这项工作是由宝来惠康（AKM）在生物医学科学事业奖，白厅基金研究资助（AKM），斯隆基金会奖学金（AKM），美国国立卫生研究院EY019277（AKM），和聂资助，视力训练补助金，EY013319（ EAK）。

<ol><li>要完整的使用双光子显微镜，一个神经元与荧光标记物标记的准备的大脑中的神经元可视化使用。这里介绍在实验中，我们使用的GFP - M的转基因小鼠线，标签5层的锥体细胞。 5层的锥体细胞投射到表层的树突状进程，使树突和树突棘的可视化深度为300微米以下的PIA水平。另一种方法是使用病毒标志物（见洛厄等 。提交给朱庇特）标签细胞。 </li><li>消毒工作区，使用70％的乙醇和高压灭菌器工具，无菌手术。支付业务用干净的敷料表面。 </li><li>麻醉与芬太尼鸡尾酒（芬太尼0.05毫克/公斤;咪唑安定5毫克/公斤; metatomadin 0.5毫克/公斤）小鼠由Avertin季度剂量使用IACUC核准程序（0.0075毫克/毫升）。预处理小鼠的镇痛，丁丙诺啡（0.5毫克/公斤）皮下注射（SQ）。麻醉平面显示器的水平测试反应尾/脚趾捏。 </li><li>保持动物在37.5 ° C加热毯使用带有附加直肠温度计。 </li><li>眼药膏眼睛，防止眼睛干燥。用一张小纸罩的眼睛，保护眼睛在手术过程中光源。 </li><li>用剪刀，去除头发从后脑勺（背后耳朵）眼睛。 </li><li>清洁动物的连续应用70％乙醇的头顶部，其次是优碘擦洗和优碘解决方案。 </li><li>背后的耳朵和向上的L型切割的切口，沿中线（取决于哪个半球成像）对眼睛的右边或左边。折叠皮肤远离手术的部位。不要清除皮肤成像会议将缝合皮肤。 </li><li>用细镊子轻轻拉动皮肤，和远离你感兴趣的领域。 </li><li>远离暴露头骨的面积用干净的镊子或显微刀片轻轻刮去骨膜。使用棉签，申请了10％三氯化铁少量颅骨骨膜膜完全干燥，并确保它已完全去除。重要的是，头骨是完全干的胶水，以确保正确的坚持（见11）。使用产钳或显微刀片轻轻刮去干骨膜膜。 </li><li>应用周围氰基丙烯酸酯胶的一层薄薄的不锈钢封头板（见图1），并加盖颅骨外板成像窗口。 </li><li>用棉花拭子棒木，适用于胶成像窗口内缝线;务必填写颅骨成​​像板和曲率之间的所有空隙。 </li><li>将下降到窗口的胶水加速器。快速擦去多余的胶水加速器，用棉签或金擦拭。胶水应完全凝固钻前。 </li><li>开始使用牙钻一个0.7毫米的不锈钢毛刺贴在成像窗口颅骨薄。备用钻井偶尔应用0.9％的无菌生理盐水，以避免产生过多的头骨上的热。 </li><li>薄的整个头骨表面的牙钻使用小中风的头骨上一个4x4毫米的窗口。测试与钝端钳薄的头骨。头骨表面缩进稍微温和的压力。颅骨成像的最佳厚度为10至30微米。 </li><li>当颅骨薄，窗口清理所有杂物，放置在头骨生理盐水。安装解剖范围的数码相机拍摄的成像窗口。脑血管摄影和使用，将有助于定位的位置放置后续成像会话成像板。 </li><li> 2光子钻机运输动物。动物应该保持在整个成像会议上加热毯（直肠探头）。注：而下的镇静，动物的核心体温会在一个加热造成皮质的损害或可能死亡的单位的情况下波动很大。 </li><li>一个与埋汰10W的最大功率和修改后的奥林巴斯Fluoview共焦单位的固态激光器，泵的双光子显微镜使用。深部组织成像系统优化和外部探测器接近客观，尽可能地让大脑最大的荧光检测，安装。奥林巴斯LUMPlan FL / IR 20X/0.95NA物镜是用来。 0.9％生理盐水用于在客观淹没成像。 </li><li>使用这种显微镜，低数码变焦（X1）确定一个区域，包含明亮标神经元。 </li><li>使用数码相机，贴在显微镜目镜，成像区域的照片。另外，勾勒出血液血管格局。这是有必要重新在随后的时间点，打开相同的成像位置。 </li><li>通过整个大脑树突分枝的程度（通常是跨越〜200微米）的可见程度，获取一个低倍率栈（X20目标，512x512像素; 5微米的一步）。