作者要感谢毕业后的学者计划办公室和诺华医学研究所作为一个整体为这项研究提供资金。

1. 细胞的制备
注意: 这个协议, 包括数据生成, 使用 HEK293 细胞转基因与杆状病毒编码 NR1 和 NR2A 细胞。
<ol><li>种子适当数量的 HEK293 细胞, 并添加 NR1 和/或 NR2A 病毒在适当的最终浓度 (1.00 µL 每个)。对于一个384井板, 使用1万细胞/以及在最后的体积30µL。</li>
	<li>或者, 种子 HEK293-NR1-NR2A 细胞在适当的细胞数量和容量 (为384井板材, 使用1万个细胞/井在最后容量30µL)。添加四环素在2µg/毫升的浓度, 以诱导 NR2A 表达, 并添加保护化合物 (100 µM MDL105,519 或5µM CGP07066724)。</li>
	<li>注意: 杆状病毒编码 NR1 和 NR2 应该有一个近似的效价之间的 5 x 109 -10 x 109感染单位每毫升24。</li>
</ol>2. NMDA 受体活性的测定
<ol><li>在任何保护性化合物的存在下, 用 HEK293 细胞转基因与 NR1/NR2 或 HEK293-NR1-NR2A 细胞一起制备384井板。使用透明底, 黑色框架, 聚 d-赖氨酸涂层板。 注: 保护与100µM MDL105,519 是用来赋予对甘氨酸的敏感性, 而5µM CGP070667 用于赋予谷氨酸的敏感性。</li>
	<li>在37摄氏度和 5% CO2上孵育盘子16小时 (隔夜)。</li>
	<li>将盘子从孵化器中取出, 并通过减慢翻转板在兼容的 biowaste 容器上轻轻地丢弃细胞介质。在纸巾上轻轻地敲击盘子, 将介质从盘子边缘清除, 并排出剩余的介质。</li>
	<li>在10毫升的孵化缓冲器 (HBSS pH 7.5、20 mm HEPES、1 mm 氯化镁2、1 mm 丙磺舒) 中, 将一小瓶 calcium6 染料 (1 片尺寸) calcium6-dye。在37摄氏度和 5% CO2上孵化2小时的板材。</li>
	<li>从孵化器中取出盘子, 让细胞调整到室温10分钟。</li>
	<li>轻轻地倒出 calcium6-dye, 如步骤2.3 所述, 并添加30µL 的化验缓冲器 (HBSS pH 7.5, 20 毫米 HEPES, 1.8 毫米 CaCl2, 1 毫米丙磺舒)。</li>
	<li>重复步骤 2.6, 共3次洗涤。最后一次清洗后, 在细胞上留下30µL 的化验缓冲器。</li>
	<li>让细胞在室温下休息5分钟。</li>
	<li>制备配体板, 使4倍的库存400µM 甘氨酸和400µM 谷氨酸 (最终浓度100µM; 配位的最佳浓度应确定如下所述)。将至少25µL 的配体料转移到 V 底384井板中。</li>
	<li>将配体板和细胞板装入 FDSS (功能性药物筛选系统)。</li>
	<li>测量电池板的基线荧光 (Fo) 30 秒。用 FDSS 配药功能将配体的10µL 转移到细胞板中, 通过记录 calcium6-dye 荧光量5分钟来测量钙流量。</li>
	<li>通过将基线荧光 (f最大/fo) 获得的最大荧光划分, 计算最大荧光比。</li>
</ol>3. NMDAR 表达水平的优化
<ol><li>要确定使用的杆状病毒的最佳数量, 执行 NR1 和 NR2A 病毒的滴定。准备一个有 HEK293 细胞的384井板 (推荐1万个细胞/好, 30 µL 介质), 并包括步骤2.1 所述的保护化合物。</li>
	<li>在板块的不同行中添加 NR1 病毒的数量变化 (例如: 分别添加0µL、2µL、1µL、0.5 µL、0.25 µL)。为数据准确性添加重复项。</li>
	<li>在板块的不同列中添加不同数量的 NR2A 病毒 (例如: 分别添加0µL、2µL、1µL、0.5 µL、0.25 µL)。为数据准确性添加重复项。</li>
	<li>在37摄氏度和 5% CO2上孵育盘子16小时 (隔夜)。</li>
	<li>通过在 FDSS 上执行钙通量测量, 确定 NR1 和 NR2A 病毒的最佳比率和数量 (见上文)。</li>
</ol>4. 配体滴定法
<ol><li>按照步骤2.3 中的说明准备单元格。</li>
	<li>在其他配体的饱和浓度 (100 µM) 的存在下, 制备每个配体的稀释, 作为4倍的库存溶液。例如: 对于甘氨酸的10µM 最终测试浓度, 制备40µM 的甘氨酸溶液, 包括400µM 谷氨酸。</li>
	<li>按照步骤2.11 中的说明进行 FDSS 测量。</li>
</ol>5. 复合测试
<ol><li>按照步骤1中的说明准备单元格。</li>
	<li>在检测缓冲器中制备配体板, 其最终配体浓度为5倍。</li>
	<li>在检测缓冲器中, 用所需的最后浓度的4倍的库存准备化合物板。对于最终的化合物浓度为10µM 在化验缓冲器, 准备一个40µM 的库存解决方案。<ol>
<li>将二甲基亚砜 (亚砜) 常数的浓度保持在所有样品中, 方法是在亚砜中制备预稀释的化合物, 然后再稀释成测定缓冲器。</li>
		<li>对于化合物在3至30µM 的最终测定中的剂量反应, 在亚砜中制备化合物的预稀释, 从1到10毫米不等。稀释那些在化验缓冲器到4倍的存货浓度。亚砜的最终浓度不应超过0.5%。 注意: 建议在 triplicates 中测试每个样本。</li>
	</ol>
</li>
	<li>将配体、复合物和细胞板装入 FDSS。</li>
	<li>测量基线荧光30秒。</li>
	<li>添加10µL 的复合库存, 并测量荧光5分钟。</li>
	<li>添加10µL 的配体股票, 并测量荧光5分钟。</li>
	<li>在测量的最后5分钟内, 通过计算荧光比曲线下的面积来确定荧光比的积分。 注: 另外, 在配体添加后, 荧光的最大比值可用于分析。</li>
</ol>

