Name: visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing
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本研究由中国国家重点研发项目（项目代码2019YFC1709103;2018YFC1707804号）和中国国家自然科学基金（项目代码81774211;第81774432号项目代码，第81801561号）支持。

本研究经中国医学科学院针灸与莫西布西翁研究所伦理委员会批准（参考号D2018-09-29-1）。所有程序均按照《国家实验室动物护理和使用卫生指南》（1996年.C，华盛顿特区国家学院出版社）执行。这项研究使用了12只成年斯普拉格-道利雄性大鼠（体重220±20克）。动物[许可证号SCXK（JING） 2017-0005]由国家食品和药物管制研究所提供。
1. 大鼠颅杜拉母体的内向
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	<li>Kekere, V., Alsayouri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy,+Head+and+Neck,+Dura+Mater.">Anatomy, Head and Neck, Dura Mater.</a> StatPearls. , (2020).</li><li>Shimizu, T., 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=Distribution+and+origin+of+TRPV1+receptor-containing+nerve+fibers+in+the+dura+mater+of+rat.">Distribution and origin of TRPV1 receptor-containing nerve fibers in the dura mater of rat.</a> Brain Research. 1173, 84-91 (2007).</li><li>Jacobs, B., Dussor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+contributions+to+migraine:+moving+beyond+vasodilation.">Neurovascular contributions to migraine: moving beyond vasodilation.</a> Neuroscience. 338, 130-144 (2016).</li><li>Dodick, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase-by-phase+review+of+migraine+pathophysiology.">A phase-by-phase review of migraine pathophysiology.</a> Headache. 58, 4-16 (2018).</li><li>Amin, F. M., 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=Investigation+of+the+pathophysiological+mechanisms+of+migraine+attacks+induced+by+pituitary+adenylate+cyclase-activating+polypeptide-38.">Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38.</a> Brain: A Journal of Neurology. 137, 779-794 (2014).</li><li>Keller, J. T., Marfurt, 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=Peptidergic+and+serotoninergic+innervation+of+the+rat+dura+mater.">Peptidergic and serotoninergic innervation of the rat dura mater.</a> The Journal of Comparative Neurology. 309 (4), 515-534 (1991).</li><li>Messlinger, K., Hanesch, U., Baumg&#228;rtel, M., Trost, B., Schmidt, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+the+dura+mater+encephali+of+cat+and+rat:+ultrastructure+and+calcitonin+gene-related+peptide-like+and+substance+P-like+immunoreactivity.">Innervation of the dura mater encephali of cat and rat: ultrastructure and calcitonin gene-related peptide-like and substance P-like immunoreactivity.</a> Anatomy and Embryology. 188 (3), 219-237 (1993).</li><li>Lennerz, J. K., 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=Calcitonin+receptor-like+receptor+(CLR),+receptor+activity-modifying+protein+1+(RAMP1),+and+calcitonin+gene-related+peptide+(CGRP)+immunoreactivity+in+the+rat+trigeminovascular+system:+differences+between+peripheral+and+central+CGRP+receptor+distribution.">Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: differences between peripheral and central CGRP receptor distribution.</a> The Journal of Comparative Neurology. 507 (3), 1277-1299 (2008).</li><li>Eftekhari, S., Warfvinge, K., Blixt, F. W., Edvinsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+nerve+fibers+storing+CGRP+and+CGRP+receptors+in+the+peripheral+trigeminovascular+system.">Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system.</a> The Journal of Pain: Official Journal of the American Pain Society. 14 (11), 1289-1303 (2013).</li><li>Xu, D. S., 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=Characteristics+of+distribution+of+blood+vessels+and+nerve+fibers+in+the+skin+tissues+of+acupoint+"Taichong"+(LR3)+in+the+rat.">Characteristics of distribution of blood vessels and nerve fibers in the skin tissues of acupoint "Taichong" (LR3) in the rat.</a> Zhen Ci Yan Jiu. 41 (6), 486-491 (2016).</li><li>Cui, J. 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=The+expression+of+calcitonin+gene-related+peptide+on+the+neurons+associated+Zusanli+(ST+36)+in+rats.">The expression of calcitonin gene-related peptide on the neurons associated Zusanli (ST 36) in rats.</a> Chinese Journal of Integrative Medicine. 21 (8), 630-634 (2015).</li><li>Andres, K. H., von D&#252;ring, M., Muszynski, K., Schmidt, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+fibres+and+their+terminals+of+the+dura+mater+encephali+of+the+rat.">Nerve fibres and their terminals of the dura mater encephali of the rat.