这项工作是由美国国立卫生研究院（NIH）的资助DA030396支持。感谢成员的Drenan实验室的有益讨论和批评的稿件。特别感谢糜然金的技术援助和乔纳森·托马斯婷的意见，对成年小鼠的大脑切片。

1。溶液的制备脑片的制备及电<ol><li>先前描述解制备脑片3，4。制备N-甲基D-葡糖胺（NMDG）-基切削和恢复解决方案下面的组合物（以mM计）：93 N-甲基D-葡糖胺，2.5氯化钾，1.2的NaH 2 PO 4，30的NaHCO 3，20 HEPES，25葡萄糖，5 Na +的抗坏血酸盐，2硫脲，3的Na +丙酮酸，10用MgSO 4·7H 2 O，0.5的CaCl 2·2H 2 O。调整为300-310毫渗NMDG。 10 N的盐酸调节pH值至7.3-7.4。 <ol class="lalpha"><li>微孔水硫酸镁4•7H 2 O和CaCl 2·2H 2 O溶解在2 M解决方案的每个股票。 </li><li>称取其它组分按列出的顺序和使用Millipore水加至部分填充的容量瓶中，同时搅拌以溶解。一旦一切都被添加，填充使用Millipore水的容量瓶中。 </li><li>测量pH值，并用10 N HCl调整。 </li><li>测量的渗透压与NMDG必要时进行调节。 </li></ol></li><li>准备HEPES保持人工脑脊液（人工脑脊液）下面的组合物（以mM计）：92的NaCl，2.5氯化钾，1.2的NaH 2 PO 4的NaHCO 3，30，20 HEPES，25葡萄糖，5 Na +的抗坏血酸，2硫脲，3钠+丙酮酸，2硫酸镁4•7H 2 O 2的CaCl 2·2H 2 O。调整到与蔗糖的300-310毫渗。用1N HCl或NaOH调节pH值至7.3-7.4。 <ol class="lalpha"><li>称出组件中列出的顺序和使用Millipore水加至部分填充的容量瓶中，同时搅拌以溶解。一旦一切都被添加，填充容量瓶中，用Millipore水。 </li><li>测量pH值，如果需要的话，用NaOH或HCl调节。 </li><li>测量必要时与蔗糖的渗透压和调整。 </li></ol></li><li>准备标准的人工脑脊液记录下面的组合物的溶液（以mM计）：124的NaCl，2.5氯化钾，1.2的NaH 2 PO 4，24的NaHCO 3，12.5葡萄糖，2用MgSO 4·7H 2 O，2个的CaCl 2·2H 2 O。调整到与蔗糖的300-310毫渗。用1N HCl或NaOH调节pH值至7.3-7.4。 </li></ol> 2。急性脑片的制备<ol><li>脑切片的切割和恢复完成后，如先前描述的3，4。气泡2晶体菜肴填充与NMDG回收溶液与95％氧/ 5％CO 2气体（Carbogen增），在冰上的一盘，在33℃下</li><li>气泡一个晶体培养皿填充用HEPES与Carbogen增在室温下保持人工脑脊液。 </li><li>麻醉与Na +戊巴比妥钠（200毫克/千克，IP）的成年小鼠（年龄2个月至12个月）。继续进行到脚趾，捏动物有没有反应的过程。 </li><li>如果有必要，切尾的组织样本后genotypING的动物。 </li><li>打开胸腔，暴露心脏，夹住降主动脉。割伤右心房。 </li><li> Transcardially灌注动物用5-10毫升0-4°C NMDG恢复解决方案。 </li><li>斩首动物，取出大脑，并将其放置在4℃NMDG回收溶液，持续1分钟。 </li><li>在冰 - 冷却的金属板，在所需的取向（冠状，矢状窦，水平等）修剪大脑包括感兴趣的区域。 </li><li>词缀大脑块与强力胶的干燥阶段表面振动的限幅器（DSK-零1;土坂），大脑块浸入4℃下NMDG回收溶液，并不断泡沫溶液与Carbogen增。 </li><li>切割片的大脑区域的利益。切片厚度为200-400微米，取决于特定的大脑的感兴趣区域，动物的年龄，和实验以进行。 </li><li>切割后，立即孵育所需的片在carbogenated的，33°C NMDG恢复的解决方案整整12分钟，然后转移到carbogenated，室温HEPES最初的60分钟内解决方案。这孵育60分钟后，切片可用于记录。片被保持，直到它们被用于记录整个一天保持溶液在HEPES。 </li></ol> 3。膜片钳记录神经元在大脑切片<ol><li>拉一个标准的补丁微量移液器与一个可编程的火焰山 - 布朗拉出器（萨特P-97或等效垂直拉拔器）。千兆欧姆形成密封的最佳选择移液器，通常有4-6MΩ的电阻</li><li>一个片传输到记录室（RC-27L;华纳仪器）上的一个直立显微镜（尼康FN-1）阶段。