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作者没有利益冲突, 也没有什么可透露的。

这种化验的成功主要取决于使用的 HEK 细胞的健康状况。需要使用指数增长和低通道数的细胞。这种分析涉及许多转移和补充的解决方案, 所以使用谨慎将确保更高的准确性, 其结果。化合物和所有其他试剂的浓度也应进行交叉检查, 以尽量减少错误。当用测定缓冲液取代细胞培养基进行钙通量测定时, 应轻轻倾斜板, 使细胞不分离。最后, 在本协议中使用聚 d-赖氨酸涂层板增加了细胞对板的附着。
根据 HEK293 细胞在转导时的通道数和适应性, NMDAR 表达和亚单位表达所需的病毒数量可能存在差异。此外, 杆状病毒的保质期短, 从六月到一年不等。因此, 病毒的质量是该检测的一个重要参数。在进一步实验24之前, 标准免疫荧光检测可用于检测和确认 NMDAR 单位的表达水平和定位。其他细胞系可用于研究 NMDA 受体使用该协议。然而, 使用杆状病毒的 NMDARs 的耐受性和表达必须在继续执行该协议之前得到确认;否则, 可能会发生意外的结果。此外, 据报道, 拓扑异构酶 II 抑制剂等化合物提高了杆状病毒表达29的功效。这些替代方案可以根据用户的喜好进行测试和优化。
与原细胞类型不同的细胞类型的生物过程研究在科学界引起问题。本试验的局限性是利用肾细胞系 (HEK 293) 作为模型系统研究离子型谷氨酸受体, NMDAR。HEK 细胞因其偏振光膜电位与偏振光神经元相似而被使用。此外, 我们的补充研究证实了 NMDARs 在大脑和模型中的相似性。积极的构调制器的活动, 如 GNE-8324, 负调节剂如 ifenprodil, 和渠道阻滞药, 如氯胺酮, 以及 NMDAR 的电压依赖性, 都证实了我们的模型22,23 ,24。此外, 我们调整 NR1 和 NR2A 亚基病毒数量的能力确保了最佳的功能表达水平。
由于效率低、成本高、成功率极小, 目前在低吞吐量的手工膜片钳分析22、23中对单个受体及其独特药理学进行了表征。另一方面, 当使用稳定转染的细胞时, 兴奋仍然是一个主要障碍, 因为功能性 NMDARs 的过度表达对于被释放到培养基18中的配体 (谷氨酸和甘氨酸/d-丝氨酸) 对细胞有害。作为一个更强的替代品, 我们的检测采用杆状病毒介导的功能 NMDARs 表达, 克服兴奋和产生快速, 滴定表达的受体。这种检测方法不受通道阻滞剂粘滞性的限制, 也可以利用弱配体结合部位的拮抗剂。这些拮抗剂与培养基内的内源配体竞争, 保护配位结合部位。因此, 在拮抗剂被清除后, 对谷氨酸和甘氨酸/d-丝氨酸结合部位的外部添加配体的敏感性恢复。
这种检测结合了 CRISPR 等分子生物学的最新进展, 将有助于对 NMDAR 表达和功能的新调控者进行靶向和表征。同时, 这项化验将有助于识别小分子, 它们可能直接或间接调节 NMDAR 活动, 而不需要产生稳定的细胞系, 从而提供与遗传和小分子的屏幕的兼容性。