注：这种低倍率的图像将被用来定位在随后的时间点成像区域的血管和树突保持其在青少年和成年动物的结构。 </li><li>使用图像处理软件（Fluoview，诉5.0，FV300），增加8倍数字​​放大倍率。调整X，Y坐标（坐标平移）如果有必要，并收集了高倍率栈，它会显示详细的树突状形态，包括地点和树突棘的结构。每个堆栈收集，即泛坐标，收集，Z -步长和步骤在您的堆栈数量范围进行详细的注释。这些债券将返回这个树突或轴突在随后的时间点时使用。 </li><li>成像后，取出轻轻分开头骨表面的板头板。使用镊子，取出头骨其余任何残胶，头骨表面，并用0.9％无菌生理盐水擦拭。在头骨一起使用＃6缝合线缝合皮肤。用70％乙醇擦拭面积。为了减轻在手术后的恢复疼痛，管理0.1毫克/公斤丁丙诺啡镇痛，SQ的。 </li><li>将动物热灯，直到它被唤醒，是流动的，在清洁笼子。返回到原来的笼子里的动物。 </li><li>要重新映像在稍后的时间点（我们的成像时间点，可以在任何地方从2天，除了许多个月）的动物，去除缝线，或重新使用上述无菌方法对头部皮肤。使用脑血管（第一次手术日期）的数字图像，找到原来的成像位置。 Reaffix不锈钢磁头板（步骤10-12）。使用显微刀片轻轻薄了成像时间点之间的任何可能重新种植骨。如有必要，牙钻薄。碎片的清晰成像窗口申请下降0.9％无菌生理盐水。 </li><li>携带动物双光子显微镜，并找到原始影像领域。使用援助的第一次成像会议期间采取的数码照片。 </li><li>打开Fluoview方案和开原的低倍率的图像。贴上投影胶片，在计算机屏幕上和草图的主要血管的模式使用的透明度钢笔。 </li><li>打开实时图像，并重新调整血液血管的透明胶片上勾画图案。 </li><li>为了重新映像结构中的8X放大，开辟从以前的成像会话中收集了所需的栈。画出结构（即轴突，树突，神经胶质）到一个透明的薄膜，贴在计算机屏幕上。 </li><li>变焦8X从现场影像和设置泛坐标，第一天的成像参数。根据调整低倍率的位置的准确性，它可能是必要的，还可以调整角度的图像略有。一旦找到所需的结构，您勾勒的高放大倍率的模式匹配结构。收集相同的步骤大小和Z - Stack的图像，供后续分析，Z - Stack的。 </li><li>重复步骤29和30，直到所有的高放大倍率的图像重新收集。 </li><li>当成像完成后，重复步骤22-24。可反复重新成像在2个独立的时间点。 </li><li>动物实验动植物公园和罗切斯特大学医学和牙科学院的实验室动物医学部规定的准则和法规的规定进行全面评审，由实验动物评估和认可协会，国际（AAALAC），并在遵守国家法律，联邦法律和美国国立卫生研究院政策。 </li></ol>代表性的成果 GFP - M的表达小鼠，第5层的锥体细胞和相应的树突状进程的一个子集可以很容易地可视化，使用双光子激光扫描显微镜（2PLSM）1。在这里，我们展示了一个薄的头骨的准备，其中超过所需的成像位置的头骨变薄〜10-30微米。正确执行，我们的协议使我们清楚看到皮层的树突和完整的视觉皮层的急性或慢性的分析（图2）刺。 <img src="/files/ftp_upload/2060/2060fig1.jpg" alt="图1" /> 图1。自定义头和底板 （一）定制的头和颅骨变薄，手术前准备用于底板的顶视图。 （二）定制的头和底座的侧视图。 （C，D）头板的详细原理和形象。 <img src="/files/ftp_upload/2060/2060fig2.jpg" alt="图2" /> <stro吴图2。代表结果从2PLSM以下薄头骨准备（一）树突体内成像在视觉皮层的转基因GFP - M的使用双光子显微镜的鼠标。 Image是一个单一的形象从PIA获得每5微米到175微米的最终深度的投影。在装箱的面积放大的区域，在（B）和（C）0（D0）和四个​​天后（D4），天成像。 （D，E）的放大倍率，树突分支，表现出稳定的刺（白色箭头头），在B和C盒装丢失/回缩棘（红色箭头头）和新棘（绿色箭头头）。比例尺= A 200微米，B，C 20微米，D，E 5微米。

