小灵通格兰特（MH51393）支持这项工作。 ，我们使用的一些海兔海兔的下RR10294迈阿密大学的研究资源，美国国立卫生研究院的国家中心提供的国家资源。

1。准备<ol><li>麻醉的动物注射75-100毫升等渗氯化镁溶液。我们使用成像的海兔一般在150-200克，并从马里努斯科学。 </li><li>针麻醉动物蜡盖菜。注射器针头的工作以及用于此目的，无菌操作技术是没有必要的。总产钳和标准剪刀做一个切口在动物的脚和暴露颊质量。找出颊神经节。使用春风似剪刀和细镊子，小心地切割颊神经节。 </li><li>删除的节，并将其放置在一个Sylgard的涂层菜含有人工海水。将一些昆虫引脚通过颊神经，稳定神经节。 Desheath使用超细钳和春风似剪刀，以免暴露的神经元细胞内记录的口腔神经节。然后重新针神经节，约15细虫（分utien）引脚。节必须牢牢掌握在地方举行的任何运动将在成像过程中的问题。 </li></ol> 2。准备电极<ol><li>拉使用玻璃毛细管用灯丝电极（例如WPI TW100F-4）和一个牵拉。应设置单独创建所需的电阻的电极。当填充有3 M醋酸钾（乙酸钾）我们的电极电阻通常为约10兆欧姆，如果电极是倾斜的，或约5兆欧姆如果斜切步骤被省略。 </li><li>填充的溶液含有3 M KAc/30 mM KCl中使用的注射器和microfil的针电极的一组。这些电极将被用于电生理学。 </li><li>填充与钙指示剂染料的第二组电极，通过浸渍染料中的电极的后端，先前与大约重组。 40微升蒸馏水为500μg染料。当在电极的前端已填充，回填土约1/4英寸用200mM的KCl，以确保良好的电接触的电极的端部。 </li><li>锥电极是有利的，因为这有利于染料注射和时间造成的伤害由多个膜贯穿。我们使用自定义的倒角，从而生成流的盐水/氧化铝粉悬浮液。电极被安装在一个支架内，和它的端部进入的流在45°的角度。电极电阻的连续监测和终止斜切当阻力达到约5兆欧为醋酸钾充满的电极时。的染料填充电极斜切一个相等的时间量，并典型地具有15-20兆欧姆的电阻。 </li></ol> 3。载入中钙指示剂染料<ol><li>将准备上电钻机，直观地定位在突触前神经元的利息。我们的实验已经完成了一个确定的感觉神经元，利用在喂养过程中，B21 5，6。相对固定positio的N，尺寸（约100微米），B21的长形的胞体，它很容易找到。 </li><li>穿刺含有钙指示剂染料与电极的神经元的利益。刺穿突触后神经元B8，B21的身份进行验证。去极化的B21〜8 nA电流触发峰值，并在促进抽水蓄能电站B8 7的结果。 </li><li>注入染料在突触前神经元的利益，通过超极化脉冲（通常为-15 nA的，1赫兹，75％的占空比），约30分钟。如果染料加载成功，神经元的胞体将获得一个淡淡的粉红色。中取出的含染料的电极通过缓慢地撤回从神经元。 </li><li>发布准备约30分钟，以使染料扩散到精细的工序，使从动神经元突触接触。 </li></ol> 4。钙成像和电生理记录<ol><li>将准备的成像力显微镜氧化聚乙烯。与Permagum密封线件，不同的是，粘土，仍然粘湿的时候冷却平台上保持与该制剂的菜。固定台显微镜主要由移动目标。阶段保持静止，它允许连接磁铁安装机械手。作为高倍率使得振动（特别是横向运动）的一个问题，整个成像设置应放置在隔振平台。 </li><li>与含有正常电解质溶液的电极穿刺突触前和突触后神经元。 </li><li>选择一个过滤器模块适用于图像钙橙色（激发波长549 nm，发射576纳米）和调整摄像头。让研究者选择最科学的CCD相机的CCD读出的细胞的一个子集。此外，细胞可以组合（离散化）。选择一个较小的子集的增长速度，而分级提高了灵敏度。这两个设置，一起曝光时间，确定帧速率，即可以每秒采集多少张图像。合适的设置将图像的面积为580×350微米，500×300像素（使用10倍镜头和0.5倍的相机适配器）。添加中性密度过滤器的光路调整的照明强度的最小量，提供正确的曝光的曝光时间&lt;25毫秒。这些设置导致大约30Hz的帧速率-在海兔是速度不够快，图像的单穗诱发钙的改变。最后，关闭，以便只有被照亮的成像区域的场光圈。 </li><li>在成像软件，你要测量神经元的区域标记。 AR软件中的元素，这是在“时间测量”部分，通过把地区的利益（投资回报率）。 B21是一个双极性神经元，和以前的工作已经证明，其横向过程联系人感兴趣的9突触后神经元。我们一般图像小学，中学，第三胸罩通过放置一个ROI在每个部分的神经元中的横向过程nches。旁边的染料填充神经元的一个额外的投资回报测量提供一种购买]是从所有其它的数据点中减去背景值。 </li><li>指导电生理采集（在这些实验中穗II）等待相机“帧读出”信号提供一个触发信号。启动图像采集（元素AR），穗II将自动开始录音时，相机将拍摄第一帧。您还可以使用TTL输出功率1401上的LED安装在显微镜的相机端口暂时关闭。这将提供一个信号，随后同步成像和电生理数据。 </li><li>刺激突触前神经元通过注入一系列简要的去极化电流脉冲在所需的频率。重要的是要监视每个脉冲（如刺激触发示波器），以确定是否一个动作电位是successfully产生。 ，而钙橙色是非常相比，其他染料光稳定性，这是一个好主意，记录无刺激的对照试验，检查多少染白化现象。 </li><li>在实验中，细胞内钙离子浓度的被操纵，药物如EGTA引入突触前神经元，并重复上述的刺激和数据采集步骤。 </li></ol> 5。有代表性的数据分析<ol><li>它写入到一个文本文件中导出的成像数据（测得的强度值列表中的每个投资回报率）。在穗II自定义脚本可以导入的文本文件。该脚本还带的各个探针的区的归一​​化值，并减去的值的背景探针。的数据点，然后绘制作为“虚拟RealWave”通道旁边的电生理学数据。 “道工序/时移功能在穗II”是用来精确地对准成像数据POI个核苷酸，使用LED的信号作为参考。 </li><li>要获取值的百分比变化，分 ​​析成像数据计算的相对变化荧光DF / F O =（FF O）/ F o的。 F o的代表在刺激之前的刺激和F的荧光的荧光水平。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/3907/3907fig1.jpg" alt="图1" /> 图1。总体方案的实验。在海兔神经节准备进行实验。细胞内钙的变化进行成像用荧光染料，这是离子电渗疗法的突触前神经元引入。可以诱导和监控，使突触传递和突触后神经元，然后用锋利的电极刺穿。 <img src="/files/ftp_upload/3907/3907fig2.jpg" alt="图2" /> <strONG&gt;图2。钙成像和海兔口腔神经节神经元细胞内记录。 （A）照片的成像侧枝神经元B21。的方块表示测得的地区的利益。 （B1）的B21细胞内刺激唤起了动作电位（底部的迹线）和促进突触后电位（私人参建居屋计划）跟随神经元的突触后B8（中跟踪）造成的，也火了动作电位。增加突触前细胞钙离子荧光显示在顶部曲线。 （B2）单突触型长期促进突触前EGTA的影响。 EGTA细胞内注入B21〜15分钟内刺激。 EGTA它被认为是一个缓慢作用的钙离子螯合剂，它在低浓度时抑制突触传递速度不够快。注意的广泛钙信号和相应的减少在PSP振幅减少。 （C）的的PSP幅度与突触前calciu的米的信号。 <a href="http://www.jove.com/files/ftp_upload/3907/3907fig2large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
