HSV-Mediated Transgene Expression von chimären Konstrukten Behavioral Funktion von GPCR Heteromeren bei Mäusen zu studieren
Summary
Dieser Artikel beschreibt, wie virale Vektoren in die Maus frontalen Kortex zu injizieren Verhaltenstests zu testen, die GPCR heteromeren Bildung erfordern.
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
Halluzinogene wie LSD, Psilocybin und Meskalin zu erheblichen Veränderungen im menschlichen Bewusstsein, Kognition und Emotion 7-9. Die Inaktivierung von Serotonin 5-HT 2A – Rezeptor – Signal entweder durch genetische oder pharmakologische Ansätze verursacht deutlich Verhaltensreaktionen auf Halluzinogene in beiden Tiermodellen 3,10 und Menschen 11 gedämpft. Obwohl Halluzinogene anderen Rezeptor – Subtypen binden , 8, die 5-HT 2A -Rezeptor ist als die für die einzigartige Verhaltensaktivität dieser Chemikalien betrachtet.
Gruppe II metabotropen Glutamatrezeptoren (dh., MGlu2 und mGlu3) haben das Ziel beträchtlicher Aufmerksamkeit in Bezug auf den molekularen Mechanismus von Halluzinogenen und deren integrale Rolle Psychose zugrunde liegenden 12. Zuvor wurde gezeigt, dass Mäuse ohne Expression mGlu2 Protein (mGlu2-KO-Mäuse) unempfindlich sind, auf die zelluläre und Verhaltenswirkungen von hallucinogens 5. Es wurde auch vorgeschlagen, dass der 5-HT 2A -Rezeptoren und die mGlu2 einen bestimmten heteromeren Komplex bilden , durch den Serotonin und Glutamat – Liganden um das Muster der G – Protein – Kopplung in lebenden Zellen 1,2 modulieren.
Strukturell Trans (TM) Domänen 4 und 5 der mGlu2 eine fundamentale Rolle in heteromeren Bildung spielen mit dem 5-HT 2A -Rezeptor – 5. Zusätzlich zeigten weitere Untersuchungen , daß drei Reste am intrazellulären Ende TM4 von mGlu2 angeordnet sind notwendig , um die 5-HT 2A -Rezeptor -mGlu2 Hetero in lebenden Zellen 6 zu bilden.
Auf Basis dieser Erkenntnisse in heterologen Expressionssystemen beobachtet, beschreiben wir die Verwendung von HSV-vermittelte Expression von Wildtyp mGlu2 und mGlu2 / mGlu3 chimären Konstrukten im frontalen Kortex von mGlu2-KO – Mäuse , ob heteromeren Bildung zu testen , zwischen 5-HT 2A und mGlu2 ist notwendig, dass dieKopf-Twitch – Verhalten von halluzinogenen 5-HT 2A -Rezeptor – Agonisten induziert.
Protocol
Representative Results
Discussion
Zusammen mit früheren Ergebnissen in mGlu2-KO – Mäusen 5 sind die Ergebnisse mit und mGlu2 mGlu2 / mGlu3 chimären Konstrukten , die nicht bilden schlagen die 5-HT 2A Rezeptor – Komplex -mGlu2 in kultivierten Zellen, die die 5-HT 2A -mGlu2 heteromeren Rezeptor – Komplex in Maus frontalen Kortex ist erforderlich Kopfzucken Verhalten von LSD-ähnlichen halluzinogene 5-HT 2A -Rezeptor – Agonisten zu induzieren. Eine Einschränkung dieses Verfahrens ist, dass es nicht zu einem s…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 beteiligte sich an der Finanzierung dieser Studie. Wir möchten Drs danken. Yasmin Hurd und Scott Russo am Mount Sinai School of Medicine für die Spende von Mäusen und die Verwendung ihrer Operation und Verhalten Einrichtungen während der Dreharbeiten zu dieser Arbeit.
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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