随着目前医学的进步, 预期寿命显著增加;然而, 与年龄相关的疾病的流行率也是如此。中枢神经系统疾病 (CNS), 如精神分裂症, 肌萎缩侧索硬化症, 阿尔茨海默氏病, 帕金森病等, 也不例外, 并预计将增加在未来十年1,2,3. 被称为 n-甲基-d-天门冬氨酸受体 (NMDAR) 的离子型谷氨酸受体的故障与阿尔茨海默病、精神分裂症、外伤性脑损伤、中风、糖尿病和青光眼等有联系, 这就保证了需要研究他们的生物学为发展有效, 疾病修改疗法4,5,6,7。
NMDARs 由四单体或亚基4,8,9组成。NMDAR 的结构构成在脑子7,10显示发展和区域可变性。NMDARs 参与突触可塑性, 认知和节奏的世代为呼吸和运动11,12,13。作为电压门控通道, 它主要是在静止膜电位 (-70 mV) 不导电, 并被镁堵塞, 以防止离子进一步渗透。该通道是由两个配体, 谷氨酸和甘氨酸/d-丝的结合激活, 并在突触膜 AMPA 受体介导的, 另一子类离子型谷氨酸受体的同时去极化。退极化去除 NMDAR 镁堵塞, 使阳离子的汇集, 特别是钙14,15,16。尽管激活 NMDAR 是细胞存活的必要条件, 但过度活化可能导致细胞死亡17,18,19通过兴奋。这, 除了受体的复杂性质, 使其具有挑战性的研究开发有效的治疗所必需的。
对 NMDAR 的研究开发了不同的方法。然而, 每一个都有相应的警告。例如, 一种广泛使用的技术是一种基于荧光的检测方法, 用于测量在四环素诱导启动子 (NMDAR) 控制下稳定细胞系内细胞内钙的变化.然而, 在这个系统中, 所需配体的 supramaximal 浓度和 NMDAR 抑制剂在测量过程中存在的要求, 几乎不可能检测到竞争对手的活动。在其他类似的系统中, 功能受体的表达导致毒性, 要求通道阻滞剂, 如氯胺酮21,22保存细胞培养。这些通道阻滞剂坐在受体的核心, 很难洗出来, 尤其是以平板为基础的格式, 所以他们干扰的功能研究的受体。最后, 在电生理测量, 如补丁夹紧, 有有限的吞吐量, 和大规模的研究是非常昂贵的23。尽管如此, 上述系统对甘氨酸刺激不敏感;因此, 研究 NMDAR 的甘氨酸依赖性活动成为一项挑战。
在这里, 我们描述了一个新的方法来研究 NMDAR, 克服了讨论的局限性。我们的技术是利用杆状病毒表达系统来表达受体在功能水平上的最佳比例的亚基在16小时。此外, 杆状病毒的使用允许一种简单的组合方法, 它提供了一个广泛的描述不同的重组 NMDAR 亚型。与其他化验不同, 该协议不需要通道阻滞剂, 因为使用弱拮抗剂。该方法的最大优势是, 在弱拮抗剂的冲刷后, 受体对个体甘氨酸和谷氨酸结合部位的调制敏感, 除了甘氨酸/d-丝氨酰胺和谷氨酸配体结合的双重调制网站。该检测概括已知的 NMDAR 受体药理作用及其已知的阳性和阴性调节剂的影响。最后, 这种体外细胞检测的产生克服了过量钙流入引起的细胞毒性, 允许高通量的受体功能研究, 从而加速 NMDAR 调制器的发现。在疾病状态。

