我们感谢整个Koshy实验室进行有益的讨论。我们感谢侯佩岑Jansma和亚利桑那州神经科学系的大学征求意见，并与成像帮助。我们也感谢波雷卡实验室使用他们的vibratome的。这项研究是由健康的美国国家研究院​​（NIH NS065116，AAK）的支持。

注：小鼠饲养，并保持在一个温度和湿度控制的房间12小时逆转食物和水可用自由采食光/暗周期在亚利桑那大学。实验下的指引和亚利桑那大学的机构动物护理和使用委员会批准进行的。所作的所有努力，尽量减少痛苦。该Cre的报告小鼠是在C57BL / 6背景11和可商购。 1.鼠标感染注：小鼠感染弓形虫下面所描述的方法已被用于在先前公布的10项研究- 12。 <ol style="margin-left: 40px;"><li>生长弓形虫菌株在人包皮成纤维细胞（HFFS）的Dulbecco氏高糖培养的改良的Eagles培养基（DMEM），补充有10％FBS，100U / ml青霉素，100mg / ml的链霉霉素，和2mM L-谷氨酰胺（cDMEM）的T-25烧瓶内的5％的CO 2培养箱中，直到寄生虫已形成大虫空泡。 </li><li>通过从烧瓶底部刮的细胞和使所得到的溶液通过一个25号针头连接到3毫升注射器的2倍的烧瓶内，则所有溶液转移至5ml注射器壳体连接到27号针头的注射器释放寄生虫已被置于一个15毫升的锥形管内上下颠倒。柱塞连接到注射器，并通过该寄生虫溶液进入锥形管。 </li><li>离心管在300×g下10分钟。吸出上清液，然后重悬沉淀在4-6毫升无菌USP级1×磷酸盐缓冲盐水（PBS）pH 7.4的（原液）。 </li><li>装入10微升原液血球，等待5-10分钟的寄生虫定居然后计算寄生虫的数目，中间大正方形的四角和中心方块。 </li><li>第稀网站的100K / 200微升合适的接种物大小的1X PBS，然后用200微升的总体积注射小鼠腹膜内（IP）。 注：在此过程中，小鼠接种100K型III（CEP）13速殖子200μl的1X PBS。当感染小鼠弓形虫的其它菌株，接种体浓度可能需要进行调整以考虑毒力的水平。 </li></ol> 2.灌注和脑收获注意：此协议是第21天感染后（dpi）的进行，因为这代表了时间点被发现在小鼠大脑囊肿峰的数字，但也可以使用其它的时间点。大小，数量，位置，以及存在包囊的存在与否，取决于很多因素，包括小鼠品系，寄生虫菌株类型以及接种量7,14 - 17。研究者将需要单独确定合适的接种物为鼠标和Toxoplas马应变，他/她使用。 <ol style="margin-left: 40px;"><li>设置所有手术收获的工具（ 图1）。 </li><li>对于每个小鼠，制备并保持在冰上：20ml注射器填充有0.9％的氯化钠（NaCl）中+ 10单位/毫升肝素在1×无菌USP级的PBS（肝素/盐水）; 20毫升注射器填充有4％多聚甲醛（PFA）;闪烁小瓶用15毫升的4％的PFA。对于多个小鼠，制备填充有所需的所有小鼠，然后制定一个新鲜注射器充满每个灌注溶液各溶​​液的全部量的烧杯中。 </li><li>麻醉小鼠IP用200μl/ 25克体重的氯胺酮/甲苯​​噻嗪混合物（24毫克/毫升与4.8毫克/毫升，分别）在感兴趣的日子。通过检查脚趾捏反射监视鼠标麻醉水平。继续协议只有一次鼠标显示了一个坚定的脚趾捏没有反应。 