这项工作是支持R01 - HL87290补助，R01 - HL87290 - S1和美国国立卫生研究院R01 - AI082610 LJMC。

1。开颅手术需要提前1如前所述，除了一个钛棒是不是动物的头放在一个开颅手术在8到10周大的老鼠。慢性颅窗口是一个稳定的准备允许甚至几个月后，被植入的软脑膜微循环检查。通常情况下，我们执行2-3周后颅窗口植入我们的研究。 2。活体显微镜<ol><li>开颅手术后两三个星期，体温检查，然后鼠标轻轻麻醉与异氟醚（归纳为4％，维持1-2％）。浅麻醉是用来防止动物在实验过程中的压力和不适。 </li><li>动物俯卧位，然后放置在一个立体的框架，并仔细担保使用耳酒吧头。使用左，右杠杆水平调整。使用电热垫保持核心体温。由于小鼠脑型疟疾的发展低温，温度设定需要调整每个动物。 </li><li>颅窗口盖玻片，轻轻地用棉签蘸用矿物油和立体显微镜和一台数码相机（尼康Coolpix 995，日本）的帮助下采取的一个窗口下的船只的全景照片，清洗。 ，图像打印，确定并注明日期，并将于血管直径和红细胞（RBC）的速度测量的地图。 </li><li>鼠标，然后转移到一个活体显微镜阶段（定制徕卡麦克贝恩，加利福尼亚州圣迭戈）。一滴水是利用牙科丙烯酸形成颅窗口，重点是调整。测量是使用20X（LUMPFL“世界投资报告”，数值孔径0.6，奥林巴斯）水浸泡的目的。遵循相同的船只，这样可以允许小样本人群更强大的统计，其基准水平的直接比较。用一个数字低光高速摄像机（滨松C9300 - 221，日本），或低光模拟摄像机（4815 COHU，圣迭戈，CA）连接到录像机的磁带（JVC，日本），时间戳记（显微视频拍摄的图像系统，Boyertown，PA）和彩色显示器（派尔高，克洛维斯，CA）。磁带录制开始前，正确地识别和时间戳允许记载了这样的事件，在此期间，被重新编码的时间内查明事件。 </li><li>的船只进行检查以评估该制剂的质量，如果血液中流动的所有船只。然后，选择要测量的船只，它应该包括不同直径的静脉和动脉（在我们的小鼠软脑膜的准备，大多数船只20至80μm的范围内），并覆盖窗口暴露的面积内的不同地点。动脉和静脉，可以很容易地通过检查在该船只的后果的血流量（是否分发或收集血液）的方向分化。在我们的研究中，测量在12艘。每次测量现场的确切位置是在软脑膜血管的图片注释。 </li><li>对于每一个部位，血管直径测量使用图像剪切设备（图像剪切，Vista的电子，圣迭戈，CA）2。一旦当场被选中时，该船只的形象是一致的垂直位置和图像剪切，直到反对极端对齐和阅读记录。这个值可以被翻译成微米，由先前的校准为每个特定的使用光罩校正阶段微米（埃德蒙光学公司，巴林顿，新泽西州）的放大倍率。 </li><li>每一个点是在至少30秒红细胞跟踪记录。的血液样本进行跟踪，收集，和红细胞荧光标记的carbocyanine染料DIL（Molecular Probes公司，卡尔斯巴德，CA），并通过尾静脉注入，以获得一个在体内的浓度约为 0.4-0.5％ 3。在与疟原虫 ANKA（PBA）表达绿色荧光蛋白（捐赠的疟疾研究和参考试剂资源中心，弗吉尼亚州马纳萨斯MR4，PBA - GFP的;由CJ Janse和AP水域沉积）感染的动物，没有输液的标记细胞是必要的。在每秒150帧的视频图像记录。这个比率是获得一至六个月的细胞图像上一个视频帧的速度决心，至8毫米/秒视频图像的数字化与个人使用的Adobe总理4.0软件的计算机，每个细胞图像的XY坐标数据得到使用SigmaScan Pro 4.0的软件（SPSS芝加哥，IL） 4。细胞职位手动决心，而不是通过图像分析，一个受过训练的观察者的眼睛被证明是一个一个细胞的中心，位置好估计一般h对应的大多数细胞方向观察到的最大荧光的位置。多个位置和速度的确定是为每个单元，平均每场获得平均红细胞速度。 </li><li>当红细胞流速测量离线执行，每个鼠标的总观测时间不超过10-15分钟，尽量减少麻醉的cardiodepressive效果。 </li><li>一旦血管直径和RBC的速度测量，计算在每艘船舶的血流量，可以使用公式：Q = V Xπ（D / 2）2，其中Q =血流量，V =红细胞速度和D =血管直径。 </li><li>随着时间的推移，这些程序可以重复。例如，在脑型疟疾的小鼠模型，我们进行感染的第4天开始每天测量。获得每天为每个鼠标的值是0天（前感染），这被认为是100％归。 </li><li>除了血管直径和血流测量，可以进行评估，例如改进的灌注和软脑膜血管中的白细胞和/或血小板滚动和坚持的分析评估。这些测量与荧光标记的标记帮助。疟疾的情况下，我们还可以通过使用PBA菌株，表达GFP的检测循环或附着寄生虫。 </li><li>对于灌注的评估，我们使用的解决方案FITC -标记的白蛋白（Sigma公司，圣路易斯，密苏里州50μg/mouse），量化白细胞粘附我们使用对泛白细胞标记与德克萨斯红（TXR）标记CD45抗体的软脑膜血管（ Invitrogen公司，卡尔斯巴德，CA4μg/mouse）。对于这一点，白蛋白FITC（2mg/mL）和抗CD45 - PE抗体20μL（200μg/mL）混合，并在预热尾静脉注射25μL。鼠标就可以立即成像，但是，如果泄漏测量白蛋白FITC标记允许分发了15分钟。使用阿尔法生动：XF100 - 2（欧米茄光，伯瑞特波罗（Brattleboro），VT）和抗CD45 - TXR荧光（615nm）退出和白蛋白- FITC和GFP（PBA - GFP PRBC）发出绿色荧光（518nm）被捕获捕获一个生动的标准：XF42。 </li><li>荧光标记白蛋白允许改进的血管网络，包括穿透血管的可视化，是有用的，特别是在疾病状态，如脑型疟疾检查非灌注或灌注下的船只。它也可以用来测量血管渗漏5。 </li><li>荧光标记的抗CD45抗体可以很容易识别和量化白细胞滚动和软脑膜血管粘连。为了避免在量化的偏见，量化滚动和粘附在预先定义的测量血流量的相同点。白细胞粘附的量化是由白细胞计数100μm的血管长度。滚动量化计算的行驶速度明显高于在同一100μm的长度在30秒，血流速度慢的白细胞数量。 </li></ol> 3。代表性的成果<img src="/files/ftp_upload/2184/2184fig1.jpg" alt="图1" /> 图1。 （步骤2.6）鼠标软脑膜血管网络，通过颅窗口访问的图片。 <img src="/files/ftp_upload/2184/2184fig2.jpg" alt="图2" /> 图2。 （步骤3.1-3.5）示意图显示设置小鼠软脑膜微循环活体显微镜。 1：活体显微镜; 2：20X水浸泡的目标; 3：光源; 4A：数字低光高速摄影机; 4B：模拟摄像机; 5：鼠标在立体定位框架; 6：电脑显示器; 7：模拟采煤机监测显示，图像剪切（箭头）如何做是为了测量血管直径。 <img src="/files/ftp_upload/2184/2184fig3.jpg" alt="图3" /> 图3。 （步骤3.6-3.8）微血管的红血细胞的细胞追踪的速度测量，从高速荧光录像。图片一到F是一个软脑膜血管的序列图像，呈现出一个单一的移动红细胞过路相机微观领域的分隔。手动确定由15个或更多的细胞，过路的领域，其预校准的距离帧帧的立场，允许在每艘船舶平均红细胞速度计算与Excel电子表格的帮助。 <img src="/files/ftp_upload/2184/2184fig4.jpg" alt="图4" /> 图4。 （步骤3.9）一个具有代表性的软脑膜血流量的变化显示在疟原虫 ANKA（PBA）（N = 5）感染小鼠的时间和感染的对照组小鼠在实验数据（N = 5） 。而在对照组小鼠软脑膜血流量相对稳定，随着时间的推移，PBA感染小鼠的血流量明显减少在脑型疟疾的发展（6天）的时间。数据的平均值± SEM。 <img src="/files/ftp_upload/2184/2184fig5.jpg" alt="图5" /> 图5。 （步骤4.3），抗CD45 -得克萨斯州的红色荧光抗体染色显示，大量白细胞粘附感染伯氏疟原虫ANKA鼠标的软脑膜血管。

