Name: in vivo imaging of fully active brain tissue in awake zebrafish larvae and juveniles by skull and skin removal
Uploaded: 2021-02-10T14:00:00
Description: Watch this Scientific Journal Video about In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal at JoVE.com

我们特别感谢蒂莫·弗里奇出色的动物护理，赫尔曼·德林、穆罕默德·艾尔赛、索尔·波塞-门德斯、雅各布·冯·特罗塔、科马利·瓦利谢蒂和芭芭拉·温特给予我们帮助的支持。我们还感谢科斯特实验室的所有其他成员的反馈。该项目部分由德国研究基金会（DFG，KO1949/7-2）项目241961032（到RWK）和德国联邦委员会（BMBF）资助。时代网神经NII CIPRESS项目01EW1520到JCM）得到认可。

此处描述的所有动物工作均符合法律规定（欧盟指令 2010/63）。养护和处理鱼类已得到地方当局和布劳恩施魏格技术大学动物福利代表的批准。
1. 准备人工脑脊液（ACSF）、低熔化藻和锋利的玻璃针
<ol><li>通过溶解蒸馏水中的以下浓度溶解所列化学品，准备 ACSF。134 m M NaCl （58.44 克/摩尔）， 2.9 mM KCl （74.55 克/摩尔）， 2.1 m CaCl2 （110.99 克/摩尔）， 1.2 mM MgCl2 6...

