我们感谢他们的支持以下内容：英国联邦奖学金委员会资助（惠普），EPSRC的（惠普），BBSRC的（最惠国待遇）和欧盟的玛丽·居里行动（最惠国）。

1。矢状切片制备 1.1解剖大脑半球所有的动物实验应遵循当地的伦理审查和国家法规。在这里描述实验的情况下，这项工作符合英国的动物（科学规程）行动1986年。我们经常使用颈椎脱位，没有麻醉安乐死之前消除大脑的老鼠。或者鼠标可以末期麻醉，但在这种情况下，它可能是必要的，以确定麻醉的选择，影响神经的属性。 从鼠标的大脑，并立即在寒冷的地方（4-8°）切割人工脑脊液（学联）（解决方案的组成见表1）冒泡carbogen（95％O2，5％CO 2）饱和。 最多3分钟后，小心地取出大脑从Cutting学联用压舌板轻轻地放置在直立位置（背侧朝上）在滤纸上，已蘸切割学联（ 图1A）。 为了便于准确安装在半球，用剃刀或解剖刀删除，而不影响迈克（位于大脑的尾极端）为的小脑尽可能多和冠状面切片，消除大脑延髓第三（ 图1B）。 Hemisect的大脑，照顾节完全沿中线（ 图1C）的垂直平面。 返回半球冒泡的切割学联为一分半钟。 1.2安装在vibratome半球在安装之前，确保在20度从横向的角度，的vibratome刀片的尖端（ <s的阮富仲&gt;图1D）。在安装面上，以vibratome刀片浅强力胶平行条状，约一个半球的宽度，长到足以容纳两个半球月底至年底。 照顾，以尽量减少对身体的影响，用锅铲和位置的切割学联从每个半球，其内侧，使依赖于铲，背程度面临着对切片机的刀片。轻轻地滑入强力胶带，每个半球，照顾，以确保每个半球的内侧表面是平行的切片基地。我们发现，每个半球的背表面，以取得最佳效果应该是平行，和面临的vibratome的刀片（ 图1D）。 1.3切片的准备过程中的维护下列安装立即被淹没在冷切割学联（4 - 8℃）的半球。保持的。温度e和carbogen整个切片过程中的饱和度。如果直接冷却和冒泡的切割室的解决方案是不实际的，在香港总商会定期补充新鲜冷冻和冒泡的解决方案的切割学联解决方案。 1.4切段使用一个vibratome，从矢状面两半球皮质，直到你确定（通常从侧面〜1mm的下）（ 图1F）最外侧的MEC的程度。 之间削减抬起〜的vibratome的刀片200微米，以避免拖动刀背和损坏裸露的组织。从两个半球同时减少MEC的内侧程度，直到达到400微米的矢状部分（如果他们排成如图1D他们都将在同一时间被削减）。这可确定由厚厚的白色带的情况下，周围的T腹，尾的曲线他海马（外部胶囊），其喙边界的非凸形状的角尾后背成“角”。 图1E说明海马在矢状面的圆形外观，MEC的外侧。 图1F-G的说明如何在MEC的部分出现在不同侧的内侧位置时，切片。注意逐步海马豆状出现在更内侧的位置。每个半球通常会产生两个或三个400微米厚的片MEC的。 1.5孵育片的每个切割后立即放置在的carbogen饱和标准学联水浴保持在35°C片让片学联约15分钟后在35°C孵育切片是完整的。 从水浴中取出切片持有人，并继续在室温下的carbogen为在l起泡向东45分钟。 2。例如矢状窦切片实验使用该制剂是一个典型的实验，使MEC的第二层星状细胞电生理记录。 2.1优化光学在录制之前，确保冷凝器是在切片平面（克勒光照）的重点和中心下的高倍率（例如40倍）客观。 2.2细胞识别的利益使用低倍率（如4X）在MEC的（ 图2A-B的目标识别）近似录音区域。切换到高放大倍率的目标，以确定这一区域内的活细胞（ 图2C-V）。 作为一个例子，假定II层星状细胞在视觉上类似的直径确定他们的多角形或卵圆形的形状和多个主树突之三，并没有一个单一的大直径顶树突2,13,14。他们是可靠的I / II层的边界，边缘上确定，他们是丰富，往往出现在2,9小组（ 图2C-V）。也应该是可识别的interneurons和锥体细胞，其他类型的细胞，如。 