连续superfuse（1.5 - 2.0毫升/分钟）的切片与carbogenated，标准记录人工脑脊液加热并保持在32℃下另外，切片可以被保持在室温下。 </li><li>兴趣使用的大脑区域的定位和中心10倍的空中目标（尼康计划福陆公司的10倍，NA 0.3，WD：16毫米）。 </li><li>可视化单个神经元，使用40倍的近红外（IR）的水浸泡目标（尼康APO 40XW; NA：0.80; WD：3.5毫米）连接到红外微分干涉对比（DIC）的光学和近红外的一个高速电荷耦合器件（CCD）摄像机（滨松C-7500）。在IR-DIC观察，健康的神经细胞呈现出光滑，浅灰色的质膜。 </li><li>填写微量合适的细胞内记录解决方案。对于大多数的应用中，我们使用下面的解决方案（以mM计）：135葡糖酸钾，5 EGTA，0.5的CaCl 2，2的MgCl 2，10 HEPES，2 Mg的ATP，和0.1 GTP（调节pH至7.25，Tris碱，渗透压调整至290毫渗用蔗糖）。建立一个可视化的神经元的全细胞记录。 </li></ol> 4。药物局部应用神经元片<ol><li>拉动上述标准的膜片钳微吸管。运用如萨特P-97，产生两个相同的“姐姐”的提示，在同一块玻璃移液管牵引是有利的，当在同一单元中要使用多种药物的解决方案。提示的大小，如果他们被填充有上述的细胞内的解决方案，将有一个电阻〜5MΩ都是最优的。 </li><li>回填的微量吸移管在carbogenated，标准记录脑脊液稀释药物的溶液。尖端指向向下，挥药物填充微量移液器被困在列的解决方案，以消除任何气泡。 </li><li>挂载填充药物的微量吸移管与管连接到一个合适的压力喷射系统，例如作为一个Picospritzer III（一般阀门有限公司）中的吸液管保持。吸管架应安装在相同的分辨率显微操作机械手常用的膜片钳记录（例如，萨特MP-285）。此外，操纵器应该允许斜向运动的流入和流出的切片。曼ually喷射到微管中的压力的​​3至4倍，以建立的列后面的流体的足够的压力。 </li><li>使用微操作机器人控制，降低药物的吸液管片，而IR-DIC光学可视化的提示下。理想情况下，一个应该可视地识别和中心的神经元被记录，随后定位药物吸移管附近的细胞之前降低记录微吸管到切片。定位药物吸移管可以在周边所记录的细胞，可能会破坏的记录，如果药物移液管移入位置后建立gigaohm密封引起的组织的运动。 </li><li>使用IR视频，定位药物微量吸移管的顶面处的组织切片。执行一个压力喷射和显示器1）的前端附近的运动有关喷射的碎片，和2）的微量吸移管前端的尖端被堵塞或阻塞的任何迹象。如果笔尖被封锁/堵塞，收回pipette和它与一个新的更换。 </li><li>接近记录的单元格从斜切片的顶部这样的，在最终位置时，填充药物的吸移管的前端是1）在相同的焦平面（或稍低于）作为记录的细胞和在记录电极的前端，和2）从所记录的小区10至40微米。如果药物填充的移液管尖的是上面的记录单元的力从压力喷射可能扰乱gigaohm的的密封。 </li><li>钳位电压模式或电流钳模式中的单元格，记录所需的细胞免疫应答。我们经常在记录乙酰胆碱和/或尼古丁引起的细胞电流，同时在电压钳模式下的神经元。虽然压力喷射可以手动触发使用Picospritzer III时，更具有可重复性的结果，最好是使用的采集系统（轴突Digidata 1440和膜片钳pClamp 10.3）与数字TTL脉冲触发的压力喷射。 </li></ol> 5。控制药物填充微管与一个压电翻译<ol><li>如果可能的话，装载的药物填充的吸液管保持到单维压电翻译（ 例如 Piezojena PA-100或伯利PZM-150），其能够接受一个模拟电压输入（再次，从采集系统），这是然后安装在高精密显微操作器（的萨特MP-285）。一种压电转换器是非常有用的，流入和流出的薄片，包括入口/出口的时间和速度，因为运动的药物填充移液器可以更容易地控制和再现从实验的实验。在研究的nAChRs，脱敏尼古丁泄漏的影响，从填充药物的吸移管，他们应该发生，被最小化的吸移管时只移动靠近细胞具有压电翻译的压力喷射的时刻。 </li><li>使用电位器手动控制，的压电电压控制放大器，请拨打电压到其最大值。 </li><li>使用显微操纵器，位置在切片的药物填充吸管移液管将退出其内容上录制的细胞，以及收回移液管通过手动的电压降低到零上的压电电压控制放大器。 </li><li>使用从记录采集系统的模拟输出信号，将药物填充吸管到切片中超过250毫秒300毫秒开始的压力喷射TTL脉冲发生之前的期间。退移液管超过250毫秒的一段时间，起始TTL脉冲结束后的50毫秒。 </li></ol>