<ol>
	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-51 (2000).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Majewska, A. K., Newton, J. R., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+synaptic+structure+in+sensory+cortical+areas+in+vivo.">Remodeling of synaptic structure in sensory cortical areas in vivo.</a> J Neurosci. 26 (11), 3021-3029 (2006).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat Protocols. 5 (2), 201-208 (2010).</li><li>Xu, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nat Neurosci. 10 (5), 549-551 (2007).</li></ol>

慢性成像，利用双光子显微镜技术正成为一个日益流行的技术，研究过程中的可塑性2-5引发的形态学变化。在这里，我们展示了一个薄的头骨的准备工作，按照不同的成像天在完整的小鼠大脑中确定的树突棘。在这个协议中，头骨是原封不动的，造成最小损害的皮质和neuroinflammation非常低的水平，这可能会改变大脑功能6。这使得动物成像后，立即在第一次手术，然后随后重新制作...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fentanyl Citrate</td>
<td>&nbsp;</td>
<td>In-house pharmacy</td>
<td>&nbsp;</td>
<td>Fentanyl Cocktail: Anesthesic Cocktail; 
IP Dose: 0.0125 ml/g
 Final cocktail conc.: 0.05 mg/kg</td>
</tr>
<tr>
<td>Medetomindine Hydrochloride</td>
<td>&nbsp;</td>
<td>In-house pharmacy</td>
<td>&nbsp;</td>
<td>Fentanyl Cocktail: Anesthesic Cocktail; 
IP Dose: 0.0125 ml/g
 Final cocktail conc.: 0.05 mg/kg</td>
</tr>
<tr>
<td>Midazolam HCL</td>
<td>&nbsp;</td>
<td>In-house pharmacy</td>
<td>&nbsp;</td>
<td>Fentanyl Cocktail: Anesthesic Cocktail; 
IP Dose: 0.0125 ml/g
 Final cocktail conc.: 0.05 mg/kg</td>
</tr>
<tr>
<td>2,2,2-tribromoethanol </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Avertin Cocktail: Anesthetic; 
IP Dose: 0.0075 cc/ g Final cocktail conc.: 1%
</td>
</tr>
<tr>
<td>2-methyl-2-butanol (Final concentration: 0.775%)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Avertin Cocktail: Anesthetic; 
IP Dose: 0.0075 cc/ g Final cocktail conc.: .775%
</td>
</tr>
<tr>
<td>TC-1000 Temp. Control System for Mice</td>
<td>&nbsp;</td>
<td>CWE, Inc.</td>
<td>TC-1000 Mouse</td>
<td>Body Temp. Regulation</td>
</tr>
<tr>
<td>Toberadex</td>
<td>&nbsp;</td>
<td>In-house pharmacy</td>
<td>&nbsp;</td>
<td>Eye Ointment</td>
</tr>
<tr>
<td>10% Ferric Chloride</td>
<td>&nbsp;</td>
<td>Ricca Chemical, Inc.</td>
<td>3120-32</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Microsurgical Blade</td>
<td>&nbsp;</td>
<td>Sable Industries</td>
<td>S-6400</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Cyanoacrylate 404,401</td>
<td>&nbsp;</td>
<td>Loctite</td>
<td>P/N 46551</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Cyanoacrylate 401</td>
<td>&nbsp;</td>
<td>Loctite</td>
<td>P/N 40140</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Zip Kicker Glue Accelerator</td>
<td>&nbsp;</td>
<td>Pacer Technology</td>
<td>PT-29</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Micro Drill Steel Burrs 0.7mm tip diameter</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>19008-07</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>Microtorque Control Box and Tech2000 Handpiece</td>
<td>&nbsp;</td>
<td>Ram Products, Inc.</td>
<td>TECH2000ON/OFF</td>
<td>Thin-skull preparation</td>
</tr>
<tr>
<td>#6-0 (0.7 metric ) silk suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>K8894H</td>
<td>Taper C-1</td>
</tr>
<tr>
<td>Eye Dressing Forceps, 10cm, tip width 0.5mm, curved</td>
<td>Surgical Tools</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm</td>
<td>Surgical Tools</td>
<td>Fine Science Tools</td>
<td>14084-08</td>
<td>&nbsp;</td>
<tr>
<td>Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm</td>
<td>Surgical Tools</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chronic imaging of mouse visual cortex using a thinned-skull preparation