<ol>
	<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. (15), (2008).</li><li>Intaglietta, M., Tompkins, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+measurements+by+video+image+shearing+and+splitting.">Microvascular measurements by video image shearing and splitting.</a> Microvasc. Res. 5, 309-312 (1973).</li><li>Briceno, J. C., Cabrales, P., Tsai, A. G., Intaglietta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+displacement+of+red+blood+cells+during+hemodilution+and+the+effect+on+arteriolar+oxygen+profile.">Radial displacement of red blood cells during hemodilution and the effect on arteriolar oxygen profile.</a> Am. J. Physiol. Heart Circ. Physiol. 286, 1223-1228 (2004).</li><li>Bishop, J. J., Nance, P. R., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+erythrocyte+aggregate+size+and+flow+rate+in+skeletal+muscle+venules.">Relationship between erythrocyte aggregate size and flow rate in skeletal muscle venules.</a> Am. J. Physiol. Heart Circ. Physiol. 286, H113-120 (2004).</li><li>Baldwin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+hemoglobins+produce+venular+interendothelial+gaps+and+albumin+leakage+in+the+rat+mesentery.">Modified hemoglobins produce venular interendothelial gaps and albumin leakage in the rat mesentery.</a> Am. J. Physiol. Heart Circ. Physiol. 277, H650-H659 (1999).</li><li>Cabrales, P., Zanini, G. M., Meays, D., Frangos, J. A., Carvalho, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cerebral+malaria+is+associated+with+a+vasospasm-like+microcirculatory+dysfunction,+and+survival+upon+rescue+treatment+is+markedly+increased+by+nimodipine.">Murine cerebral malaria is associated with a vasospasm-like microcirculatory dysfunction, and survival upon rescue treatment is markedly increased by nimodipine.</a> Am. J. Pathol. 176 (3), 1306-1315 (2010).</li></ol>

