这项工作是由美国国家神经紊乱和中风研究所（NS - 19108），偏头痛研究基金会，和白理查德P. Kraig基金会的赠款。玛西娅体育Kraig女士的协助下，准备文化传媒和维护切片文化。我们感谢她的意见，并在本文的最后版本的修订叶莲娜Grinberg。

<ol>
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两个极其重要的概念，重要的是参与冷预处理神经保护作用的细胞因子信号系统的划分是在图6和7所示。首先，细胞因子的浓度极低，在正常脑组织中的信号分子。然而，生理细胞因子浓度的变化有一个巨大的可能改变大脑结构和功能（即表型），因为他们有能力改变基因的表达（图6）。此外，细胞因子是高度冗余和多效性，多种细胞因子，可以有类似的效果，和一个单一的细胞因子可以变量的影响（图7）。因此，要准确地建立先天的神经保护作用的细胞因子冷预处理（或其他生理预处理刺激），必须确定相关的信令变量的综合分析基地。这是通过复用检测策略。这将建立的“签名”冷预处理神经保护作用的细胞因子。 <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="图6" /> 图6。脑细胞因子信号的电源 。插图传达巨大的信号的脑细胞因子的生理浓度的力量，相比其他公认同行的浓度。浓度表示为距离的倒数，作为参考点的单猫须开始。钠（10 -1 m的一粒胡椒说明）和钾（10 -3 M说明了一个西红柿）是在约150个和3毫米，分别的水平间质性脑空间，并有公认角色神经细胞的电生理功能。同样，pH值（即〜10 -7 m层的氢离子和780米沿芝加哥的湖畔所示）和钙（即〜10 -8看到7.8公里，从谷歌卫星图像沿着芝加哥的麦考密克湖畔看到的水平地点在海德公园附近的芝加哥大学岬点）。此外，这些浓度间质空间释放的神经递质影响当地的大脑区域的活动。与此相反，细胞因子（从地球到火星的距离显示），可以改变浓度的脑功能少十几万次。 <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="图7" /> 图7。互动信号与生俱来的细胞因子的途径。细胞因子是高度冗余，多种细胞因子多效可以有类似的效果，可以有一个单一的细胞因子变量的影响。这种多样性源于复杂的交互信号配体，受体和磷酸化水平发生。事实上，大脑的不同类型的细胞，每个先天细胞特异的细胞因子，受体，和相关的磷酸化变化的能力，组成更加复杂源于。这里说明的目的，只有天生的细胞因子信号转导通路（从免疫细胞研究中派生）所示。为了简便起见，绘制大脑是作为一个单一的细胞显示出与生俱来的细胞因子的潜在的相互作用（白线）（IL -1α和IL -1β（这里​​称为IL -1α/β），肿瘤坏死因子-α，IL - 6，干扰素γ和IL - 10），受体（IL - 1R1，TNFR1，IL-6/gp130，IFNγR，和IL - 10R）和磷酸化（即，P38（P38 - MAPK）和JNK激酶ERK1 / 2）和转录因子（ATF - 2，NFκB，和STAT3）。例如，TNF -α信号通过TNFR1 JNK，P38 MAPK和ERK1 / 2，从而引发通过ATF - 2基因的表达。 TNF -α也可直接通过改变基因表达NFκB的激活。总之，这些转录因子的激活唤起增加（箭头）和下降（钝端）所示的细胞因子及其受体的表达。更重要的是，这些途径表明，在一个细胞因子的变化（如TNF -α）的影响其他细胞因子（如IL -1β）的生产。因此，建立准确的冷预处理，必须确定相关的信令变量的综合分析的细胞因子神经保护基地。这将建立先天的“签名”冷预处理的细胞因子。 （图片编制的数据参考＃25。）

