这项工作是由国防部批准PT074620（SLJ）和美国国立卫生研究院资助R01NS245014（SLJ）的支持。

1。试剂准备<ol><li>解冻冷冻基质胶，湿冰实验前（不加快积极变暖的过程中）留下足够的时间。基质胶是用来建立一个3D的环境极为相似利于细胞外基质细胞迁移。 它提供了与基板和机械包裹的细胞 。 基质胶是储存在-20 ° C冷冻在使用之前，它应该是逐渐长大的温度0-4 ° C，即在湿冰或在冰箱解冻。基质胶不可逆转的设置，形成凝胶在孵化温度（37℃）。因此，这是不建议加快解冻浸泡在温水中的容器 。 </li><li>准备从产后当天0（P0），鼬的大脑皮层的总蛋白提取物等电聚焦（IEF）的蛋白质组分。分离后，分数是对20MM的Tris -盐酸PH7.2透析过夜清洁聚焦缓冲成分，这可能是有毒的组织中提取。 </li><li> 从大脑皮层中提取的蛋白质交付系统。蛋白质被吸附在荧光乳胶珠，然后慢慢释放的蛋白质environment.We发现，荧光功能有助于在微球的视觉处理，并允许从网站容易发现即使是最小的外溢珠存款时，荧光显微镜下观察。 <ol class="lalpha"><li>添加荧光珠加入10μl的蛋白质溶液100 -200μL </li><li>在室温下孵育一个摇床在黑暗30分钟</li><li>最大速度为1小时的台式离心机旋转珠</li><li>去除上清液</li><li>重新暂停使用新鲜培养液中加入10μl的旋涡</li><li>暂停添加基质胶30μl，拌匀，置于冰上。 </li></ol></li><li>缓冲器和媒体<ol><li>准备和过滤消毒人工脑脊液（ACSF 124毫米氯化钠，碳酸氢钠3 26MM的NaH 2 PO 4 1.2MM，3.2MM氯化钾，硫酸镁4 1.2MM， 氯化钙 2.4毫米，葡萄糖10MM）。搪塑冻结学联事前，所以准备在组织剥离时，为了使它充满无菌学联在-80 ° C冰箱，放置一个塑料瓶 。 监视器冻结每个所以很多时候，所以当了一层冰建立的内壁上，打破它，并与流体混合 。 重复，直到烧瓶中主要含有冰融雪。缓冲区准备这样不仅支持组织的生存，而且还将低温，比矮胖的冰块组织机械危害较小 。 </li><li>准备Neurobasal培养基与N - 2和B - 27补充剂补充说，根据制造商的建议，以及1mg/100ml庆大霉素，2毫米L -谷氨酰胺和0.6％葡萄糖。通过0.22μm的膜过滤消毒。注意保暖，在37℃，直到使用C。 </li></ol></li><li>培养板<ol class="lalpha"><li>由点睛每口井用少量冷基质胶使用20μL枪头（约2μL），在它的中心，并把每下降一个蒸压一轮18毫米玻璃盖玻片的文化品位塑料六孔板准备。无论是板块，也不是盖玻片需要，否则文化对待。 </li></ol></li></ol> 2。组织编制<ol><li>获得的脑组织<ol class="lalpha"><li>深深Euthasol动物麻醉后，取出大脑，并将其放置在冰冷的ACSF </li><li>单独用手术刀半球和切片冠在500微米，使用的首选设备，收集到一个小含有冰冷学联菜片</li></ol></li><li>植（使用解剖显微镜） <ol class="a"><li>识别片含有神经节金榜（GE）和皮质镶边地区可能会卷曲通过GE和干扰进一步的步骤</li><li>按哈里斯单核孔在该地区靠近心室表面的GE，主要是一个含有脑室区收回的核心，它驱逐到冰冷的介质（图1）柱塞三到四个0.5植毫米直径可以从GE在单P0深挖大脑切片。请注意，在老鼠和雪貂，融合在这个年龄段的内侧和外侧GES。如果一个核心的片或停留在被套牢的塑料盘底部，调动喷出学联与一个枪头，用移液器转移到包含冷Neurobasal培养基的菜 。 保持湿冰盘。 </li><li>从中长期转移一批外植体放置在一个单独的盘的大幅回落到一个冰冷的基质胶（约0.2​​50.5毫升），照顾有植完全沉浸和避免泡沫。保持湿冰盘。一个批次的大小取决于工作流程下游的速度。 </li></ol></li></ol> 3。文化设置（使用解剖显微镜） <ol><li>始终保持基质胶和含冰基质胶混合，以避免固化。 </li><li>混音珠悬挂和1 -2μL或珠悬浮在冰冷的基质胶在以前放置在培养板以及使用20μL吸管尖玻璃盖玻片中心的地方。让它一点点，避免干燥。 </li><li>使用20μL枪头拿起基质胶下降2植珠存款附近（至少1毫米远离存款边缘）两侧对称。