Name: monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc aplysia
Uploaded: 2012-07-15T14:00:00
Description: Watch this Scientific Journal Video about Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia at JoVE.com

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...

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	<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., ...

Research

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Neuroscience

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

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