Name: local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices
Uploaded: 2012-10-29T14:00:00
Description: Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

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p...

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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

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