</a> Anatomy and Embryology. 175 (3), 289-301 (1987).</li><li>Leng, C., Chen, L., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+P2X1-6+receptor+expression+in+retrograde+Fluorogold-labeled+DRG+neurons+from+rat+chronic+neuropathic+pain+model.">Alteration of P2X1-6 receptor expression in retrograde Fluorogold-labeled DRG neurons from rat chronic neuropathic pain model.</a> Biomedical Reports. 10 (4), 225-230 (2019).</li><li>Huang, T. 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=Factors+influencing+the+retrograde+labeling+of+retinal+ganglion+cells+with+fluorogold+in+an+animal+optic+nerve+crush+model.">Factors influencing the retrograde labeling of retinal ganglion cells with fluorogold in an animal optic nerve crush model.</a> Ophthalmic Research. 51 (4), 173-178 (2014).</li><li>Huang, T. L., Chang, C. H., Lin, K. H., Sheu, M. M., Tsai, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+protective+effect+of+local+administration+of+triamcinolone+or+systemic+treatment+with+methylprednisolone+against+damages+caused+by+optic+nerve+crush+in+rats.">Lack of protective effect of local administration of triamcinolone or systemic treatment with methylprednisolone against damages caused by optic nerve crush in rats.</a> Experimental Eye Research. 92 (2), 112-119 (2011).</li><li>Tsai, R. K., Chang, C. H., Wang, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+recombinant+human+granulocyte+colony-stimulating+factor+(G-CSF)+in+neurodegeneration+after+optic+nerve+crush+in+rats.">Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in neurodegeneration after optic nerve crush in rats.</a> Experimental Eye Research. 87 (3), 242-250 (2008).</li><li>Iyengar, S., Ossipov, M. H., Johnson, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcitonin+gene-related+peptide+in+peripheral+and+central+pain+mechanisms+including+migraine.">The role of calcitonin gene-related peptide in peripheral and central pain mechanisms including migraine.</a> Pain. 158 (4), 543-559 (2017).</li><li>Russell, F. A., King, R., Smillie, S. J., Kodji, X., Brain, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitonin+gene-related+peptide:+physiology+and+pathophysiology.">Calcitonin gene-related peptide: physiology and pathophysiology.</a> Physiological Reviews. 94 (4), 1099-1142 (2014).</li><li>Kou, Z. Z., 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=Alterations+in+the+neural+circuits+from+peripheral+afferents+to+the+spinal+cord:+possible+implications+for+diabetic+polyneuropathy+in+streptozotocin-induced+type+1+diabetic+rats.">Alterations in the neural circuits from peripheral afferents to the spinal cord: possible implications for diabetic polyneuropathy in streptozotocin-induced type 1 diabetic rats.</a> Frontiers in neural circuits. 8, 6 (2014).</li><li>Alarcon-Martinez, 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=Capillary+pericytes+express+&#945;-smooth+muscle+actin,+which+requires+prevention+of+filamentous-actin+depolymerization+for+detection.">Capillary pericytes express &#945;-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.</a> eLife. 7, 34861 (2018).</li><li>Wang, 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=A+new+approach+for+examining+the+neurovascular+structure+with+phalloidin+and+calcitonin+gene-related+peptide+in+the+rat+cranial+dura+mater.">A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater.</a> Journal of Molecular Histology. 51 (5), 541-548 (2020).</li><li>Liu, Y., Broman, J., Edvinsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+sensory+innervation+of+the+rat+superior+sagittal+sinus.">Central projections of sensory innervation of the rat superior sagittal sinus.</a> Neuroscience. 129, 431-437 (2004).</li><li>Liu, Y., Broman, J., Edvinsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+the+sensory+innervation+of+the+rat+middle+meningeal+artery.">Central projections of the sensory innervation of the rat middle meningeal artery.</a> Brain Research. 1208, 103-110 (2008).</li><li>Schmued, L. C., Fallon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoro-Gold:+a+new+fluorescent+retrograde+axonal+tracer+with+numerous+unique+properties.">Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties.</a> Brain Research. 377 (1), 147-154 (1986).</li></ol>

