マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現
Summary
この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。
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
そのようなLSD、サイロシビンやメスカリンなどの幻覚剤は、人間の意識、認知と感情7-9に大きな変化を引き起こします。セロトニンの不活性化は、遺伝的または薬理学的のいずれかの方法により、5-HT 2A受容体のシグナル伝達は、両方の齧歯類モデル3,10、およびヒト11で幻覚剤に著しく減衰行動反応を引き起こします。幻覚剤は、他の受容体サブタイプ8に結合するが、5-HT 2A受容体は、これらの化学物質の固有の行動活性に必要と考えられています。
グループII代謝型グルタミン酸受容体（ すなわち 、mGlu2およびmGlu3）は幻覚剤の分子機構に関するかなりの注目の対象と精神病12の基礎となる彼らの重要な役割となっています。これまでは、mGlu2タンパク質の発現なしでのマウス（mGlu2-KOマウス）はHALの細胞や行動への影響に鈍感であることが実証されていますlucinogens 5。また、5-HT 2AおよびmGlu2受容体は、セロトニンおよびグルタミン酸リガンドが生細胞1,2のGタンパク質結合のパターンを調節し、それを通して、特定のヘテロマー複合体を形成することが示唆されています。
構造的には、膜貫通（TM）ドメイン4およびmGlu2の5は、5-HT 2A受容体5とヘテロマー形成に重要な役割を果たしています。さらに、さらなる調査がmGlu2のTM4の細胞内の端部に位置する3つの残基が、細胞を6生体内5-HT 2A -mGlu2レセプターヘテロを形成するのに必要であることを実証しました。
異種発現系で観察されたこれらの知見に基づいて、ここでは、5-HT 2Aとの間のヘテロマー形成するかどうかをテストするために、野生型mGlu2およびmGlu2-KOマウスの前頭皮質におけるmGlu2 / mGlu3キメラ構築物のHSV媒介表現の使用を記載していますそして、mGlu2はのために必要です幻覚5-HT 2A受容体アゴニストによって誘導される頭部攣縮挙動。
Protocol
Representative Results
Discussion
一緒にmGlu2-KOマウス5の前の所見と、培養細胞において5-HT 2A -mGlu2受容体複合体を形成しないmGlu2およびmGlu2 / mGlu3キメラ構築物を用いた結果は、5-HT 2A -mGlu2ヘテロマー受容体複合体ことを示唆していますマウスの前頭皮質は、LSDのような幻覚5-HT 2A受容体アゴニストによって頭部攣縮挙動を誘発するために必要とされます。この方法の制限は、それが天然の組織内…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894は、この研究の資金調達に参加しました。我々は、博士に感謝したいと思います。マウスの寄付と、この作品の撮影時に、その手術や行動施設の利用のためのマウントサイナイ医科大学でヤスミンHurd氏とスコット・ルッソ。
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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