注：如之前达到麻醉的适当水平的麻醉的其它方法也可以使用，只要在继续进行协议。 </li><li>鼠标放置在有盖的泡沫平台上仰卧位。 PIN所有四肢出在脚上，然后喷洒胸部和腹部用70％乙醇（乙醇）（ 图2A）。另外的鼠标下几个无菌纸巾可以用来帮助吸收灌注流体的溢出。 </li><li>正下方用钳子胸骨抬起皮肤，然后用剪刀做一个切口。拉回来的皮肤暴露胸部（ 图2B）。 </li><li>提起剑突，使刚刚膈下腹部切口，暴露出这是钝性分离离膈肌肝脏。使两个切口，一个在左，一个在通过隔膜和右肋的底部。延伸的左，右的切口大约1 / 2-2 / 3的方式向上胸腔，然后将其反射回以打开胸腔（ 图2C）。 注意：小心不要削减任何器官或主要血管杜响此过程，因为这样做会损害灌注质量。 </li><li>用10-20毫升肝素/盐其次是10-20毫升4％PFA灌注transcardially。 注意：如果灌注得当，肝脏会从红色变成棕褐色（ 图2D）。如果没有正确完成，血管将可见（ 图2E）。 </li><li>把鼠标放在其上腹部，重脚脚再喷头部和颈部用70％乙醇。从颈部的底座抬起皮肤，使一个切口。去除皮肤暴露的头骨（ 图2F）。斩首头在肩的水平。取出颅骨，轻轻取出大脑，并放入闪烁瓶中装满4％PFA（ 图2G-H）。 注意：如果灌注良好，大脑会出现白色无可见的血管（ 图2I）。如果大脑没有得到很好的灌注，血管就会可见（ 图2J）。 </li><li>将火花化小瓶大脑在4％PFA在4℃过夜，涵盖了从光。冲洗脑部非USP级1×PBS中，并转移到一个新的闪烁小瓶填充有冷却的1×PBS。存储大脑在1×PBS中，在4℃，涵盖了从光。 注：成功清算和成像已经完成与存储在1×PBS中进行长达一个月的部分，但最好是部分在几天之内为长时间储存​​在PBS将扭转PFA固定和大脑可能导致低于最佳的图像。自体荧光和增加的模糊已注意到在一些路段储存后成像在1×PBS中一个月或更长的时间。 </li></ol> 3.脑切片<ol style="margin-left: 40px;"><li>从1 PBS取出大脑，并把它变成一个大脑基质（ 图3A），以便更精确，通过大脑甚至削减。剪掉小脑和嗅球，如果存在的话，然后切成冠脑部成2个或3段。 <b转/&gt;注：切边和脑切割可以不经大脑基质的帮助完成。正在使用的切割冠将取决于的vibratome的切割距离段数。大脑可以分段在任何方向，虽然矢状面和冠状节最常用于神经解剖鉴定。 </li><li>贴上各脑切片上与氰基丙烯酸酯样品安装块。加盖的背后支持的脑切片（ 图3B）的5％琼脂糖凝胶的小块。 注：其它支持机制都可以使用，但不提供支持，所述的vibratome可推动对脑造成不均匀的切片。 </li><li>切割组织成厚的切片用的vibratome设置为4的速度和9振幅为160μm（用于成像物镜的工作距离）。 注：在速度和幅度的设置可以根据特定的机器上进行调整所使用，以获得均匀，平滑的部分。如果设置不正确，结果可能是不均匀的部分，组织或振痕撕裂。 </li><li>收集组织切片成闪烁小瓶填充有新鲜，冷冻的1×PBS，如果他们打算一个星期内，以被清除。如果段不打算在一周内被清除，放置部分成1.5ml微量管中填充有在-20℃冷冻保存液溶液和存储。 </li></ol>脑组织切片4.光学结算注：此协议已被修改从先前公布的方法8。 <ol style="margin-left: 40px;"><li>在1×PBS + 2％吐温-20（GL / PBST）中制备25％，50％，75％和90％体积/体积甘油。 </li><li>从1×PBS中移除部分，或者，如果在冷冻保存液，冲洗在1×PBS中并转移到含有10毫升25％的GL / PBST闪烁瓶中。地方小瓶在振荡器上在4℃下，盖从暴露于光，12小时。 </li><li>确认所有部分已经沉没到微博TTOM小瓶。吸出25％的GL / PBST中，只留下足以覆盖部分然后填充在振荡器上在小瓶用10毫升50％的GL / PBST和地点在4℃下，盖从暴露于光，12小时。对于75％GL / PBST那么90％GL / PBST重复此过程。留在90％GL / PBST路段24小时达到最高结算。在结算过程中的过程中，逐步观察光透明（ 图4）。 注意：如果解决方案更改后立即路段已经沉没到小瓶的底部这个过程可能会加快。在75％与90％的溶液，部分不会下沉一路小瓶的底部，但将在中间浮动。 </li></ol> 5.安装脑组织切片<ol style="margin-left: 40px;"><li>切1.5号盖玻片到5个创建间隔。用尺子和金刚石尖铅笔得分和打破盖玻片沿图5A中所示的虚线。丢弃吨他围绕一块，安排4件外的一个普通的载玻片，以创建一个窗口，然后刷透明指甲油到接缝坚持间隔的碎片在正确的配置（ 图5B）的幻灯片。 注：该垫片将被用于创建载玻片和盖玻片用于安装清除部分之间的必要的空间。盖玻片可被切割以不同的方式来创建所需的任何尺寸的窗口。预切割泡沫衬垫也可商购。为了让指甲油完全干燥，最好是安装部分，至少提前一天进行幻灯片与垫片。 </li><li>使用具有尖端的塑料移液管转移到节幻灯片切断。请务必填写周边地区有90％GL / PBST。小心地放下盖玻片在确保不引入任何气泡的部分。多余的GL / PBST可吸走了无绒擦拭。 </li><li>图像GFP +含RFP元+ <em&gt;弓形虫包囊用共聚焦显微镜立即（ 图7A-B），然后存储幻灯片平坦和覆盖从光在4℃。 注意：可替换地，密封滑动完全围绕所有边缘来存储和成像在以后的时间。使用的透明指甲油密封滑动是可选的，并且可以使之更难以除去盖玻片如果一个人以除去从滑动部分在稍后的日期，以便进一步处理。 </li></ol> 6.成像注意：任何显微镜，可以使用具有获得高解析度的z栈的能力。 <ol style="margin-left: 40px;"><li>在最初发现的焦平面的10倍，变为20倍的目标，并通过部分扫描识别位于GFP +细胞内囊肿。在视场中心的感兴趣区域（ROI）的区域。 </li><li>更改为40X的目标。注重上下贯通整个投资回报率，以确保整个细胞和CYST是可见的。 注：图片均使用共焦显微镜用40X / 1.30石油DIC M27目标获得。 </li><li>使用安装的成像软件，得到Z堆叠包括与囊肿整个单元。用于获取图像的平均设置示于图6中 。 注：设置可能需要根据每个研究者的组织切片和显微镜的能力进行调整。 </li><li>由突起的数目设置为1。由突起的数目设定为64获得完整的旋转视图获取Z堆叠的最大投影图像。 注：其它软件包可用，并且可以被用于获得最大突出，旋转以及Z堆叠图像的其它视图。 </li><li>分析图像与图像分析软件。 </li></ol>