它设置为不超过5分钟，在室温。 适当时机的步骤B和C是关键。如果时间太短，它会导致不完整的胶凝和文化安装的组件将移动时，他们的最后一层基质胶覆盖，如果时间过长，可能会导致过度干燥的凝胶和创造物理屏障，阻碍细胞的自由流动。 </li><li>与平层约30μl基质胶盖珠和外植体，超越植（图2）。让孵化器为约15分钟。 </li><li>添加补充Neurobasal中等2毫升。 </li><li>孵育37℃，5％的CO 2 / 95％的O 2每天监测候鸟活动。 </li></ol> 4。实时成像<ol><li> 3-4天的潜伏期后，审查培养皿，并选择感兴趣的盖玻片。外植体，应该产生的细胞呈放射状均匀的云。更换到一个新的培养板将用于实时成像选择盖玻片。 </li><li>让盖玻片在新盘在夜间坐在。放在显微镜成像板之前，在细胞培养罩，使用无菌压舌板移动盖玻片，以释放任何盖玻片和细胞培养板收集之间的微泡。气泡的干扰会毁了图像。将盖玻片以及中心。 </li><li>为了防止蒸发，填补与水井之间的空间。 </li><li>小心地转移板块从细胞培养罩显微镜不动以及中心的盖玻片。 </li><li>定义成像计划，利用图像处理软件。 由于文化设置的3D性质的细胞也有垂直移动，因此重要的是收集Z型飞机，包括全面深入的迁移云。 成像面积的XY程度上也应设置足够大的预测是在录制过程中，细胞迁移，人口的地区和含珠的存款，以记录外植体细胞迁移的角度方向和相对的蛋白质来源。还可以考虑更多的成像z平面，或更多的面积覆盖，它需要更多的时间记录在一特定时点上一个周期，迫使细胞追踪的时间分辨率的损失和样品之间的间隔较长时间。 在我们的例子中，使用了10倍的目标，最优方案是一个Z型飞机每20微米，间隔为30分钟的间隔，每组10只的3x3瓦的记录。 我们发现，30分钟的间隔时间最长的，允许在连续的时间点，这是可靠跟踪他们的行踪的关键细胞的明确识别。 </li><li>启动时间的推移成像。 </li><li>所需的时间后，取出板和固定在4％的磷酸盐缓冲多聚甲醛的文化。它可用于进一步的形态学及免疫细胞化学分析。 </li></ol> 5。运动分析<ol><li>转换成的格式进行进一步的处理最方便，TIFF格式最便携的图像记录的时间序列。 </li><li>跟踪单个细胞随着时间的推移，录音XY坐标的运动，这是可以做到像ImageJ（NIH）免费提供软件使用手动跟踪插件，如ImagePro，Volicity或AutoQuant或任何其他的商业应用。 </li><li>使用Excel或内部开发的软件绘制的轨道。 </li><li>每个跟踪的单元格计算动力学参数，如平均车速，路径的曲率，随着时间的推移定向变异，分支，走向的蛋白质来源偏差等计算，可以在MS Excel或任何同等的电子表格。 我们的经验表明，然而，这是得多生产力发展内部的脚本（使用R，MATLAB，使用Python，Perl或其他，大多是开源语言），因为他们比那些更灵活的个性化和更复杂的分析方法列入允许在安装的Excel的基本 。 </li></ol> 6。代表性的成果： <img src="/files/ftp_upload/2583/2583fig1.jpg" alt="图1"/> 图1。的解剖原稿图N外植体。这是一个绘制了一个hemisected揪出脑冠状切片。灰色圆圈表示的地区，采取从外植体。 <img src="/files/ftp_upload/2583/2583fig2.jpg" alt="图2"/> 图2图文化设置。珠存款的蛋白质来源;，外植体放置到任何一方的所有元素都与基质胶盖。 <img src="/files/ftp_upload/2583/2583fig3.jpg" alt="图3"/> 图3。通讯地块象限（箭头指向点珠存款）彩色细胞轨道。右边的单元格的曲目对分数D，这似乎吸引细胞迁移;左侧的细胞走向的一小部分，这似乎浅水湾细胞迁移的迁移。 <img src="/files/ftp_upload/2583/2583fig4.jpg" alt="图4"/> 图4。 A）系列呈现出越来越多的外植体迁移的细胞云从选定的时间点的图像。由箭头指示，例如细胞，是通过时间跟踪，如下所示（比例尺为200μm）。此外植体是38小时实时成像与可视化这里展示的是从开始录制的图像最终图像B）细胞中的一部分显示在次表示离开的外植体细胞的图像。该单元格显示重复的分支（比例尺为50μm）。

