作者要感谢Neurotar有限公司提供技术援助，CIMO基金会和FGSN提供财政支持。

涉及动物主题手续已批准的全国动物实验局，芬兰。 1，动物准备成像<ol><li>由intraperitional（IP）注射氯胺酮（每体重0.08毫克）和甲苯噻嗪（每体重0.01毫克），麻醉鼠标。检查与后脚趾捏反射麻醉。 </li><li>沉浸动物的眼睛滴眼液（，涉及的），以保护眼睛免受脱水。 </li><li>把鼠标放在一个加热垫（仕霸）在37℃，以防止体温过低。 </li><li>清洁指定用于成像，用70％乙醇的足垫。 </li><li>添加一滴水在脚下垫皮和玻璃盖之间的耦合浸泡的皮肤。 </li><li>把塑料包装材料在被定位在一个金属环与盖类的后肢。 </li><li>调整塑料包装材料的厚度，以弄平皮肤。 </li><li>把一滴水封面上的GLASS表示玻璃盖和物镜之间的浸没。 </li></ol> 2，金属环固定器的制备<ol><li>对于皮肤的稳定利用该金属固定器。我们使用社区设计保护两翼固定器与金属环（礼貌Neurotar有限公司，芬兰）。填充注射器强力胶，把小滴强力胶，通过针环上，轻轻地传播胶水环表面均匀覆盖。关键是要放的是只需要为均匀的覆盖胶的最小量，因为胶水过多量可以减少视图的字段和阻碍后续的成像。 注:在金属条（从下方）和标准显微镜玻片上（作为盖）的可替换的组合可以被用来压平的皮肤，并确保在组件14的适当的光学耦合。两个纸夹可用于固定玻璃和金属条表面之间的爪（ 图4 </s仲&gt;）。 </li><li>覆盖环显微镜盖玻片（直径5 mm，电子显微镜学）。 </li><li>拧上环固定器是拼接而成的电动显微镜阶段的刚性金属条。 </li></ol> 3，影像学检查<ol><li>在使用THY1-YFP-H的小鼠中选择的荧光灯的光谱的蓝色部分的可视化的神经纤维。查找在萤光模式感兴趣的神经和聚焦。 </li><li>写下纵向成像的点的坐标。使用腕垫作为参考点。在接下来的成像会议，第一次检测的腕垫，然后找到同样的上游的大神经纤维，回去使用保存的坐标。至关重要的是定位在同一方向上的脚垫垂直于金属棒保持的基准相同的系统。 </li><li>打开的双光子模式。为THY1-YFP-H老鼠，它适合于使用激光辐射的950纳米波长的可视化的神经纤维。 </li><li>选择激光波长和发射信道（从450至480纳米的二次谐波生成的检测，从520到550nm的YFP标记的神经成像和从580到630纳米的检测YFP的荧光和头发的自发荧光的），开始低激光功率（3-5％），以防止光损伤和漂白。在光电倍增管的典型电压为600-700 V对于这种成像。 </li><li>设置所需的分辨率（512×512或640×640为快速事件的测量，800×800或1024×1024的形态学成像），轴向步骤（1-3微米），将样品的厚度和时间间隔（1-10分）。曝光时间是在每1个像素的1毫秒的顺序。 </li><li>第一标记中的软件的成像体积的上边界和下边界。 </li><li>获取参考图像堆栈病变前的基准。当需要时，可以在几分钟的时间来记录的基线。 </li><li&gt;重复（至少每10-15分钟）检查动物的呼吸率和反射，在需要时应用麻醉剂的额外量。 </li><li>后成像清洁用纸巾垫，把鼠标在恢复中，在36°C，直到它完全清醒。 注意:通常情况下，一个成像会话结合激光诱导损伤的持续时间（见下文）不超过1小时。我们建议您确认该动物是深度麻醉每次10-15分钟;这不应该随着影像本身的干扰，但同时，实验步骤，计划应予以考虑。单氯胺酮/ xylazyne剂量效果的持续时间大约是30到40分钟，因此我们建议申请额外四分之一后25-30分钟½剂量。 注意:它应该被认为是很好的时候试验计划的成像会话的单个动物的频率不应该超过1次每2天，避免了重新不利影响petitive麻醉。 </li></ol> 4，激光损伤<ol><li>获取参考图像堆栈后，用漂白协议，使微小病变。 </li><li>增加激光功率为100％。 </li><li>增加曝光时间，以每感兴趣（100-1,000x超过了标准的情况下成像）区域100-1,000毫秒。 </li><li>概述了病变区域。 </li><li>通过在漂白切换使病变。 </li><li>切换回普通拍摄模式，并继续延时录像。 </li></ol> 5，数据处理和分析<ol><li>使用混合光谱中的插件ImageJ的8混溶继续（约阿希姆·沃尔特<a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html</a> ）。 </li><li>打开图像堆栈和分裂的通道。 </li><li>发现游离的神经或头发的背景测量的面积。把一个区域Ø˚F的兴趣在这一领域。 </li><li>小心地勾划出发，其中它是由神经清晰可辨的一部分。 </li><li>封装在所述图像的一部分，它是独立于头发的神经。 </li><li>保存解混矩阵。 </li><li>整个堆栈应用实测解混矩阵。 </li><li>保存为*。TIF格式，新的图像处理堆栈。 </li></ol>

