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>

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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>High sucrose cutting solution</td>
			<td></td>
			<td></td>
			<td>in mM: 206 sucrose, 10.0 MgCl2, 11.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 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 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 2.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 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>100x15mm culture dish</td>
		</tr>
		<tr>
			<td>Small culture dish</td>
			<td>Falcon</td>
			<td align="right">353001</td>
			<td>35x10mm 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&#8211;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&#160; 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 365nm exitation, XF2001 400nm dichroic, XF3080 510nm 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

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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

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

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

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

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

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 ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

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 ...