Name: methods for the discovery of novel compounds modulating a gamma-aminobutyric acid receptor type a neurotransmission
Uploaded: 2018-08-16T13:00:00.000+00:00
Duration: 7 min 16 s
Description: Watch this Scientific Journal Video about Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission at JoVE.com

作者感谢朱蒂丝兰格, 玛丽亚 Karg, Grégoire Friz, 雷切尔哈伯历和玛丽克莱尔 Pflimlin 从罗氏和 Tifany 马丁·舍尔和黛博拉保卢奇从 HiQScreen 为他们出色的技术援助。

爪蟾被安置和处理根据日内瓦小行政区动物指南。
1. 放射配基装订
<ol><li>
测定板的制备
	<ol>
<li>用5毫米氯化钾、1.25 毫米 CaCl2、1.25 毫米氯化镁2、120毫米氯化钠和15毫米三种方法制备 1.5 L 的测定缓冲器;用 HCl 调整 pH 值为7.4。</li>
		<li>准备在50.76 µM (例如, 5 µL 化合物10毫米到980µL 化验缓冲区在离心管) 测试的化合物。用涡流机在高速2–5的情况下混合。</li>
		<li>吹打1µL 的化合物 (10 毫米) 到1970µL 的检测缓冲剂, 制备参考化合物马西尼的预稀释。用涡流机在高速2–5的情况下混合。</li>
		<li>稀释 [3H] 马西尼股票溶液与化验缓冲器在4°c 到4毫微米在50毫升聚丙烯管。</li>
		<li>吸管50µL/井的化验缓冲区进入1和3–11的96井微板块, 如图 2所示的板块布局显示。</li>
		<li>吸管73.2 µL 的化合物稀释在50.76 µM (见步骤 1.1.2) 到12栏的板块。</li>
		<li>稀释每个化合物, 使用几何级数超过9步 (1E-10 -1E-06米), 23.12 µL 传输量和填充柱3–11。彻底混合稀释。每次稀释后都要改变笔尖。</li>
	</ol>
</li>
	<li>
稀释细胞膜
	<ol>
<li>解冻先前从 HEK293 overexpressing 人 GABAA受体 (0.025–0.15 毫克/毫升) 中分离出来的细胞膜, 在室温下获得80毫升, 并将悬浮液转移至4摄氏度的化验缓冲器。</li>
		<li>并用重悬膜溶液仍然在4°c 与组织均质体为 30–40 s 在 10,000–12,000 rpm。</li>
	</ol>
</li>
	<li>
稀安定用于非特定结合 (NSB) 控制
	<ol>
<li>将4毫米安定的溶液稀释成5毫升的浓度为40µM 的测定缓冲液。</li>
	</ol>
</li>
	<li>
开始反应
	<ol>
<li>吸管50µL 4 毫微米 [3H] 马西尼入96井板材的每个井并且保持它在一个冰水容器。</li>
		<li>添加100µL 的 GABAa受体亚型膜制备有 0.5mg/毫升的蛋白质浓度。</li>
		<li>吸管50µL 40 µM 安定入专栏2。</li>
		<li>在冰上孵化1小时的盘子。</li>
		<li>在96井滤板的每个井中添加50µL, 用于后续膜分离和放射性测量, 随后停止反应。</li>
	</ol>
</li>
	<li>
停止反应
	<ol>
<li>准备一个洗涤缓冲器与50毫米三 HCl, pH 值7.4。</li>
		<li>使用96井细胞收割机过滤解决方案。用300µL 的冷水洗涤缓冲液将板材洗净3x。</li>
		<li>用塑料薄膜封住盘子的底部, 并添加40µL 的闪烁鸡尾酒, 用于每个井中的液体闪烁计数, 并用乙醇70% 擦拭。</li>
		<li>在 RT 中轻轻摇动盘子至少1小时。让它至少在 RT 上再坚持一个小时。</li>
		<li>通过将盘子放在闪烁计数器上测量放射性 (在 CPM 中)。允许测量每井3分钟。</li>
	</ol>
</li>
	<li>
数据分析
	<ol>
<li>确定8复制非特定绑定 (NSB) 和总绑定 (TB) 以及样本的重复或 triplicates 的平均值。根据以下公式计算每个样本的平均值的% 特定绑定 (SB): <img alt="Equation 1" src="/files/ftp_upload/57842/57842eq1.jpg"> 这里 总某人= 平均 TB 减去平均 NSB。</li>
		<li>在横坐标中, 在调节抑制剂浓度时, 将其与对照。使用单一站点竞争分析公式拟合数据: <img alt="Equation 2" src="/files/ftp_upload/57842/57842eq2.jpg"> 这里 y = 某人的%, 最小的y, B = 最大y, C = IC50, X = 竞争化合物浓度的对数10 , D = 曲线的斜率 (一个小山系数)。</li>
		<li>用半最大抑制浓度 (IC50) 计算结合亲和性 (基), [3h] 马西尼在各自膜9上的离解常数 (Kd) 和 [3h] 的浓度马西尼在化验中根据以下数据使用等式: <img alt="Equation 3" src="/files/ftp_upload/57842/57842eq3.jpg">
