The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

以下协议符合动物保护的加拿大议会的指引，并已获得医药动物保健委员会的教授在多伦多大学。 1，准备好所需的试剂中的说明表1 <ol><li>添加蛋白酶和磷酸酶（如果需要的话）的抑制剂，以所有的蔗糖溶液，缓冲液和DDH 2 O的以表1所列的浓度。重要：执行所有的步骤在4℃和预冷所有在实验前的起始试剂和设备。标签所有管道，包括超速离心管，用记号笔。 </li></ol> 2，解剖相应的脑区<ol><li>颈椎脱位或断头牺牲动物。众议院和安乐死的动物，根据动物保健机构和政府的政策。 </li><li>快速去除日ê大脑从冷冻解剖块头骨和地点。 </li><li>从新鲜组织解剖相应的大脑区域，并继续执行步骤3。 </li></ol> 3，组织匀浆用电机驱动玻璃特氟隆均质机<ol><li>将解剖组织50-100毫克（为SPM）或100-200毫克（为PSD），在含有4毫升的0.32M HEPES缓冲蔗糖溶液标记13毫升聚丙烯管。将样品转移到15毫升锥形玻璃铁氟龙组织研磨机/均质器，设置电机驱动器900转（设置7），均质12招在30秒时段的样本。使用不同的玻璃匀浆器特氟龙样品之间，或用清水冲洗样本之间的冷蒸馏水的均质机，用的Kimwipe擦干。注：最多4个克组织的可匀浆于4毫升的0.32M HEPES缓冲蔗糖溶液，但最好是使用较少的组织，以确保适当的分级分离。 </li><li>均匀化的样本传送回吨Ø同13毫升聚丙烯管。储备100微升匀浆并存储在-80℃用于随后的蛋白质定量和总蛋白级分的免疫印迹分析。 </li></ol> 4，低速离心去除细胞核部分（收益率上清S1） <ol><li>离心机在4℃下将匀浆物在一个固定角转子，在900×g离心10分钟。上清液（S1）转移到一个新的，标为13毫升管和悬浮细胞核部分沉淀（P1），在500微升的0.32M HEPES缓冲蔗糖。注：（P1）的部分可被存储在-80℃下，并用于核级分的免疫印迹分析。 </li></ol> 5，丰富的粗突触体部分的（球团产量P2） <ol><li>离心上清液（S1）以10,000 xg离心15分钟，在4℃下。取出上清液（S2）和储备500μL的微量离心管中保存在-80℃，随后蛋白定量ification和胞浆/轻膜部分免疫印迹分析。重悬剩余的粗突触体部分沉淀（P2）在1ml的0.32M HEPES缓冲蔗糖溶液，然后添加另一毫升0.32的3M HEPES缓冲蔗糖溶液。 </li><li>离心机以10,000×g进行15分钟，在4℃下。取出上清液（S2&#39;），并储备500μL的微量离心管中保存在-80℃，随后蛋白定量和胞浆/轻膜部分免疫印迹分析。保存洗涤的粗突触体丸粒（P2&#39;）的聚丙烯管中。 </li></ol>在原油突触体部分的6裂解（低渗休克） <ol><li>裂解的粗突触体丸粒（P2&#39;）的聚丙烯管中再悬浮是在1ml的DDH 2 O的添加另一个3毫升DDH 2 O的，转移到玻璃铁氟龙组织匀浆，并经均质手3招。注意：这是很重要的以尽可能快地执行此步骤，并迅速进行到步骤6.2。 </li><li>从均化器放回同一13毫升聚丙烯管迅速转移样品调整样本回至4毫米的HEPES和16微升的1M HEPES溶液，颠倒混合。 </li><li>转动样品，在4℃下进行30分钟以确保完全裂解。 </li></ol> 7，丰富的突触膜部分（P3） <ol><li>离心机以25,000×g离心20分钟，在4℃下。除去粗泡状部分上清液（S3），并保留其在5毫升管存放于-80℃，随后蛋白定量和Western blot分析。重悬的突触体膜部分（P3）在1ml的0.32M HEPES缓冲蔗糖溶液。 </li></ol> 8，制备连续蔗糖梯度<ol><li>吸管准确3.5毫升的1.2M HEPES缓冲蔗糖溶液和准确3.0毫升每1.0摩尔和0.8摩尔HEPES-缓冲蔗糖溶液为单独6毫升卡口盖管。注意：这是为了预先测量，用于使蔗糖梯度的缓冲液的体积。