这项工作是支持的，战斗的儿童癌症基金会，阿拉巴马州伯明翰大学综合癌症中心的放射肿瘤学，Gabrielle的天使基金（ESY。）系的影响力大奖。

1。细胞培养<ol><li>的文化神经细胞和治疗自己的需要。 </li><li>收获细胞成15毫升管抽吸介质，清洗用磷酸盐缓冲液（PBS，钙和镁免费的），加入胰蛋白酶，收集在15毫升的试管中胰蛋白酶与适当的含药血清媒体。 </li><li>附带在1,000 xg离心5分钟。 </li><li>吸介质。 </li><li>在PBS中重悬细胞。 </li><li>附带在1,000 xg离心5分钟。 </li><li>吸媒体和新鲜的PBS重悬细胞。 </li><li>使用血球计数器或您首选的细胞计数器计数细胞。 </li><li>细胞样品立即开始检测之前，应准备。 </li><li>所有样品应处理在暗或黄色的光，以防止DNA免受紫外线伤害。 </li></ol> 2。彗星试验 1。幻灯片准备<ol><li>熔体1％琼脂糖（1克/ 100毫升的1X Tris碱，硼酸，EDTA，结核E）在微波3分钟，直到所有的颗粒消失。 </li><li>熔化的琼脂糖和DIP滑入边擦拭干净与kimwipe。琼脂糖允许空气干燥的透明膜。可以被存储，这是可以做到，预先和幻灯片。 </li></ol> 2。中性彗星实验<ol><li>冰寒裂解缓冲液（2.5M氯化钠，100mM的EDTA pH值为10，10mM的Tris碱，1％月桂基肌氨酸钠，和1％Triton X-100，pH为10）在4℃下，在使用之前的至少1小时。 </li><li> 2.2.2。熔体1％的低熔点琼脂糖（1克/ 100毫升在1X Tris碱，硼酸，EDTA）在微波炉中3分钟，直至所有的颗粒消失。琼脂糖的需要在水浴中冷却至37℃，以避免人工诱导彗尾。理想的情况下，需要被冷却为半小时后再使用琼脂糖。 </li><li> 2.2.3。稀释细胞悬浮液，使每毫升有100,000个细胞。结合与低熔点琼脂糖（在37℃下）的细胞悬浮液的比例为1:10（体积/体积），涡简要，并立即移液50微升到彗星幻灯片。使用移液管尖的侧的细胞悬浮液均匀地传播，在样品区。每个治疗组中应是至少在一式三份。 </li><li>地点滑动平面在冰箱中30分钟，直到出现一个圆形的外周中的幻灯片。 </li><li>预冷的裂解溶液和淹没幻灯片在该溶液中30分钟，在黑暗中在4℃（或在冰箱中）。 </li><li>倒出或吸液的裂解缓冲液，并添加1X中性电泳缓冲液（Tris碱，硼酸，EDTA 1X TBE缓冲液）。留在这个缓冲区在冰箱中半小时的幻灯片。 </li><li>加入预冷1X中性电泳缓冲液（TBE），电泳室，电泳滑动托盘的地方幻灯片。对齐，使它们从电极等距离的幻灯片。 </li><li> 1X中性电泳缓冲液倒入0.2英寸以上幻灯片。过量的缓冲区间FERE电泳。 </li><li>将电源电压，以每平方厘米1 V（测量电极，电极），并在4°C（或冷水机房）在黑暗中运行30分钟。 </li></ol> 3。碱性彗星试验<ol><li>冰寒裂解缓冲液（2.5M氯化钠，100mM的EDTA pH值为10，10mM的Tris碱，1％月桂基肌氨酸钠，和1％Triton X-100，pH为10）在4℃下，在使用之前的至少1小时。 </li><li>熔体1％低熔点琼脂糖（1克/ 100毫升在1X Tris碱，硼酸，EDTA）在微波炉中3分钟，直至所有的颗粒消失。然后冷却在37℃水浴中至少30分钟。 </li><li>结合细胞在1×105 /毫升用熔融低熔点琼脂糖（在37℃下）在一个比为1:10（体积/体积），涡旋简单地说，并立即移液50微升到彗星幻灯片。使用移液管尖的侧的细胞悬浮液均匀地传播，在样品区。每个治疗组中应是至少在一式三份。 </li><li>地点下滑上课平在冰箱中30分钟，直到出现一个圆形的外周中的滑动。 </li><li>在预冷的裂解液浸泡幻灯片和离开在4°C，1小时在黑暗中过夜。 </li><li>从裂解液中取出幻灯片，沥干的幻灯片和冲洗一次用冷的中和缓冲液5分钟，以除去残留的清洁剂和它的盐，碱 - 开绕工序之前。 </li><li>将幻灯片中的凝胶电泳室充满了预冷的新鲜电泳缓冲液（300毫米1毫米的NaOH，EDTA，pH值&gt; 13）不超过0.5厘米以上的幻灯片。对齐，使它们从电极等距离的幻灯片。 </li><li>让幻灯片坐的碱性缓冲液中，在黑暗中30分钟，以允许解除碱负责损害的DNA和表达。 </li><li>电源电压，以每平方厘米1 V（测量电极，电极），运行30分钟，在4°C（或冷水机房）。 </li></ol> 4。固定和站矿业细胞<ol><li>沥干多余的电泳缓冲液</li><li>浸入滑动预冷的蒸馏水，在室温5分钟。 </li><li>在RT下的5分钟的预冷冻的70％乙醇中的浸入滑动。 </li><li>干样品过夜。不要让幻灯片明亮的光线。样品可以得分在这个阶段之前，在室温下存放数月。 </li><li>通过浸渍在SYBR GREEN（1X在PBS中稀释）在冰箱中的20分钟染色幻灯片。 </li><li>删除幻灯片，并允许他们在黑暗中完全干燥。琼脂糖完全干燥时也会变得透明。 </li></ol> 4。图像采集和分析<ol><li>采集图像，使用荧光显微镜的绿色过滤器“（蔡司AxioVision）和分析利用彗星分析软件（感知工具）。 </li><li>点击“彗头”（核）和软件计算出的平均尾矩和DNA量的参数，包括核。 </li><li>分析至少200个细胞，每个处理。 </li><li>将数据导出到Microsoft Excel。 </li><li>计算平均每个治疗组和绘图数据，适当尾矩。 </li></ol> 5。代表性的成果对神经元细胞的彗星试验分析的一个例子在图2和图3所示。在这种情况下，照射的神经元细胞诱导的DNA损伤。由于细胞受到的电场，该DNA迁移由于它随后被使用彗星试验检测软件分析差异大小以不同的速率。更多的DNA损伤，更远的DNA迁移出的核心。这将减少在细胞核中的荧光强度，它随后被拾起在更高的尾矩由软件和结果表1示出了代表性的表从彗星试验分析和图4示出了代表中对准焦点图比较神经细胞的DNA损伤的，辐射测量彗星试验。 <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="图1"/> 图1。流程图彗星试验。 <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="图2"/> 图2代表的神经元细胞的图像（A）的不和（B）与彗尾。 <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="图3"/> 图3。彗星试验分析，使用彗星试验软件。 （A）代表屏幕截图使用卡尔蔡司荧光显微镜获得的图像和彗星试验分析检测软件。 （B）的细胞核或“彗星头”（绿色）点击产生的荧光图和图（黄色）和（C）数据表（红色圆圈）。 <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="图4"/> 图4代表通过绘制通过分析在使用中性彗星实验的照射和非照射的神经细胞的DNA损伤的平均尾矩而获得的曲线图。正如预期的那样，在照射神经细胞由更高的平均尾矩3Gy辐射（X-射线），诱导DNA损伤所描绘。图中所示为平均尾矩（+ / - 标准差），** P &lt;0.01。 <img src="/files/ftp_upload/50049/50049table1.jpg" alt="表1"/> 表1中。 </STRONG&gt;代表辐照与未辐照的神经细胞彗星试验分析表。 <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">点击这里查看大图</a> 。

