키메라를 구축의 HSV-중재 형질 전환 유전자의 발현은 마우스의 GPCR Heteromers의 행동 기능을 연구하는
Summary
이 문서는 GPCR 이종 형성을 요구하는 행동 분석을 테스트 할 마우스 정면 피질로 바이러스 벡터를 주입하는 방법을 설명합니다.
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
이러한 LSD, 실로시 빈과 메스와 같은 환각제는 인간의 의식,인지와 감정 7-9에 상당한 변화를 일으킬. 유전 또는 약리학 중 접근 방식에 의해 세로토닌 5-HT 2A 수용체 신호의 불 활성화는 크게 두 설치류 모델 3,10에서 환각제에 대한 행동 반응을 감쇠 11을 인간 원인. 환각제 다른 수용체 아형 8 바인딩 있지만, 5-HT의 2A 수용체는 이러한 화학 물질의 고유 한 행동 활동에 필요로 간주됩니다.
그룹 II 대사성 글루타메이트 수용체 (예., mGlu2 및 mGlu3)는 환각제의 분자 메커니즘과 정신병 (12)의 기초가 자신의 필수적인 역할에 대해 상당한 관심의 대상이되고있다. 이전에는 mGlu2 단백질 (mGlu2-KO 마우스)없이 식을 마우스 복도의 셀룰러 및 행동 효과에 영향을받지 것으로 입증되었다lucinogens 5. 또한, 5-HT (2a)와 mGlu2 수용체와 세로토닌 글루타메이트 리간드 1,2- 살아있는 세포에서 G 단백질 커플 링 패턴을 변조되는 특정 이종 복합체를 형성하는 것이 제안되었다.
구조적으로, 횡단 (TM) 영역 (4) 및 mGlu2 5는 5-HT의 2A 수용체 (5)과 이종 체 형성에 중요한 역할을한다. 또한, 추가 조사는 mGlu2의 TM4의 세포 내 끝에있는 세 가지 잔류 물이 세포 6 살아있는에서 5-HT (2A)의 -mGlu2 수용체 헤테로을 형성 할 필요가 있음을 보여 주었다.
이종 발현 시스템에서 관찰 이러한 연구 결과를 바탕으로, 우리가 있는지 여부를 테스트하기 위해 야생 형 mGlu2 및 mGlu2-KO 마우스의 전두엽 피질에서 mGlu2 / mGlu3 키메라 구조의 HSV-매개 표현의 사용을 설명하는 5-HT (2A) 사이의 이종 형성 및 mGlu2가 필요하다환각 5-HT 2A 수용체 작용제에 의해 유도 머리 트 행동.
Protocol
Representative Results
Discussion
함께 mGlu2-KO 마우스 (5) 이전의 결과와 함께 배양 된 세포에서의 5-HT 2A의 -mGlu2 수용체 복합체를 형성하지 않는 mGlu2 및 mGlu2 / mGlu3 키메라 구조와 결과가 제안하는 5-HT의 2A -mGlu2에서 이종 체 수용체 복합체 마우스 전두엽 피질 LSD 같은 환각 5-HT 2A 수용체 작용제에 의해 헤드 트 동작을 유도하는데 필요하다. 이 방법의 한계는 기본 조직의 세포 내 수준에 가까운 분자 ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894이 연구의 자금 조달에 참여했다. 우리는 박사에게 감사의 말씀을 전합니다. 마우스의 기부금이 작품의 촬영 기간 동안 자신의 수술과 행동 시설의 사용에 대한 의학의 마운트 시나이 학교에서 야스민 허드와 스콧 루소.
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 |
Riferimenti
- Fribourg, M. et al. Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs. Cell. 147 (5), 1011-1023 (2011).
- Gonzalez-Maeso, J. et al. Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature. 452 (7183), 93-97 (2008).
- Gonzalez-Maeso, J. et al. Hallucinogens Recruit Specific Cortical 5-HT(2A) Receptor-Mediated Signaling Pathways to Affect Behavior. Neuron. 53 (3), 439-452 (2007).
- Gonzalez-Maeso, J. et al. Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex. J Neurosci. 23 (26), 8836-8843 (2003).
- Moreno, J. L., Holloway, T., Albizu, L., Sealfon, S. C., & Gonzalez-Maeso, J. Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists. Neurosci Lett. 493 (3), 76-79 (2011).
- Moreno, J. L. et al. Identification of Three Residues Essential for 5-HT2A-mGlu2 Receptor Heteromerization and its Psychoactive Behavioral Function. J Biol Chem. 287 44301-44319 (2012).
- Geyer, M. A., & Vollenweider, F. X. Serotonin research: contributions to understanding psychoses. Trends Pharmacol Sci. 29 (9), 445-453 (2008).
- Nichols, D. E. Hallucinogens. Pharmacol Ther. 101 (2), 131-181 (2004).
- Hanks, J. B., & Gonzalez-Maeso, J. Animal models of serotonergic psychedelics. ACS Chem Neurosci. 4 (1), 33-42 (2013).
- Fiorella, D., Rabin, R. A., & Winter, J. C. Role of 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. II: Reassessment of LSD false positives. Psychopharmacology (Berl). 121 (3), 357-363 (1995).
- Vollenweider, F. X., Vollenweider-Scherpenhuyzen, M. F., Babler, A., Vogel, H., & Hell, D. Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport. 9 (17), 3897-3902 (1998).
- Moreno, J. L., Sealfon, S. C., & Gonzalez-Maeso, J. Group II metabotropic glutamate receptors and schizophrenia. Cell Mol Life Sci. 66 (23), 3777-3785 (2009).
- Kurita, M. et al. HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity. Nat Neurosci. 15 (9), 1245-1254 (2012).
- Kurita, M. et al. Repressive Epigenetic Changes at the mGlu2 Promoter in Frontal Cortex of 5-HT2A Knockout Mice. Mol Pharmacol. 83 (6), 1166-1175 (2013).
- Rives, M. L. et al. Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration. Embo J. 28 (15), 2195-2208 (2009).
- Milligan, G. The Prevalence, Maintenance and Relevance of GPCR Oligomerization. Mol Pharmacol. Epub ahead of print (84), 158-169 (2013).
- Ferre, S. et al. G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol Rev. 66 (2), 413-434 (2014).
- Gonzalez-Maeso, J. GPCR oligomers in pharmacology and signaling. Mol Brain. 4 (1), 20 (2011).
- Gonzalez-Maeso, J. Family a GPCR heteromers in animal models. Front Pharmacol. 5 226 (2014).
- Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., & Gambhir, S. S. Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects. Proceedings of the National Academy of Sciences of the United States of America. 108 (29), 12060-12065 (2011).
- Calebiro, D. et al. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci U S A. 110 (2), 743-748 (2013).
- Fonseca, J. M., & Lambert, N. A. Instability of a class a G protein-coupled receptor oligomer interface. Mol Pharmacol. 75 (6), 1296-1299 (2009).
- Hern, J. A. et al. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc Natl Acad Sci U S A. 107 (6), 2693-2698 (2010).
- Hlavackova, V. et al. Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1. Sci Signal. 5 (237), ra59 (2012).
- Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature. 495 (7442), 534-538 (2013).
- Calebiro, D., Nikolaev, V. O., Persani, L., & Lohse, M. J. Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci. 31 (5), 221-228 (2010).
- Celada, P., Puig, M. V., Diaz-Mataix, L., & Artigas, F. The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs. Biol Psychiatry. 64 (5), 392-400 (2008).
- Imad, M., Mladenovic, L., Gingrich, J. A., & Andrade, R. Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex. Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).