</li>
	</ol>
</li>
</ol>2.爪蟾卵母细胞的受体表达和记录
<ol><li>卵巢采集与卵母细胞的制备
	<ol>
<li>制备无菌 Barth 溶液, 88 毫米氯化钠, 1 毫米氯化钾, 2.4 毫米 NaHCO3, 10 毫米 HEPES, 0.82 毫米 MgSO4. 7H2o, 0.33 毫米钙 (NO3)2. 4H2o, 0.41 毫米 CaCl2. 6H2o, pH 值7。4, 辅以100单位/毫升青霉素, 100 µg/毫升链霉素和0.25 µg/毫升两性霉素 B。</li>
		<li>准备 1x OR2 培养基 (no CaCl2) 与88.5 毫米氯化钠, 2.5 毫米氯化钾, 5 毫米 HEPES, 1 毫米氯化镁2. 6H2O, pH 值7.4。</li>
		<li>在冷却的 Tricaine 甲磺酸 (浓度为150毫克/升, 调整在 pH 7.4) 和碳酸氢钠 (300 毫克/升) 后, 以20分钟的深度麻醉, 牺牲一只爪蟾女性, 然后斩首。</li>
		<li>用干净的剪刀和镊子迅速收获卵巢, 并将其放置在2培养皿 (10 厘米), 填充40毫升 1x Barth 溶液和抗生素/防霉。</li>
		<li>将非游离卵巢存放在 Barth 溶液中长达2周, 达到4摄氏度。</li>
		<li>为离解, 切开卵巢与干净剃刀刀片在小组分 (3) 并且孵化他们在50毫升 OR2 培养基没有 CaCl2 (OR2-noCaCl2) 包含0.2% 个胶原酶 (I 型) 在17–19°c 在100毫升整流罩瓶与缓慢的搅拌第4-5 h. 验证磁条是否放置在微调瓶底部约3–5厘米, 以避免粉碎卵母细胞。</li>
		<li>在第4-5 h 之后, 验证大多数卵母细胞是从卵泡中释放出来的 (也就是说, 它们单独游动)。用200毫升的 OR2 1.8 毫米 CaCl2洗净他们的5x。</li>
		<li>将 defolliculated 卵母细胞转移到培养皿 (10 厘米), 填充 Barth 溶液和抗生素/antimycotics, 并使它们至少在17摄氏度前一夜间在 cDNA 或 mRNA 注射之前。</li>
		<li>第二天, 在双目下选择一个健康的卵母细胞, 它呈现出一种清晰而独特的动物与植物极 (用于植物极的暗褐色与淡黄色)。使用干净的巴斯德吸管, 并将卵母细胞一个一个地处理成无菌的锥形96注入井板。 注: 在放置卵母细胞时, 不需要特别注意注射;而对于 cDNA 注射来说, 将卵母细胞定向到向上的动物极是必不可少的。</li>
	</ol></li>
	<li>mRNA 或 cDNA 自动注射
	<ol>
<li>根据推荐的说明, 使用商业上可用的工具包合成基因。用 RNase 清洁仪器和桌子, 戴上适当的防护手套。 注意: mrna 的质量是决定表达良好的决定性因素, 在注射过程中防止 mrna 降解是必不可少的。</li>
		<li>使用商用套件准备基因, 并在 bidistilled 水中溶解所需量, 浓度为0.2 µg/µL。</li>
		<li>注射10–50的 mRNA 溶液浓度为0.2 µg/µL, 或 10 nL 的 cDNA 溶液在浓度范围从0.02–0.2 µg/µL, 最好是分批95卵母细胞。</li>
		<li>对于需要多个亚基的膜蛋白 (即异型缝隙连接α1β2γ2氨基丁酸A受体), 将相应的基因或基因混合在所需比例 (即1:1: 1 或 1:10:1)。</li>
		<li>将卵母细胞保持在17摄氏度, 以防止卵母细胞对热休克蛋白的表达;将微板块存储在一个温度控制的存储区域中。</li>
		<li>在0.1–1,000 µM OR2 的结合试验中, 溶解测试的化合物, 用于电生理记录, 并在96井平底聚丙烯板上处理。</li>
	</ol></li>
	<li>表达的质粒
	<ol>
<li>遵循商业可用的试剂盒的指导手册, 为 mRNA 合成与细菌 T7 或 T6 助剂, 并使20µL 的解决方案在0.2 µg/µL 在 RNase 无水。</li>
		<li>准备至少20µL 的溶液, 其中含有真核细胞表达载体的 cDNA 表达, 通常在 bidistilled 水中溶解的0.2 µg/µL。</li>
	</ol></li>
	<li>卵母细胞显微注射及其质量的可视化
	<ol>
<li>注射含有质粒的溶液的 10–50 nL (见步骤2.3.1 或 2.3.2), 使用玻璃微注射针, 顶端直径可达100µm 安装在机器人装有压力弹射系统或自动注射系统。 注: 在这里讨论的例子中, 卵母细胞由95批次注射, 其自动化系统适合用于 cDNA 或 mRNA。</li>
		<li>用1µL 的染料 (如亚甲基蓝) 填充注射针 1%, 并使用标准程序 (无论是在细胞核中的 cDNA 或细胞质中的 mRNA) 注射卵母细胞。</li>
		<li>将注射卵母细胞在沸水中释放约1分钟。在双目下用剃刀刀片将卵母细胞切成两半。 注: 染料保持本地化, 如图 3E所示, 允许对注射进行精确观察。</li>
	</ol></li>
	<li>双电极电压钳记录
	<ol>
<li>将含有卵母细胞的井板放在自动系统上, 使用图 4所示的原理。 注: 与图 5所示的膜片钳系统相比, 该电池的真实膜电位是由电压电极读取的。</li>
		<li>使用基于图标的界面编程自动记录系统, 例如,图 6所示的方案确定浓度活动关系。</li>