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作者什么都没有透露。

由于在感染宿主细胞的细胞变化都与疾病结果在感染细胞内的其他生物如HIV，狂犬病，和衣原体18,19，我们开发出一种技术，使我们研究CNS之间发生亲密互动宿主细胞和弓形虫 。这里介绍的方法实现这一目标，通过使慢性感染神经细胞的有效成像。在此之前的方法的发展，这样的成像是耗时的，昂贵的，或者是不可能的。 现在，我们可以使用这种技术来回答有...

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>. The ordering of the authors was inverted. The order has been updated from:
 Anita A. Koshy, Carla M. Cabral
to:
Carla M. Cabral, Anita A. Koshy

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>.

Erratum: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

该方法的总的目标是要获得高的分辨率，被感染的专性细胞内寄生虫弓形虫单个神经元的三维图像。 弓形体通常被认为是由于其较大的中间宿主范围，包括人类和啮齿类动物中最成功的寄生虫之一。在人类和啮齿类动物，通过污染的食物或水摄入的急性感染后， 弓形虫能够通过从它的快速复制的形式（将速殖子）转化成其慢复制和encysting形式引起的中枢神经系统的持续性感染（在缓殖子）。在免疫活性个体，这种潜中枢神经系统感染被认为是相对无症状的，但在免疫受损的个体，如爱滋病患者或移植者，寄生虫的复发可导致致命弓形虫脑炎1,2。此外，最近的研究公顷已经表明，隐性感染弓形虫会导致行为的变化在啮齿类动物3,4，尽管机制仍不清楚。 令人惊讶的是，尽管这些数据突出了CNS- 弓形虫相互作用的重要性，相对知之甚少这种关系，特别是在细胞和分子水平。研究脑寄生虫互动，甚至简单的方面的能力受到了阻碍部分由TECHNOLOGIC限制。例如，大多数的工作表明神经元的细胞，其中包囊坚持已经完成，电子显微镜（EM）5,6。虽然EM提供高分辨率，这是费时，劳动强度大，而且价格昂贵。免疫荧光（IF）测定法最近已结合使用共聚焦显微镜来确认由EM 7所做的工作。中频测定在技术上易于实施和相对便宜的，但使用这些技术来underst和囊肿和感染的神经元之间的空间关系，需要串行重建，这是耗时的，在技术上困难，并且可能会导致有价值的信息丢失。因此，我们开发了可与中枢神经系统弓形虫病的小鼠模型中使用，并允许我们图像感染的神经元的全部未经EM或免疫组织化学（IHC）的方法。通过开发这样的技术，我们可以开始探索以相对快速和廉价的方式感染细胞和囊肿之间的蜂窝式关系。 我们开发的方法，结合了用于光学结算和成像厚脑切片通过共聚焦显微镜8较新的技术与系统，它标志着已经注射了寄生虫蛋白9,10 体内细胞。在这个系统中，我们感染表达绿色荧光蛋白（GFP）的Cre-报告小鼠仅后的Cre介导的重组11 弓形虫 </em&gt;表达红色荧光蛋白（RFP）和Cre重组酶注入到宿主细胞中的9株。这种组合可以让我们收获了感染小鼠的大脑后，中枢神经系统感染建立后，切成厚脑切片，并迅速查明相关领域的图像通过查找RFP +囊肿。要注意，作为绿色荧光蛋白的宿主细胞中表达完全取决于注射的Cre的寄生虫，而不是在感染，一些将GFP +细胞不含有寄生虫10是很重要的。作为该协议的目的是为了能够将图像的整体感染的神经元，重点是仅在GFP +神经元还含有RFP +囊肿，但该协议也可以用于图像的GFP + / RFP -神经元。 一旦感染脑收获并切片，该切片是由甘油结算呈现透明。部的适当的区域，然后用共焦显微镜，人成像感染的宿主细胞，并在其整体的成囊寄生虫降脂前所未有的可视化。在这里，我们提供了一个完整的协议识别，光学结算和成像感染神经元。

<table border="0" cellpadding="0" cellspacing="0" width="1877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Name of Reagent/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:1008px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Series 1000 Sectioning System</td>
			<td>Technical Products International, Inc.</td>
			<td></td>
			<td>Other vibratomes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>VWR</td>
			<td>48393 251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond Scriber</td>
			<td>VWR</td>
			<td>52865-005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 510 Meta confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketaject&#174; Ketamine HCl Inj., USP 100mg/ml</td>
			<td>Western Medical Supply, Inc.</td>
			<td>4165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed&#174; Injection Xylazine 20mg/ml</td>
			<td>Lloyd Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZsGreen Mice</td>
			<td>Jackson Laboratories</td>
			<td>7906</td>
			<td>B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical equipment</td>
			<td></td>
			<td></td>
			<td>Thumb forceps; Fine scissors-angled to side, sharp-sharp; Sharp-sharp scissors; Kelly hemostats; Mayo scissors; Micro spatula.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Foreskin Fibroblasts (HFF) cells</td>
			<td></td>
			<td></td>
			<td>These are primary cells from human foreskins.&#160; We make these in-house but they may be purchased from outside vendors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s High Glucose Modified Eagles Medium (DMEM)</td>
			<td>HyClone</td>
			<td>SH30081.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Penicillin Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">200mM L-alanyl-L-glutamine</td>
			<td>Corning</td>
			<td>25-015-Cl</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:469px;">25cm2 Canted neck flask</td>
			<td>Fisher Scientific</td>
			<td>1012639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>VWR</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 10X, USP Sterile Ultra Pure Grade</td>
			<td>amresco</td>
			<td>K813-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Fetal Bovine Serum</td>
			<td style="width:265px;">Gibco</td>
			<td style="width:135px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bright-Line Hemocytometer</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Brain Slicer Matrix</td>
			<td>Zivic Instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td style="width:135px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td style="width:135px;">H3393-100KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Paraformaldehyde</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">20ml Disposable Scintillation Vials</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">FS74500-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol, Ethyl, 95%, 190 Proof</td>
			<td style="width:265px;">In-house</td>
			<td>17212945</td>
			<td>This product is purchased from an in-house stockroom.&#160; Other companies are compatible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Imaris Software</td>
			<td style="width:265px;">Bitplane</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Clear nail polish</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Other brands are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">10ml Syringe with Luer-Lok</td>
			<td style="width:265px;">VWR</td>
			<td>BD309604</td>
			<td>Other syringes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Three-way Stopcock</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hypodermic needle</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible - used to pin down mouse.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cell Scraper</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G x 12&#34; Tubing, Safety Blood Collection Set, with Luer Adapter</td>
			<td style="width:265px;">Greiner Bio-One</td>
			<td style="width:135px;">450099</td>
			<td>Other brands are compatible</td>
		</tr>
	</tbody>
</table>