可以在这一点上进行实验，例如采用全细胞膜片钳记录膜电位或确定神经元膜电流，电气或optogenetic方法激活记录神经元的突触输入。可以验证记录神经元的电生理特性及在细胞内的解决方案，包括荧光标记的神经元的身份。 3。测量沿背腹轴的位置 3.1感兴趣的图片的位置和周边片到确定一个recor的位置DED沿背腹轴的神经元，先用低倍物镜形象片及周边地区的MEC的地区。标记所感兴趣的位置，例如，包括图像（ 图3A）或下台领域虹膜隔离开利息重复图像的位置，周围明亮的圆的记录电极。然后可以重复记录的位置和它的周围（ 图3B）的初始图像叠加在图像精确定位记录的位置。多达3个独立的低倍率（4X）的图像可能会被要求支付MEC的背边境从腹侧记录的位置，面积。这些图像，然后可以使用图像处理软件，提供了一个可以从距离测量（ 图3）的图像拼接在一起。神经元的身份和位置的进一步核实后可以进行录音，包括生物胞素或Alexa染料在细胞内的解决方案，然后使用适当的组织处理，录音2，如无效的标记。 3.2建立背边境的MEC MEC的背边界提​​供了一个方便的地标来衡量背腹位置。 MEC的腹侧边境是没有得到很好的界定。 图4说明了如何在尼氏染色片的定义在不同的内侧，外侧位置MEC的细胞标志出现DIC的光照下。 图4显示了圆形的海马（ 图4（I））和缺乏一个成层（ 图4（四））与矢状切片含横向嗅皮层（LEC）parasubicular突。 图4公元前显示包含典型的ME片C.突出暗背嗅/ Parasubicular在DIC的照片边界地区的背部分包含地区的parasubiculum。 Nissl染色在列（三）相应的图像清楚地表明parasubiculum面积。 MEC的（4B和4C列（四）黑色箭头）背边境腹侧，远成层（4B和4C列（四）红色箭头）突出parasubicular细胞组。 比较图4B和4C列（ii）及（三）表明，在尼氏节迈克背边界对应的位置是在DIC片背边缘的黑暗嗅/ Parasubicular边界的地区腹侧（黑色箭头）。可以预计从DIC的图像和比较参考图像边界的位置（见文献14）。未来验证的背与分子标记MEC的边界，将改善这一估计的准确性。我们注意到THAţ染色参考部分，形态识别标记的神经元，处理的部分，可能会受到相当大的收缩。在DIC和参考部分的地标，相对的绝对距离的比较，因此首先需要收缩的测量和校正（如文献2）。 3.3校准和测量距离为了方便容易测量图像的距离，使用相同的低倍率目标图像参考网格建立一个像素的距离转化率。测量像素的距离，从背MEC的边境利益沿轮廓使用图形程序迈克的位置和转换像素微米的距离（ 图5A）的距离。如果充满如生物胞素标记神经元，然后就可以记录神经元的位置也可以通过适当的处理回收（见如文献2）。 4。再版esentative结果 图5A显示了一个例子低倍率（4X）录制后矢状切片，记录神经元和测量指南叠加的位置，复合图像。从显着的背，腹星状细胞的录音显示在图5B。这些记录有助于建立细胞的身份，并说明如何MEC II层星状细胞的电性能，在背部和腹部的位置不同。 <img alt="图1" src="/files/ftp_upload/3802/3802fig1.jpg" /> 图1。矢状窦片的制备。一个全脑背面临向上侧滤纸上休息蘸切割学联，B </s阮富仲&gt;脑额叶皮质，C用于安装半横断脑小脑和延髓第三取出后，D安装胶条半球平行切割边缘vibratome刀片淹没前切割学联和切片，E含LEC的部分外观“横向”的一部分，MEC的去除外侧组织切片后的程序，尚未得到充分的标准矢状迈克片的内侧，F取出后进一步组织400微米（表面上是400微米，内侧所示的外观（F））G的外观“内侧”400微米的矢状切片后MEC的一部分已被切断您好示意图EF的解剖标志，C：分别表示。小脑，L：横向嗅皮层（LEC），M：内侧嗅皮层（MEC）H：海马，橙色：外部胶囊，蓝色：胼胝体，青色：齿状回GR的EEN：CA3区和CA1区。