	</ol></li>
	<li>曲线拟合
	<ol>
<li>使用适当的软件分析结果, 使用图示的浓度活化曲线, 将电流振幅绘制为激动剂浓度对数的函数。</li>
		<li>观察 sigmoidal 曲线, 随后可以在形式上与经验性的希尔方程配合使用: <img alt="Equation 4" src="/files/ftp_upload/57842/57842eq4.jpg"> 这里 Y= 诱发电流的分数, 欧共体50 = 集中为50% 激活, x = 化合物的浓度, nH = 小山系数或明显的协同。</li>
		<li>通过将每个响应的振幅除以最高浓度记录的值来分析从一系列单元获得的数据, 将电流正常化为统一。</li>
		<li>通过标准可用软件, 确定平均误差和标准偏差, 实现曲线拟合。</li>
	</ol></li>
</ol>3. 脑切片的电生理记录
注: 海马大鼠切片是根据国家和机构指南编写的。
<ol><li>
海马切片的制备
	<ol>
<li>准备解剖人工脑脊髓液 (dACSF) 与124毫米氯化钠 124, 2.5 毫米氯化钾, 1.25 毫米的 CaCl 2PO4, 2 毫米 MgSO4. 7H2o, 2.5 毫米2 2H2o, 26 毫米 NaHCO3, 10 毫米葡萄糖和4毫米蔗糖在瓶子里掺入 95% O2和 5% CO2的混合物。</li>
		<li>准备记录人工脑脊髓液 (rACSF), 124 毫米氯化钠, 5 毫米氯化钾, 1.25 毫米的2PO4, 2 毫米 MgSO4. 7H2o, 2.5 毫米 CaCl2 2H2o, 25 毫米 NaHCO3, 10 毫米葡萄糖在瓶子里掺入 95% O2和 5% CO2的混合物。</li>
		<li>麻醉大鼠使用2.5% 异氟醚和纯氧的混合物, 并斩首它们。</li>
		<li>用一把细剪刀剪下中线的头皮。沿中线切开头骨, 不损害底层组织。用镊子取出头骨, 用细铲铲出大脑。</li>
		<li>将大脑放在 RT 的毒气 dACSF 溶液中, 用细铲解剖左海马的形成。</li>
		<li>将横片 (400 µm 厚) 从海马体的中等部位切割成组织斩波器, 并用画笔将其转移到录音室。保持45分钟的 RT 切片。</li>
		<li>灌注的切片与毒气 rACSF 在35摄氏度和以1.5 毫升/分钟的速度. 气泡溶液与 95% O2和 5% CO2的混合物。 注意: 在这一步之后, 切片已经准备好进行电生理实验。</li>
	</ol>
</li>
	<li>
单种群穗记录
	<ol>
<li>将脑切片放入显微镜下的腔室。</li>
		<li>灌注的速度为3毫升/分钟与 rACSF。</li>
		<li>拉一杯硼硅酸盐玻璃微与具有 2 MΩ电阻的吸管拉出器。 注: 此微用作记录电极, 并与放大器电连接。</li>
		<li>用含有2米氯化钠的溶液填充微, 将其放入放大器的吸管支架中。</li>
		<li>用右机器人将记录微定位在海马片 CA1 区 pyramidale 地层中。 注意:图 7中的位置以示意图表示。</li>
		<li>将绝缘双极铂/铱电极置于左侧机器人的支架上。 注: 此电极用作刺激电极, 并与刺激发生器电连接。</li>
		<li>用右机器人将刺激电极定位在海马片 CA1 区的谢弗络中。 注意:图 7中的位置以示意图表示。</li>
		<li>每三十年代使用刺激发生器将电流脉冲传递给刺激电极 (100 µs 持续时间, 从10µA 开始), 并逐渐增加刺激强度直到出现人口峰值 (PS)。</li>
		<li>调整刺激强度以唤起与可获得的最大振幅的45% 对应的 PS。PSs 被过滤在2.4 赫和数字化在20赫使用信号放大器。</li>
	</ol>
</li>
	<li>
配对脉冲抑制
	<ol>
<li>准备脑切片 (步骤 3.1) 并按照前面的描述进行 (步骤 3.2. 1–3.2 7)。</li>
		<li>使用刺激发生器, 每三十年代交付两个电流脉冲 (100 µs 持续时间在20毫秒间隔) 到刺激电极。设置刺激强度来唤起一个对应于最大振幅的45% 的 PS。</li>
	</ol>
</li>
	<li>
复合测试
	<ol>
<li>使稀释的化合物在毒气 ACSF 中进行测试, 使亚砜的最终浓度不高于0.1%。</li>
		<li>在与复合溶液相同浓度的控制溶液中添加亚砜。</li>
		<li>记录单个 (步骤 3.2) 或配对脉冲 (步骤 3.3) PS 诱发的谢弗间接刺激每三十年代至少30分钟。PS 形状在这个基线期间应该是稳定的。</li>
		<li>准备一个装有含毒气 rACSF 的烧杯, 以测试该化合物的固定浓度。</li>
		<li>灌注海马切片与这个解决方案, 而仍然记录单或配对脉冲 PS。</li>
		<li>通过灌注片与无化合物的加毒气 rACSF 来评价复合效应的恢复。 注: 对于可逆效果, PS 还原为在复合应用程序之前的基线期间观察到的初始形状。</li>
	</ol>
</li>
	<li>
数据分析
	<ol>
<li>使用数据分析软件在4块实验过程中记录的 PS 痕迹平均值。</li>
		<li>测量平均迹线的 PS 振幅。</li>
		<li>将振幅正常化为在灌注化合物之前记录10分钟的基线值的百分比。</li>
		<li>将数据表示为平均值。</li>
	</ol>
</li>
</ol>