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动物使用：所有涉及动物的程序，按照USUHS和美国国立卫生研究院的体制指引USUHS IACUC批准。

在这里，我们提出了一个简单的系统研究和分析化学信号的神经细胞的迁徙行为。体外迁移实验已用于识别影响运动细胞的胞外分子。我们的范式，确保每一个人的细胞研究具有3维（3D）环境与互动。文化细胞外植体的迁移，这是一个相当大的优势，被迫D -聚赖氨酸包被的盖玻片平坦的表面上移动，从而泯灭的相互作用与细胞外基质（如希尔施贝格等人&#39;10 ; 8。倚天等。&#39;07）。然而，跟踪细胞迁移的规模较大，它仍然二维，细胞水平的自由相比，在底部的玻璃盖玻片和基质胶，这是比较薄的顶面之间的空间垂直约束迁移。这样的设置可以容易的运动分析，仍然基本上是2维。以前类似的3D范例组成的GE植或嵌入在胶原或基质胶GE细胞重新聚集，就业，利用细胞的聚集，表达一个特定的信号蛋白作为一种化学信号源（ 如Liodis等人&#39;07; Martini等人&#39;09。Nóbrega -佩雷拉等 &#39;08; Wichterle 等&#39;03）。坚持蛋白乳胶珠的形式，这里介绍的方法使用一个缓慢释放系统，以便在当地环境的持续的水平，这是比蛋白质直接添加到中期，使系统特别适合时可用金额蛋白质是有限的或复杂的蛋白质混合物进行测试时。珠的选择是有限的，以乳胶为基础的产品，包括其他材料，例如琼脂糖珠，各有不同的特点。为了进一步提高检测的密度，我们使用两个植apposed两侧的珠存款。要确保原产地的具体面积，我们作为一个细胞来源的神经节隆​​起植。我们评估了在内侧和外侧的神经节隆​​起部分的差异，但没有看到任何的差异。我们应该指出，这项研究使用的雪貂，在这个物种的内侧和外侧的神经节金榜保险丝比早些时候在啮齿类动物中（Poluch等&#39;08） 。在文化作了时间（P0）的内侧和外侧的神经节金榜融合，即使许多填充大脑皮层的细胞仍在生成和迁移。但是，如果真正的同质源是首选的细胞，细胞悬液，可以从解剖组织编制，并直接与基质胶混合，或者降速，并产生沉淀打了个“植”。组织消化酶的使用，准备细胞悬液气馁，甚至残留的蛋白水解酶活性，可以在实验过程中液化基质胶。此外，尽职调查已经被应用到文化设置的时间。虽然基质胶需要一段时间的升温，以聚合，甚至一个暴露的基质胶小滴（在本议定书中所用的）的空气会导致干燥和一个，是坚不可摧的细胞迁移和它可以不被擦除的shell的结果略有延长期随后用新鲜的基质胶覆盖。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</td><th>评论（可选） </th></tr><tr><td> Neurobasal培养基（1X），液体</td><td> Invitrogen公司</td><td> 21103-049 </td><td></td></tr><tr><td> N - 2个补充协议（100X），液体</td><td> Invitrogen公司</td><td> 17502-048 </td><td></td></tr><tr><td> B - 27补编（50X） </td><td> Invitrogen公司</td><td> 0080085 - SA </td><td></td></tr><tr><td>屋宇署基质胶基底膜矩阵</td><td> BD公司</td><td> 354234 </td><td></td></tr><tr><td>哈里斯单核，0.5尺寸</td><td>电子显微镜科学</td><td> 69036-05 </td><td>打孔孔</td></tr><tr><td>绿色Retrobeads IX </td><td> Lumafluor，公司</td><td></td><td>乳胶微球</td></tr></tbody></table>

