Name: modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging
Uploaded: 2015-09-18T14:00:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.

所有的程序都在伊利诺伊大学批准的机构动物护理和使用委员会。所有动物饲养在批准的实验室动物护理的认可美国协会动物护理设施。每试图尽量减少所使用的动物的数量，并减少痛苦在研究的所有阶段。 1.准备并清除脑小鼠切片<ol><li>准备灌注和切片孵化。 <ol><li>大约切片前30分钟，制备高蔗糖裁剪解决方案和低钙学联的前记录或成像切片的培养。 </li><li>奠定了所有必要的工具（大钝头剪刀，小弹簧剪刀，镊子解剖，大剪刀​​或断头台，虹膜剪刀，镊子珠宝商10毫升注射器使用27G英寸x 1/2英寸寸毫针，小片1厘米×2cm的过滤器纸，和一破放倒巴斯德吸管于sl冰传输）灌注和切片，并准备灌注托盘。 </li><li>建立孵化室含氧学联在32 摄氏度的水浴和一个培养皿中充满了切削液。 </li></ol></li><li>准备切割阶段切片。 <ol><li>切一小片3％琼脂的大约1cm 3作为逆止对大脑和1.5厘米×1.5毫米×3毫米到用于凸块，以用于支撑所述集成电路。 </li><li>胶逆止到与氰基丙烯酸酯类粘合剂的阶段以及在右侧的逆止使得它们形成一个80ø角度的凸起。 </li></ol></li><li>取出大脑。 <ol><li>深深麻醉为P12-P20（理想p14-18）鼠标氯胺酮100 mg / kg和xylaxine 3毫克/千克（或类似的伦理委员会批准的程序）。 注:经确认缺乏应对脚趾捏适当的麻醉水平。当动物是在麻醉状态下简要地，眼药膏是不必要的。 </li><li>使用钝器结束剪刀，从剑突到颈部暴露胸腔。 <ol><li>找到剑突，并进行了约1.5厘米的横向切断。 </li><li>请从以前的水平切口的两端的垂直切到肩膀，约1.5厘米。 </li></ol></li><li>通过在隔膜和肋软骨结切割以暴露心脏。 </li><li>随着小弹簧剪刀，做一个小的，2-5毫米切开右心房，从腹侧背心脏的一侧。 </li><li>使用10毫升注射器用27G英寸x 1/2英寸英寸针，注入左心室，并迅速灌注高蔗糖切削溶液的动物。 </li><li>一旦气血运行清晰，使用较大的剪刀去除头。 注:我们还没有系统地评估灌注的效用。然而，根据我们的经验，在小鼠这个年轻transcardiac灌注可近100％的成功完成，并且确实有消除红BL的好处洪水细胞，它可以发出荧光，干扰成像。 </li><li>切开皮肤下的正中线，露出颅骨。 </li><li>使用弯曲虹膜剪，两眼之间的头骨，然后从眼睛的切割，由前精心切割沿中线缝合，注意不要下损伤大脑后侧开始。 注:通常硬脑膜与颅骨脱落。如果需要的话，前仔细去除脑部取出硬脑膜。 </li><li>使用珠宝商钳，撬打开颅骨，小心地取出大脑，然后将大脑中切割解决方案。请注意，以避免损坏皮质。 </li></ol></li></ol> 2.准备的大脑切片<ol><li>准备的大脑，用于安装在vibratome阶段。 <ol><li>使用在 90标有两行的滑动，并在17 O从左上到右下的顶部的对角线，相交，两条线的满足，将大脑背侧，使用刀片，去除2-4毫米的喙端大脑创造一个平坦的表面上。 </li><li>放置脑尾侧上新创建的平坦面，并对准脑的背侧表面与水平和脑与垂直线的中线。 </li><li>对齐刀片与17 O线，倾斜剃刀以30°角和在一个双对角切口（17 O从水平面和60度从所述冠）除去约3毫米的右皮质。 </li></ol></li><li>安装脑上vibratome阶段。 <ol><li>放置一小片滤纸1厘米×2厘米的脑的腹侧，使得长尺寸垂直于中线。 </li><li>小心将少量氰基丙烯酸酯类粘合剂的该地区中的逆止（和凸块的左侧）的前面。 </li><li>将双对角切脑面子上的胶水，使脑和后脑的尾部分是PRopped经凸点和右脑的是针对逆止。 </li><li>微妙按下上脑，保证整个表面与切片室接触。 </li></ol></li></ol> 3.获取Colliculo，丘脑片<ol><li>迅速采取切割阶段，发生在vibratome，填补了舞台切割解决方案。 </li><li>对准脑的顶部（腹侧）的刀片。 </li><li>从大脑的顶部除去1-1.5毫米，除去300-500微米切片和除去后评估深度。 注:当IC的MGB，和外侧膝状体（LGN）都是可见的，而齿状回形成一个"C"形持续一到两个600微米的薄片。参见图3A。 </li><li>将切片加热（32℃）储存室。 </li></ol> 4.成像切片<ol><li>准备黄素蛋白自发荧光成像。 <ol> &lt;李&gt;在标准电生理记录室准备脑脊液为切片的灌注，气泡脑脊液用95％ 的 O 2/5％CO 2。 </li><li>拉玻璃电极的刺激。注意，多个不同的刺激电极和配置（玻璃，钨，碳纤维，单极，双极）所有的工作非常好，在与黄素蛋白自发荧光成像结合使用。 </li><li>打开电脑，相机，显微，光源，刺激软件，图像采集软件。 </li><li>灌注录音室含氧学联，将自定义的桥（在补充材料提供设计），以提高切片室。 注:灌注率可与任何个人设置而有所改变， 5-15毫升/分钟在这里使用。 </li><li>将切片室，降低液体水平，在室内，防止片从升降桥下车，同时将专门建造的竖琴过片，同时注意避免将琴弦国米的途径EST（ 图1A）。增加液面上述切片大约1-2毫米，以保持上面和下面片脑脊液流动。 注意:如果重新定位是必要的，使用断头巴斯德吸管或允许脑脊液水平上升，使用镊子格外小心。 </li><li>插入氯化银电极为充满脑脊液玻璃电极，插入玻璃电极到显微操纵并连接到刺激隔离器，并小心地在集成电路/白质，导致丘脑玻璃电极（ 见图 1A）。 </li></ol></li><li>黄素自体荧光图像采集。 <ol><li>收集在4赫兹图像（使用无限远校正2X宏物镜（NA 0.13））和相机105秒而电刺激（每次刺激包括1秒40赫兹2毫秒脉冲组织中的0.05赫兹五次）而照亮该组织与蓝光470-490 nm和捕捉以上515纳米。确保图像既不是Ø黑暗，也没有吹出来见图3左边一列，以供参考。 注:收集时间，收集频率和刺激频率是可以改变的，以适应该实验中，包括在补充材料自定义程序可以被修改为这样。 </li><li>出口图像Matlab的，并使用在补充材料中可用的定制编写程序来分析图像的频谱功率在0.05赫兹。由此产生的图像将显示已连接通路。 注:自定义程序使用在时间序列的快速傅里叶变换的象素值，以产生图像。 </li></ol></li><li>准备钙成像。 <ol><li>使用3厘米的培养皿，并提出培养膜，以允许脑脊液到在加热垫上达到顶部和切片的底部，并置于保持温度在大约32℃。制备小孵育室</li><li>填写小孵化室温暖学联，慢慢地泡有95％氧气2/5％CO 2。 </li><li>混合2μl的普朗尼克F-127酸和50的Fura-2AM溶解于48微升的DMSO微克。 </li><li>在凸起膜，小心使用微量小孵育室地方添加片染色混合物的MGB正上方（或其它感兴趣的结构）。 </li><li>覆盖片防止漂白前成像，并且允许切片孵育45分钟 - 1小时。 </li><li>取出切片加热保温室10-15分钟，以洗去多余的材料。 </li><li>按照步骤4.1.1到4.1.6的准备，以刺激和形象colliculo - 丘脑片。 </li></ol></li><li>钙图像采集。 <ol><li>在10 Hz（使用20X水浸泡的目的），持续25秒采集图像，而在0.2赫兹电刺激（每次刺激包括一个2毫秒的脉冲）组织的五倍，同时照射组织365纳米的光，并捕获上述510纳米的荧光。 </l我&gt; <li>出口图像Matlab和使用在补充材料中可用的定制编写程序来分析图像的频谱功率在0.2赫兹。由此产生的图像将显示出活跃的细胞。 </li></ol></li></ol>