<ol>
	<li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+epidermal+nerves+in+diabetic+neuropathy.">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li><li>Wu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morton's+interdigital+neuroma:+a+clinical+review+of+its+etiology,+treatment,+and+results.">Morton's interdigital neuroma: a clinical review of its etiology, treatment, and results.</a> J. Foot Ankle Surg. 35, 112-119 (1996).</li><li>Lauria, G., Lombardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy:+a+new+tool+for+diagnosing+peripheral+neuropathy.">Skin biopsy: a new tool for diagnosing peripheral neuropathy.</a> BMJ. 334, 1159-1162 (2007).</li><li>Cheng, C., Guo, G. F., Martinez, J. A., Singh, V., Zochodne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+plasticity+of+axons+within+a+cutaneous+milieu.">Dynamic plasticity of axons within a cutaneous milieu.</a> J. Neurosci. 30, 14735-14744 (2010).</li><li>Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Leukocyte+Homing+and+Migration+in+vivo.">Non-invasive Imaging of Leukocyte Homing and Migration in vivo.</a> J Vis Exp. (46), e2062 (2010).</li><li>Sacconi, L., O'Connor, R. P., Jasaitis, A., Masi, A., Buffelli, M., Pavone, F. 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作者宣称，他们没有竞争的经济利益。

在这个视频协议，我们证明了该方法的单神经末梢的非侵入性的纵向双光子成像。 皮肤神经支配的动力学在这些疾病如牛皮癣和外周神经病变2，并且在创伤性损伤9受到影响。双光子成像允许神经纤维结构中的胶原基质的详细分析。利用转基因小鼠的报道中，有助于避免与神经纤维的染色的问题。 THY1-YFP-H菌株似乎是形态学数据分析10足够坚固，而大鼠Thy1-mitoCFP小鼠可提供在神经纤维11线粒体动力学的功能性研究的机会。在YFP-16 / ICR线可用于观察迈斯纳机构12。的另一种方法可以是病毒注射到脊髓背根神经节的特定神经元群体的选择跟踪13。 但值得注意的是生产的胶原蛋白基质中选择性病变的强烈受阻相比于皮肤的上层。这就是为什么有时候曝光时间为神经纤维的夹层可以不同高达十倍，而荧光用于成像的强度不同，在10-20％的范围内。以保持恒定浸没耦合必须保持图象的质量好。 图像的分辨率通常依赖于收购所需的速度。典型的生产时间30 frames-栈，分辨率800×800像素的是1分钟，这样可以降低分辨率或成像区域的厚度检测的快速变化或增加罚款形态特征的研究解决。 足垫的固定应紧够不使图像的漂移，但同时它不应该影响血液循环的叔他的皮肤。对于该过程的优化，血管示踪剂注射剂（ 例如 ，TexasRed标记的70kDa的葡聚糖）是可能的，从而允许一个通过监测血流来调节塑料包装材料的压力。在该协议中所描述的制备的运动伪影是不可能的，因为该动物深度麻醉，爪本身被牢固地附连到固定组件，并且因为心跳和呼吸运动不直接传递到爪子。然而，轻微的运动是可能的特别是如果进行高倍率成像。在这种情况下，我们可以建议您使用设计的，以补偿运动伪影的ImageJ插件。 相同区域的重复成像可以通过使用这样的参考点作为腕垫14，或在注射情况下，在足垫注射点可以作为一个划时代6某些物质来实现。另一种可能是皮肤的纹身。