<table><tbody><tr><td>HEK-293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>Human NMDA (NR1/NR2A) Receptor Cell Line</td><td>ChanTest Corporation</td><td>CT6120</td><td></td></tr><tr><td>pFastBac Dual Expression Vector</td><td>ThermoFisher Scientific</td><td>10712-024</td><td></td></tr><tr><td>Corning 384-well Clear Flat Bottom Microplate</td><td>Corning Life Sciences</td><td>3844</td><td></td></tr><tr><td>FLIPR Calcium 6-QF Assay Kit</td><td>Molecular Devices</td><td>R8192</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>49621</td><td></td></tr><tr><td>D-serine</td><td>Sigma-Aldrich</td><td>S4250</td><td></td></tr><tr><td>L701,324</td><td>Tocris</td><td>907</td><td></td></tr><tr><td>HEPES Buffer</td><td>Boston Bio Product</td><td>BB-103</td><td></td></tr><tr><td>Magnesium Chloride Solution</td><td>Sigma-Aldrich</td><td>63069</td><td></td></tr><tr><td>Calcium Chloride</td><td>VWR</td><td>E506</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Probenecid</td><td>ThermoFisher Scientific</td><td>P36400</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX media</td><td>ThermoFisher Scientific</td><td>10565018</td><td></td></tr><tr><td>MDL105,519</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>NVP-AAM077</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>CGP070667</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr></tbody></table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

n-甲基-d-天门冬氨酸 (NMDA) 受体 (NMDAR) 被归类为离子型谷氨酸受体, 在学习和记忆中具有重要作用。NMDAR 的故障, 表现为过度或活动不足造成的突变, 改变表达, 贩运, 或本地化, 可能导致许多疾病, 特别是在中枢神经系统。因此, 了解受体的生物学以及促进发现化合物和小分子, 对于目前防治神经系统疾病的努力至关重要。目前研究该受体的方法有限制, 包括低吞吐量, 高成本, 并无法研究其功能能力, 由于必要存在的渠道阻滞剂, 以防止 NMDAR 介导的兴奋。此外, 现有的检测系统只对谷氨酸的刺激很敏感, 对 NMDAR 的另一种共配体甘氨酸的刺激缺乏敏感性。在这里, 我们提出了第一个以板块为基础的高通量的检测方法, 研究一种对同配体、谷氨酸和 d-丝氨酸/甘氨酸有敏感性的 NMDA 受体。这种方法允许研究不同的 NMDAR 亚基组成, 并允许功能研究的受体在甘氨酸和/或谷氨酸敏感模式。此外, 该方法不需要在测量过程中存在抑制剂。通过本试验可以检测到正、负构调节剂的作用, 并在本系统中复制了 NMDAR 的已知药理学。该技术克服了现有方法的局限性, 具有很高的成本效益。我们相信, 这项新的技术将加快发现治疗 NMDAR 介导的病理。