<ol>
	<li>Llano, D. A., Slater, B. J., Lesicko, A. M., Stebbings, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auditory+colliculothalamocortical+brain+slice+preparation+in+mouse.">An auditory colliculothalamocortical brain slice preparation in mouse.</a> J. Neurosci. 111, 197-207 (2014).</li><li>Agmon, A., Connors, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamocortical+responses+of+mouse+somatosensory+(barrel)+cortex+in+vitro.">Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro.</a> Neuro. 41, 365-379 (1991).</li><li>Cruikshank, S. J., Rose, H. J., Metherate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+Thalamocortical+Synaptic+Transmission+In+Vitro.">Auditory Thalamocortical Synaptic Transmission In Vitro.</a> J Neurophysiol. 87, 361-384 (2002).</li><li>Lee, C. C., Sherman, 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=Topography+and+physiology+of+ascending+streams+in+the+auditory+tectothalamic+pathway.">Topography and physiology of ascending streams in the auditory tectothalamic pathway.</a> PNAS. 107, 372-377 (2010).</li><li>MacLean, J. N., Fenstermaker, V., Watson, B. O., 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=A+visual+thalamocortical+slice.">A visual thalamocortical slice.</a> Nat meth. 3, 129-134 (2006).</li><li>Crabtree, J. W., Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+intrathalamic+pathways+allowing+modality-related+and+cross-modality+switching+in+the+dorsal+thalamus.">New intrathalamic pathways allowing modality-related and cross-modality switching in the dorsal thalamus.</a> J. Neurosci. 22, 8754-8761 (2002).</li><li>McAlonan, K., Cavanaugh, J., Wurtz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Modulation+of+Thalamic+Reticular+Neurons.">Attentional Modulation of Thalamic Reticular Neurons.</a> J. Neurosci. 26, 4444-4450 (2006).</li><li>Weese, G. D., Phillips, J. M., Brown, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Orienting+Is+Impaired+by+Unilateral+Lesions+of+the+Thalamic+Reticular+Nucleus+in+the+Rat.">Attentional Orienting Is Impaired by Unilateral Lesions of the Thalamic Reticular Nucleus in the Rat.</a> J. Neurosci. 19, 10135-10139 (1999).</li><li>Zikopoulos, B., Barbas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Projections+to+the+Thalamic+Reticular+Nucleus+form+a+Unique+Circuit+for+Attentional+Mechanisms.">Prefrontal Projections to the Thalamic Reticular Nucleus form a Unique Circuit for Attentional Mechanisms.</a> J. Neurosci. 26, 7348-7361 (2006).</li><li>Kalatsky, V. A., Stryker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Paradigm+for+Optical+Imaging:+Temporally+Encoded+Maps+of+Intrinsic+Signal.">New Paradigm for Optical Imaging: Temporally Encoded Maps of Intrinsic Signal.</a> Neuron. 38, 529-545 (2003).</li><li>Llano, D. A., Turner, J., Caspary, 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=Diminished+cortical+inhibition+in+an+aging+mouse+model+of+chronic+tinnitus.">Diminished cortical inhibition in an aging mouse model of chronic tinnitus.</a> J. Neurosci. 32, 16141-16148 (2012).</li><li>Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+studies+on+auditory+development+in+mammals+in+relation+to+higher+nervous+system+functioning.">Behavioural studies on auditory development in mammals in relation to higher nervous system functioning.</a> Acta otolaryngol. 99, 31-40 (1985).</li><li>Mikaelian, D., Ruben, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hearing+in+the+normal+CBA-J+mouse:+correlation+of+physiological+observations+with+behavioral+responses+and+with+cochlear+anatomy.">Development of hearing in the normal CBA-J mouse: correlation of physiological observations with behavioral responses and with cochlear anatomy.</a> Acta. 59, 451-461 (1965).</li></ol>