<ol>
	<li>Embryology. Zebrafish Development Available from: <a target="_blank" href="https://embryology.med.unsw.edu.au/embryology/index.php/Zebrafish_Development">https://embryology.med.unsw.edu.au/embryology/index.php/Zebrafish_Development</a> (2020)</li><li>Sassen, W. A., K&#246;ster, R. 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+molecular+toolbox+for+genetic+manipulation+of+zebrafish.+Advances+in+Genomics+and+Genetics.">A molecular toolbox for genetic manipulation of zebrafish. Advances in Genomics and Genetics.</a> Dove Medical Press. 2015 (5), 151-163 (2015).</li><li>Singh, A. P., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+stripes+as+a+model+for+vertebrate+colour+pattern+formation.">Zebrafish stripes as a model for vertebrate colour pattern formation.</a> Current Biology. 25 (2), 81-92 (2015).</li><li>Kalueff, A. V., 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=Time+to+recognize+zebrafish+'affective'+behavior.">Time to recognize zebrafish 'affective' behavior.</a> Brill: Behaviour. 149 (10-12), 1019-1036 (2012).</li><li>Karlsson, J., von Hofsten, J., Olsson, P. -. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+transparent+zebrafish:+a+refined+method+to+improve+detection+of+gene+expression+during+embryonic+development.">Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.</a> Marine Biotechnology. 3, 522-527 (2001).</li><li>Bohnsack, B. L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+development+to+changes+in+retinoic+acid+and+IGF+signaling.">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling.</a> PloS One. 6, 22991 (2011).</li><li>Elsalini, O. A., Rohr, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+disrupts+thyroid+function+in+developing+zebrafish.">Phenylthiourea disrupts thyroid function in developing zebrafish.</a> Development Genes and Evolution. 212, 593-598 (2003).</li><li>Baumann, L., Ros, A., Rehberger, K., Neuhauss, S. C. F., Segner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+disruption+in+zebrafish+(Danio+rerio)+larvae:+Different+molecular+response+patterns+lead+to+impaired+eye+development+and+visual+functions.">Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions.</a> Aquatic Toxicology. 172, 44-55 (2016).</li><li>White, R., 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Antinucci, P., Hindges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crystal-clear+zebrafish+for+in+vivo+imaging.">A crystal-clear zebrafish for in vivo imaging.</a> Scientific Reports. 6, 29490 (2016).</li><li>Burr, S. A., Leung, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curare+(d-Tubocurarine).">Curare (d-Tubocurarine).</a> Encyclopedia of Toxicology (3rd Edition). , 1088-1089 (2014).</li><li>Gesler, H. M., Hoppe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3,6-bis(3-diethylaminopropoxy)+pyridazine+bismethiodide,+a+long-acting+neuromuscular+blocking+agent.">3,6-bis(3-diethylaminopropoxy) pyridazine bismethiodide, a long-acting neuromuscular blocking agent.</a> The Journal of Pharmacology and Experimental Therapeutics. 118 (4), 395-406 (1956).</li><li>Furman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Alpha Bungarotxin. Reference Module in Biomedical Sciences. , (2018).</li><li>Attili, S., Hughes, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetic+tricaine+acts+preferentially+on+neural+voltage-gated+sodium+channels+and+fails+to+block+directly+evoked+muscle+contraction.">Anaesthetic tricaine acts preferentially on neural voltage-gated sodium channels and fails to block directly evoked muscle contraction.</a> PLoS One. 9 (8), 103751 (2014).</li><li>Namikawa, 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=Modeling+neurodegenerative+spinocerebellar+ataxia+type+13+in+zebrafish+using+a+Purkinje+neuron+specific+tunable+coexpression+system.">Modeling neurodegenerative spinocerebellar ataxia type 13 in zebrafish using a Purkinje neuron specific tunable coexpression system.</a> Journal of Neuroscience. 39 (20), 3948-3969 (2019).</li><li>Hennig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+models+of+synaptic+short+term+plasticity.">Theoretical models of synaptic short term plasticity.</a> Frontiers in Computational Neuroscience. 7 (45), (2013).</li><li>Wang, Y., 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=Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis.">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li><li>Hobro, A., Smith, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li><li>Knogler, L. D., Kist, A. M., Portugues, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+context+dominates+output+from+purkinje+cell+functional+regions+during+reflexive+visuomotor+behaviours.">Motor context dominates output from purkinje cell functional regions during reflexive visuomotor behaviours.</a> eLife. 8, 42138 (2019).</li><li>Hsieh, J., Ulrich, B., Issa, F. A., Wan, J., Papazian, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+development+of+Purkinje+cell+excitability,+functional+cerebellar+circuit,+and+afferent+sensory+input+to+cerebellum+in+zebrafish.">Rapid development of Purkinje cell excitability, functional cerebellar circuit, and afferent sensory input to cerebellum in zebrafish.</a> Frontier in Neural Circuits. 8 (147), (2014).</li><li>Scalise, K., Shimizu, T., Hibi, M., Sawtell, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+cerebellar+Purkinje+cells+during+fictive+optomotor+behavior+in+larval+zebrafish.">Responses of cerebellar Purkinje cells during fictive optomotor behavior in larval zebrafish.</a> Journal of Neurophysiology. 116 (5), 2067-2080 (2016).</li><li>Harmon, T. C., Magaram, U., McLean, D. L., Raman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+responses+of+Purkinje+neurons+and+roles+of+simple+spikes+during+associative+motor+learning+in+larval+zebrafish.">Distinct responses of Purkinje neurons and roles of simple spikes during associative motor learning in larval zebrafish.</a> eLife. 6, 22537 (2017).</li><li>Zehendner, C. 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=Moderate+hypoxia+followed+by+reoxygenation+results+in+blood-brain+barrier+breakdown+via+oxidative+stress-dependent+tight-junction+protein+disruption.">Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption.</a> PLoS One. 8 (12), 82823 (2013).</li><li>Dhabhar, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short-term+stress+response+-+mother+nature's+mechanism+for+enhancing+protection+and+performance+under+conditions+of+threat,+challenge,+and+opportunity.">The short-term stress response - mother nature's mechanism for enhancing protection and performance under conditions of threat, challenge, and opportunity.</a> Frontiers of Neuroendocrinology. 49, 175-192 (2018).</li></ol>

提出的方法提供了一种替代方法，大脑隔离或斑马鱼幼虫的治疗与药物抑制色素干扰记录高分辨率图像的神经元在其 体内 环境。使用这种方法记录的图像质量可与从外生大脑中拍摄的图像相媲美，但在自然条件下。
此外，避免了荧光强度的损失，因为没有必要用固定剂18治疗。此外，这种方法不像隔离大脑那样具有侵入性，它只包括可能导致失败的?...