3-d imaging and analysis of neurons infected in vivo with toxoplasma gondii

弓形虫是一种专性细胞内寄生虫具有广泛的宿主范围，包括人类和啮齿类动物。在人类和啮齿类动物， 弓形虫建立在大脑终身持续感染。虽然这种脑部感染是无症状的，在大多数人的免疫功能，在发育中的胎儿或免疫功能低下的个体如获得性免疫缺陷综合征（AIDS）患者，这种偏爱与执着在大脑中可能会导致灾难性的神经系统疾病。因此，很显然，献计弓形虫相互作用是由弓形虫产生的症状的疾病的关键，但我们对中枢神经系统（CNS）和寄生虫的细胞之间的细胞或分子的相互作用的了解甚少。在中枢神经系统的小鼠模型弓形体病已经知道了30多年的神经元中的寄生虫仍然存在的细胞，但信息很少哪些神经元的一部分，通常感染（体细胞，树枝状，轴突），如果菌株之间的这种蜂窝关系变化。在某种程度上，这是缺乏二次成像的难度，从动物整体可视化感染神经元。这些图像通常需要连续切片和组织通过电子显微镜或免疫染色后，激光共聚焦显微镜成像的拼接。通过组合多种技术，这里所描述的方法使得能够使用厚的切片（160微米），以确定和包含包囊图像的完整细胞，允许三维可视化和个人，慢性感染的神经元的分析，而不需要免疫染色，电子显微镜，或连续切片和拼接。使用这种技术，我们可以开始理解寄生虫和感染的神经元之间的蜂窝式关系。

图7包括两个GFP +神经元从两个不同的160微米厚的切片，以及来自囊肿至细胞体为图7B的距离的代表测量结果。 图含有囊肿-7的A和 B示出了这个新的代表图像协议允许在其全部被感染的神经元的可视化。 图7C示出了与该成像技术，现在有可能量化囊肿和胞体（了Imaris 7.7）之间的距离。 图7D是图的一个?...

Watch this Scientific Journal Video about 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii at JoVE.com

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

使用该协议，我们能够从感染寄生虫弓形虫 ，使可视化和的encysting寄生虫和感染的神经元之间的空间关系分析的小鼠图像160微米厚的脑切片。

神经元的3-D成像和分析感染<em&gt;在体内</em&gt;与<em&gt;弓形虫</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

3-d-imaging-analysis-neurons-infected-vivo-with-toxoplasma

Research

JoVE Journal

Neuroscience

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

11.4K 次观看.  University of Arizona. 使用该协议，我们能够从感染寄生虫弓形虫 ，使可视化和的encysting寄生虫和感染的神经元之间的空间关系分析的小鼠图像160微米厚的脑切片。 

视频: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

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 ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.