Summary

HSV-medieret transgene ekspression af kimære konstruktioner at undersøge adfærdsmæssige funktion GPCR heteromerer i mus

Published: July 09, 2016
doi:

Summary

Denne artikel beskriver, hvordan at injicere virale vektorer i musen frontale cortex at teste adfærdsmæssige analyser, der kræver GPCR heteromere formation.

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

Hallucinogener, såsom LSD, psilocybin og mescalin medføre betydelige ændringer i den menneskelige bevidsthed, kognition og emotion 7-9. Inaktivering af serotonin 5-HT 2A receptor signalering ved enten genetiske eller farmakologiske metoder forårsager markant svækket adfærdsmæssige reaktioner på hallucinogener i begge gnavermodeller 3,10 og mennesker 11. Selvom hallucinogener binder andre receptorsubtyper 8 er 5-HT2A-receptoren betragtes som nødvendig for den unikke adfærdsmæssige aktivitet af disse kemikalier.

Gruppe II metabotrope glutamatreceptorer (dvs.., MGlu2 og mGlu3) har været mål for betydelig opmærksomhed om den molekylære mekanisme af hallucinogener og deres integrerende rolle underliggende psykose 12. Tidligere er det blevet vist, at mus med ingen ekspression af mGlu2 protein (mGlu2-KO-mus) er ufølsomme over for de cellulære og adfærdsmæssige virkninger af hallucinogens fem. Det er også blevet foreslået, at 5-HT 2A og mGlu2 receptorer danner en specifik heteromerkompleks hvorigennem serotonin og glutamat ligander modulere mønster af G-protein kobling i levende celler 1,2.

Strukturelt transmembrane (TM) domæner 4 og 5 i mGlu2 spiller en fundamental rolle i heteromere formation med 5-HT 2A receptor 5. Derudover yderligere undersøgelse viste, at tre rester placeret i den intracellulære ende af TM4 af mGlu2 er nødvendige for at danne 5-HT 2A -mGlu2 receptor heterokompleks i levende celler 6.

Baseret på disse resultater observeret i heterologe ekspressionssystemer, Her beskrives anvendelsen af HSV-medieret ekspression af vildtype mGlu2 og mGlu2 / mGlu3 kimære konstruktioner i den frontale cortex hos mGlu2-KO-mus at teste, om heteromer dannelse mellem 5-HT 2A og mGlu2 er nødvendig forhead-twitch opførsel induceret af hallucinogene 5-HT 2A receptor-agonister.

Protocol

BEMÆRK: Alle procedurer for dyreavl og bekymringer blev udført i overensstemmelse med Institutional Animal Care og brug Udvalg (IACUC) regulering af Icahn School of Medicine på Mount Sinai. Sørg for at bruge sterile handsker under hele proceduren. 1. Drug og Virus Forberedelse Drug Preparation Forbered 15,0 ml ketamin / xylazin bedøvelsesmiddel ved opløsning 1,35 ml 100 mg / ml ketamin og 0,75 ml 20 mg / ml xylazin i 12,9 ml 0,9% saltvandsopløsning. Bland løsning. </l…

Representative Results

Tidligere resultater viser, at head-spjæt murine adfærdsmæssig reaktion er pålideligt og robust fremkaldt af hallucinogener, og det er fraværende i 5-HT 2A -KO mus 3. Endvidere er det blevet vist, at hovedet-twitch respons fremkaldt af hallucinogene 5-HT2A agonister DOI og LSD blev signifikant reduceret i mGlu2-KO-mus 5. Men selv om tidligere resultater overbevisende, at 5-HT 2A og mGlu2 er samlet som en heteromerkompleks in…

Discussion

Sammen med tidligere resultater i mGlu2-KO-mus 5, resultaterne med mGlu2 og mGlu2 / mGlu3 kimære konstruktioner, der ikke danner 5-HT 2A -mGlu2 receptorkomplekset i dyrkede celler tyder på, at 5-HT 2A -mGlu2 heteromere receptorkomplekset i muse frontale cortex er nødvendig for at inducere head-twitch adfærd ved LSD-lignende hallucinogene 5-HT 2A receptor-agonister. En begrænsning ved denne metode er, at den ikke måler tæt molekylære lighed på et subcellulært niveau i…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

NIH R01MH084894 deltog i finansieringen af ​​denne undersøgelse. Vi vil gerne takke Drs. Yasmin Hurd og Scott Russo på Mount Sinai School of Medicine ved donation af mus og brugen af ​​deres kirurgi og adfærd faciliteter under indspilningen af ​​dette arbejde.

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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Citazione di questo articolo
Holloway, T., Moreno, J. L., González-Maeso, J. HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice. J. Vis. Exp. (113), e53717, doi:10.3791/53717 (2016).

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