近几十年来，斑马鱼（Danio rerio）已发展成为胚胎和幼虫发育研究中最受欢迎的脊椎动物模型生物之一。斑马鱼雌性的巨大雌性，加上胚胎 的快速前子宫 发育及其在早期胚胎发育阶段的透明度，只是使斑马鱼成为解决发育问题的强大模型有机体的几个关键因素。分子遗传技术的进步与体内成像研究 的高 分辨率相结合，使得解决细胞生物学机制基础发育过程2。特别是在神经元分化、生理学、连通性和功能领域，斑马鱼以前所未有的细节揭示了分子动力学、大脑功能和有机体行为的相互作用。
然而，由于神经系统组织的透明度逐渐丧失，这些研究大多局限于发育第一周的胚胎和早期幼虫阶段。在这些阶段，脑组织被高分辨率显微镜方法阻止进入，从而被头骨分化和色素沉着3所屏蔽。
因此，神经元分化、成熟度和可塑性等关键问题，如神经元连接性或突触缩放的细化，都难以研究。这些细胞过程对于定义细胞机制驱动非常重要，例如，社会行为、决策或基于动机的行为，斑马鱼对数周幼虫的研究最近根据行为研究贡献了关键发现。
药理学方法抑制斑马鱼幼虫色素的色素消耗几个星期是几乎不可行，甚至可能造成有害影响5，6，7，8。具有特定色素沉着缺陷的双突变株（如casper9或晶体10）已成为极其有价值的工具，但在育种方面非常费力，提供很少的后代，并且由于近亲繁殖过度而造成遗传畸形的积累危险。
在这里，提供最小侵入性程序作为替代，适用于任何斑马鱼菌株。这个程序是从电生理学研究中改编而来，记录活的和清醒的斑马鱼幼虫的神经元活动。在电传中，皮肤和柔软的头骨软骨可以通过微皮仔细去除这些层，因为它们没有与大脑血管紧密交织。这允许暴露含有神经元和轴突的脑组织而不造成损伤，并记录神经元形态，包括突触结构及其分子含量，这反过来又包括观察生理变化，如Ca2+ 瞬态或细胞内传输事件长达数小时。此外，除了描述性特征外，直接接触脑组织还可以通过神经药物管理和光遗传学方法对成熟的神经元功能进行审讯。因此，使用这种大脑暴露策略，可以在幼斑马脑中揭示出真正的结构功能关系。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride</td>
			<td>Roth</td>
			<td>A119.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Glucose</td>
			<td>Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Tubocurare</td>
			<td>Sigma-Aldrich</td>
			<td>T2379-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary type 1</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary type 2</td>
			<td>Harvard Apparatus</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>deltalab</td>
			<td>D102424</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen (Thermo Fischer)</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Normapur</td>
			<td>26764298</td>
			<td></td>
		</tr>
		<tr>
			<td>LM-Agarose</td>
			<td>Condalab</td>
			<td>8050.55</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (Hexahydrate)</td>
			<td>Roth</td>
			<td>A537.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Leica</td>
			<td>DFC9000 GTC</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle-Puller type 1</td>
			<td>NARISHIGE</td>
			<td>Model PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle-Puller type 2</td>
			<td>Sutter Instruments</td>
			<td>Model P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur-Pipettes 3ml</td>
			<td>A.Hartenstein</td>
			<td>20170718</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>P029.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Normapur</td>
			<td>28244262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricain</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Phoenix Instrument</td>
			<td>WB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm petri dish</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm petri dish</td>
			<td>Sarstedt</td>
			<td>821473001</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo imaging of fully active brain tissue in awake zebrafish larvae and juveniles by skull and skin removal

了解大脑发育和成熟过程中发生的短暂变化需要在空间和时间上以细胞和亚细胞分辨率进行详细的高分辨率成像。分子和成像技术的进步使我们能够对透明斑马鱼胚胎中大脑发育的细胞和分子机制获得许多详细的见解。最近，在受精几周后的幼虫阶段发生的神经元连接的细化过程，例如控制社会行为、决策或动机驱动行为，已经进入研究重点。在这些阶段，斑马鱼皮肤的色素沉着会干扰光线渗透到脑组织，胚胎阶段的解决方案，例如，药理抑制色素沉着，已经不可行了。
因此，提供微创手术解决方案，用于通过微观镜进入清醒斑马鱼的大脑，该解决方案源自电生理方法。在电传中，皮肤和柔软的头骨软骨可以通过微皮这些层，暴露潜在的神经元和轴向而不损坏仔细去除。这允许记录神经元形态，包括突触结构及其分子含量，以及观察生理变化，如Ca2+ 瞬态或细胞内传输事件。此外，通过药理抑制或光遗传学操作来审讯这些过程是可行的。这种大脑暴露方法提供有关神经元结构和生理变化的信息，以及这些事件在几分钟或几小时内在活脑组织中的相关性和相互依存性。该技术适用于斑马鱼幼虫在受精后长达30天的体内脑成像，这是迄今为止测试的最新发育阶段。因此，它提供了诸如突触细化和缩放、轴突和树突运输、细胞骨骼货物突触靶向或当地活动依赖表达等重要问题。因此，可以预见这种安装和成像方法的广泛应用。

图3A， C 显示转基因线 Tg [-7.5Ca8：GFP] 的 14 dpf 幼虫bz12]15] 头骨仍然完好无损覆盖皮肤中的色素细胞分布在头部，并干扰感兴趣的区域（这里，小脑）的荧光信号。在这种情况下，幼虫不可能获得大脑的高分辨率图像。 图 3B 在进行开放性头骨手术后，显示同一转基因线的幼虫。现?...