这里描述的活体显微镜方法，提供了一个在小鼠软脑膜微循环的详细观察独特而强大的工具。它允许挑出个别动脉和静脉和测量的一些参数如血管直径，红细胞速度，血流量，坚持和白细胞，血小板和其他血液成分，血管渗漏，组织pH值及PO2滚动的变化和潜在的许多其他应用。 在体内的血管反应，可及时进行评估后，如药品监督管理局的干预，或在病理过程。此外，微循环的行为，可以动?...

<!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>Isoflurane</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>FDG9623</td>
<td>&nbsp;</td>
<tr>
<td>Carbocyanine dye Dil </td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>D306</td>
<td>&nbsp;</td>
<tr>
<td>Albumin-FITC</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A9771</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD45-TxR Ab</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>MCD4517</td>
<td>&nbsp;</td>
<tr>
<td>P. berghei ANKA-GFP</td>
<td>&nbsp;</td>
<td>MR4</td>
<td>MRA-865</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

intravital microscopy of the mouse brain microcirculation using a closed cranial window

这一实验模型的目的是评估在急性和慢性，生理和病理的血 ​​流动力学，炎症和代谢的条件下小鼠软脑膜微循环 ，在体内荧光显微镜使用。左parieto枕的老鼠的大脑皮层放置在一个封闭的颅窗口。通过使用EPI和荧光照明，测量血管直径和红细胞（RBC）的执行速度的窗口局部微循环是实时记录。使用实时交叉相关性和/或荧光标记的红细胞，红细胞流速测量。白细胞和血小板粘附到软脑膜血管灌注和血管渗漏的评估与荧光标记的标记，如白蛋白- FITC和抗CD45 - TxR还抗体帮助。可以反复录像在几天微。我们用于第一次关闭窗口脑活体显微镜研究软脑膜微循环的动态变化，并跟随在安卡疟原虫感染小鼠当然，显示CM的表达与微循环功能障碍的特点是由血管收缩，减少血液中的深刻流，并最终血管崩溃。

Watch this Scientific Journal Video about Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window at JoVE.com

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

活体显微镜，按照时间和空间在软脑膜微循环血流动力学和炎症事件。

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

intravital-microscopy-mouse-brain-microcirculation-using-closed

Research

JoVE Journal

Neuroscience

17.2K 次观看.  University of California, San Diego. 活体显微镜，按照时间和空间在软脑膜微循环血流动力学和炎症事件。

视频: Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

研究脑机接口的闭环性能的实验平台

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

科研

神经科学

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

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

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

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

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

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

灭绝培训在彭兰霞窗口无法还原的恐惧

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated thinning of the skull. Cranial window surgery is typically performed with a high speed rotary drill, and many investigators find it challenging to prevent the drill from damaging the delicate dura and blood vessels. Extensive training and practice is required to remove the bone without damage to underlying tissue and thus cranial window surgery can be difficult, time consuming, and produce tissue damage. Piezoelectric surgery, which is extensively used for maxillofacial and dental surgery, utilizes ultrasonic vibrations to remove bone without damaging soft tissues. We have developed a method applying piezoelectric surgery to improve cranial window surgery in mice in preparation for multiphoton imaging. Comparisons within our lab find that the method requires less surgery time and has a lower average rate of complications due to dural bleeding than cranial window surgery with a rotary drill.

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Piezoelectric surgery has led to improvements in human maxillofacial and dental surgery. We have developed a protocol to optimize piezoelectric surgery for cranial window surgery in mice.

压电高精度手术骨切除 (相量): 改进小鼠颅骨窗手术的技术

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...