作者感激地承认特林·W·米尔森太太的熟练援助，莉莎M.夫人配件，并在Påskehøjgaard员工。丹麦医学研究委员会，Lundbeck公司基金会和诺和诺德基金会提供资助的研究。

动物麻醉和euthanesia是按照（NIH出版号86-23，1985年修订版）和丹麦议会动物研究伦理认可的"实验动物管理的原则"进行。 1.仪器<ol><li>收集的视频介绍和材料表中所列的仪器。 </li></ol> 2.斩首注:麻醉诱导通过5mL的咪达唑仑（5毫克/毫升）和5毫升氯胺酮（25毫克/毫升）的肌内注射。 5-10分钟后，当动物被深深地镇静剂，耳静脉插管，戊巴比妥钠致死过量（100毫克/千克体重）（200毫克/毫升）中的溶液经静脉内给予。为了确保将动物安乐死完全，叉指疼痛反射测试如图Ettrup 等。 （2011）20。如中所述确保完成安乐死伦理语句上述和后跟一个穿心灌注用5L等渗盐水，由Ettrup 等人所证明。 （2011）20。所有证明过程进行尸检，排除必要的长期麻醉和术后生存福利预防措施的需要。 <ol><li>通过高圆形颈部切口斩首猪，使用外科手术刀，只是下颌角（ 图1A）的下方。 </li><li>仍与外科手术刀，向前继续切口通过颈部，包括喉和食管的软组织，直到达到骨脊柱，大致在颅颈交界的水平。 </li><li>从颅颈交界的前侧的外科手术刀推进切割，寰椎前弧的上方，并且通过前寰枕膜，由此暴露椎管和脊髓（图1B）。同时让助手从猪头拉猪身上移开，缓解颅底和第一颈椎之间的访问。 </li><li>继续通过硬膜囊和脊髓（ 图1B）的手术切口。应特别小心，以确保脊髓完全横截面实现。 注意:如果不执行上述步骤可导致在断头过程的以下步骤期间脊髓和大脑不需要的牵引力。 </li><li>有力地延伸在所述区间电平（ 图1C）的cranocervical结。与此同时，使用外科手术刀部的剩余寰枕韧带以释放枕骨髁和图谱的上铰接过程之间的关节运动。从身体分离的猪脑袋。 </li></ol><img alt="图1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /> 图1:小型猪断头。 （A）颈部切口（箭头，下颌角）。 （; OC，枕髁C1，寰椎前弧）通过寰枕韧带和在颅颈交界硬脑膜包围的脊髓（SC）（B）切口。 （C）的寰枕关节的后部分是通过在部分水平延伸有力（箭头）释放。 <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">请点击此处查看该图的放大版本。</a> 3.骷髅开幕<ol><li>猪头上表中的位置。 </li><li>使背侧纵向切口穿过皮肤和从炉鼻的后面，在头的顶点底层软组织的外科手术刀，上下贯通的后部分枕骨区域。 </li><li>通过去除位于横向于初始切口用外科手术刀软组织暴露颅骨的背壳和后部分。 </li><li>从头骨（ 图2A）与外科手术刀双边释放颞肌。确保后枕骨清洁软组织。 </li><li>使用枕骨大孔的后部入口为与Kerrison骨冲头和骨骨钳取下枕骨和暴露硬脑膜覆盖的小脑（ 图2B）。 </li><li>回到头骨的暴露前侧，然后在额骨一个切入点，就在眼前。在这点上，使用骨凿用锤穿透颅骨并进入额窦（ 图2C）。 </li><li>使用额窦的程度进一步与骨咬骨钳或骨冲头dorsoposterior除去外颅骨椎板和露出内，薄骨颅骨薄层覆盖大脑（ 图2D）。 </li><li>轻轻向前打开内骨颅骨薄层用锤子和凿子骨以暴露硬脑膜覆盖大脑（ 图2E）。 </li><li>继续骨去除以释放头骨的最终dorsoposterior部分，位于硬脑膜覆盖大脑和小脑（ 图2F）的已曝光部分之间横向使用骨凿和骨咬骨钳通过时间和顶骨。 注意:它往往是可能的，在此过程中的最后一步，用凿子从一个侧面破开后剩余的颅骨，就像一个打开一扇门。 </li></ol><img alt="图2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> 图2:微型猪颅骨开口。 （A）的dorsoposterio的曝光ř颅骨表面，包括去除枕骨和颞肌的。枕骨（B）去除（CB，硬脑膜覆盖小脑; OB，枕骨; OC，枕骨髁;和SC，脊髓）。 （C）一种锤子和凿子骨被用于向前穿透颅骨和在眼睛的水平进入额窦。 （D）额窦（FS）的程度被用于去除外部厚颅骨（1），露出的内薄骨薄层（2），其覆盖大脑。薄骨薄片（E）去除，露出硬脑膜覆盖大脑（箭头）。 （f）最后，锤子和凿子骨被用来横向连接的前部和后部颅骨开口。 <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">请点击此处查看该图的放大版本。</a> 4.脑祛瘀人<ol><li>使用手术用钳子抬起硬脑膜，并使用精细的外科手术刀（ 图3A）温柔的切口接近静脉上矢状窦。 </li><li>使用微型剪刀或硬膜刀进一步打开覆盖在大脑的背表面硬膜。 注意:必须特别注意去除对应于小脑幕（图3B）硬膜时，应考虑，因为这硬脑膜叶的保存将防止随后的脑的去除。 </li><li>猪头垂直（ 图3C）的位置。 </li><li>使用骨凿或解剖器通过从所述颅腔（ 图3D）的硬脑膜覆盖的地板嗅球的钝器解剖以释放ventroanterior大脑。 </li><li>使用精细外科手术刀部露出的视交叉（ 图3E）。揭露和部分垂体柄和动眼神经。 </li><li>通过切片的LO释放脑干腹侧WER颅神经（ 图3F）用细外科手术刀。确保硬膜小脑幕完全切开（ 图3B），因为这硬脑膜叶将通过脑干中的释放过程以其他方式切割。 </li></ol><img alt="图3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /> 图3:小型猪的大脑取出。 （A）硬脑膜开口与手术镊子和硬脑膜刀。 （B）必须小心以完全切开硬脑膜叶（箭头），位于大脑和小脑之间。 （C）的猪头颅骨基部结构的更好的可视化，并且为了对重力协助在大脑的预期位移垂直定位。 （D）甲解剖器或骨凿被用于通过从硬脑膜-covere钝部，以减轻嗅球ð颅底。 （E）所述的夹层继续沿着颅底的视交叉（箭头），漏斗柄，和动眼神经的曝光和切片后方向。 （F）的大脑释放与下颅神经的部分完成的，因为它们从脑干的腹面出发（III，动眼神经; IV，滑车神经; V，三叉神经;和VI中，外展神经）。 <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">请点击此处查看该图的放大版本。</a> 5.垂体去除<ol><li>识别垂体柄并在颅骨地板（ 图4A）及其周边硬脑膜叶（在鞍diapragma）。 </li><li>切开使用精细外科手术刀（ 图4B）硬脊膜叶横向于垂体柄。 </li><li>使用剥离释放坑uitary和将其取出垂体窝（ 图4C）的。 </li></ol><img alt="图4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /> 图4:微型猪垂体切除。 （A）的垂体窝（*）在颅骨地板被识别（1，嗅球; 2，视交叉;和PF，后颅窝）。 （B）硬脑膜覆盖（鞍diagphragm，（箭头））被横向切开。 （C）的垂体（箭头）被释放与解剖器和提升出垂体窝。比例尺（AC）= 10毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">请点击此处查看该图的放大版本。</a>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+substantia+nigra+pars+compacta+of+the+minipig:+An+anatomical+and+stereological+study.">The substantia nigra pars compacta of the minipig: An anatomical and stereological study.</a> Brain Struct Funct. (4-5), 481-488 (2009).</li><li>S&#248;rensen, J. C., Bjarkam, C. R., Simonsen, C. Z., Danielsen, E., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+sectioning+of+irregular+tissue+blocks+in+relation+to+computerized+scanning+modalities.+Results+from+the+domestic+pig+brain.">Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities. Results from the domestic pig brain.</a> J Neurosci Methods. 104, 93-98 (2000).</li><li>Bjarkam, C. R., Pedersen, M., S&#248;rensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+embedding,+orientation+and+sectioning+of+small+brain+specimens+enable+direct+correlation+to+MR-images,+brain+atlases,+or+use+of+unbiased+stereology.">New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.</a> J Neurosci Methods. 108, 153-159 (2001).</li><li>Bjarkam, C. R., S&#248;rensen, J. C., Geneser, F. A. <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+morphology+of+serotonin-immunoreactive+axons+in+the+hippocampal+region+of+the+New+Zealand+white+rabbit.+I.+Area+dentata+and+hippocampus+proper.">Distribution and morphology of serotonin-immunoreactive axons in the hippocampal region of the New Zealand white rabbit. I. Area dentata and hippocampus proper.</a> Hippocampus. 13 (1), 21-37 (2003).</li><li>Bjarkam, C. R., Glud, A. N., Orlowski, D., S&#248;rensen, J. C., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telencephalon+of+the+minipig,+cytoarchitecture+and+cortical+surface+anatomy.">The telencephalon of the minipig, cytoarchitecture and cortical surface anatomy.</a> Brain Struct Funct. , (2016).</li><li>Boltze, J., Nitzsche, B., Geiger, K. D., Schoon, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+investigation+of+different+MCAO+modalities+and+impact+of+autologous+bone+marrow+mononuclear+cell+administration+in+an+ovine+stroke+model.">Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model.</a> Transl Stroke Res. 2, 279-293 (2011).</li><li>Jortner, B. 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+return+of+the+dark+neuron.+A+histological+artifact+complicating+contemporary+neurotoxicologic+evaluation.">The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.</a> Neurotoxicology. 27, 628-634 (2006).</li></ol>

作者什么都没有透露。

大多数实验研究神经科学在小动物物种，如小鼠和大鼠，在那里访问CNS由薄skull-和硬脑膜厚度容易进行。然而，在较大的实验动物如猪1，4，8，羊32，以及非人类灵长类动物中，相当大的厚度这些结构需要使用坚固的仪器（材料表）和适当的入口点头骨骨去除（ 图的2）。需要限制硬?...

