HSV-mediata Transgene Espressione di chimerici costrutti di studio comportamentale funzione di GPCR Heteromers nei topi
Summary
Questo articolo viene descritto come iniettare vettori virali nel mouse corteccia frontale per testare test comportamentali che richiedono la formazione eteromerico GPCR.
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
Allucinogeni, come l'LSD, psilocibina e mescalina provocano significativi cambiamenti nella coscienza umana, cognizione ed emozione 7-9. L'inattivazione della serotonina 5-HT 2A recettore segnalazioni da approcci sia genetici o farmacologici cause notevolmente attenuato le risposte comportamentali a allucinogeni in entrambi i modelli di roditori 3,10 e gli esseri umani 11. Anche se allucinogeni legano altri sottotipi di recettori 8, il recettore 5-HT 2A è considerato come necessario per l'attività comportamentale unica di queste sostanze chimiche.
Gruppo II recettori metabotropici del glutammato (es., MGlu2 e mGlu3) sono stati oggetto di considerevole attenzione per quanto riguarda il meccanismo molecolare di allucinogeni e il loro ruolo fondamentale sottostante psicosi 12. In precedenza, è stato dimostrato che topi con nessuna espressione di proteine mGlu2 (topi mGlu2-KO) sono insensibili agli effetti cellulari e comportamentali di hallucinogens 5. È stato anche suggerito che la 5-HT 2A e recettori mGlu2 formano un complesso specifico eteromerico attraverso cui serotonina e glutammato ligandi modulano il modello dell'accoppiamento proteina G nelle cellule viventi 1,2.
Strutturalmente, transmembrana (TM) domini 4 e 5 di mGlu2 giocano un ruolo fondamentale nella formazione eteromerico con la 5-HT 2A recettore 5. Inoltre, ulteriori indagini hanno dimostrato che tre residui si trovano alla fine intracellulare di TM4 di mGlu2 sono necessari per formare il 5-HT 2A -mGlu2 recettore eterocomplesso nelle cellule viventi 6.
Sulla base di questi risultati osservati in sistemi di espressione eterologa, qui si descrive l'uso di espressione HSV-mediata di tipo selvaggio mGlu2 e mGlu2 / mGlu3 costrutti chimerici nella corteccia frontale di topi mGlu2-KO per verificare se la formazione eteromerico tra 5-HT 2A e mGlu2 è necessario per ilcomportamento testa contrazione indotta da allucinogeni 5-HT 2A agonisti del recettore.
Protocol
Representative Results
Discussion
Insieme con i precedenti risultati in mGlu2-KO topi 5, i risultati con mGlu2 e mGlu2 / mGlu3 costrutti chimerici che non formano la 5-HT complesso recettoriale 2A -mGlu2 in cellule in coltura suggeriscono che la 2A 5-HT -mGlu2 eteromerico complesso recettoriale in topo corteccia frontale è necessario per indurre comportamenti testa contrazione da allucinogeni 5-HT 2A agonisti del recettore LSD-like. Una limitazione di questo metodo è che non misura prossimità molecolare a l…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 partecipato al finanziamento di questo studio. Vorremmo ringraziare il Dott. Yasmin Hurd e Scott Russo al Mount Sinai School of Medicine per la donazione di topi e l'uso dei loro chirurgia e comportamento strutture durante le riprese di questo lavoro.
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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