Kimerik Yapılar HSV-aracılı Transgen İfade Farelerde GPCR Heteromers Davranışsal Fonksiyon Eğitim için
Summary
Bu makalede, GPCR heteromerik oluşumunu gerektiren davranış testleri test etmek için fare frontal korteks içine viral vektörlerin enjekte açıklamaktadır.
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
LSD, psilosibin ve meskalin olarak halüsinojenler, insan bilinci, biliş ve duygu 7-9 önemli değişikliklere neden olmaktadır. Genetik ya da farmakolojik ya yaklaşımlarla serotonin 5-HT2A reseptörü sinyallemesinin inaktivasyonu belirgin iki kemirgen modellerinde 3,10 bölgesindeki halüsinojenler davranışsal tepkileri zayıflatılmış ve 11 insanlarda neden olur. Halüsinojenler, diğer reseptör alt-tipleri 8 bağlanan, ancak 5-HT2A reseptörü, bu kimyasal maddelerin benzersiz davranış aktivite için gerekli olarak kabul edilir.
Grup II metabotropik glutamat reseptörleri (yani., MGlu2 ve mGlu3) halusinojenler moleküler mekanizması ve psikoz 12 altında yatan onların ayrılmaz rolüne ilişkin önemli ilgi hedef olmuştur. Daha önce, mGlu2 protein (mGlu2-KO fareleri) herhangi bir ifade ile farenin hal hücresel ve davranışsal etkilere karşı duyarsız olduğu gösterilmiştirlucinogens 5. Ayrıca, 5-HT2A ve mGlu2 reseptörleri serotonin ve glutamat ligandlan, canlı hücreler 1,2 G protein bağlama modelini modüle geçtiği belirli bir heteromerik kompleks oluşturduğunu ileri sürülmüştür.
Yapısal olarak, zar-ötesi (TM) etki 4 ve mGlu2 5 5-HT2A reseptörü 5, heteromerik oluşumunda önemli bir rol oynamaktadır. Buna ek olarak, daha fazla araştırma mGlu2 bölgesinin TM4 hücre içi ucunda yer alan, üç kalıntıları hücreler 6 canlı 5-HT2A reseptörü -mGlu2 heterokompleks oluşturmak için gerekli olduğunu göstermiştir.
Heterolog ifade sistemlerinde görülen Bu bulgulara dayanarak, burada test etmek için, vahşi tip mGlu2 ve mGlu2-KO farelerde frontal kortekste mGlu2 / mGlu3 Şimerik yapıların HSV-aracılı ifade kullanımını tarif 5-HT2A ile heteromerik oluşumu ve mGlu2 için gerekli olanhalüsinojenik 5-HT2A reseptörü agonistleri tarafından neden kafa seğirme davranışı.
Protocol
Representative Results
Discussion
Birlikte mGlu2-KO farelerde 5 mi olacak biçimde kültürlenmiş hücrelerde 5-HT2A -mGlu2 reseptör kompleksini oluşturmayan mGlu2 ve mGlu2 / mGlu3 kimerik yapıları ile sonuçlar, 5-HT2A -mGlu2 in heteromerik reseptör kompleksi fare frontal korteks LSD benzeri halüsinojenik 5-HT2A reseptörü agonistleri tarafından baş seğirme davranışını uyarmak için gereklidir. Bu yöntemin bir sınırlaması yerli dokuda hücre içi düzeyde yakın moleküler yakınlığı öl…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
NIH R01MH084894 Bu çalışmanın finansmanı katıldı. Biz Dr teşekkür etmek istiyorum. farelerin bağış ve bu çalışmanın çekimleri sırasında cerrahi ve davranış tesislerin kullanımı için Mount Sinai Tıp Okulu Yasmin Hurd ve Scott Russo.
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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