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作者弗雷德里克·帕西 Knoflach 和玛丽亚-克莱门的雇员是霍夫曼-罗氏股份公司, 4070 瑞士巴塞尔, 一家制药公司。作者丹尼尔. HiQScreen Sàrl 6, Compois, 1222 Vésenaz 瑞士日内瓦, 为制药公司提供甄别设施的雇员。

在配体-门控离子通道上活性的新型化合物的开发, 如 GABA 受体需要使用多种方法。通常, 第一步通常是使用结合分析进行的;然而, 这种测量不足以描述化合物的生理活动或其精确的药理学。特别是, 与受体结合的化合物可以增强或抑制其功能, 甚至不产生功能性影响 (即, 在不改变其功能的情况下绑定到受体)。因此, 一个完整的复合特性需要一个功能测试。
装订化验: 结合试验的主要优点是, 它可以有效地进行大量的样品, 范围达数以千计, 它为活性分子提供了具有约束力的亲和力。如本文所述, 第一步包括建立一个评估所需受体亚型的方法。也就是说, 虽然结合可以对本地受体从分数或整个大脑, 这样的方法将防止确定的亲和力在特定的受体亚型。因此, 有必要使用重组系统在所需目标上筛选分子。目前, 在细胞系中对所需受体亚单位基因的瞬态或稳定转染提供了一种有效而可靠的方法来表达感兴趣的受体亚型 (如GABAa α5β3γ2)。传统的结合检测利用 radioligands, 但现在的技术可以避免处理放射性的不便 (例如, 定量荧光基配体结合)。
主机系统中的函数表达式: 为了表达宿主系统中的基因, 必须将感兴趣的编码序列插入包含适当助剂的质粒中。通常, 对于体外mRNA 合成, 使用细菌 T7 或 T6 助剂。对于 cDNA 表达, 感兴趣基因的转录必须由真核细胞助剂 (巨细胞病毒) 或等效物来调节。现在, 有几个质粒, 包括两个助剂 (即pUNIV, pCMV-6AC, psf-CMV/T7, pCI-新, pcDNA), 可以允许在体外合成 mRNA 或 cDNA 表达式, 如图 15所示。氨基丁酸受体的表达可以忠实地获得在细胞系或爪蟾卵母细胞。后者的主要优点是缺乏内源性受体表达、操作的简单性以及用于微注射和录音的自动化系统的有效性。本协议中描述的程序说明了自动系统的效率, 允许在大约7分钟内注射96只卵母细胞, 不需要微操作系统技能。记住, 在10,000–30,000 卵母细胞之间的一个单一的卵巢从爪蟾包含, 很明显, 大量的细胞测量可以进行一个单一的准备。此外, 作为一个表达可以使用 cDNA 注射, 相同的质粒含有的基因, 为结合实验开发的兴趣, 可以用于功能表征, 而无需进一步的分子生物学操作。
由于它的大面积和高表面表达, 一个单一的卵母细胞产生了一些受体, 大约相当于35毫米培养皿中的汇合细胞。然而, 卵母细胞的制备和注射是细胞系成本的一小部分, 因为实验不需要特定的培养基和无菌操作。一个单一的 GABA 的激活通道产生一些 picoamperes (10-12) 和电流范围高达十 microampere (10-6) 很容易记录在一个单一的卵母细胞, 这证实了伴随的活动, 数以百万计的受体在一个单一的反应。
在配体-门控离子通道的表征中, 第一步通常是受体的明显亲和性的测定。需要进行的实验包括应用一系列短暂的激动剂测试脉冲和绘制诱发电流的振幅, 或者在某些情况下, 将曲线下的区域作为激动剂浓度对数的函数。由于在同一批卵母细胞中 microinject 不同质粒浓度和比值是很容易的, 这种表达系统特别适合于这种特征。此外, 批对批次的比较显示, 在不同的 EC50s 的高度稳定性。亚单位组成对受体的明显亲和性的影响是容易形象化使用蜘蛛情节, 如图 10所示。
在爪蟾卵母细胞中获得的双电极电压钳的结果往往与使用膜片钳电极的录音进行比较。虽然它将超出这项工作的范围, 深入到这两个系统之间的详细比较, 有几个点可以审查。虽然细胞中的膜片钳为生物物理特性的快速药物应用提供了优势, 但它的要求比爪蟾卵母细胞的双电极记录更复杂。第一个和不可忽略的困难是一个特定的蛋白质的表达与细胞培养的必要性, 瞬变或稳定的转染, 以及表达所需结构的细胞的标识。此外, 研究细胞内源表达的可能贡献也是必不可少的。此外, 膜片钳记录要求有技能的人在高放大显微镜下进行微操作系统, 而该人也需要在实验期间进行充分的分析, 以评估, 例如, 密封的质量,接触电阻等, 双电极电压钳记录, 特别是使用自动电生理系统, 不需要任何具体技能, 可以由实验室技术员进行。
在获得电生理设置时, 成本考虑无疑是需要仔细分析的一个重要因素。例如, 虽然自动化系统的成本相对较高, 但更接近的比较表明, 安装完整的电生理平台包括购买设备, 如良好的并联, 双目透镜, 放大器, 灌注系统, 以及一个反振动表, 同时也获取数据和执行分析。两电极电压钳设置与膜片钳设置之间的成本比较显示出更大的差异。此外, 自动化系统还提供了全自动设备的优点, 日夜无人值守。
化合物的药理性质由受体的作用方式决定。例如, 竞争性抑制剂是分子, 可以进入相同的捆绑口袋, 或 orthosteric 网站, 因为激动剂本身导致竞争。非竞争性抑制剂是抑制受体的化合物, 在另一地点相互作用, 在配体-门控离子通道的情况下, 可以进入和阻断离子孔隙, 例如。这将超出这项工作的范围, 以审查不同的互动机制, 但进一步的信息可以获得从药理教科书和/或其他工作, 如22。
在构调制器的情况下, 化合物绑定在一个与 orthosteric 站点不同的站点上, 但分子的存在改变了活动状态和其他状态之间的能量屏障。正构调制器 (PAMs) 是一种能将能量屏障从静止状态减少到活性态的化合物, 因此增强了激动剂的作用。为了描述可能的 PAM 效应, 因此, 有必要确定接触到化合物是否增强了对激动剂的反应, 如果是这样, 那么浓度。图 11和图 14所示的实验说明了可以成功用于 PAMs 在 GABAA受体上的特性的协议。
在某些情况下, 单独接触 PAM 可能足以激活受体。此类活动可以通过对此处提出的实验协议稍加修改来确定。即, 在接触氨基丁酸的过程中, 不使用调制器的联合应用, 可以对该协议进行修改, 以单独使用该化合物, 然后在 gaba 的存在下进行。直接激动剂活动的特征将由化合物本身诱发的反应幅度的测定和与 GABA 诱发电流的比较来进行。
在脑切片中进行的实验, 如图 7所示, 用于确定抑制回路中的原生受体的复合活动, 然后再进一步的动物模型和沿路径的临床试验。相对简单的数据记录和分析协议, 通过评估其对 PS 振幅的差分调节效应, 可以对多个化合物进行排序。也可以检测与 GABA 系统无关的化合物的非特异效应 (例如, 当观察到 PS 形状的变化时)。直接, GABAergic 神经传递可以评估这些情况下, 通过测量化合物对抑制性突触后电流 (IPSCs) 使用全细胞膜片钳技术8。

