Name: presynaptically silent synapses studied with light microscopy
Uploaded: 2010-01-04T17:00:00
Description: Watch this Scientific Journal Video about Presynaptically Silent Synapses Studied with Light Microscopy at JoVE.com

这项工作是由美国国立卫生研究院拨款DA018109 MH78823，和美国国立卫生研究院神经科学蓝图的核心格兰特P30NS057105华盛顿大学的支持。

文化准备<ol><li>准备从出生后1 0到3动物大鼠或小鼠海马细胞分离细胞培养。我们的神经元坚持一个基本的星形胶质细胞单层，这反过来又坚持一个0厚度盖玻片上传播的胶原蛋白层。板的神经元密度约每平方厘米500细胞。 </li><li>允许的文化成长，让突触的发育和成熟10-14天。治疗在体外4天的文化与抗有丝分裂ARAC逮捕进一步胶质增长。饲料在体外5 Ne​​urobasal中加B27的补充，以提高神经元...

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	<li>Mennerick, S., Que, J., Benz, A., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+and+synaptic+properties+of+hippocampal+neurons+grown+in+microcultures+and+in+mass+cultures.">Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures.</a> J. Neurophysiol. 73, 320-332 (1995).</li><li>Moulder, K. L., Jiang, X., Taylor, A. A., Olney, J. W., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+activity+depresses+synaptic+function+through+an+effect+on+vesicle+priming.">Physiological activity depresses synaptic function through an effect on vesicle priming.</a> J. Neurosci. 26, 6618-6626 (2006).</li><li>Moulder, K. L., Jiang, X., Chang, C., Taylor, A. A., Benz, A. M., Conti, A. C., Muglia, L. J., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+role+for+Ca2+-dependent+adenylyl+cyclases+in+recovery+from+adaptive+presynaptic+silencing.">A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.</a> J. Neurosci. 28, 5159-5168 (2008).</li><li>Sara, Y., Mozhayeva, M. G., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+vesicle+recycling+supports+neurotransmission+during+sustained+stimulation+at+hippocampal+synapses.">Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses.</a> J. Neurosci. 22, 1608-1617 (2002).</li><li>Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 809-817 (1999).</li><li>Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, N., Rhee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+vesicle+priming+and+short-term+plasticity+by+Munc13+isoforms.">Differential control of vesicle priming and short-term plasticity by Munc13 isoforms.</a> Neuron. 33, 411-424 (2002).</li><li>Altrock, W. D., tom Dieck, S., Sokolov, M., Meyer, A. C., Sigler, A., Brakebusch, C., Fassler, R., Richter, K., Boeckers, T. M., Potschka, H., Brandt, C., Loscher, W., Grimberg, D., Dresbach, T., Hempelmann, A., Hassan, H., Balschun, D., Frey, J. U., Brandstatter, J. H., Garner, C. C., Rosenmund, C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+inactivation+of+a+fraction+of+excitatory+synapses+in+mice+deficient+for+the+active+zone+protein+bassoon.">Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.</a> Neuron. 37, 787-800 (2003).</li><li>Ting, J. T., Kelley, B. G., Lambert, T. J., Cook, D. G., Sullivan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+overexpression+depresses+excitatory+transmission+through+both+presynaptic+and+postsynaptic+mechanisms.">Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.</a> Proc. Natl. Acad. Sci. USA. 104, 353-348 (2007).</li></ol>

意义<ol><li>突触通常被认为是释放与一个可衡量的概率的发射机操作。自己和他人的工作，使得它显然有些突触难治性释放神经递质，尽管水泡神经递质转运和其他关键的突触前标记2，6-8全套。以前我们已经表明，在基底神经元网络活动是至关重要的，以保持这种不活跃的突触的人口。 </li></ol>关键步骤<ol><li>列入一个500微米Advasep - 7的简短洗显着增强，FM1 -​​ 43FX染...

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		<th>Material Name</th>
		<th>Type</th>
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<td>FM1-43FX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-35355</td>
<td>A red-shifted FM4-64FX is also available, but has not proven as amenable to assays of presynaptic silencing.</td>
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<td>Advasep-7</td>
<td>&nbsp;</td>
<td>CyDex</td>
<td>AR-0A7-001</td>
<td>&nbsp;</td>
<tr>
<td>vGluT-1</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB5905</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 647-conjugated anti-GP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21450</td>
<td>&nbsp;</td>
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<td>Fluoromount-G</td>
<td>&nbsp;</td>
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presynaptically silent synapses studied with light microscopy

突触可塑性的神经系统的学习和记忆能力，否则，防止成为神经毒性损害的侮辱，也可能代表一个适应性的可能的基础。我们一直在研究一种突触前可塑性，是有趣的部分，因为它是一个数字切换和关闭突触前终端的能力，包含神经递质谷氨酸释放囊泡表示。在这里，我们展示了一个可视化的文化准备从啮齿类动物海马突触前在分离细胞终端的活动状态的协议。该方法依赖于检测用的可修复形式的苯乙烯基染料FM1 -​​ 43常用标签突触小泡，染色积极突触。针对囊泡膜谷氨酸转运体1（vGluT 1），染色设计无论激活状态标签的所有谷氨酸突触的抗体相比，同一终端的免疫染色轮廓。我们发现，去极化刺激诱发突触前的沉默。突触是无声的基准条件下的人口可以被激活电气长时间沉默，或通过cAMP信号通路的激活。

Watch this Scientific Journal Video about Presynaptically Silent Synapses Studied with Light Microscopy at JoVE.com

Presynaptically Silent Synapses Studied with Light Microscopy

谷氨酸突触主动模式可以切换到无声模式。我们证明，在啮齿类动物神经细胞的分离文化突触前的活动状态的可视化使用一个可修复的FM1 -​​ 43染料形式活跃的突触形象化vGluT - 1抗体免疫可视化所有谷氨酸突触。

光镜研究沉默突触的突触前

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