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-HT_2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis^1,2. Consequently, investigation of structural details of the interaction between 5-HT_2A 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-HT_2A knockout (KO) mice^3,4. Additionally, by conditionally expressing the 5-HT_2A receptor only in cortex, it was demonstrated that 5-HT_2A 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-HT_2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT_2A-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-HT_2A-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 doe....

Protocol

LET OP: Alle procedures voor het fokken van dieren en zorgen werden uitgevoerd volgens de Institutional Animal Care en gebruik Comite (IACUC) regulering van Icahn School of Medicine op de berg Sinaï. Zorg ervoor dat u steriele handschoenen te gebruiken tijdens de gehele procedure.

1. Drug en Virus Voorbereiding

1. drug Voorbereiding
   1. Bereid 15,0 ml ketamine / xylazine anesthesie door het oplossen van 1,35 ml 100 mg / ml Ketamine en 0,75 ml 20 mg / ml xylazine in 12,9 ml 0,9% zoutoplossing. Meng oplossing.
2. viruspreparaat
   1. Kloon de mGlu2 en mGlu2ΔTM4N constructen in een bicistronische....

Results

Eerdere bevindingen tonen aan dat het hoofd-kramp murine gedragsreactie betrouwbaar en robuust opgewekt door hallucinogenen, en het is afwezig bij _5-HT2A -Ko ³ muizen. Verder is aangetoond dat het hoofd-spierrespons opgewekt door hallucinerende _5-HT2A agonisten DOI en LSD significant verlaagd in mGlu2-KO muizen ^5. Hoewel eerdere bevindingen overtuigend aangetoond dat 5-HT _2A en mGlu2 geassembleerd als een heteromeer complex in vi.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution
Ketamine	Vedco	KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06	1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% 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 0.9% saline solution to the concentration of 2.0 mg/kg