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.

NOT: Hayvan ıslahı ve bakımları için tüm prosedürler Sina Dağı&#39;nda Tıp Icahn Okulu Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) yönetmeliğine göre yapılmıştır. prosedür boyunca steril eldiven kullandığınızdan emin olun. 1. İlaç ve Virüs Hazırlık <ol><li> İlaç Hazırlama <ol><li> 12.9 ml% 0.9 tuzlu su çözeltisi içinde 100 mg / ml Ketamin 1.35 ml ve 20 mg / ml ksilazin 0.75 ml eritilmesi ile 15.0 mi, ketamin / ksilazin anestezik hazırlayın. Iyice çözüm karıştırmak. </li></ol></li><li> Virüs hazırlanması <ol><li> MGlu2 Clone ve mGlu2ΔTM4N standart protokoller daha önce 6 açıklanan aşağıdaki bir bicistronic herpes simpleks virüsü (HSV) vektörü içine oluşturur. Daha önce tarif edildiği gibi 6,13,14 viral partikülleri paketleyin. Kalıntıların Değişiklik Ala-677 4.40, mGlu2 Ala-681 4.44 ve Ala685 4.48 Ser686 4.40, Ph içinE690 4.44 ve Gly-694 4.48 mGlu3 (HA-mGlu2ΔTM4N) &#39;de daha önce 6 tarif edilmiştir. Not: Bu, daha önce kimerik yapısı, HA-mGlu2ΔTM4N sağlam G-protein-bağlı 6 Sinyal plazma membranında eksprese edildiği gösterilmiştir. </li><li> viral vektörlerin saklayın -80 ° C kullanılmadığı zaman. Daha sonra çözülme viral buz üzerinde vektör ve 10 ul kısma bölmeyin. cerrahi işlem için, buz üzerinde tutmak. </li></ol></li></ol> 2. Cerrahi <ol><li> cerrahi Hazırlık <ol><li> (1.1.1 ayrıntılar için bkz) fare tartılır ve ketamin / ksilizin kokteyl uygun dozda ile fare enjekte. </li><li> Düzgün anestezi olmadığını görmek bir tepki ortaya çıkarmak için yapamaz fare düzgün anestezi ise, ağrı yanıt ayak ve kuyruk sıkmak için fareyi kontrol edin. </li><li> makası kullanarak burun ucu kafatası tabanından fare kafasını tıraş. Fare af oftalmik jel uygulayınfare körlüğü önlemek için terward. </li><li> stereotaksik çerçeve üzerine her şırınga yükleyin. onlar 10 derece uzakta normal böylece daha sonra stereotaksik çerçeve her kolun dik kısmını yatırın. iğneler birbirine bakacak şekilde, kollar eğik olduğundan emin olun. </li><li> % 70 etanol ile iğne doldurarak her bir şırınga temizleyin. Şırınga temiz olduğundan emin olmak için iğne en az üç kez doldurun. </li><li> İğne temizlendikten sonra, çift distile H2O ile iğne doldurarak iğne floş Bir kez kızardı, çift distile H2O 1.3 ul her iğne doldurun Çift distile H2O 0.3 ul serbest bırakmak için şırınga pistonu Büküm İğnenin ucunda su boncuklar, dikkatlice su uzakta silin. hiçbir şey şırınga çıkarsa, aşağı tamamen pistonu itin ve şırınga temizlik tekrarlayın. </li><li> Su ile doldurduktan sonra, daha sonra şırınga ile dolum şırınga yukarı çekinHavanın 0.5 ul. </li><li> Hava ve su şırınga kez, dikkatle virüs çözümü 1.3 ul şırınga doldurun. Bu noktada şırınga toplam hacim 2.8 ul olduğundan emin olun. Yine virüsün 0.3 ul serbest bırakmak için şırınga ucu bükün. İğnenin ucundaki sıvı boncuklar, dikkatle sıvıyı uzak silin. hiçbir şey şırınga çıkarsa, aşağı tamamen pistonu itin ve şırınga temizlik tekrarlayın. </li></ol></li><li> cerrahlık <ol><li> Kafatası seviyesi ve düz olacak şekilde stereotaksik çerçeve ayarlamak için emin olun, stereotaksik çerçeve için fareyi