蜜蜂匀浆中磷脂酶 C 活性的检测
Summary
为了检验药理学药物对蜜蜂脑不同区域磷脂酶 C (plc) 的抑制作用, 我们提出了一种生化检测方法来测量这些地区的 plc 活性。该方法可用于比较组织中的 PLC 活动, 以及不同行为的蜜蜂之间的对比。
Abstract
蜜蜂是一种评估复杂行为和更高的大脑功能的模型有机体, 如学习、记忆和分工。蘑菇体 (MB) 是一个更高的大脑中心, 被建议成为复杂蜜蜂行为的神经基质。尽管以前的研究发现了在 MBs 和其他脑区表达差异的基因和蛋白质, 但每个区域的蛋白质的活动尚未完全理解。为了揭示这些蛋白质在大脑中的作用, 药理学分析是一种可行的方法, 但首先必须确认药理操作确实改变了这些脑区的蛋白质活动。
我们以前发现在 MBs 中, 编码磷脂酶 C (PLC) 的基因的表达比其他脑区更高, 药理评估了 PLC 参与蜜蜂行为的情况。在这项研究中, 我们生化测试了两个药理剂, 并证实他们降低了在 MBs 和其他大脑区域的 PLC 活动。在此, 我们详细介绍了如何检测蜜蜂脑匀浆中的 PLC 活性。在该检测系统中, 由不同脑区产生的组织匀浆与合成荧光基板反应, 并对由 PLC 活动引起的荧光进行量化, 并对脑区进行比较。我们还描述了我们对某些药物对 PLC 活动的抑制作用的评估, 使用相同的系统。虽然该系统可能受到其他内源荧光化合物和/或测定成分和组织的吸收, 使用该系统的 PLC 活动的测量比使用传统的检测更安全和容易, 这需要核素基板。通过简单的程序和操作, 我们可以检查在不同社会任务中参与的蜜蜂大脑和其他组织中的 PLC 活动。
Introduction
欧洲蜜蜂 (api 蜜蜂L) 是一种群居昆虫, 雌性蜜蜂显示种姓依赖的生殖和年龄依赖性的分工。例如, 在被称为 “工人” 的蜜蜂的无菌种姓中, 年轻的个体喂养巢, 而年长的人则在蜂巢1外觅食花蜜和花粉。学习和记忆能力在蜜蜂的生命中至关重要, 因为觅食必须反复往返于食物来源和巢穴之间, 然后通过舞蹈将好的食物来源的位置传达给他们的 nestmates。通信1。以前的研究表明, MB, 一个更高的大脑中心, 昆虫, 参与的学习和记忆能力的蜜蜂2,3,4。不同的表达基因和蛋白质已经在蜜蜂的各个脑区被发现5,6,7,8,9,10 ,11, 表明它们与每个脑区的独特功能有关。虽然药物的药理抑制或活化蛋白的兴趣是一个良好的方法来揭示蛋白质的功能在蜜蜂行为12,13,14, 它是未知的是否所有药物在蜜蜂大脑的不同区域有功能效应。这种药物的功能验证将加强行为药理学研究的结论。
在这里, 我们专注于 PLC, 其中一项涉及老鼠认知的酶15,16,17,18。PLC 通过将磷酸肌醇 45-bisphosphate (PIP2) 降解为肌醇14、5-trisphosphate (IP3) 和甘油 (达格·哈马舍尔德)19、20、21, 从而触发钙信号。ip3在内质网 (er) 上打开 ip3受体, 导致钙离子从 ER 中释放出来。释放钙激活钙/钙调素依赖性蛋白激酶 II (CaMKII) 与钙调素和蛋白激酶 C (PKC) 在存在的达格·哈马舍尔德。两种蛋白激酶都参与学习和记忆22,23, 符合 PLC 参与这个过程。PLCs 分为子类型, 包括 PLCβ、PLCγ和 PLCε, 其结构为20。每个 PLC 子类型在不同的上下文20中激活, 编码这些亚型的基因在不同的组织中表现出差异。我们以前证明, 蜜蜂 MBs 表达基因编码 PLCβ和 PLCε亚型在较高的水平比其余的大脑区域24, 和两个 pan-PLC 抑制剂 (edelfosine 和新霉素硫酸 [新霉素]) 降低 plc 活动不同的大脑区域, 事实上, 影响蜜蜂的学习和记忆能力24。
传统上, PLC 的酶活性已被测量使用核素 PIP225, 这需要适当的培训, 设备和设施。目前, 已建立了 plc 的合成荧光基板26, 便于对标准实验室的 plc 活动进行评估。在这里, 我们提出了一个详细的协议, 以检测在不同的大脑区域的蜜蜂使用荧光基质, 并随后测试 edelfosine 和新霉素对 plc 在这些组织的抑制效应。因为该协议只需要基本的操作, 它可能适用于对不同的社会任务分配给蜜蜂的其他组织或脑区的 PLC 活动的研究。
Protocol
Representative Results
Discussion
蛋白质活性的生化检验对于理解大脑中的分子信号是非常重要的, 因为酶的活性受各种分子 (如基质和抑制剂) 的影响, 因此可以随动物行为 (例如, 学习和记忆)5。在蜜蜂研究中, 据报道, 在不同的脑区, 基于循环安培依赖性蛋白激酶 A、循环 GMP 依赖性蛋白激酶、PKC、磷酸化 CaMKII 和腺苷酸酶的酶有差异表达。免疫组化5,10,<s…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
图 4B – 4D从 Suenami等修改。24以生物开放的允许。作者感谢出版商的许可。这项工作得到了人类前沿科学计划 (RGY0077/2016) 的支持, 索塔 Suenami 和宫崎骏。
Materials
| Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23227 | The reagent kit for measurement of protein concentration |
| Pierce Bovine Serum Albumin Standard Ampules 2mg/mL | ThermoFisher Scientific | 23209 | The standard samples used in BCA assay |
| Paraffin wax | GC | 13B1X00155000141 | Dental wax used as dissection stage |
| Insect pin | Shiga | No. 0 | Stainless, solid head |
| PLCglow | KXT Bio | KCH-0001 | A fluorogenic substrate of PLC |
| 384-well microplate | Corning | 4511 | Low-volume, round-bottom plate in black color |
| Gemini EM microplate reader | Molecular Devices | ||
| Edelfosine | Santa Cruz Biotechnology | sc-201021 | pan-PLC inhibitor |
| Neomycin sulfate | Santa Cruz Biotechnology | sc-3573 | pan-PLC inhibitor |
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