在体内成像，利用双光子激光扫描显微镜（2PLSM）允许在完好的大脑活体细胞和神经元突起的研究。这里介绍的技术，允许在几个时间点与微观的决议，允许这是代表多数在中枢神经系统的兴奋性突触后网站的一小结构的树突棘的跟踪（慢性成像）的大脑同一区域的成像。能够清楚地解决以上几个时间点的优良的皮质结构有很多优点，特别是在大脑的可塑性，在突触和电路重塑的形态学变化可能有助于解释底层机制的研究。在这个视频和补充材料，我们将展示一个长期在体内的完整使用一个头骨变薄，准备的脑成像协议。薄头骨准备是一种微创的方法，从而避免了潜在损害的硬脑膜和/或皮质，从而减轻炎症反应的发生。当执行这个协议是正确的，它可以清楚地监测树突棘的特点，在经过一段时间的长期完整的大脑变化。

Watch this Scientific Journal Video about Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation at JoVE.com

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

我们在这个视频和补充材料，表现出对慢性协议<em&gt;在体内</em&gt;完好使用一个头骨变薄，准备的脑成像。

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 ...

chronic-imaging-mouse-visual-cortex-using-thinned-skull

Research

JoVE Journal

Neuroscience

13.7K 次观看.  University of Rochester. 我们在这个视频和补充材料，表现出对慢性协议在体内完好使用一个头骨变薄，准备的脑成像。

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

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 ...

科研

神经科学

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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 ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed as candidate sites for misrouting. Since a correlation between the number of lateral geniculate nucleus (LGN) relay neurons and LGN size has been shown, and based on previously reported reductions in LGN volumes in human albinism, we suggest that fiber projections from LGN to the primary visual cortex (V1) are also reduced. Studying structural differences in the visual system of albinism can improve the understanding of the mechanism of misrouting and subsequent clinical applications. Diffusion data and tractography are useful for mapping the OR (optic radiation). This manuscript describes two algorithms for OR reconstruction in order to compare brain connectivity in albinism and controls.An MRI scanner with a 32-channel head coil was used to acquire structural scans. A T1-weighted 3D-MPRAGE sequence with 1 mm3 isotropic voxel size was used to generate high-resolution images for V1 segmentation. Multiple proton density (PD) weighted images were acquired coronally for right and left LGN localization. Diffusion tensor imaging (DTI) scans were acquired with 64 diffusion directions. Both deterministic and probabilistic tracking methods were run and compared, with LGN as the seed mask and V1 as the target mask. Though DTI provides relatively poor spatial resolution, and accurate delineation of OR may be challenging due to its low fiber density, tractography has been shown to be advantageous both in research and clinically. Tract based spatial statistics (TBSS) revealed areas of significantly reduced white matter integrity within the OR in patients with albinism compared to controls. Pairwise comparisons revealed a significant reduction in LGN to V1 connectivity in albinism compared to controls. Comparing both tracking algorithms revealed common findings, strengthening the reliability of the technique.

This manuscript describes deterministic and probabilistic algorithms for white matter (WM) reconstruction, used to examine differences in optic radiation (OR) connectivity between albinism and controls. Although probabilistic tractography follows the true course of nerve fibers more closely, deterministic tractography was run to compare the reliability and reproducibility of both techniques.

在人类白化病的初级视途径弥散张量成像和测量束成像连通

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

大鼠慢性植入的外周神经刺激电极的制备

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...