卷的精确测量是非常重要的，以确保所得到的梯度将在超速离心平衡。 </li><li>使用玻璃巴斯德吸管和灯泡，转移的1.2M HEPES缓冲蔗糖溶液的12毫升异质同超速离心管。小心层上的1.2M HEPES缓冲蔗糖溶液的顶部的1.0M HEPES缓冲蔗糖溶液。最后，层上的1.0M HEPES缓冲蔗糖溶液的顶部的0.8M的HEPES缓冲蔗糖溶液。使用新鲜的巴斯德吸管每个蔗糖溶液，并注意不要打扰蔗糖梯度。从板凳漩涡或离心振动会干扰梯度的完整性。 </li><li>使用巴斯德吸管，层上的准备连续蔗糖梯度顶部的悬浮突触膜部分（P3）。确保该梯度是通过称量它们的水平​​转头的水桶内平衡。如果水桶是不均衡的，使用P200移液管小心地添加的0.32M HEPES缓冲蔗糖梯度的顶部和平衡转子轮叶。 </li></ol> 9分馏的突触质膜（SPM） <ol><li>超速离心机的水平转头150000 XG 2小时，4℃。小心地从超速离心桶取出蔗糖梯度。用18G的针和1ml注射器，穿刺管在1.0米/ 1.2M的HEPES缓冲蔗糖溶液相间的底部，并撤回突触质膜层（SPM）。 </li><li>记每个SPM样品占据的1ml注射器中的体积。将收集到的SPM层在3.5mL厚壁超速离心管中，添加恰好2.5体积的4 mM的HEPES调节从1.2M的蔗糖浓度为0.32 M。一旦各样品已经调整编到0.32M蔗糖，平衡管和0.32M的HEPES缓冲蔗糖溶液。 </li><li>超速离心机在一个固定角转头200000×g离心30分钟，在4℃。拆下并丢弃上清液。悬浮突触质膜颗粒（SPM）的300微升50毫米肝素钠/ 2毫米的EDTA溶液。直到用于免疫印迹商店的样品在-80℃。 </li></ol> 10，准备突触后密度的分数（PSD） <ol><li>解冻的SPM样品在冰上。 </li><li>让0.54％的Triton X-100的50毫米肝素钠/ 2毫米EDTA溶液的解决方案。 </li><li>在5ml的聚苯乙烯管中，结合2.7的Triton X-100 / HEPES / EDTA溶液和300μl的SPM分数毫升，旋转样品15分钟，在4℃下</li><li>转移样品至3.5毫升厚壁超速离心管并离心样品在32,000 xg离心20分钟，在4℃下。 </li><li>保留上清液（曲拉通X-100可溶组分[TS]）​​，并储存在-80;°℃。注：在此阶段，将沉淀表示“PSD-1T”分数。用洗衣粉第二次治疗将产生一个“PSD-2T”部分。如果PSD-1T部分需要，继续执行步骤10.6。 <ol><li>以产生一个PSD-2T馏分，重悬在3ml 0.5％的Triton粒料X-100的50mM HEPES / 2mM EDTA的溶液中。旋转样品15分钟，在4℃下。以32,000×g离心20分钟，在4℃下离心样品，并继续执行步骤10.6。 </li></ol></li><li>弃上清，重悬突触后（PSD）颗粒在50毫米肝素钠/ 2 mM的EDTA溶液。注：该悬浮量可取决于颗粒的大小;建议50-75微升的体积。由于PSD的分数是洗涤剂不溶部分，通常需要加入1-2微升0.5％SDS中以使蛋白质的完整的再悬浮。直到用于免疫印迹商店的样品在-80℃。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+scaffolds+for+nicotinic+receptors+on+neurons.">Postsynaptic scaffolds for nicotinic receptors on neurons.</a> Acta Pharmacol Sin. 30, 694-701 (2009).</li><li>Cella, N., Cornejo-Uribe, R. R., Montes, G. S., Hynes, N. E., Chammas, 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+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