制备切片文化<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> Millicell小CM组织培养插入（30毫米） </td><td>赛默飞世尔</td><td> PICM0 - 3050 </td><td></td></tr><tr><td>基础培养基鹰</td><td> Invitrogen公司</td><td> 21010 </td><td></td></tr><tr><td>厄尔s平衡盐溶液</td><td>西格玛</td><td> E2888 </td><td></td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen公司</td><td> 35050 </td><td></td></tr><tr><td>庆大霉素</td><td> Invitrogen公司</td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen公司</td><td> 15295 </td><td></td></tr><tr><td> D -葡萄糖</td><td>西格玛</td><td> G8769 </td><td></td></tr><tr><td> BSL - 1通风柜</td><td>面包师傅</td><td></td><td></td></tr><tr><td> McIlwain组织菜刀</td><td>布林克曼</td><td></td><td></td></tr><tr><td>特氟隆磁盘</td><td>布林克曼</td><td> 023-49-310-1 </td><td></td></tr><tr><td>抹刀</td><td>赛默飞世尔</td><td> 14 - 373 - 25A </td><td></td></tr><tr><td>弧形光圈剪刀</td><td>精细科学的工具</td><td> 14091-09 </td><td></td></tr><tr><td>长直剪</td><td>精细科学的工具</td><td> 14002-14 </td><td></td></tr><tr><td>长钳</td><td>赛默飞世尔</td><td> 13-812-40 </td><td></td></tr><tr><td>斜角钳</td><td>精细科学的工具</td><td> 11808-02 </td><td></td></tr><tr><td>短钳</td><td>赛默飞世尔</td><td> 13-812-38 </td><td></td></tr><tr><td> ＃5 Inox公司钳</td><td>精细科学的工具</td><td> 11254-20 </td><td></td></tr><tr><td> 2虹膜铲刀</td><td>精细科学的工具</td><td> 10094-13 </td><td></td></tr><tr><td> 150 × 15毫米培养皿</td><td>赛默飞世尔</td><td> 08-757-148 </td><td></td></tr><tr><td> 60 × 15毫米培养皿</td><td>赛默飞世尔</td><td> 08 - 757 - 100B </td><td></td></tr><tr><td>野生M8的体视显微镜</td><td>野生Heerbrugg </td><td></td><td></td></tr><tr><td> Gey的平衡盐溶液</td><td>西格玛</td><td> G9779 </td><td></td></tr><tr><td> Blak -射线短波紫外线测量仪</td><td> UVP </td><td> J - 225 </td><td></td></tr><tr><td> 6猎鹰培养皿</td><td>赛默飞世尔</td><td> 08 - 772 - 1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen公司</td><td> 21103</td><td></td></tr><tr><td> B27的补充</td><td> Invitrogen公司</td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td>双子座生物制品</td><td> 400-160-010 </td><td></td></tr><tr><td>抗坏血酸</td><td>西格玛</td><td> A4544 </td><td></td></tr><tr><td> CO 2分析仪</td><td>巴哈拉赫</td><td> ＃2820 </td><td></td></tr></tbody></table>预检片文化的活力<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> Sytox绿</td><td> Invitrogen公司</td><td> S7020 </td><td></td></tr><tr><td> DMIBRE倒置显微镜</td><td>徕卡</td><td></td><td></td></tr></tbody></table>冷预处理<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> BSL - 2通风柜</td><td> NuAire </td><td></td><td></td></tr></tbody></table>兴奋毒性损伤<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> CoolSnap FX CCD相机</td><td> Photometrics </td><td></td><td></td></tr><tr><td> Fluoroscein </td><td> Invitrogen公司</td><td> F36915 </td><td></td></tr><tr><td> MetaMorph软件</td><td>分子器件</td><td>五，04年7月5日</td><td></td></tr><tr><td> 100微米深hemacytometer </td><td> Hausser科学</td><td></td><td></td></tr><tr><td> N -甲基- D -天冬氨酸</td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat软件</td><td> SYSTAT软件公司</td><td>诉3.5 </td><td></td></tr></tbody></table>冷预处理应用的联合治疗<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> sTNFR1 </td><td> R＆D系统</td><td> 425 - R1 - 050 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A - 4503 </td><td></td></tr></tbody></table> RNA分离<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> RNAL ATER </td><td> Ambion公司</td><td> AM7021 </td><td></td></tr><tr><td>微的RNeasy试剂​​盒</td><td> Qiagen公司</td><td> 74004 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>费舍尔</td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1.5 RNase的免费杵</td><td>南泽西精密工具和模具公司</td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega公司</td><td> N261A </td><td></td></tr><tr><td>三羟甲基]氨基甲烷</td><td>西格玛</td><td> T3069 </td><td></td></tr><tr><td> EDTA（乙二胺四乙酸二钠水合物</td><td>西格玛</td><td> ＃E7889 </td><td></td></tr></tbody></table> RNA定量<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> RiboGreen含量</td><td> Invitrogen公司</td><td> R11491 </td><td></td></tr><tr><td>酵母tRNA </td><td> Invitrogen公司</td><td> 15401-011 </td><td></td></tr><tr><td> 1.5 mL离心管</td><td>美国科学</td><td> 16155500 </td><td></td></tr><tr><td> 96荧光检测板</td><td> NUNC </td><td> DK - 4000 </td><td></td></tr><tr><td> 96孔荧光酶标仪</td><td> TECAN </td><td>沙费尔二</td><td></td></tr></tbody></table>定量PCR <table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> iScript反向转录试剂盒</td><td> Bio - Rad公司</td><td> 170-8891 </td><td></td></tr><tr><td> SYBR GREEN </td><td> Invitrogen公司</td><td> S7563 </td><td></td></tr><tr><td>白金Taq聚合酶</td><td> Invitrogen公司</td><td> RNA的Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler热循环仪</td><td> Bio - Rad公司</td><td> iCycler光模块</td><td></td></tr><tr><td> iCycler系统软件</td><td> Bio - Rad公司</td><td>诉3.1 </td><td></td></tr></tbody></table>定量PCR微阵列<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td>核糖核酸酶客队 </td><td>分子生物产品</td><td> ＃7000 </td><td></td></tr><tr><td> RT 2第一链套件</td><td> SABiosciences </td><td> C - 03 </td><td></td></tr><tr><td>基于SYBR GREEN - PCR混合物</td><td> SABiosciences </td><td> PA - 011 </td><td>特定iCycler </td></tr><tr><td> RT 2探查PCR阵列</td><td> SABiosciences </td><td> PARN - 011A </td><td></td></tr></tbody></table>蛋白质组学的含量<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号</td><td> 评论 </td></tr><tr><td>细胞裂解试剂盒</td><td> Bio - Rad公司</td><td> 171-304011 </td><td></td></tr><tr><td>离心</td><td> Fisher Scientific则</td><td>微型7 </td><td></td></tr><tr><td> BSA的蛋白质股票</td><td> Bio - Rad公司</td><td> 500-0007 </td><td></td></tr><tr><td> BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td><td></td></tr><tr><td>免疫96微孔板</td><td> NUNC </td><td> 449824 </td><td></td></tr><tr><td>密封胶带</td><td> Bio - Rad公司</td><td> 2239444 </td><td></td></tr><tr><td> 96孔板读者</td><td> Perkin Elmer公司</td><td> 1420 - mulilabel柜台</td><td></td></tr><tr><td> Bioplex大鼠肿瘤坏死因子-α细胞因子的测定</td><td> Bio - Rad公司</td><td> X0000001T </td><td></td></tr></tbody></table>免疫染色<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> 16％多聚甲醛</td><td>阿法埃莎</td><td> 43368 </td><td></td></tr><tr><td>赖氨酸</td><td>西格玛</td><td> L - 6001 </td><td></td></tr><tr><td>磷酸钠</td><td> Fisher Scientific则</td><td> S374 - 1 </td><td></td></tr><tr><td>高碘酸钠</td><td>西格玛</td><td> S - 1878年</td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S - 2002 </td><td></td></tr><tr><td>过氧化氢（30％） </td><td>西格玛</td><td> H1009 </td><td></td></tr><tr><td>山羊血清</td><td>科罗拉多州的血清公司</td><td> CS 0920 </td><td></td></tr><tr><td> TRITON X - 100 </td><td>西格玛</td><td> T - 8787 </td><td></td></tr><tr><td>染色菜</td><td>特德佩拉公司</td><td> 14511 </td><td></td></tr><tr><td>板振动筛</td><td>实验室线仪器公司</td><td> 3520 </td><td></td></tr><tr><td> ＃1滤纸（185毫米） </td><td>瓦特曼</td><td> 1001-185 </td><td></td></tr><tr><td> 3,3 - 二氨基</td><td>西格玛</td><td> D8001 </td><td></td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D8418 </td><td></td></tr><tr><td>乙醇</td><td> Pharmco - AAPER </td><td> 111000200 </td><td></td></tr><tr><td>二甲苯</td><td> Fisher Scientific则</td><td> X3S - 4 </td><td></td></tr><tr><td> Permount安装媒体</td><td> Fisher Scientific则</td><td> SP15 - 100 </td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td>厘米3 050小号</td><td></td></tr><tr><td>组织Tek的</td><td>特德佩拉公司</td><td> 2705​​0 </td><td></td></tr><tr><td>细尖的油漆刷</td><td>特德佩拉</td><td> 11806 </td><td></td></tr><tr><td>人民行动党堵笔</td><td>特德佩拉公司</td><td> 22309 </td><td></td></tr><tr><td> SFX信号增强器</td><td> Invitrogen公司</td><td> A31631 </td><td></td></tr><tr><td>延长抗淬灭</td><td> Invitrogen公司</td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