在测试小分子的影响之前, 你必须确定 NMDARs 的最佳表达水平以及最佳配体浓度。如所述, 在384井板中, HEK293 细胞每井1万个细胞, 在5µM CGP060667, 然后转基因的 NR1 和 NR2A 病毒数量不等。在一夜之间孵化后, 配体诱导的钙通量被测量 (图 1)。这些实验的结果显示了每一个亚基使用的病毒的最佳数量和比率 (图 1, 黄色盒)。
确定了 NR1 和 NR2A 转导的最佳用量和配比后, 采用配体滴定法测定 NMDARs 活化的最佳配体浓度。为了达到这一目的, 在固定的、饱和浓度的其他 co 配体 (图 2A-2B) 中添加了一个配位体。类似的结果也获得了其他亚单位组合的 NMDAR, 如 NR1/NR2B 和 NR1/NR2D (图 2C), 表明该检测的适用性, 其他 NMDAR 家庭成员24。在确定最佳表达水平和配体浓度后, 可以对 NMDAR 调节剂在测定中的作用进行测试。
与已发表的报告一致, 这一以平板为基础的试验研究 NMDARs 对甘氨酸和谷氨酸的敏感性, 在大尺度上再现了 NMDAR14,25,26的已知属性的数据。在这里, 我们使用两个化合物的活动, [(R)-[(S)-1-(4-溴苯基)-乙氨基]-(23-dioxo-12, 34-tetrahydroquinoxalin 5 基) 甲基] 膦酸 (NVP-AAM077), 谷氨酸部位拮抗剂, [7-氯-4-羟基-3-(3-苯氧基)苯基-2 (H) 喹诺酮) (L701,324), 甘氨酸站点拮抗剂, 以例证27,28的预测结果 (图 3)。最后, 对每个化合物进行了试验, 在 EC80 各自的天然配体的结合点。配体浓度的降低预期会增加拮抗剂的效力, 而预期对非竞争性抑制剂或孔隙阻滞剂的效力没有影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58160/58160fig1.jpg"> 图 1: NR1 和 NR2A 亚基的滴定法.不同数量的病毒编码 NMDAR 亚基 NR1 和 NR2A 是滴定, 以确定所需数量的最佳功能活动的受体。1:1 (v/v) 比 NR1:NR2A 被观察作为最佳的比率与1µL 每 (黄色箱子)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58160/58160fig2.jpg"> 图 2: 配体滴定法.a. 在固定浓度的甘氨酸 (100 µM), 不同的谷氨酸浓度滴定和钙通量记录。误差线表示标准偏差。谷氨酸保持在100µM 的浓度, 滴定对不同浓度的 d-丝氨酸和甘氨酸, 并测定钙通量。甘氨酸钙通量与 d-丝氨酸之间无显著差异。误差线表示标准偏差。用100µM 谷氨酸、d-丝氨酸或100µM d-丝氨酸的不同浓度、不同浓度的谷氨酸和钙通量来刺激细胞转基因 NR1 和 NR2D 杆状病毒。误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58160/58160fig3.jpg"> 图 3: 配体结合部位拮抗剂的表征活性.利用预定的最佳配体和亚单位表达式, 对谷氨酸结合部位拮抗剂、NVP-AAM077 和甘氨酸结合部位拮抗剂 L701,324 的影响, 以 NR1/NR2A 的钙通量测定为特征。浓度。在加入 NVP-AAM077 和 L701,324 后, 在其他配体的饱和浓度 (100 µM) 的存在下, 浓度等于甘氨酸或谷氨酸的 EC80。误差线表示标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

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

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

本议定书的目的是促进对 NMDA 受体 (NMDAR) 的研究, 使之更大规模地进行, 并允许检查小分子的调节作用及其治疗应用。

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

a-high-throughput-calcium-flux-assay-to-study-nmda-receptors-with

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.2K 次观看.  Novartis Institutes for BioMedical Research. 本议定书的目的是促进对 NMDA 受体 (NMDAR) 的研究, 使之更大规模地进行, 并允许检查小分子的调节作用及其治疗应用。

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

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

谷氨酰胺通量成像使用基因编码的传感器

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

科研

生物工程

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

神经科学

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

生物化学

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

血液中 n-甲基-d-天门冬氨酸 (NMDA) 受体自身抗体的简易细胞免疫荧光检测

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...