HSV mediada por la expresión transgénica de constructos quiméricos para estudiar la función del comportamiento de los GPCR heterómeros en ratones
Summary
En este artículo se describe cómo inyectar vectores virales en la corteza frontal del ratón para probar los ensayos de comportamiento que requieren la formación heteromérica GPCR.
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
Alucinógenos, como el LSD, psilocibina y la mescalina causan cambios significativos en la conciencia humana, cognición y la emoción 7-9. La inactivación de la señalización del receptor de serotonina 5-HT 2A mediante enfoques ya sea genéticos o farmacológicos hace que atenuó marcadamente las respuestas de comportamiento a los alucinógenos, tanto en modelos de roedores y seres humanos 3,10 11. A pesar de los alucinógenos se unen otros subtipos de receptores 8, el receptor 5-HT 2A se considera necesario para la actividad de comportamiento único de estos productos químicos.
II receptores de glutamato metabotrópicos del grupo (es decir., MGlu2 y mGlu3) han sido objeto de considerable atención en relación con el mecanismo molecular de alucinógenos y su papel integral psicosis 12 subyacente. Anteriormente, se ha demostrado que los ratones con ninguna expresión de la proteína mGlu2 (ratones mGlu2-KO) son insensibles a los efectos celulares y de comportamiento de hallucinogens 5. También se ha sugerido que el 5-HT2A y los receptores mGlu2 forman un complejo heteromérico específico a través del cual los ligandos de la serotonina y el glutamato modulan el patrón de acoplamiento de proteínas G en las células vivas 1,2.
Estructuralmente, transmembrana (TM) dominios 4 y 5 de mGlu2 desempeñan un papel fundamental en la formación heteromérica con el 5-HT 2A receptor 5. Además, una mayor investigación demostró que tres residuos situados en el extremo intracelular de TM4 de mGlu2 son necesarios para formar la 5-HT heterocomplejo receptor 2A -mGlu2 en las células 6 viviente.
Sobre la base de estos hallazgos observados en sistemas de expresión heterólogos, aquí se describe el uso de la expresión mediada por HSV de tipo salvaje mGlu2 y mGlu2 / mGlu3 construcciones quiméricas en la corteza frontal de ratones mGlu2-KO para probar si la formación heteromérica entre el 5-HT 2A y mGlu2 es necesario para lael comportamiento de cabeza de contracción inducida por alucinógenos agonistas de los receptores 5-HT 2A.
Protocol
Representative Results
Discussion
Junto con los resultados anteriores en ratones mGlu2-KO 5, los resultados con mGlu2 y mGlu2 / mGlu3 construcciones quiméricas que no forman el complejo receptor 2A -mGlu2 5-HT en células cultivadas sugieren que el 2A 5-HT -mGlu2 complejo receptor heteroméricos en se necesita corteza frontal del ratón para inducir un comportamiento de contracción cabeza por alucinógenos agonistas de los receptores 5-HT 2A LSD. Una limitación de este método es que no mide la proximidad m…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 participó en la financiación de este estudio. Nos gustaría agradecer a los Dres. Yasmin Hurd y Scott Russo en el Mount Sinai School of Medicine de la donación de los ratones y el uso de sus instalaciones de la cirugía y el comportamiento durante el rodaje de este trabajo.
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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