片包含LEC的海马延髓边界的外观是凹（H 中黑色箭头），而在片只包含迈克的边界可以是线性的（红色箭头）或凹（红色箭头） 在 J。彩色解剖标志近似的形状，在艾伦脑图谱（注释<a href="http://mouse.brain-map.org/atlas/ARA/Sagittal/browser.html">http://mouse.brain-map.org/atlas/ARA/Sagittal/browser.html</a> ）。 <img alt="图2" src="/files/ftp_upload/3802/3802fig2.jpg" /> 图2。 DIC的光照下识别MEC II层细胞。例如复合图像一个准<html>矢状脑切片在低倍率（4倍）。单独的图像已对齐和混合删除分心的边缘和暗角。重要的具有里程碑意义的地区H：海马，M：。迈克B从一个单独的低倍率（4X）图像包含MEC的，右手边缘附近的暗竖条纹可见ÇMEC II层第二层在高倍作物（与DIC照明的40倍）。形象是几个图像对齐和混合的复合材料。密集的中央“一栏中的”细胞层II D单高倍率DIC的形象，呈现出健康假定星状细胞和第二层的interneurons。如下所示，星状细胞经常在组I / II层的边界上发生。锥体细胞往往可以找到更接近第二层/第三边境。交流虚线轮廓显示BD的程度分别。比例尺：在A和B 500微米，C和D 100微米。 图3“SRC =”/ files/ftp_upload/3802/3802fig3.jpg“/&gt; 图3。测量感兴趣的地点背腹的位置。一个不结盟的低倍率（4X）的图像，包括整个背腹之间的利益的位置（标记记录电极尖端）和黑暗的嗅/ Parasubicular边界的地区，具有里程碑意义的（用于建立背边境的MEC的程度迈克-见图4）乙在A，但与场可变光圈（降低不透明覆盖），而不是记录电极标记感兴趣的位置停了下来，包括叠加图像。 <img alt="图4" src="/files/ftp_upload/3802/3802fig4.jpg" /> 图4。估算DIC的矢状切片图像MEC的背边境。交流矢状窦旁路段外侧向内侧嗅皮层（）。 DIC和尼氏的部分是从不同的麦克风E 交流栏（i）：全矢状片（400微米厚）（低倍率（4X）图像对齐和混合材料）AC列（二）：特写与特征黑暗嗅/ Parasubicular边界的地区内嗅皮质每个图像的顶部区域。（三）从不同的鼠标AC列尼氏染色切片（40微米厚）与交流（二）图像对齐。较深，细胞致密层是第二层。比较列（ii）及（三）尼氏染色细胞出现在DIC图像显示了如何交流专栏（四）：尼氏染色切片的细节。 B和C列（四）包括从parasubiculum（黑暗的嗅/ Parasubicular边界的地区背在DIC图像的一部分）和从背迈克细胞的细胞。 （四）在B和C（四）parasubicular细胞扩展到层深大背补丁，我是很容易可见光（红色箭头）。这些补丁的腹侧边缘对应于背博MEC的rder（黑色箭头）。 （四）parasubicular补丁缺席，表明是太为标准矢状迈克的片制备的横向切片。在所有面板，黑色箭头表示的MEC背边境。灰色箭头表示MEC的近似腹边境。 <img alt="图5" src="/files/ftp_upload/3802/3802fig5.jpg" /> 图5。具有代表性的结果。一个图像混合和裁剪图3中的一部分， 一个一个背细胞和腹侧细胞的立场是分别由蓝色和绿色实心圆圈表示。黑色箭头标志着估计MEC的背边境和白色虚线延长到深层。白色实线显示MEC的背边境距离，位于腹侧的细胞像素测量的轮廓路径，沿着它是一个指导。比例尺： 乙电500微米。从细胞的生理痕迹表明在A，从左至右 - 阈下的反应，目前用来计算输入电阻的步骤，扣球一个大的积极的一步，穗细节。