	<li>Zucker, R. S., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+synaptic+plasticity.">Short-term synaptic plasticity.</a> Annu. Rev. Physiol. 64, 355-405 (2002).</li><li>Eberhard, M., Erne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+to+fluorescent+calcium+indicators:+Calcium+green,+calcium+orange+and+calcium+crimson.">Calcium binding to fluorescent calcium indicators: Calcium green, calcium orange and calcium crimson.</a> Biochem. Biophysical Res. Comm. 180, 209-215 (1991).</li><li>Escobar, A. L., Velez, P., Kim, A. M., Cifuentes, F., Fill, M., Vergata, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+properties+of+DM-nitrophen+and+calcium+indicators:+rapid+transient+response+to+flash+photolysis.">Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.</a> Eur. J. Physiol. 434, 615-631 (1997).</li><li>Ivanov, A. I., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+spike-mediated+synaptic+transmission+by+presynaptic+background+Ca2++in+leech+heart+interneurons.">Modulation of spike-mediated synaptic transmission by presynaptic background Ca2+ in leech heart interneurons.</a> J. Neurosci. 23, 1206-1218 (2003).</li><li>Rosen, S. C., Miller, M. W., Evans, C. G., Cropper, E. C., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+connections+between+peptidergic+radula+mechanoafferent+neurons+and+neurons+in+the+feeding+system+of+Aplysia.">Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia.</a> J. Neurophysiol. 83, 1605-1620 (2000).</li><li>Ludwar, B. C. h., Evans, C. G., Jing, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+mechanisms+mediate+potentiating+effects+of+depolarization+on+synaptic+transmission.">Two distinct mechanisms mediate potentiating effects of depolarization on synaptic transmission.</a> J. Neurophysiol. 102, 1976-1983 (2009).</li><li>Evans, C. G., Ludwar, B. C. h., Askansas, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+holding+potential+on+the+dynamics+of+homosynaptic+facilitation.">Effect of holding potential on the dynamics of homosynaptic facilitation.</a> J. Neurosci. 31, 11039-11043 (2011).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Handbook. , (2005).</li><li>Borovikov, D., Evans, C. G., Jing, J., Rosen, S. C., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proprioceptive+role+for+an+exteroceptive+mechanoafferent+neuron+in+Aplysia.">A proprioceptive role for an exteroceptive mechanoafferent neuron in Aplysia.</a> J. Neurosci. 20, 1990-2002 (2000).</li><li>Goldberg, J. H., 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=Chapter+38:+A+practical+guide:+Two-photon+calcium+imaging+of+spines+and+dendrites.">Chapter 38: A practical guide: Two-photon calcium imaging of spines and dendrites.</a> Imaging in Neuroscience and Development. , (2005).</li></ol>

