HSV-Mediated Transgene Expressie van Chimere constructen tot Behavioral functie van GPCR heteromeren in Muizen Studie
Summary
In dit artikel wordt beschreven hoe u virale vectoren te injecteren in de muis frontale cortex gedragsproblemen assays die GPCR heteromere vorming nodig te testen.
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
Hallucinogenen zoals LSD, psilocybine en mescaline leiden tot significante veranderingen in het menselijk bewustzijn, cognitie en emotie 7-9. Inactivering van serotonine 5-HT2A receptor signalering door hetzij genetische of farmacologische benaderingen veroorzaakt aanzienlijk verzwakt gedragsreacties hallucinogenen zowel diermodellen 3,10 en 11 mens. Hoewel hallucinogenen binden andere receptorsubtypen 8, wordt de 5-HT2A receptor geacht voor de unieke gedragsactiviteit van deze stoffen.
Groep II metabotrope glutamaatreceptoren (bijv., MGlu2 en mGlu3) hebben het doelwit van veel aandacht met betrekking tot de moleculaire mechanisme van hallucinogenen en hun integrale rol onderliggende psychose 12 geweest. Eerder is aangetoond dat muizen zonder expressie van mGlu2 eiwit (mGlu2-KO muizen) ongevoelig zijn voor de cellulaire en gedragseffecten van hallucinogens 5. Ook is gesuggereerd dat het 5-HT2A en mGlu2 receptoren vormen een specifiek heteromeer complex waardoor serotonine en glutamaat liganden moduleren het patroon van G-eiwit koppelen in levende cellen 1,2.
Structureel, transmembraan (TM) domeinen 4 en 5 van mGlu2 speelt een fundamentele rol in heteromerische formatie met de 5-HT 2A receptor 5. Bovendien, verder onderzoek aangetoond dat drie residuen die aan het intracellulaire eind TM4 van mGlu2 noodzakelijk om de 5-HT2A receptor -mGlu2 heterocomplex in levende cellen 6 vormen.
Op basis van deze bevindingen waargenomen bij heterologe expressiesystemen Hier beschrijven we het gebruik van HSV-gemedieerde expressie van wildtype mGlu2 en mGlu2 / mGlu3 chimere constructen in de frontale cortex van mGlu2-KO muizen te testen of heteromere vorming tussen 5-HT2A mGlu2 en is nodig voor dehead-twitch gedrag geïnduceerd door hallucinogene 5-HT 2A receptor agonisten.
Protocol
Representative Results
Discussion
Overzicht van eerdere bevindingen in mGlu2-KO muizen 5, de resultaten mGlu2 en mGlu2 / mGlu3 chimere constructen die de 5-HT2A -mGlu2 receptorcomplex in gekweekte cellen vormen suggereren dat de 5-HT2A -mGlu2 heteromere receptorcomplex muis frontale cortex is nodig om het hoofd-twitch gedrag te induceren door LSD-achtige hallucinogene 5-HT 2A receptor agonisten. Een beperking van deze methode is dat het niet dicht moleculaire omgeving heeft gemeten op een subcellulair niveau n…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 deel aan de financiering van deze studie. We danken Drs. Yasmin Hurd en Scott Russo op Mount Sinai School of Medicine voor de donatie van muizen en het gebruik van hun operatie en het gedrag faciliteiten tijdens het filmen van dit werk.
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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