migratory behavior of cells generated in ganglionic eminence cultures

细胞迁移是一个共同的过程，导致脊椎动物的中枢神经系统的发展和成熟（阿唐，&#39;99）。包括两个基本的神经类型：兴奋和抑制的大脑皮层。这些细胞出现在不同的地区，迁移到皮质，沿不同路线（ 珀尔曼等，&#39;98） 。抑制interneurons迁移切线方向从皮质来源，大多来自不同区域的神经节金榜（Gelman 等 ，&#39;09;徐等 ，&#39;04）。。他们的运动，需要精确的时空控制，对环境线索，以便为建立适当的细胞结构和大脑皮层连接（Caviness Rakic​​，&#39;78，阿唐，&#39;90; Rakic​​，&#39;90）。要学习的迁徙行为产生的细胞增殖区的神经节金榜公司（GE） 在体外新生雪貂，我们使用一个在BD基质胶基质的三维文化的安排。文化设置包括两个GE植和从大脑皮层中提取和定位之间的植（Hasling 等 ，&#39;03，谜语等 ，&#39;97）。荧光乳胶Retrobeads第九吸附测试蛋白质的来源。经过2-3天文化，细胞开始出现在边缘外植体，呈现出倾向留在径向方向的组织。实时成像允许迁徙模式的观察，没有标签或标记的细胞的必要性。当暴露等时深挖皮质中提取的蛋白质获得分数，GE细胞显示个别细胞的定量动力学分析判断，不同的行为。

Watch this Scientific Journal Video about Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures at JoVE.com

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

时间失效成像的三维组织培养研究，单个细胞对原产于大脑皮层反应蛋白提取物分割神经节隆起的迁徙行为。

在神经节尊贵的文化产生细胞的迁徙行为

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

migratory-behavior-of-cells-generated-in-ganglionic-eminence-cultures

Research

JoVE Journal

Neuroscience

9.9K 次观看.  Uniformed Services University. 时间失效成像的三维组织培养研究，单个细胞对原产于大脑皮层反应蛋白提取物分割神经节隆起的迁徙行为。

视频: Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

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

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

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

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

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

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

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好<em&gt;孔雀鱼latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

结合行为和脑电图研究正念冥想对情景记忆的影响

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...