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L., Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+cortex+of+the+rat:+organization+of+intrinsic+connections.">Entorhinal cortex of the rat: organization of intrinsic connections.</a> The Journal of Comparative Neurology. 398, 49-82 (1998).</li></ol>

没有透露。

为了方便调查MEC的电路特性，遵循背腹的组织，我们已经描述了在这里详细为生产矢状切片准备保留MEC的背腹的程度的过程。 关键步骤 卸下从动物大脑 。特别小心，以避免对大脑施加压力。这是比迅速清除大脑更重要。 切片。切片应牢固地粘在地板控股室。在vibratome应该有Z轴振动&lt;2微米，并应与高振幅和?...

<table border="1"><tbody><tr><td></td><td> 切割学联（毫米） </td><td> 标准学联（MM） </td><td> 内部解决方案（MM） </td><td> CASNumber </td><td> 供应商目录编号 </td></tr><tr><td>氯化钠</td><td> 86 </td><td> 124 </td><td></td><td> 7647-14-5 </td><td>西格玛S9888 </td></tr><tr><td>的NaH 2 PO 4计 </td><td> 1.2 </td><td> 1.2 </td><td></td><td> 13472-35-0 </td><td>西格玛71505 </td></tr><tr><td>氯化钾</td><td> 2.5 </td><td> 2.5 </td><td> 10 </td><td> 7447-40-7 </td><td>西格玛P3911 </td></tr><tr><td> 碳酸氢钠 </td><td> 25 </td><td> 25 </td><td></td><td> 144-55-8 </td><td>菲舍尔S/4240 </td></tr><tr><td>葡萄糖</td><td> 25 </td><td> 20 </td><td></td><td> 50-99-7 </td><td>西格玛G5767 </td></tr><tr><td>蔗糖</td><td> 75 </td><td></td><td></td><td> 57-50-1 </td><td>西格玛S5016 </td></tr><tr><td>钙CL 2 </td><td> 0.5 </td><td> 2 </td><td></td><td> 10043-52-4 </td><td>厂商VWR 190464K </td></tr><tr><td> MgCl 2的 </td><td> 7 </td><td> 1 </td><td> 2 </td><td> 7786-30-3 </td><td>西格玛63020 </td></tr><tr><td> ķ葡萄糖</td><td></td><td></td><td> 130 </td><td> 299-27-4 </td><td>西格玛G4500 </td></tr><tr><td>肝素钠</td><td></td><td></td><td> 10 </td><td> 7365-45-9 </td><td>西格玛H3375 </td></tr><tr><td> EGTA </td><td></td><td></td><td> 0.1 </td><td> 67-42-5 </td><td>西格玛E4378 </td></tr><tr><td>那2个 ATP </td><td></td><td></td><td> 2 </td><td> 34369-07-8 </td><td>西格玛A7699 </td></tr><tr><td>钠2的GTP </td><td></td><td></td><td> 0.3 </td><td> 36051-31-7 </td><td>西格玛G8877 </td></tr><tr><td> NaPhospho，肌酸</td><td></td><td></td><td> 10 </td><td> 19333-65-4 </td><td>西格玛P7936 </td></tr><tr><td>生物胞素（可选） </td><td></td><td></td><td> 2.7 </td><td> 576-19-2 </td><td&gt;西格玛B4261 </td></tr></tbody></table> 表1。切割学联，学联标准的K-葡萄糖的内部解决方案食谱。

preparation of parasagittal slices for the investigation of dorsal-ventral organization of the rodent medial entorhinal cortex

在大脑的计算依赖于适当的回应突触输入的神经元。神经元的不同，在他们的补充和细胞膜离子通道的分布，确定他们如何应对突触输入。然而，这些细胞的特性和动物行为的神经元功能之间的关系还不是很清楚。这个问题的办法之一，是研究地形组织的神经回路在单个神经元映射到它们编码的信息或计算的位置，他们携带1。使用这种方法的实验表明调节突触反应，感觉和认知电路2,3基本信息编码的原则。 沿背腹轴内侧嗅皮层（MEC）的空间表示地形的组织提供了一个机会，以建立细胞的机制和计算之间的关系，我空间认知mportant。啮齿类动物的MEC的第二层中的神经元编码的位置，使用网格状发射领域4-6。为MEC的背仓发现的神经元，个别的射击领域，形成了一个网之间的距离是30厘米的顺序，而这个距离在逐步位置更腹侧神经元增加至大于1米。一些研究表明MEC的，像网格射击领域之间的间距，也根据自己的背腹位置不同的第二层的神经元细胞的特性，这表明这些细胞的特性是重要的空间计算2,7-10。 在这里，我们描述程序编制和维持MEC的有利地形组织MEC的神经元的生物物理和解剖特性的调查背腹程度的脑切片的电生理记录。背腹的位置确定列印eurons相对解剖标志是难以建立与使用水平的MEC 7,8,11,12片的协议，准确，因为它是难以确定确切的背腹片的位置参考点。我们所描述的程序，使沿背腹轴的迈克，以及可视化的2,10分子梯度准确和一致的测量记录细胞的位置。成年小鼠（28天）使用的程序已经开发，并已成功受聘小鼠1.5岁。的调整，他们可以用年轻的老鼠或其他啮齿类动物。标准化体系的编制及测量系统的调查将有助于这方面的细胞和微电路的特性。

Watch this Scientific Journal Video about Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex at JoVE.com

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

我们描述了大脑切片，保持背腹轴的内侧嗅皮层（MEC）的编制和电生理记录的程序。由于定位的神经编码如下内MEC的背腹侧组织，这些程序协助调查重要的导航和记忆的细胞机制。

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

preparation-parasagittal-slices-for-investigation-dorsal-ventral

Research

JoVE Journal

Neuroscience

15.5K 次观看.  University of Edinburgh. 我们描述了大脑切片，保持背腹轴的内侧嗅皮层（MEC）的编制和电生理记录的程序。由于定位的神经编码如下内MEC的背腹侧组织，这些程序协助调查重要的导航和记忆的细胞机制。

视频: Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

情绪调节的神经关联的脑成像研究

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

科研

神经科学

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the 
investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the 
neural correlates of non-emotional memories3, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in4, 5-7). 
Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with 
event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically 
associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, 
SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. 
This protocol can be used in healthy subjects8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception 
and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)16, 17.

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

脑成像的情感记忆的促进作用调查

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

脑成像的情感对认知损害的影响调查

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

一个全面协议的内侧颞叶结构的手动分割

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备