HSV-Mediated Expression du transgène de chimériques Constructs pour étudier la fonction Behavioral des GPCR hétéromères chez la souris
Summary
Cet article décrit comment injecter des vecteurs viraux dans le cortex frontal de la souris pour tester tests comportementaux qui nécessitent la formation hétéromère 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
Hallucinogènes, comme le LSD, la psilocybine et la mescaline provoquent des changements importants dans la conscience humaine, la cognition et l' émotion 7-9. L' inactivation de la sérotonine 5-HT signalisation du récepteur 2A par des approches génétiques ou pharmacologiques soit provoque nettement atténué les réactions comportementales aux hallucinogènes dans les deux modèles de rongeurs 3,10 et humains 11. Bien que les hallucinogènes se lient d' autres sous – types de récepteurs 8, le récepteur 5-HT2A est considéré comme nécessaire à l'activité comportementale unique de ces produits chimiques.
Groupe II récepteurs de glutamate métabotropique (ie., MGlu2 et mGlu3) ont été la cible d' une attention considérable en ce qui concerne le mécanisme moléculaire d'hallucinogènes et de leur rôle essentiel qui sous – tend la psychose 12. Auparavant, il a été démontré que des souris sans expression de la protéine mGlu2 (souris mGlu2-KO) sont insensibles aux effets cellulaires et comportementaux hallucinogens 5. Il a également été suggéré que la 5-HT 2A et les récepteurs de mGlu2 forment un complexe hétéromère spécifique à travers lequel la sérotonine et de glutamate de ligands modulent le motif de G couplage des protéines dans les cellules vivantes 1,2.
Structurellement, transmembranaires (TM) , les domaines 4 et 5 de mGlu2 jouent un rôle fondamental dans la formation hétéromère avec le récepteur 5-HT 2A 5. En outre, une investigation a montré que trois résidus situés à l'extrémité intracellulaire de TM4 de mGlu2 sont nécessaires pour former la 5-HT 2A -mGlu2 hétérocomplexe de récepteur dans des cellules vivant à 6.
Sur la base de ces résultats observés dans les systèmes d'expression hétérologues, ici , nous décrivons l'utilisation de l' expression HSV-médiée de type sauvage mGlu2 et mGlu2 / mGlu3 constructions chimériques dans le cortex frontal des souris mGlu2-KO pour vérifier si la formation hétéromère entre 5-HT 2A et mGlu2 est nécessaire pour lacomportement tête contraction induite par hallucinogènes 5-HT des agonistes des récepteurs de 2A.
Protocol
Representative Results
Discussion
Ainsi que les résultats précédents en mGlu2-KO 5 souris, les résultats obtenus avec mGlu2 et mGlu2 / mGlu3 constructions chimères qui ne forment pas le complexe récepteur 2A -mGlu2 5-HT dans des cellules en culture suggèrent que la 5-HT 2A -mGlu2 complexe récepteur hétéromère en souris cortex frontal est nécessaire pour induire un comportement tête twitch par hallucinogènes 5-HT des agonistes des récepteurs 2A LSD-like. Une limitation de cette méthode est qu…
Disclosures
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 a participé au financement de cette étude. Nous tenons à remercier les Drs. Yasmin Hurd et Scott Russo au Mount Sinai School of Medicine pour le don de la souris et l'utilisation de leurs installations de chirurgie et de comportement pendant le tournage de ce travail.
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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