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Physiol. 587, 345-361 (2009).</li><li>Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptor+subtypes+involved+in+facilitation+of+GABAergic+inhibition+in+mouse+superficial+superior+colliculus.">Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.</a> J. Neurophysiol. 94, 3893-3902 (2005).</li><li>Bouzat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structural+bases+of+activation+of+Cys-loop+receptors.">New insights into the structural bases of activation of Cys-loop receptors.</a> J. Physiol. Paris. , (2011).</li></ol>

本文提出的方法具有广泛的可用于研究配体门控离子通道的功能在大脑切片的准备工作。但是，也有若干因素，这将显着影响的实验数据，利用此方法的结果的质量和可重复性。例如，诱发电流填充药物的吸移管的前端的直径是非常敏感的。小tips将导致喷射药液，并具有低电阻的大秘诀将更有可能扰乱gigaohm记录的小区和记录电极之间的密封困难。提出的方法的另外一个限制是，在10微米的记录小...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;的试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt; N-甲基-D-葡糖胺</td&gt;<td&gt; SIGMA</td&gt;<td&gt; M2004</td&gt;</tr&gt;<tr&gt;<td&gt;氯化钾</td&gt;<td&gt; SIGMA</td&gt;<td&gt; P3911</td&gt;</tr&gt;<tr&gt;<td&gt;氢化钠<sub&gt; 2</sub&gt; PO<sub&gt;</sub</td&gt;<td&gt; SIGMA</td&gt;<td&gt; S9638</td&gt;</tr&gt;<tr&gt;<td&gt;碳酸氢钠<sub&gt; 3</sub</td&gt;<td&gt; SIGMA</td&gt;<td&gt; S6014</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt; SIGMA</td&gt;<td&gt; H3375</td&gt;</tr&gt;<tr&gt;<td&gt;葡萄糖</td&gt;<td&gt; SIGMA</td&gt;<td&gt; G5767</td&gt;</tr&gt;<tr&gt;<td&gt;娜<sup&gt; +</sup&gt;抗坏血酸</td&gt;<td&gt; SIGMA</td&gt;<td&gt; A4034</td&gt;</tr&gt;<tr&gt;<td&gt;硫脲</td&gt;<td&gt; SIGMA</td&gt;<td&gt; T8656</td&gt;</tr&gt;<tr&gt;<td&gt;娜<sup&gt; +</sup&gt;丙酮酸</td&gt;<td&gt; SIGMA</td&gt;<td&gt; P2256</td&gt;</tr&gt;<tr&gt;<td&gt;硫酸镁<sub&gt;</sub&gt;•7H<sub&gt; 2</sub&gt; O</td&gt;<td&gt; SIGMA</td&gt;<td&gt; 230391</td&gt;</tr&gt;<tr&gt;<td&gt;氯化钙<sub&gt; 2</sub&gt;•2H<sub&gt; 2</sub&gt; 0</td&gt;<td&gt; SIGMA</td&gt;<td&gt; 223506</td&gt;</tr&gt;<tr&gt;<td&gt;氯化钠</td&gt;<td&gt; SIGMA</td&gt;<td&gt; S9625</td&gt;</tr&gt;<tr&gt;<td&gt;娜<sup&gt; +</sup&gt;戊巴比妥</td&gt;<td&gt; Vortech制药</td&gt;<td&gt; 76351315</td&gt;</tr&gt;<tr&gt;<td&gt;葡萄糖酸钾</td&gt;<td&gt; SIGMA</td&gt;<td&gt; G4500</td&gt;</tr&gt;<tr&gt;<td&gt; EGTA</td&gt;<td&gt; SIGMA</td&gt;<td&gt; E3889</td&gt;</tr&gt;<tr&gt;<td&gt; MG-ATP</td&gt;<td&gt; SIGMA</td&gt;<td&gt; A9187</td&gt;</tr&gt;<tr&gt;<td&gt; GTP</td&gt;<td&gt; SIGMA</td&gt;<td&gt; G8877</td&gt;</tr&gt;<tr&gt;<td&gt; DSK-1零振动切片机</td&gt;<td&gt;特德·佩拉，</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; P-97火焰山/褐色微量移液器拉马</td&gt;<td&gt;萨特</td&gt;<td</td&gt;</tr&gt;<tr&gt;<tdRC-27录音室</td&gt;<td&gt;华纳</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; TC-344B灌注热水器控制器</td&gt;<td&gt;华纳</td&gt;<td&gt; 640101</td&gt;</tr&gt;<tr&gt;<td&gt; SH-27B解决方案加热器</td&gt;<td&gt;华纳</td&gt;<td&gt; 640102</td&gt;</tr&gt;<tr&gt;<td&gt;尼康FN-1</td&gt;<td&gt;尼康</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; C-7500 CCD摄像机</td&gt;<td&gt;滨松市</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Picospritzer III</td&gt;<td&gt;阀门有限公司</td&gt;<td</td&gt;</tr&gt;<tr&gt;<tdMP-285微操作机器人</td&gt;<td&gt;萨特</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PA-100压电翻译</td&gt;<td&gt; piezosystem耶拿公司</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 12V40压电放大器</td&gt;<td&gt; piezosystem耶拿公司</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Axopatch 200B中</td&gt;<td&gt;分子设备公司</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Digidata 1440A</td&gt;<td&gt;分子设备公司</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