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

皮肤的神经末梢发生在不同的病理生理状态的动态变化。神经纤维可以通过变性和再生或重组过程中这种疾病的过程中如周围神经病变2莫顿神经瘤3。跌打损伤后，神经末梢动态皮肤的一个重要组成部分是受损区域的神经再支配。然而，对于神经末梢调查的通常的方法是在体外组织学切片，缺乏对正在进行的流程4的实时信息。用遗传编码的荧光标记物，所以能够掌握其神经末梢活体动物的皮肤，从而获得关于结构变化丰富和显著更多相关信息。皮神经末梢的调查能够使用常规荧光显微镜，然而，皮肤组织的强烈的光散射强烈破坏了质量数据的获取5。多光子显微镜能够取得在强烈散射组织中的高清晰度图像，由于只从物镜的焦点导致荧光发射的激发光的光子能量的非线性总和。这个效果导致了强劲增长的穿透深度和改善信号对噪声比的测量在皮肤组织6。使用相同的激光作为成像，所以能够产生神经纤维7的选择性清扫。在下面的协议，我们将展示在体内的皮肤神经末梢纵向成像的记者转基因小鼠结合使用市售的多光子显微镜系统选择性激光损伤的方法。

<table border="0" cellpadding="0" cellspacing="0" width="1231">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

在皮肤神经末梢参与生理过程，如感测1以及在病理过程，如神经性疼痛2。他们贴近地表的定位有利于皮肤神经末梢的生活完整的动物显微成像。使用多光子显微术，它能够获得精细图像克服的皮肤组织的强烈的光散射问题。表达的THY-1启动子在神经元（包括周感觉神经元）的控制下EYFP记者转基因小鼠是适合于个别的神经末梢在延长的时间周期的纵向研究长达数月甚至终生。此外，使用相同的飞秒激光作为用于成像，所以能够产生神经纤维的高选择性病变的神经纤维重组的研究。在这里，我们提出了一个简单而可靠的协议进行纵向多光子活体成像和基于激光显微对小鼠皮肤的神经末梢。

使用所描述的技术也能够掌握其损伤后在同一光纤，并研究了受损的神经末梢（ 图1）的降解。采集的堆栈120-150微米的厚度通常是正确的重复成像期间数天，以保持在整个纤维中的视场。 病灶通常可以简明地产生时的塑料包装材料被调整到弄平皮肤，使胶原蛋白层出现在图像上均匀的强度（ 图2）。神经末梢的胶原层之上是，以产生精确的病变最合适的。 如果皮肤不平坦的，可能会出现图像模糊（ 图3）。在这种情况下，强度仍然可以是适合于在神经纤维的形态学研究，但会在激光损伤过程<html>受到阻碍。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig1highres.jpg" src="/files/ftp_upload/51045/51045fig1.jpg" /> 的激光损伤的神经纤维表现为最大的投影图像的效果如图1时间轨道，上部图片显示纤维和病变后直接，下图显示，30分钟后和10天（左和同一纤维前右侧面板，相应的）。有非神经性的出现损伤后10天有YFP的表达结构。这些结构很可能是已知能表达大鼠Thy1基因瞬时创伤的条件下的角质形成细胞。神经纤维的YFP的荧光显示为黄色。箭头标记病灶的区域。比例尺为30μm。 <html> /ftp_upload/51045/51045fig2highres.jpg"SRC ="/文件/ ftp_upload / 51045 / 51045fig2.jpg"/&gt; 为在平坦的皮肤区域神经末梢图2最大的投影图像。神经纤维的YFP的荧光显示为黄色，在二次谐波生成的胶原蛋白显示为蓝色。比例尺为50μm。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig3highres.jpg" src="/files/ftp_upload/51045/51045fig3.jpg" /> 图3中所获得的最大z轴投影为多个的光学部分的皮肤的弯曲区域中的神经末梢。神经纤维和毛发的自体荧光的YFP的荧光显示为黄色，在二次谐波生成的胶原的示蓝色。比例尺为50μm。 ftp_upload / 51045 / 51045fig4highres.jpg"SRC ="/文件/ ftp_upload / 51045 / 51045fig4.jpg"/&gt; 图4:采用显微镜载玻片为掩护的爪子固定的程序。滑动连接到使用标准的回形针的金属条。

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

该协议的动态纵向成像和神经末梢记者在转基因小鼠中的选择性激光损伤呈现。

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

in-vivo-two-photon-microscopy-of-single-nerve-endings-in-skin

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

10.7K 次观看.  University of Helsinki. 该协议的动态纵向成像和神经末梢记者在转基因小鼠中的选择性激光损伤呈现。 

视频: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

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 ...

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 ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...