在这项研究中，我们成功地演示了CGRP免疫活性神经纤维在颅尿母体中的分布和起源，使用CGRP抗体和FG神经示踪剂的免疫荧光、3D重建和神经追踪方法，为更好地了解神经血管网络提供了组织学和化学证据。
众所周知，CGRP在偏头痛4、17的发病机因中起着至关重要的作用。结果表明，增加CGRP可导致血管扩张和神经原性炎症，导致沿三?...

颅骨母体是保护大脑的最外层，含有丰富的血管和不同种类的神经纤维1，2。许多研究表明，敏感颅杜拉母体可能是导致头痛发生的关键因素，涉及异常血管扩张和内向3，4，5。因此，颅内神经血管结构知识对于理解头痛的发病机理，特别是偏头痛的发病机理非常重要。
虽然杜拉内在研究以前与传统的免疫化学，神经纤维和血管的空间相关性在颅杜拉母体较少研究6，7，8，9。为了更详细地揭示杜拉神经血管结构，钙素基因相关肽（CGRP）和巴洛丁被选为分别用免疫荧光和荧光组织化学10染色全山颅骨神经纤维和血管的标记物。这可能是获得神经血管结构的三维（3D）视图的最佳选择。此外，氟金（FG）被应用于颅内杜拉母体中脑膜动脉（MMA）周围区域，以确定CGRP免疫活性神经纤维的来源， 并追溯到三角结节 （TG） 和颈椎 （C） 多面根神经质 （DRGs）， 而 FG 标记的神经元使用免疫荧光与 CGRP 一起进一步检查。
这项研究的目的是为研究颅内杜拉母体的神经血管结构提供一个有效的工具，用于CGRP免疫活性内分和其起源。通过利用透明的全山杜拉母校，结合免疫荧光、逆行追踪、共焦技术和3D重建，我们期望呈现颅杜拉母校神经血管结构的新3D视图。这些方法方法可以进一步用于探索不同头痛的发病机理。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse IgG (H+L)</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>A21202</td>
			<td>Protect from light; RRID: AB_141607</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>CellSens Dimension</td>
			<td>Olympus</td>
			<td>Version 1.1</td>
			<td>Software of fluorescent microscope</td>
		</tr>
		<tr>
			<td>Confocal imaging system</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogold (FG)</td>
			<td>Fluorochrome</td>
			<td>52-9400</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Fluorescent imaging system</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International&#160;GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus FV10-ASW 4.2a</td>
			<td>Olympus</td>
			<td>Version 4.2</td>
			<td>Confocal image processing software system</td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-CGRP</td>
			<td>Abcam</td>
			<td>ab81887</td>
			<td>RRID: AB_1658411</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin 568</td>
			<td>Molecular&#160;Probes</td>
			<td>A12380</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Photoshop and&#160; Illustration</td>
			<td>Adobe</td>
			<td>CS6</td>
			<td>Photo editing software</td>
		</tr>
		<tr>
			<td>Rabbit anti- Fluorogold</td>
			<td>Abcam</td>
			<td>ab153</td>
			<td>RRID: AB_90738</td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>National Institutes for Food and Drug Control</td>
			<td>SCXK (JING) 2014-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
	</tbody>
</table>

visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing

本研究的目的是利用免疫荧光、三维（3D）重建和逆行追踪技术，研究钙化物基因相关肽（CGRP）免疫感官神经纤维的分布和起源。在这里，神经纤维和血管分别使用CGRP和荧光法洛丁的免疫荧光和组织化学技术进行染色。3D重建证明了杜拉尔CGRP-免疫神经纤维和血管的空间相关性。同时，通过神经追踪技术从颅内杜拉母体中脑膜动脉（MMA）周围区域到三叉神经结节（TG）和颈椎（C）多头根神经痛（DRG）的神经追踪技术检测出CGRP免疫活性神经纤维的起源。此外，还使用双免疫荧光与CGRP一起检查了TG和DPG中FG标记神经元的化学特性。利用透明的全安装样本和3D重建，结果表明，CGRP免疫活性神经纤维和巴洛丁标记动脉一起运行或单独形成一个神经血管网络在3D视图， 而FG标记的神经元被发现在TG的眼科、最大侧和支线分支，以及C2-3 DRG ipsilateral到示踪器应用的一侧，其中一些FG标记的神经元呈现CGRP免疫活性表达。通过这些方法，我们演示了颅内杜拉母体血管周围CGRP免疫活性神经纤维的分布特征，以及TG和DPG这些神经纤维的来源。从方法论的角度看，它为理解颅骨在生理或病理条件下复杂的神经血管结构提供了宝贵的参考。

颅体母体的神经血管结构 在免疫荧光和荧光组织染色与CGRP和phalloidin后，CGRP免疫活性神经纤维和法洛丁标记的杜拉尔动脉和结缔组织在3D模式（图2C，D，E，F）的全山颅骨母体中清晰地表现出来。结果表明，厚和薄的CGRP免疫活性神经纤维与神经动脉平行运行，在血管壁周围，或血管之间（图2D，E，F?...

Watch this Scientific Journal Video about Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing at JoVE.co

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

在这里，我们提出了一个协议，可视化钙素基因相关肽（CGRP）免疫活性神经纤维和血管的空间相关性，分别使用CGRP和phalloidin的免疫荧光和荧光组织化学。此外，这些神经纤维的起源是逆行跟踪荧光神经示踪剂。

可视化与钙西通宁基因相关的肽免疫活性内活性与免疫发光和神经追踪

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...