<html>猪转化神经科学的研究在过去二十年中已经变得越来越流行。猪大脑的大尺寸使得能够使用常规临床脑成像器和直接使用和来自人诊所1，2，3，4，5，6，7，8神经外科手术和设备测试。在过去的20年里，猪，迷你猪特别是（ 例如，哥廷根小型猪），已被用于检查神经调节的治疗方式，如造血干细胞移植;病毒载体转染;和深部脑刺激针对帕金森病，肥胖症，抑郁症，和阿尔茨海默病2，6，= "外部参照"&gt; 9，10，11，12，13，14，15，16，17。这一直跟着的立体性和手术方法的发展，操纵小型猪CNS 3，18，19，20，21。的提起CNS变化在使用脑成像（PET 10，13，22，24和MR 23），膀胱测压11，12，25活体动物被评估，步态分析17，神经评价9，17，和基于组织学和体视分析14，15，17，26，27，31尸检。然而，事后分析需要曝光和去除猪脑，这不是一件容易的事，作为一个厚，骨颅骨和硬脑膜纤维覆盖包围猪脑子。 本文和视频教学的目的是描述如何在死后猪脑和垂体可在15-20分钟使用非机动手术工具进行曝光和去除的完整状态。教学视频和摄影插图显示雄性迷你猪（年龄:20-25公斤:6个月，体重），用于在小型猪脑垂体解剖研究。

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			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
			<td style="width:191px;">FST (Fine Science Tools)</td>
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			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
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			<td>Optimal for dural incision and precision work</td>
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			<td>10011-00</td>
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			<td height="21" style="height:21px;">Bone Rongeur</td>
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			<td>Must be robust, bite size 15 x 5 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
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			<td>Must be robust, bite size 25 x 15 mm&#160;</td>
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			<td height="21" style="height:21px;">Bone Chisel</td>
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			<td>67-0335</td>
			<td>The size of the chisel head should not exceed 20 mm</td>
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			<td height="21" style="height:21px;">Mallet (Hammer)</td>
			<td>Millarco</td>
			<td>5624108</td>
			<td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Scissor</td>
			<td>FST&#160;</td>
			<td>14002-14&#160;&#160;</td>
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			<td height="21" style="height:21px;">Dissector</td>
			<td>Aesculap Neurosurgery</td>
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		<tr height="42">
			<td height="42" style="height:42px;">&#160;G&#246;ttingen minipigs</td>
			<td style="width:191px;">&#160;Ellegaard G&#246;ttingen Minipigs A/S, Denmark</td>
			<td style="width:149px;"></td>
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			<td height="21" style="height:21px;">Euthanimal</td>
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			<td style="width:149px;"></td>
			<td>pentobarbital</td>
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			<td height="21" style="height:21px;">Ketamine</td>
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			<td height="21" style="height:21px;">Midazolam&#160;</td>
			<td style="width:191px;">Hameln Pharmaceuticals</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
	</tbody>
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exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

猪有大型动物平移神经科学的研究越来越受欢迎作为一个经济上和道德上可行的替代品，以非人类的灵长类动物。猪的大脑神经尺寸允许使用常规临床脑成像器和直接使用和神经外科手术和设备测试从人的诊所。进一步宏观和组织学分析，但是，需要的猪中枢神经系统（CNS）和脑随后去除死后曝光。这不是一件容易的事，因为猪的中枢神经系统是由厚，骨头骨和脊柱封装。本文和视频教学的目的是描述如何揭露和删除死后猪脑和完整状态的脑垂体，适合后续的宏观和组织学分析。

为了防止干燥的组织材料，建议去除的脑和垂体存储在填充有固定剂或已经执行宏观分析之后立即等渗盐水罐。组织材料可以被存储在固定剂多年，而存储在等渗盐水，即使在冰箱中，会导致组织随时间衰减。 除去的垂体也可以通过浸入干冰冷却液体的2-甲基丁烷中直接冷冻，而完整猪脑为直接冷冻28过大。?...

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

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

本文和视频教学的目的是描述如何揭露和删除死后猪脑和垂体处于完整状态，适合后续宏观和组织学分析。

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 ...

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15.3K 次观看.  Aalborg University Hospital. 本文和视频教学的目的是描述如何揭露和删除死后猪脑和垂体处于完整状态，适合后续宏观和组织学分析。 

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

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

科研

神经科学

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

使用功能磁共振成像和弥散加权成像脑结构与功能分析

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

一个全面协议的内侧颞叶结构的手动分割

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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 ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

驯养猪脑的提取和解剖

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

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

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

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 ...