The authors have no conflicts of interest to disclose.

还有在该过程的几个步骤，这对于一个成功的结果至关重要。在步骤3中，重要的是均质化的一致度，实现对每个样品。当均质化组织与马达驱动的均化器，以恒定速度不仅用于杵的旋转，而且还与笔划的数量。在渗透休克的温育时间应精确，因为在低渗溶液中的扩展同质或孵化将裂解线粒体和污染SPM样品。 另一个关键步骤是在试剂，尤其是蔗糖溶液的制备;如果该蔗糖溶液的...

神经元通过突触进行通信，并且该通信质量是通过在蛋白质在突触的组成改​​变调节到很大程度。特别是位于突触后密度蛋白通过密切脚手架神经递质受体与它们的信号转导系统1参与神经元沟通。此外，在突触效能的强度持久的变化是由于突触后密度1-6添加或去除受体的控制。因此，突触蛋白的分离和定量是一种必要的和有用的技术，来获得有关该神经元响应的刺激和改变突触效能7的方式。本文介绍了一种常见的技术，通过离心分离从啮齿动物脑组织中突触蛋白在连续蔗糖梯度。突触细胞膜部分可以得到丰富和孤立的基础上其在蔗糖密度，已根据经验确定，以类似于1.2M的蔗糖。 根据不同的生物学问题，亚细胞组分可以由蔗糖或珀可连续或不连续梯度进行分离。连续梯度允许蛋白质成多个组分的分离;这可以是特别有用的，以证明一个给定的级分8中的共定位的蛋白质。然而，连续梯度的制备是更费力的和是不必要的许多应用。不连续的梯度是比较容易制备和可用于蛋白分离成几，通常定义的馏分。这是由增加的摩尔浓度的3蔗糖层的不连续梯度，已被广泛用于分离与突触质膜（SPM）相关的蛋白质。本突触质膜级分可被进一步加工，以突触后密度fractioN（PSD）的洗涤剂不溶部分的去污剂处理和隔离。 当这个过程在1960年的9,10首次被描述，电子显微镜被用来证实细胞器及膜的大致限定突触质膜和突触后密度级分9-14。这些研究证明了包含前和突触后膜和在SPM分数突触小泡;洗涤剂治疗主要是电子致密后，突触后密度为可见的。在该过程中，低渗休克用于从细胞体10夹断一样处理。这个步骤需要的事实，线粒体是渗透冲击更耐和保持不动，并且因此它们在蔗糖梯度（ 图1）的底部沉淀的优点。 用同样的富集技术，对SPM和PSD分数分别为第一生化通过聚丙烯酰胺凝胶电泳和主要蛋白质组分15-17的序列来定义。随后免疫印迹分析已被用于检测和定量突触蛋白的水平，并进一步确定这些级分（ 图2）。我们已经使用该技术在我们的实验室进行量化改变多巴胺转运时Slc6a3基因被复制在小鼠18所发生的突触水平。我们也使用这种技术在NMDA受体缺陷小鼠揭示突触特异性降低的蛋白质，是在DISC1相互作用组19的一部分。 它是由免疫印迹分析了SPM的馏分含有突触小泡膜蛋白，核内体标记物，线粒体蛋白质，膜相关合成酶和信号转导分子，以及突触后密度的组成部分和突触质膜20-23明显。 ËVEN PSD级分可具有污染具有丰富的线粒体蛋白质，它可能有必要进行第二梯度沉降或额外的纯化步骤，以除去它们13。最近，定量质谱法提供了超过100个蛋白质在单独的突触后密度的列表，以及这些部件24,25的相对丰度的指示。

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preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

神经元亚细胞分离技术，允许被贩卖，并从突触蛋白的定量。如在1960年代末期最初所述，突触质膜相关蛋白，可以通过超速离心法在蔗糖密度梯度分离。一旦突触膜分离，大分子复合物被称为突触后密度可以随后，由于其不溶于洗涤剂中分离。用于分离突触细胞膜和突触后密度蛋白的技术基本上保持40年后相同的，并且被广泛应用于目前神经科学研究。本文将详细介绍与突触细胞膜及突触后密度采用不连续蔗糖密度梯度相关的蛋白质的分离。导致蛋白制剂适用于免疫印迹或2D DIGE分析。

在蔗糖密度梯度的制备方法应导致蔗糖（0.8，1.0和1.2M的蔗糖）的3摩尔的解决方案的明确分离。参见图3A为渐变的示例的蛋白质样品加入之前。如果梯度制备太多提前，或者如果它被制备的​​长凳上表面与来自其它设备的振动，梯度将受到损害，并适当分离，将无法实现。如果三种解决方案的明确分离是不可见的，最好是加入的蛋白样品之前，使一个新的梯度。参见图3B...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

本文将详细介绍与突触质膜上超速不连续蔗糖梯度相关蛋白的富集。随后的制备突触后密度蛋白也被描述。蛋白制剂适用于免疫印迹或2D DIGE分析。

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

31.0K 次观看.  University of Toronto. 本文将详细介绍与突触质膜上超速不连续蔗糖梯度相关蛋白的富集。随后的制备突触后密度蛋白也被描述。蛋白制剂适用于免疫印迹或2D DIGE分析。 

视频: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

膜电位，突触反应，神经回路，神经调节和肌肉组织学用小龙虾：学生实验室练习

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

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

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.