachment+of+NMDA+versus+AMPA+receptors.">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li><li>Harms, K. J., Tovar, K. R., Craig, A. M. <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+regulation+of+AMPA+receptor+subunit+composition+by+activity.">Synapse-specific regulation of AMPA receptor subunit composition by activity.</a> J Neurosci. 25, 6379-6388 (2005).</li><li>Xie, Z., Huganir, R. L., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+dendritic+spine+structural+plasticity+is+regulated+by+small+GTPase+Rap1+and+its+target+AF-6.">Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6.</a> Neuron. 48, 605-618 (2005).</li><li>Jones, K. 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+modulation+of+spine+morphology+by+the+5-HT2A+serotonin+receptor+through+kalirin-7+signaling.">Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling.</a> Proc Natl Acad Sci USA. 106, 19575-19580 (1957).</li><li>Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+spine+motility+in+the+mammalian+central+nervous+system.">Developmental regulation of spine motility in the mammalian central nervous system.</a> Proc Natl Acad Sci USA. 96, 13438-13443 (1999).</li><li>Lichtman, J. W., Conchello, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2, 910-919 (2005).</li><li>Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+spines+and+dendrites+distribute+dye+evenly.">Do spines and dendrites distribute dye evenly.</a> Trends Neurosci. 27, 445-446 (2004).</li></ol>

上面描述的详细定量分析树突棘形态，固定或现场初级皮层神经元的线性密度和蠕动的技术重点了解突触后的机制，可能有助于到neuropathologies影响。类似的方法可以用来量化脊柱形态或运动的任何刺的神经元，包括海马锥体，浦肯野，或中等刺神经元。 这里所描述的协议，可以适应看看突触蛋白和/或药物治疗树突棘的影响的基本性质。此外，它还可以被用来评估不等骨架蛋...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 18毫米的圆形玻璃盖号1.5 </td><td>华纳仪器</td><td> 64-0714（CS - 18R15） </td><td></td></tr><tr><td> 22平方毫米的盖玻片号1.5 </td><td>华纳仪器</td><td> 64-0721（CS - 22S15） </td><td></td></tr><tr><td>聚- D -赖氨酸</td><td>西格玛</td><td> P - 0899 </td><td> MW 70〜150 kDa的</td></tr><tr><td> Neurobasal媒体</td><td> Invitrogen公司</td><td> 21103049 </td><td></td></tr><tr><td> B27 </td><td> Invitrogen公司</td><td> 17504044 </td><td></td></tr><tr><td>谷氨酰胺</td><td> Invitrogen公司</td><td> 21051024 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140148 </td><td></td></tr><tr><td> D，L - APV（AP - 5） </td><td>攀登科学</td><td> ASC - 004 </td><td></td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668019 </td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965092 </td><td></td></tr><tr><td> HEPES </td><td> MediaTech Cellgro </td><td> 25 - 060 - C 1 </td><td> 1M，pH值7 </td></tr><tr><td>甲醛溶液</td><td>电解二氧化锰化学品</td><td> FX0415 - 5 </td><td> 36％，组织学级</td></tr><tr><td>正常山羊血清</td><td> VWR </td><td> 100188-514 </td><td>杰克逊Immunoresearch实验室</td></tr><tr><td> TRITON X - 100 </td><td> Fisher Scientific则</td><td> AC21568 - 2500 </td><td> Acros Organics公司</td></tr><tr><td>的Alexa Fluor 488山羊抗小鼠IgG（H + L）高度交叉吸附</td><td> Invitrogen公司</td><td> A - 11029 </td><td></td></tr><tr><td>的Alexa Fluor 488山羊抗兔IgG（H + L）*高度交叉吸附* </td><td> Invitrogen公司</td><td> A - 11034 </td><td></td></tr><tr><td>延长黄金抗淬灭剂</td><td> Invitrogen公司</td><td> P36934 </td><td>特殊包装</td></tr><tr><td>封闭式成像阶段室</td><td>华纳</td><td> RC - 30HV </td><td></td></tr><tr><td>温度控制器单元</td><td>华纳</td><td> TC - 344B </td><td></td></tr><tr><td> MetaMorph </td><td>通用成像</td><td></td><td></td></tr></tbody></table>

analysis of dendritic spine morphology in cultured cns neurons

树突棘是多数大脑内的兴奋连接的网站，并形成突触的突触后车厢。这些结构中的肌动蛋白丰富，并已被证明是高度动态的。在古典Hebbian可塑性以及neuromodulatory信号，树突棘可以改变形状和数量，这被认为是神经回路的细化和在大脑中的信息处理和存储的关键。树突棘之内，复杂的蛋白质网络连接允许树突棘的形态和数量的控制与肌动蛋白cyctoskeleton的细胞外信号。神经病理研究表明，从精神分裂症，自闭症谱系障碍的疾病状态，显示树突棘形态异常或数字。此外，最近的基因研究已经确定了在大量的基因编码突触的蛋白质突变，导致这些蛋白质可能会导致异常的脊柱可塑性的部分，这些疾病的病理生理基础的建议。为了研究这些蛋白质在控制树突棘形态/数量的潜在作用，培养皮层神经元的使用提供了几个优点。首先，这个系统可以在固定细胞树突棘的高清晰度成像以及时间推移活细胞成像。其次，在体外系统，这使得容易操纵蛋白质的功能表达的蛋白质，击倒的shRNA结构，或药物治疗。这些技术使研究人员开始解剖疾病相关的蛋白质的作用，并预测这些蛋白质的突变可能在体内的功能。

Watch this Scientific Journal Video about Analysis of Dendritic Spine Morphology in Cultured CNS Neurons at JoVE.com

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

许多最近的研究已经确定了在与大脑病症相关突触蛋白的基因突变。原代培养皮层神经元在研究这些疾病相关的蛋白质对树突棘的形态和运动的影响提供了极大的灵活性。

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Research

JoVE Journal

Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...