神经损伤的发病率和死亡率的常见原因，从一般的麻醉和相关手术过程中，可以有效的发展缓解，易于管理和安全的预处理治疗。我们寻求定义冷预处理手段确定疗法的发展新目标，以保护脑损伤发病前的神经免疫信号。低级亲炎症介质，信号随时间变化的冷预处理神经保护作用是必不可少的。这个信号是一致的生理调节兴奋效应的基本原理，这就需要刺激性的刺激达到足够的时间适应保护的刺激阈值的大小，才能显现。 因此，在冷预处理神经保护作用的免疫信号划分要求，生物系统和实验操作，再加上技术能力的高度重复性和敏感。我们的做法是使用作为海马脑片培养，在体外模型，密切反映他们在与多突触的神经网络，成熟的和静态的胶质细胞/小胶质细胞的影响体内同行。小胶质细胞胶质状态尤为重要，因为它们的细胞因子的主要来源，这是femtomolar范围执行。此外，切片文化，可以在体外保持几个星期，这是足够的时间来唤起激活刺激和评估的适应性反应。最后，环境条件，可以准确地控制使用切片文化，使细胞因子信号寒冷的预处理可以进行测量，模仿，并调制，剖析关键的节点方面。细胞因子信号系统的分析需要使用的敏感和可重复性复用的技术。我们使用肿瘤坏死因子-α定量PCR，实时定量PCR定量阵列筛选，评估组织范围内的细胞因子变化的小胶质细胞激活屏幕。后者是一个最敏感的和可重复性的手段来衡量多个细胞因子系统，同时信号的变化。重大变化，然后有针对性的qPCR蛋白质检测证实。我们探头组织为基础的细胞因子蛋白复微流式细胞仪检测使用Luminex公司的技术变化。确定双标免疫组化细胞特异性细胞因子的产生。两者合计，这个脑组织编制和使用的样式，再加上建议的调查战略可能是一个确定发展的新型疗法，可以模仿冷预处理的优势的潜在目标的最佳途径。

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

我们寻求定义冷预处理手段确定疗法的发展新目标，以保护脑损伤发病前的神经免疫信号。我们目前所做的这些工作需要的生物系统，实验操作，加上技术能力的高度重复性和敏感的战略。

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K 次观看.  The University of Chicago Medical Center. 我们寻求定义冷预处理手段确定疗法的发展新目标，以保护脑损伤发病前的神经免疫信号。我们目前所做的这些工作需要的生物系统，实验操作，加上技术能力的高度重复性和敏感的战略。

视频: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

脑血氧测量基于磷光氧依赖淬火

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

科研

神经科学

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...