HSV-medierad transgenexpression av chimära konstruktioner för att studera Behavioral funktion GPCR Heteromers i möss
Summary
I artikeln beskrivs hur man injicerar virala vektorer i musen frontala cortex att testa beteendemässiga analyser som kräver GPCR hetebildning.
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 och meskalin orsaka betydande förändringar i det mänskliga medvetandet, kognition och emotion 7-9. Inaktivering av serotonin 5-HT2A-receptorsignalering antingen genetiska eller farmakologiska metoder orsakar markant försvagade beteende svar på hallucinogener i båda gnagarmodeller 3,10 och människor 11. Även hallucinogener binda andra receptorsubtyper 8, är 5-HT2A-receptorn anses nödvändigt för den unika beteendeaktivitet av dessa kemikalier.
Grupp II metabotropa glutamatreceptorer (dvs., MGlu2 och mGlu3) har varit föremål för stor uppmärksamhet om den molekylära mekanismen av hallucinogener och deras viktig roll underliggande psykos 12. Tidigare har det visats att möss med ingen expression av mGlu2 protein (mGlu2-KO möss) är okänsliga för de cellulära och beteendemässiga effekter av hallucinogens 5. Det har också föreslagits att 5-HT 2A och de mGlu2 receptorema bildar en specifik heteromert komplex genom vilken serotonin och glutamatligander modulera mönstret av G-proteinkoppling i levande celler 1,2.
Strukturellt trans (TM) domänerna 4 och 5 av mGlu2 spela en avgörande roll i heterobildning med 5-HT2A-receptorn 5. Dessutom visade ytterligare undersökningar att tre rester belägna vid den intracellulära slutet av TM4 av mGlu2 är nödvändiga för att bilda 5-HT2A -mGlu2 receptorhetero i levande celler 6.
Baserat på dessa fynd som observerats i heterologa expressionssystem, här beskriver vi användningen av HSV-medierad uttryck av vildtyp mGlu2 och mGlu2 / mGlu3 chimära konstruktioner i frontala cortex av mGlu2-KO möss för att testa om heterobildning mellan 5-HT2A och mGlu2 är nödvändig för atthead-twitch beteende induceras av hallucinogena 5-HT-2A-receptoragonister.
Protocol
Representative Results
Discussion
Tillsammans med tidigare resultat inom mGlu2-KO-möss 5, resultaten med mGlu2 och mGlu2 / mGlu3 chimära konstruktioner som inte bildar den 5-HT 2A -mGlu2 receptorkomplexet i odlade celler tyder på att 5-HT 2A -mGlu2 heteromert receptorkomplex i behövs mus frontala cortex att framkalla huvud rycka beteende genom LSD-liknande hallucinogena 5-HT2A-receptoragonister. En begränsning med denna metod är att den inte mäter nära molekyl närhet vid en subcellulär nivå i nativ …
Divulgations
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 deltog i finansieringen av denna studie. Vi vill tacka Dr.. Yasmin Hurd och Scott Russo på Mount Sinai School of Medicine för donation av möss och användningen av deras kirurgi och beteende anläggningar under inspelningen av detta arbete.
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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