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
Summary
本文介绍如何注入病毒载体插入鼠标额叶皮质测试需要GPCR异形成行为检测。
Abstract
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 mGlu2ΔTM4N (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.
Introduction
致幻剂，如LSD，裸盖菇碱和麦司卡林引起人的意识，认知和情感7-9显著的变化。无论是由遗传或药物的方法羟色胺5-HT 2A受体信号的失活会导致明显减弱，以致幻剂的行为反应在两种啮齿类动物模型3,10和人类11。虽然致幻剂结合其他受体亚型8，对5-HT 2A受体被认为是必要的这些化学物质的独特的行为活动。
II组代谢型谷氨酸受体（ 即 ，mGlu2和mGlu3）一直是相当大的注意关于致幻剂的分子机制和其底层的精神病12整体作用的目标。以前，已经显示，与mGlu2蛋白（mGlu2-KO小鼠）中不表达小鼠是不敏感的HAL的细胞和行为效应lucinogens 5。还已经提出，对5-HT 2A和mGlu2受体形成一个特定的异聚复合物，通过该血清素和谷氨酸的配体调节在活细胞中的1,2- G蛋白偶联的图案。
在结构上，跨膜结构域4和mGlu2的5异的形成中发挥基础性作用与5-HT 2A受体5。此外，进一步的研究表明，三个残基位于mGlu2的TM4的细胞内端是必要的，以形成在活细胞6对5-HT 2A -mGlu2受体heterocomplex。
基于这些发现在异源表达系统中观察到，在这里，我们描述了使用野生型mGlu2和mGlu2-KO小鼠的额皮质mGlu2 / mGlu3嵌合构建的HSV介导的表达来测试是否5-HT 2A之间异形成和mGlu2是必要的通过致幻5-HT 2A受体激动剂诱导头肌的行为。
Protocol
Representative Results
Discussion
连同mGlu2-KO小鼠5以前的研究结果，与mGlu2和mGlu2 / mGlu3嵌合构建的结果不形成在培养的细胞对5-HT 2A -mGlu2受体复合表明对5-HT 2A -mGlu2在异聚受体复合需要鼠标额叶皮层由LSD样致幻5-HT 2A受体激动剂诱导头部抽动行为。这种方法的一个限制是，它不会在天然组织亚细胞水平测量接近分子的接近。此外，也有应注意各种临界点。因为将小鼠用HSV病毒载体注射，即要执行的实…
Divulgations
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894参与这项研究的经费。我们想感谢博士。优思明赫德和斯科特·鲁索在西奈山医学院学校小鼠的捐赠，这项工作的拍摄过程中使用其手术和行为的设施。
Materials
| mGlu2 bicitronic herpes simplex virus (HSV) vector | MIT Core | mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu) | |
| mGlu2ΔTM4N bicitronic herpes simplex virus (HSV) vector | MIT Core | mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu) | |
| GFP bicitronic herpes simplex virus (HSV) vector | MIT Core | mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu) | |
| xylazine | Lloyd | List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10 | 1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution |
| ketamine | Vedco | KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06 | 1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution |
| ophthalmic gel | Fisher Scientific | NC0550805 | |
| burret clips | Fisher Scientific | NC9268369 | |
| Feather surgical blade | Fisher Scientific | NC9032736 | |
| Hydrogen Peroxide | Fisher Scientific | 19-898-919 | |
| Hamilton syringe | Fisher Scientific | 14815203 | |
| Hamilton™ Small Hub Removable Needles (33 Ga) | Fisher Scientific | 14816206 | |
| Cordless Micro Drill | Fisher Scientific | NC9089241 | |
| Dermabond Dermal Adhesive | Fisher Scientific | NC0690470 | |
| (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) | Sigma-Aldrich | 42203-78-1 | Dissolved in .9% saline solution to the concentration of 2.0 mg/kg |
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