在这里, 我们提出的协议, 发现化合物活性在 GABAA受体, 从绑定到生理学和药理学。在寻找特定于氨基丁酸受体的新分子时, 至关重要的是, 尽可能精确地定义感兴趣的亚型和新确定化合物的特异性评估 (例如, 为审查, 见鲁道夫和Knoflach1或 Sieghart2)。图 1说明了药物发现的典型路径和需要实现的步骤。
有约束力的化验已经和仍然是主要用于药物发现的第一步。在氨基丁酸受体的情况下, 它们被优化, 以确定绑定到受体苯二氮结合部位的化合物, 治疗有用和安全的药物绑定。其他技术, 使用荧光成像板阅读器 (FLIPR) 膜电位的红色染料为基础的化验3, 检测化合物, 如巴比妥, 绑定到其他网站, 是不太可取的, 因为他们不需要的副作用的轮廓。此外, 使用的染料可以直接激活 GABA 的受体, 从而质疑这些化验的效用4药物发现。结合化验只能提供证据表明, 某一化合物可以绑定到特定的受体类。用细胞膜进行体外检测, 鉴定选择性人 GABA α5β3γ2受体配位。瞬时转染的 HEK293 细胞表达人 GABAA α5β3γ2, α1β3γ2, α2β3γ2和α3β3γ2受体被用来为这些化验准备膜。对配体的影响通过测量马西尼的 [3h] 的闪烁 (抑制 [3h] 马西尼结合) 来检测。这项技术的主要优点是, 在感兴趣的受体中, 快速有效地测定复合结合的亲和性。
功能性研究对评价化合物的功能活性是至关重要的, 并对化合物与受体的结合所引起的机制提出生理和药理解释。今天, 它是公认的功能 GABAA感受器起因于五个亚基的汇编在由离子孔形成的 pseudosymmetry 的轴附近和结果从五个相同亚基的汇编。大多数 GABAA受体由两个或多个不同的亚基组成。主要大脑 GABAa受体, 例如, 由α1, β2和γ2亚基组成的化学计量学 2, 2 和1分别5,6。在诸如爪蟾卵母细胞或 HEK293 的宿主系统中进行重组, 可以快速探索受体的药理特性。
然后用脑切片7中的胞外记录探讨这些化合物的药理性质。这种方法可以探索化合物对神经传递的影响, 并提供了一个有效的方法来确认在异源表达系统中确定的化合物在本机受体水平上的功能效应, 在整个神经元环境。GABAergic 神经传递还可以通过测量化合物对抑制突触后电流 (IPSCs)8的影响, 在分子水平上进行评估。但是在这里使用的协议和基于全细胞补丁钳录音的脑切片更详细, 并产生较低的吞吐量。
最后, 从不同的技术及其内在的局限性出发, 探讨了体外筛选α5β3γ2的优缺点。这项工作应提供专家和非专家在氨基丁酸受体领域的一个有用的审查结合不同的体外方法, 用于解决新的调制器, 这些配体门控离子通道的发现。