我们什么都没有透露。

我们证明，可用于同时监测细胞内钙离子浓度和评价疗效突触传递的技术。这些技术是有用的，用于确定如何短期可塑性的各种形式的介导。 用荧光显微镜，CCD摄像机的摄像进行。这些设备的要求也比较适中相比，功能最全的成像设置。该技术简单，易于学习。虽然用CCD照相机的成像允许可视化的大面积具有良好的时间分辨率，空间分辨率是有限的。因此，它是一种有用的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>钙橙色</td><td> Invitrogen公司</td><td> C-3013 </td><td></td></tr><tr><td> EGTA </td><td>西格玛</td><td> E-4378 </td><td></td></tr><tr><td>钙校准缓冲液试剂盒</td><td> Invitrogen公司</td><td> C-3008MP </td><td>用于测试的信号的灵敏度和动态范围的非常有用</td></tr><tr><td>氯化镁六水合物</td><td>西格玛</td><td> M0250 </td><td> 0.33米的解决方案中使用的麻醉动物</td></tr></tbody></table> 表1所用的试剂。 <table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 评论 </td></tr><tr><td> FN-1直立荧光显微镜</td><td>尼康仪器</td><td>与成茂ITS-FN1阶段</td></tr><tr><td> NMN-21机械手</td><td>成重</td><td>安装在舞台上与磁铁</td></tr><tr><td> CoolSNAP HQ CCD相机</td><td>光度</td><td></td></tr><tr><td> NIS元素A​​R （版本3.22） </td><td>尼康仪器</td><td>用于收购的荧光数据成像软件</td></tr><tr><td>福陆公司的目标10X/0.3w计划</td><td>尼康仪器</td><td>这水浸没透镜有很长的工作距离为3.5毫米</td></tr><tr><td>的X-Cite 120 PC金属卤化物灯</td><td> EXFO </td><td>用于荧光成像</td></tr><tr><td> LS-DWL 卤素灯</td><td> Sumica旅游</td><td></td></tr><tr><td> ET-CY3滤波器组</td><td>色度技术</td><td>; </td></tr><tr><td>电源1401 A / D转换器</td><td>剑桥电子设计</td><td>在3 kHz取样， </td></tr><tr><td>穗II （7.07版） </td><td>剑桥电子设计</td><td>软件用于收购电数据</td></tr><tr><td> SEC-10 LX放大器</td><td> NPI电子</td><td>使用10倍的探头</td></tr><tr><td> 410型放大器</td><td>布朗利精度</td><td>用于放大和滤波的信号</td></tr><tr><td> WS-4 </td><td>减去k科技</td><td>隔振成像</td></tr><tr><td>散热平台</td><td>客户定制</td><td>黄铜板上，通过该冰水以可变速率被泵</td></tr></tbody></table> 表2使用的设备。

monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc aplysia

它已被建议的改变的细胞内钙介导诱导了一些重要的形式的突触可塑性（例如，单突触型长期便利）1。这些假设可以通过同时监测细胞内钙的变化和变更，在突触效能测试。我们演示了如何这可以通过与细胞内记录技术相结合钙成像。我们的实验是进行中的软体动物海兔夜蛾的口腔神经节。该制剂具有许多实验的有利特征：Ganglia可以很容易地从海兔中除去和实验用成人的神经元，使正常突触连接，并具有一个正常的离子通道分布。由于低代谢率的动物和相对低的温度（14-16℃），是天然的海兔 ，制剂是稳定的，很长一段时间。 ENT“&gt;检测细胞内游离钙离子浓度的变化，我们将使用细胞浸透版本的钙橙2，这是很容易的神经元通过离子导入”加载“到。当这个长波长的荧光染料结合钙荧光强度增加。钙橙色快速动力学特性，不同比例染料（如探针Fura 2），不需要滤光轮的成像，这是相当照片的稳定，减少光毒性比其他染料（如，荧光-3）2,4。像所有的非比例染料，钙橙色表示钙离子浓度的相对变化。但是，因为它考虑到由于加载和扩散染料浓度的变化是不可能的，它不能被校准，以提供绝对的钙离子浓度。 一个直立的，固定的阶段，化合物使用显微镜用CCD照相机能够记录每秒30帧左右的图像神经元。在这个时间分辨率海兔是绰绰有余，即使只有一个尖峰诱导细胞内钙离子浓度的改变，来检测。同时，夏普电极用于诱导和记录确定前和突触后神经元突触传递的。在每次试验结束，自定义脚本，结合电生理和影像学资料。为了确保正确的同步，我们使用一个光脉冲从LED安装在显微镜的相机端口。操作突触前钙的水平（例如，通过细胞内EGTA注射），让我们来测试特定的假设，细胞内钙离子的作用，在调解各种形式的可塑性。

Watch this Scientific Journal Video about Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia at JoVE.com

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

我们演示了如何在细胞内游离钙离子浓度和突触效能的变化，可以同时监测神经节准备的<em&gt;海兔</em&gt;。我们的图像使用的荧光染料，钙奥兰治，用锋利的（细胞内）电极和诱导和监测突触传递的细胞内钙离子。

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

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Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

10.9K 次观看.  Mt. Sinai School of Medicine. 我们演示了如何在细胞内游离钙离子浓度和突触效能的变化，可以同时监测神经节准备的海兔。我们的图像使用的荧光染料，钙奥兰治，用锋利的（细胞内）电极和诱导和监测突触传递的细胞内钙离子。

视频: Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

科研

神经科学

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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 ...

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. ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

通过神经树桩将钙染料加载到青蛙神经末梢：青蛙神经肌肉接头中的钙瞬态登记

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.