The authors have nothing to disclose.

This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to...

在听觉系统，虽然有感官周边和下丘之间的信息实质性加工，有相当大的附加的处理之前到达听觉皮层。我们所知甚少有关如何处理已完成，因此一些关于这种转变让大脑解释传入的感觉信息。除嗅觉，每个感官具有与最初被中继高保真其中下降的信号上升到皮质外设信号非常相似的组织。皮质然后发送突起到下部结构来进一步调节所述输入信息。这种复杂的系统进行了研究，以各种方式在体内以及在一些体外制剂。在前者中，所有的连接都完好，使研究者探测任何组连接，同时控制感觉输入和测量输出在任何给定的区域中。采用这种方法，很少有无法控制的种类繁多的其他投入，包括其他感觉输入，觉醒和重视，以一种强烈的复杂的输出便出现在体外 ，大脑切片被切断捕捉到一个单套的预测，或者两个相连的脑区，这使研究人员，以刺激和评估各种传入或脑区。这些通常是要么丘脑皮层或tectothalamic片，其中任一输入到丘脑或丘脑和其输出到皮层被保留2-5。这些制剂允许各种各样的药理，电气和光遗传学操作。然而，只有两个脑区，它们主要评估的信息的传送，并且缺乏以评估信息的改造，因为它通过丘脑的能力。另外，网状丘脑突起，其可在关注调制发挥作用6-9是公关ESENT在此片。在这里，我们展示的改进在我们之前的准备1，允许各种投入到丘脑研究者控制给予的丘脑盖茨和过滤信息如何以独特的视角。我们夫妇这种新颖的切片准备与黄素蛋白自发荧光成像来评估片连接和大规模活化分析，在丘脑神经元群体分析钙成像，和单细胞记录以测量的各种输入上的单细胞水平的影响。 为了协助维护这些应用广泛的联系，我们还开发了一些垂直切片锚（又名"竖琴"）的修改举行的脑切片的地方，桥梁，以提升切片增强灌注。竖琴被设计成修改马蹄形以包围切片，并允许可定制的连接点为竖琴字符串。三字符串附，使得i）一种位于沿水平方向内侧边缘的切片，ⅱ）从IC到AC的尾部边缘的尾部边缘上的一个延伸，并且iii）一种从斜内侧缘的片的延伸到的区域喙到AC（ 见图 1A）。小凹口在帧胶合（与氰基丙烯酸酯胶）的琴弦允许有减少量的压力中的片上，以帮助保持片完整性的（参见图1B）。通过使用三维印刷，我们能够定制设计竖琴我们独特的规格，以及其允许的上方和下方的组织的人工脑脊液（ACSF）理想的流量桥。这也保持着大面积的光穿透组织的膜片钳电。