<table><tbody><tr><td>&lt;strong&gt;General equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 well cell harvester -(Filtermate 196)&amp;nbsp;</td><td>Packard</td><td></td><td>To filter membranes with bound radioligand</td></tr><tr><td>Microtiter plate liquid scintillation counter (Top Count NXT)</td><td>Packard</td><td></td><td>Microplate Scintillation and Luminescence counter</td></tr><tr><td>Vial liquid scintillation counter (Beta-Couter 2500 TR)</td><td>Packard</td><td></td><td>Vial Scintillation and Luminescence counter</td></tr><tr><td>Liquid&amp;nbsp; handler (Biomek 2000)</td><td>Beckman coulter</td><td></td><td>To prepare compound dilutions</td></tr><tr><td>Vortex (Vortex Genie 2)</td><td>Scientific industries Inc.</td><td></td><td>To mix compound stock solutions</td></tr><tr><td>Tissue homogenizer (Polytron PT1200E)</td><td>Kinematica AG</td><td></td><td>To resuspend the membrane preparations</td></tr><tr><td>XLfit5 software</td><td>Microsoft Excel&lt;br/&gt; &amp;nbsp;add-on by IDBS</td><td></td><td>Curve fitting software</td></tr><tr><td>Smart Pull</td><td>UniPix</td><td>-</td><td>Electrode Puller</td></tr><tr><td>RoboInject</td><td>MultiChannel Systems</td><td>-</td><td>Automated Injection (Xenopus Oocytes)</td></tr><tr><td>HiClamp</td><td>MultiChannel Systems</td><td>-</td><td>Automated Voltage Clamp (&lt;em&gt;Xenopus&lt;/em&gt; Ooocytes)</td></tr><tr><td>DataMining</td><td>MultiChannel Systems</td><td>-</td><td>Data Analysis Software</td></tr><tr><td>DataMerger</td><td>MultiChannel Systems</td><td>-</td><td>Data Processing Software</td></tr><tr><td>Digidata 1550</td><td>Molecular Devices</td><td></td><td>Low noise data acquisition system</td></tr><tr><td>CyberAmp 380 or equivalent</td><td>Axon Instruments</td><td></td><td>Programmable Signal Conditioner</td></tr><tr><td>pClamp program suite</td><td>Molecular Devices</td><td></td><td>Software for data acquisition and analysis software</td></tr><tr><td>Antivibraton table&amp;nbsp;</td><td>TMC</td><td></td><td>To avoid microelectrode vibrations&amp;nbsp;</td></tr><tr><td>Patchstar Micromanipulators</td><td>Scientifica</td><td></td><td>To accurately position microelectrodes in brain slices</td></tr><tr><td>Faraday Cage&amp;nbsp;</td><td>Sutter Instruments</td><td></td><td>To isolate recordings from noise</td></tr><tr><td>McIlwain Tissue Chopper</td><td>Campden Instruments LTD&amp;nbsp;</td><td></td><td>To prepare brain slices</td></tr><tr><td>Borosilicate glass micropipette&amp;nbsp;</td><td>Warner Instruments</td><td>GC150TF-10</td><td>To record extracellular potentials in brain slices</td></tr><tr><td>Twisted pair, platinum iridium wires stimulation electrode</td><td>World Precision Instruments</td><td></td><td>To stimulate the brain slices with current</td></tr><tr><td>Stimulus generator for current and voltage driven stimulation</td><td>MultiChannel Systems</td><td>STG4000</td><td>Delivers the current to the stimulation electrode</td></tr><tr><td>&lt;strong&gt;Plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microtiter plate round bottom</td><td>Corning</td><td>#3365</td><td>Used for the binding assay</td></tr><tr><td>GF/C glassfibre filter-bottom 96-well microplate with 1.2 &amp;micro;m poresize, for cell harvesting assays using a vacuum manifoldwell filter&amp;nbsp;</td><td>Packard&amp;nbsp;</td><td>#6005174</td><td>used for the binding assay</td></tr><tr><td>50 mL Tubes</td><td>Falcon&amp;nbsp;</td><td>#352070</td><td>used for the binding assay</td></tr><tr><td>Safe-lock