HSV-Mediated expressão do transgene da quiméricos Construções para estudar a função comportamental de GPCR heterómeros em Ratos
Summary
Este artigo descreve como para injetar vetores virais no córtex frontal do rato para testar ensaios comportamentais que exigem formação 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 o LSD, a psilocibina e mescalina causar mudanças significativas na consciência humana, cognição e emoção 7-9. Inactivação de sinalização do receptor de serotonina 5-HT2A por abordagens genéticas ou farmacológicas quer marcadamente atenuado provoca respostas comportamentais ao alucinógenos em ambos os modelos de roedores e seres humanos 3,10 11. Embora alucinógenos ligar outros subtipos de receptores 8, o receptor 5-HT 2A é considerado como necessário para a actividade comportamental única destes produtos químicos.
Grupo II receptores de glutamato metabotrópicos (ie., MGlu2 e mGlu3) têm sido alvo de considerável atenção sobre o mecanismo molecular de alucinógenos e seu papel fundamental subjacente a psicose 12. Anteriormente, foi demonstrado que os ratinhos com nenhuma expressão de proteína de mGlu2 (ratinhos de mGlu2-KO) são insensíveis aos efeitos celulares e comportamentais de Hallucinogens 5. Também tem sido sugerido que a 5-HT 2A e os receptores de mGlu2 formar um complexo heteromérico específico através do qual a serotonina e glutamato ligandos modular o padrão de acoplamento de proteínas G em células vivas 1,2.
Estruturalmente, transmembranares (TM) domínios 4 e 5 do mGlu2 desempenham um papel fundamental na formação heteromérica com o receptor 5-HT 2A 5. Adicionalmente, investigação adicional demonstrou que três resíduos localizados na extremidade intracelular de TM4 de mGlu2 são necessários para formar o heterocomplexo do receptor A2A -mGlu2 5-HT 6 em células vivas.
Com base nestes resultados observados em sistemas de expressão heterólogos, aqui descreve-se o uso da expressão de HSV-mediada de tipo selvagem de mGlu2 e mGlu2 / mGlu3 construções quiméricas no córtex frontal de ratos mGlu2-KO para testar se a formação heteromérica entre 5-HT 2A e mGlu2 é necessário para ocomportamento cabeça de contração induzida por agonistas dos receptores 5-HT2A alucinógenas.
Protocol
Representative Results
Discussion
Juntamente com os resultados anteriores em ratinhos KO-mGlu2 5, os resultados com mGlu2 e mGlu2 / mGlu3 construções quiméricas que não formam o complexo receptor 2A -mGlu2 5-HT, em células cultivadas sugerem que o 5-HT 2A -mGlu2 complexo receptor heteromérico em rato córtex frontal é necessário para induzir o comportamento cabeça de contração por agonistas dos receptores 5-HT2A alucinógenos semelhantes ao LSD. Uma limitação deste método é que ele não mede pro…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 participou no financiamento deste estudo. Nós gostaríamos de agradecer Drs. Yasmin Hurd e Scott Russo no Mount Sinai School of Medicine para a doação de ratos eo uso de suas instalações de cirurgia e comportamento durante as filmagens deste trabalho.
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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