<table><tbody><tr><td>High sucrose cutting solution</td><td></td><td></td><td>in mM: 206 sucrose, 10.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 11.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 0.5 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>Low calcium aCSF</td><td></td><td></td><td>in mM: 126 NaCl, 3.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>aCSF</td><td></td><td></td><td>in mM: 126 NaCl, 2.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 2.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>Stimulus Isolator</td><td>World Precision Instruments</td><td>A360</td><td></td></tr><tr><td>DMSO</td><td>Life Technologies</td><td>D12345</td><td>Lot: 1572C502</td></tr><tr><td>Fura-2AM</td><td>Life Technologies</td><td>F1201</td><td>Lot: 144912</td></tr><tr><td>Pluronic F-127</td><td>Life Technologies</td><td>P3000MP</td><td>Lot: 1499369</td></tr><tr><td>Large culture dish</td><td>Fisherbrand</td><td>08-757-13</td><td>100 x 15 mm culture dish</td></tr><tr><td>Small culture dish</td><td>Falcon</td><td>353001</td><td>35 x10 mm culture dish</td></tr><tr><td>Raised culture membrane</td><td>Millicell</td><td>PICMORG50</td><td>Used to maintain oxygenated fluid perfusion on both sides of slice.</td></tr><tr><td>Flavoprotein imaging fluorescence cube</td><td>Olympus</td><td>UMNIB</td><td>470&amp;ndash;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&amp;nbsp; We have found that virtually any green fluorescence protein filter cube will work here.</td></tr><tr><td>Calcium imaging fluorescence cube</td><td>Omega Optical</td><td>BX-18</td><td>XF1005 365 nm exitation, XF2001 400 nm dichroic, XF3080 510 nm emission</td></tr><tr><td>Agar for blocking brain</td><td></td><td></td><td>3% by weight in water</td></tr><tr><td>Viper si Stereo Lithography Apparatus</td><td>3D Systems</td><td></td><td></td></tr></tbody></table>

modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

在P15小鼠获得colliculo-丘脑皮层小鼠脑切片的一个例子示于图2中 ，理想的切片将包含四个主要脑和前脑听觉结构集成电路，MGB，TRN，和AC，这些当IC被激发的所有激活（图2A）。使用傅立叶分析，光谱功率的测量是在电刺激频率，以显示活性为周期性和夹带在刺激频率10连接的大脑区域。该协议允许对信息尤其是在丘脑的脑和皮质之间的关系上升流的研究。而在生?...

Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

利用多角度切割鼠标小狗大脑，我们在提高先前描述的急性脑切片捕捉之间最重要的听觉脑和前脑结构的连接。

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

modification-colliculo-thalamocortical-mouse-brain-slice

Research

JoVE Journal

Neuroscience

8.2K 次观看.  University of Illinois Urbana-Champaign. 利用多角度切割鼠标小狗大脑，我们在提高先前描述的急性脑切片捕捉之间最重要的听觉脑和前脑结构的连接。 

视频: Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

行为学

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 ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

一种从介观尺度到微观尺度的神经元成像组织清除方法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

上丘的双光子和宽场钙成像

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

作者聚焦:揭示上丘的神经编码和视觉处理机制 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

摘要

探索更多视频

此视频中的章节

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

成年鼠脑铸造三维成像和形态分析

多层皮质Ca

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

小鼠大脑皮层细胞组织的大尺度三维成像

一种从介观尺度到微观尺度的神经元成像组织清除方法

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像