tubes 1.5 mL</td><td>Eppendorf</td><td>#0030 120086</td><td>used for the binding assay</td></tr><tr><td>Safe-lock tubes 2 mL</td><td>Eppendorf</td><td>#0030 120094</td><td>used for the binding assay</td></tr><tr><td>Shipping tubes 180 mL&amp;nbsp;</td><td>Semadeni Europe AG&amp;nbsp;</td><td>#3722</td><td>used for the binding assay</td></tr><tr><td>Pony vial Polyethylene 6 mL&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>#6013329</td><td>used for the binding assay</td></tr><tr><td>Top Seal&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>#60051859</td><td>used for the binding assay</td></tr><tr><td>Spinner Flask</td><td>Bellco</td><td>BELLCO # 1965-00100</td><td>Spinner Flask</td></tr><tr><td>96&amp;nbsp; Well Plate (Conical)</td><td>Thermo Scientific Milian</td><td>56368</td><td>Injection plate for the oocytes</td></tr><tr><td>96&amp;nbsp; Well Plate (Flat bottom)</td><td>Corning (Vitaris Switzerland)</td><td>3364-cor</td><td>Compound Plate for the HiClamp</td></tr><tr><td>Glass capillary&amp;nbsp;</td><td>Hilgenberg (Germany)</td><td>-</td><td>borosilicate glass 1.2 O.D. 0.76 I.D. with filament</td></tr><tr><td>&lt;strong&gt;RNA preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ambion mMessage mMachine</td><td>Thermo Fisher Scientific</td><td></td><td>for the &lt;em&gt;in vitro&lt;/em&gt; synthesis</td></tr><tr><td>Chemicals</td><td></td><td></td><td></td></tr><tr><td>Assay buffer: KCl 5 mM; CaCl&lt;sub&gt;2&lt;/sub&gt; 1.25 mM; MgCl&lt;sub&gt;2&lt;/sub&gt; 1.25 mM; NaCl 120 mM; Tris 15 mM pH adjust with HCl to 7.4; store up to 3 months at 4 &amp;deg;C.</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>Washing buffer: Tris 50 mM -HCl pH 7.4; store at 4 &amp;deg;C.</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>Stock solution of test compounds 10 mM in DMSO</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>Flumazenil (RO0151788) 10 mM in DMSO</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>Diazepam 4 mM in DMSO</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>[&lt;sup&gt;3&lt;/sup&gt;H]-Flumazenil 60-85 Ci/mmol in Ethanol; store at -20 &amp;deg;C</td><td></td><td></td><td>Used for binding assay</td></tr><tr><td>Microscint 40&amp;nbsp;</td><td>Packard&amp;nbsp;</td><td>#6013641</td><td>Used for binding assay</td></tr><tr><td>Ultima Gold&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>#6013329</td><td>Used for binding assay</td></tr><tr><td>MS-222</td><td>Sigma</td><td>Sigma # A5040</td><td>MS-222</td></tr><tr><td>AB/AM</td><td>Sigma</td><td>Sigma # A5955</td><td>antibiotics / antimycotics</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>Sigma # C1030</td><td>Collagenase Type I</td></tr><tr><td>Rnase</td><td>Sigma</td><td>Sigma # R2020-250 mL</td><td>RNaseZAP</td></tr><tr><td>EndoFree Plasmid maxi Kit</td><td>Qiagen</td><td>Qiagen # 12362</td><td></td></tr><tr><td>Salts for artificial cerebrospinal fluid (NaCl, KCl, etc.)</td><td>Sigma</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Membranes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Frozen membrane preparations from transient or stably transfected HEK293 overexpressing different human GABA&lt;sub&gt;A&lt;/sub&gt; receptor subtypes: &amp;alpha;1&amp;beta;3&amp;gamma;2, &amp;alpha;2&amp;beta;3&amp;gamma;2, &amp;alpha;3&amp;beta;3&amp;gamma;2, &amp;alpha;5&amp;beta;3&amp;gamma;2. See Ballard &lt;em&gt;et al.&lt;/em&gt; 2009 for detailed methods.</td><td></td><td></td><td>Used for binding assay</td></tr></tbody></table>