烟草使用导致许多健康问题，包括癌症，心脏疾病，肺气肿和中风。吸烟成瘾是一种普遍的神经精神疾病，源于尼古丁对整个中枢神经系统的烟碱型乙酰胆碱受体（nAChRs）细胞生物物理和行动。理解不同胆碱受体亚型中存在的对尼古丁上瘾有关的脑区是一个重要的优先事项。 实验，采用如全细胞膜片钳或两电极电压钳记录的电生理技术是有用的药理特性的兴趣的nAChRs。细胞表达的nAChRs，如哺乳动物组织培养细胞或非洲爪蟾卵母细胞，在物理上隔离的，因此容易使用现代药理学工具研究。已经取得了很大的进展，特别是当使用这些技术的目标受体已知一个第二异位表达很容易实现。然而，通常情况下，有必要在他们的母语环境中学习的nAChRs：从实验室小鼠或大鼠急性收获大脑切片的神经元在。 ，例如，小鼠表达“过敏性”胆碱受体亚基如的α4L9&#39;A小鼠1和α6L9&#39;S的小鼠2，允许用于明确识别它们的功能表达的特定的胆碱受体亚单位的基础上的神经元。虽然全细胞膜片钳技术记录神经元在大脑切片的由熟练的电生理常规的做法，它是具有挑战性的局部应用药物，如乙酰胆碱或尼古丁的记录细胞内的脑切片。药物稀释成灌流（沐浴应用）是不可逆的迅速，和不容易适应U型管系统与大脑切片。 在本文中，我们描述了一种方法，迅速将胆碱受体激活的神经元的药物在成人米乌斯大脑切片。标准全细胞的记录，则在切片中的神经元，并填充与感兴趣的药物的第二微吸管被操纵到所记录的小区附近的位置。加压的空气或惰性填充药物的吸移管的入氮气的注入会导致少量药液要喷射到记录小区从移液管。胆碱受体介导的电流是使用这种方法，能够解决的问题与毫秒的精度。药物的应用时间可以很容易地被改变，和填充药物的吸移管可以缩回和替换一个新的移液管，使浓度 - 反应曲线，以创建为一个单一的神经元。虽然描述的胆碱受体神经生物学的上下文中，这种技术应该是有用的用于研究许多类型配体门控离子通道或受体的神经元从大脑切片。

在我们的实验中，我们经常记录多巴胺（DA）神经元的腹侧被盖区（VTA）和黑质致密部（SNC）。在电压钳模式下，压力应用乙酰胆碱或尼古丁这些细胞通常会导致在一个快速，向内阳离子电流到达100-200毫秒（ 图1A-B）内的峰。通过扩散作用部位的药物从很大程度上取决于电流衰减，在该切片中的酶是否是本代谢成无活性形式的药物。例如，乙酰胆碱是位于细胞表面的脑区DA神经元5?...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

在本文中，我们描述了一个有用的方法来研究神经元的急性分离的大脑切片配体门控离子通道的功能。这种方法涉及使用的药物填充微量移液器为本地应用程序使用标准的膜片钳技术记录神经元的药物。

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

local-application-drugs-to-study-nicotinic-acetylcholine-receptor

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Pharmacology

Neuroscience

Unit 1

Drugs Acting on Autonomic Nervous System: Cholinergic Agonists and Antagonists Agents

SCIENTISTS IN ACTION

18.9K 次观看.  Purdue University. 在本文中，我们描述了一个有用的方法来研究神经元的急性分离的大脑切片配体门控离子通道的功能。这种方法涉及使用的药物填充微量移液器为本地应用程序使用标准的膜片钳技术记录神经元的药物。

视频: Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

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 ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

粉扑诱导的全细胞电流在脑切片中的应用

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

利用可活性可选位的激光光致发光体研究小鼠脑片中的烟碱乙酰胆碱受体功能

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...