Watch this Scientific Journal Video about In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal at JoVE.com

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

在这里，我们提出了一种方法，以图像斑马鱼胚胎大脑在体内到幼虫和幼年阶段。这种微创程序，根据电生理学方法改编，提供成熟神经元的细胞和亚细胞细节，并可与光遗传学和神经药理学研究相结合，以描述大脑功能和药物干预。

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

这种方法允许斑马鱼大脑在以后的幼虫阶段的可视化，允许以以前不可能的非常详细的方式观察神经元结构。色素细胞，在后来的幼虫发育过程中出现，防止大脑清晰成像，是没有问题的，因为这种方法只是删除。使用该方法，应该可以研究在大脑幼虫发育过程中的后期发生的过程，影响突触可塑性、神经元退化或其再生的过程。在开始实验之前，使用微管拔拔器从玻璃毛细血管中制备锋利、薄薄的玻璃针头。接下来，使用塑料巴斯德移液器将幼虫收集到直径为90毫米的培养皿中，其中含有适当的溶液。将选定的幼虫转移到一个直径35毫米的培养皿中，内含ACSF，并将一个24乘24毫米的方形玻璃盖滑入培养皿盖。然后将实验所需的ACSF体积计算成与卡博根氧合的合适噬菌体。要嵌入幼虫，请使用巴斯德移液器将麻醉幼虫转移到立体显微镜下的安装室。如果任何幼虫仍然能够移动，不要使用鱼做实验，直到鱼完全不能移动。当幼虫无法移动时，小心地去除多余的介质，并立即在幼虫身上加入至少一毫升的低熔化藻。将斑马鱼定向，使多面体区域朝上，尽可能靠近海藻表面。如果用倒置显微镜对幼虫进行成像，在凝固后，将含有幼虫的阿加罗斯修剪成一个小的幼虫块。为了暴露大脑成像，在必要时修剪大脑感兴趣的区域多余的气喘，并用玻璃针在靠近但未超过感兴趣的区域时，通过皮肤进行小切口，而不会深入到组织中。在皮肤表面下几乎不移动针头，继续小心地在感兴趣的区域周围进行非常小的切口，直到感兴趣的区域的皮肤可以移除或推到一边。当组织从所有胚胎中取出时，在倒置显微镜先前准备好的成像室中加入一小滴低熔化藻类，然后用一个小铲子将180度的黄藻糖块翻转到成像室的阿加罗斯落差上。当藻子凝固后，用新鲜的ACSF填充成像室，并开始对幼虫进行成像。在这里，可以观察到一个完整的14天后受精转基因幼虫与头骨仍然完好无损。如图所示，覆盖皮肤内的色素细胞分布在头部，干扰感兴趣的区域的荧光信号。开放颅骨手术后，感兴趣的区域可以自由访问，进行详细的高分辨率成像。皮肤、头骨或血脑屏障也阻碍物质进入大脑，这些脑细胞中仅在开放性头骨手术后观察到的强力核染料染色就说明了这一点。这些优势在施肥后30天内也观察到。在含有荧光脑血管的转基因鱼类中，由于红荧光蛋白的表达，mCherry在内皮细胞中，图像堆栈深度值的颜色编码表明，即使是深埋在大脑内近250微米的血管，仍然清晰可见和可追溯。执行此程序时，请始终记住，这是活体动物的手术。因此，它需要尊重和专注的头脑。在此程序之后，神经元形态的记录，包括突触结构，生理和细胞内传输事件可以在受精后7天以上的幼虫中进行。通过这项技术，我们希望提供一种方法，允许研究大脑中在幼虫晚期发生的细胞过程，并参与建立诸如社会行为或决策等复杂行为。

Research

JoVE Journal

Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

猪中枢神经系统的组织学分析的曝光：一个手册斩首，颅骨打开，并去除脑

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...