methods for the discovery of novel compounds modulating a gamma-aminobutyric acid receptor type a neurotransmission

这篇手稿提出了一个分步的协议, 筛选化合物在伽玛-丁酸 a (GABAa) 受体和它的用途, 以鉴定新分子活性的临床前化验方法从体外重组受体对他们的药理作用在本机受体在啮齿动物脑切片。对于受体的苯二氮部位的化合物结合, 第一步是建立一个主要的屏幕, 其中包括发展放射配基结合化验的细胞膜表达主要 GABAa亚型。然后, 利用鼠和人 GABA 的异种表达, 在 爪蟾卵母细胞或 HEK 293 胞内的受体, 可以在电生理检测中探讨不同受体的生理特性亚型和所鉴定化合物的药理性质。爪蟾卵母细胞系统将在这里提出, 从分离的卵母细胞和他们的显微注射与不同的基因, 由药理学表征使用双电极电压钳。最后, 在啮齿类动物脑切片中进行的录音将被描述为一种二次生理试验, 用于评估在定义良好的神经元回路中的分子在其本机受体中的活性。利用多神经元的种群反应进行细胞外记录, 并与药物应用相结合。

Watch this Scientific Journal Video about Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission at JoVE.com

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

在这里, 我们提出的协议, 以发现活性化合物在 GABAA受体, 从绑定到生理学和药理学。

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

methods-for-discovery-novel-compounds-modulating-gamma-aminobutyric

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13.5K 次观看.  Roche Innovation Center Basel. 在这里, 我们提出的协议, 以发现活性化合物在 GABAA受体, 从绑定到生理学和药理学。

视频: Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

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

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

在大鼠脑片的单通道和补品GABA激活电流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

生物化学

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA<sub&gt;一个</sub&gt;受体

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

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 ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

一种基于板的测定法，用于测量急性脑切片中内源性单胺释放

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

摘要

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此视频中的章节

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

在大鼠脑片的单通道和补品GABA激活电流

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

谷氨酸和GABA快速微离子导入：一个有用的工具，调查突触整合

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA

粉扑诱导的全细胞电流在脑切片中的应用

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

一种基于板的测定法，用于测量急性脑切片中内源性单胺释放

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

摘要

探索更多视频

此视频中的章节

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

在大鼠脑片的单通道和补品GABA激活电流

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA

粉扑诱导的全细胞电流在脑切片中的应用

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

一种基于板的测定法，用于测量急性脑切片中内源性单胺释放

谷氨酸和GABA快速微离子导入：一个有用的工具，调查突触整合