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没有利益冲突的声明。

彗星试验分析单个细胞具有独特的能力。这是有利的鉴定表明微分响应的细胞毒性剂的细胞亚群。一个必须考虑一些实际的限制。的细胞数，可以单独评估，可能会有所不同，这取决于个人。样本大小需要增加内的人口在DNA损伤，如果有方差。可行的单细胞悬液，此测定是至关重要的，因为优势坏死或凋亡的细胞的存在下，将加至错误。的细胞凋亡，小的DNA片段（小于50KB），线粒体DNA的裂解和电...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论（可选） </td></tr><tr><td> 1.5 ml离心管</td><td>圣克鲁斯生物技术公司</td><td> SC-200271 </td><td></td></tr><tr><td> 10X TBE（Tris碱，硼酸，EDTA） </td><td> Fisher Scientific则</td><td> BP13331 </td><td></td></tr><tr><td> 15 ml管</td><td> Fisher Scientific则</td><td> 0553851 </td><td></td></tr><tr><td>琼脂糖</td><td>西格玛</td><td> A5093 </td><td></td></tr><tr><td>铝箔</td><td> Fisher Scientific则</td><td> 01213101 </td><td></td></tr><tr><td>烧杯</td><td> Fisher Scientific则</td><td> FB102300 </td><td></td></tr><tr><td>离心分离</td><td> Thermo Scientific的</td><td> 75004261PR </td><td></td></tr><tr><td>彗星试验检测软件</td><td>彗星试验IV图像分析系统</td><td></td><td></td></tr><tr><td>气缸</td><td> Fisher Scientific则</td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td>电泳室</td><td> Thermo Scientific的</td><td> 09528101 </td><td></td></tr><tr><td>乙醇</td><td> Fisher Scientific则</td><td> A407P4 </td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司Axio的愿景</td><td></td><td>荧光显微镜绿色过滤器就足够了</td></tr><tr><td>血球</td><td> Fisher Scientific则</td><td> 0267152 </td><td></td></tr><tr><td>低熔点琼脂糖</td><td> Promega公司</td><td> V2111 </td><td></td></tr><tr><td>微波</td><td>西尔斯</td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水，无钙，镁</td><td>胎牛血清</td><td> SH3025601 </td><td></td></tr><tr><td>电源</td><td> BioRad公司</td><td> 1645050 </td><td></td></tr><tr><td>冰箱</td><td>西尔斯</td><td></td><td></td></tr><tr><td>统治者</td><td>斯台普斯</td><td></td><td></td></tr><tr><td>滑动</td><td> Fisher Scientific则</td><td> 12550143 </td><td></td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S7653 </td><td></td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> S5881 </td><td></td></tr><tr><td>月桂基肌氨酸钠</td><td> Fisher Scientific则</td><td> S529 </td><td></td></tr><tr><td> SYBR绿色</td><td> Invitrogen公司</td><td> S7585 </td><td></td></tr><tr><td>托盘</td><td>金融机构她的科学</td><td> 15242B </td><td></td></tr><tr><td>三基地</td><td>西格玛</td><td> T6066 </td><td></td></tr><tr><td>的Triton-X 100的</td><td>西格玛</td><td> T8787 </td><td></td></tr><tr><td>胰蛋白酶</td><td>胎牛血清</td><td> SH3023601 </td><td></td></tr><tr><td>涡流</td><td> Fisher Scientific则</td><td> 02216108 </td><td></td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

一些药物的目标是DNA修复途径，通过建立DNA损伤诱导细胞死亡。因此，直接测量DNA损伤的进程已经得到了广泛的评估。传统的方法是费时的，昂贵的，资源密集型的时间，需要复制的细胞。与此相反，彗星试验，单细胞凝胶电泳法，是更快的，非侵入性，价格低廉，直接和敏感的DNA损伤和修复措施。所有形式的DNA损伤以及DNA修复可以使用这个强大的技术在单细胞水平的可视化。 彗星试验的原则是高度有序的，完整的DNA，而DNA损伤，破坏了这种组织。受损的DNA的琼脂糖基质渗入和电场进行时，带负电荷的DNA是带正电的向阴极迁移。大的未损坏的DNA链是不能够迁移远离原子核。 DNA损伤创建更小的DNA片段更远较完整的DNA。彗星试验检测，图像分析软件，措施和比较的整体在细胞核中的DNA的荧光强度与已迁移出细胞核的DNA。从迁移的DNA的荧光信号是正比于DNA损伤。更长的明亮的DNA尾着增加DNA损伤。某些测量的参数，该参数是尾矩，它是衡量两者的DNA的量和分布在尾部，尾部长度的DNA和DNA的百分比在尾部。此法可以测量DNA修复以及修复已经发生DNA损伤的决议表示。灵敏度的极限是约50的链断裂每二倍体哺乳动物细胞1,2。与任何DNA损伤剂，如鬼臼乙叉甙处理的细胞，也可以使用作为阳性对照。因此，彗星试验是一种快速，有效的方法来衡量DNA损伤。

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

彗星试验是检测单和双链断裂，包括碱不稳定的的网站和DNA-DNA/DNA-protein交联在所有细胞中的DNA，包括海马神经元的一种有效方式。由于DNA损伤的量的差异，在电场中的方法，利用差DNA迁移。

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K 次观看.  University of Alabama-Birmingham. 彗星试验是检测单和双链断裂，包括碱不稳定的的网站和DNA-DNA/DNA-protein交联在所有细胞中的DNA，包括海马神经元的一种有效方式。由于DNA损伤的量的差异，在电场中的方法，利用差DNA迁移。

视频: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

含量测定β-淀粉样蛋白的毒性，使用转基因 C。线虫模型

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

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. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人希马神经前体细胞的乳化电路特定调节

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...