该协议可能为理解颅骨母体在生理或病理状况上的复杂神经血管结构提供可变的方法参考。利用透明的全坐骑杜拉母体，在3D视野中可以证明杜拉尔CGRP免疫活性神经纤维和血管的痉挛相关性。对大鼠实施安乐死后，用100毫升的0.9%正常盐水进行心肌增补，然后在0.1摩尔磷酸盐缓冲液中注入250至300毫升4%的副甲醛。完成后，取出头部皮肤并打开头骨，露出大脑的杜拉母体和后侧。沿着脑系统解剖颅骨，以整个安装模式将颅骨灯泡分解到嗅球上。在 0.1 摩尔磷酸盐缓冲区中冲洗颅骨杜拉母体约一分钟，并在阻塞溶液中孵育。将杜拉母体在一夜之间转移到鼠标抗CGRP抗体中。第二天，用0.1摩尔磷酸盐缓冲器洗三次杜拉母校。在驴抗鼠标 Alexa Fluor 488 二次抗体和 0.1 中的 568 的混合溶液中孵育杜拉母体。在室温下含有1%普通驴血清和0.5%Triton X-100的磷酸盐缓冲器，在室温下1.5小时。然后，在 0.1 摩尔磷酸盐缓冲器中洗三次杜拉母校，修剪边缘，并将其安装在显微镜滑梯上。在观察前用50%甘油盖上盖片。用荧光显微镜拍摄整个杜拉母校，然后使用共聚焦显微镜拍摄杜拉母校中 CGRP 免疫活性神经纤维和巴洛丁标记血管的图像。用电动剃须刀剃掉老鼠的头，然后把钝耳棒放在老鼠身上，放在立体声装置上。将口腔支架放置，并在眼睛上涂抹眼药膏。使用 75% 乙醇清洁头部皮肤的手术侧，并在头皮中线进行切口。使用无菌棉尖贴施用器从头骨上模糊地去除围产层和肌肉组织。使用毛刺钻孔钻一个小孔，在中间脑膜动脉上方的左侧切口和时间骨骼上使用圆尖位。用牙科硅酸盐水泥在孔周围建造一个银行，用10微升微注射器将两微升氟金加入中间脑膜动脉周围的孔中。用一小块止血海绵盖住洞。在孔上放一块石蜡膜，用骨蜡封住边缘。用无菌螺纹缝合伤口。将老鼠关在温暖的区域，直到它们完全康复。然后把老鼠送回笼子里。存活7天后，用0.9%的正常盐水灌注这些大鼠，然后在0.1摩尔磷酸盐缓冲液中注入4%的副甲醛。解剖出TG和颈椎一到四个DPG，然后在下垂方向的低温微原子系统上以30微米的厚度切割它们。并将部分安装在硅烷涂层玻璃滑梯上。用组织化学笔将各部分圈起来，并在阻塞溶液中孵育30分钟。将样品转移到兔子抗氟金和小鼠抗CGRP抗体的溶液中，并在四摄氏度的夜间孵化。第二天，用0.1摩尔磷酸盐缓冲器清洗三次。将洗涤部分孵化在驴防兔亚历克萨氟 594 和驴反鼠标 Alexa Fluor 488 次要抗体的混合溶液中。然后在 0.1 摩尔磷酸盐缓冲区清洗三次，并涂抹盖片，用 50% 甘油进行观察。使用荧光显微镜拍摄 TG 和 UV 照明下 DRG 中标有荧光的神经元图像。然后，捕获 TG 和 DRG 中标记的氟金和 CGRP 标记神经元的图像。免疫荧光和荧光组织染色与CGRP和phalloidin后，CGRP免疫活性神经纤维和标有杜拉尔动脉和结缔组织的phalloidin在整个坐骑颅骨母体中以3D模式清晰可视化。厚和薄的CGRP免疫性神经纤维与血管壁周围的神经动脉或血管之间平行运行。在中脑膜动脉和大鼠颅状杜拉母体区域应用氟金应用7天后，在示踪剂应用的ipsilathal侧的TG和颈椎DPG中检测到荧光标记神经元，在荧光显微镜的紫外线照射下直接观察到。在TG的所有三个分支中都发现了氟罗戈尔德标记神经元，眼科和最大细分部的浓度较高，在支气管分裂上的浓度较低。在宫颈两到三个DPG中也观察到了一些标有氟金标签的神经元。这些双免疫荧光代表照片显示TG和DMG中的感官神经元。带有FG、CGRP以及FG和CGRP的标记神经元通过双免疫荧光可视化。在尝试此协议时，请记住完全解剖颅面 dura 母校，并将其粘贴在整个安装模式的幻灯片上。

Research

JoVE Journal

Neuroscience

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

科研

神经科学

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

调查的意识到，不知道用功能磁共振成像的恐惧记忆的神经机制

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Viral Tracing of Genetically Defined Neural Circuitry

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...