takın. maruz kafa derisi povodine-iyot uygulayın. Bir neşter kullanılarak, maruz traş alanı içinde kafatası orta hat boyunca sagittal bir kesi yapmak. Sonra kafatası maruz kalır emin olmak için kesi yerinde cilde büret kelepçeleri takın. </li><li> Ve dikişler maruz periost uzak çözmek için H 2 O 2 kullanınkafatası. Şimdi Bregma ve dikişler görünür olduğundan, kafatası seviyesi olduğundan emin olmak için stereotaksik çerçeve ayarlamak için emin olun. </li><li> bregma ile şırınga iğne uçları hizalayın ve bregma koordinatlarını kaydetmek. iğneler takılı olacak koordinatlarını hesaplamak. <ol><li> Rostral-Cauldal (RC) düzlem için, kaydedilen RC Bregma koordinatları (bregmadan 1,6) 1.6 mm ekleyin. Dorsal-ventral (DV) düzlem için, kaydedilen DV Bregma koordinatları (bregmadan -2.4) 2.4 mm çıkarın. </li><li> Son olarak, Medial Lateral (ML) düzlem için, kaydedilen ML Bregma koordinatları (bregmadan 2,6) 2.6 mm ekleyin. Bu ikili bir enjeksiyon olduğu gibi tüm koordinatlar için, hem sol ve sağ koordinatlarını kaydetmek için emin olun. </li></ol></li><li> İstenilen koordinatlarına iğne getir. iğneler sokulur ve bir matkap ile işaretlenmiş alanların matkap olacak nereye yerleri işaretleyin. </li><li> Bir pamuk uçlu aplikatör wi ileherhangi bir aşırı kan ya da kemik parçasını uzağa pe. </li><li> iğne uçları beynin yüzeyine dokunmadan kafatasına iğneler getirin. Sonra yavaş yavaş onları düşürücü istenilen koordinatlara iğneler indirin. </li><li> iğne istenen koordinatlara olduğunda, yavaş (toplam 0.5 ul) 5 dakika boyunca dakikada iğne 0.1 ul pistonu bükerek enjekte edilir. </li><li> Enjeksiyon yapıldıktan sonra başka bir 5 dakika kortekste şırınga bırakın. </li></ol></li><li> Up / kapama Bakımı <ol><li> sürekli ve yavaş yavaş fare korteks iğne çıkarın. Sonra stereotaksik çerçeve fare kaldırmak. </li><li> forseps cilt flep kapmak ve birlikte koyun ile daha sonra kesi deri taban kanatlarına siyanoakrilat (dermal yapıştırıcı) uygulayın ve. </li><li> siyanoakrilat kurumasını bekleyin. ısıtma yastığı Surg eğer isteğe bağlıdır (bir ısıtma yastığı üzerinde kafes içinde fare yerleştirinnik paketi 37 ° C oda sıcaklığında altında tutulur - Aksi gerekli değildir). yatak cerrahi bölgeye bağlı olmadığından emin olmak için bir kağıt havlu üzerine fare koyun emin olun. </li><li> İşlemden sonra 60 dakika - cerrahi işlemin süresine bağlı olarak, fareler 30 içinde anestezi dışında olduğundan emin olun. Ameliyatı takiben, hayvan kendi kafes yerleştirilir ve kurtarmak için kendi odasına dönmeden önce bilinçli hale gelene kadar izlenir. Onlar beyin biyokimyasal yollar bazı değiştirerek bizim deneylerin sonuçlarını değiştirebilir çünkü hiçbir analjezikler, postoperatif kullanılmaktadır. Ancak ameliyat günü anestezi kurtarmak kadar, hayvanlar izlenir, ve enfeksiyon ve ağrı / sıkıntı değerlendirilmesi belirtileri günlük operasyon sonrası. </li></ol></li></ol> 3. Kafa Seğirme Tepki Deneyi <ol><li> Kurmak <ol><li> 3 gün stereota sonra - 2 10:00 ve 02:00 PM arasındaki tüm davranış testleri yürütmekViral parçacıklar CTIC enjeksiyonu. </li><li> Çözündürün (±) -1- (2,5-dimetoksi-4-iyodofenil) 2.0 mg / kg bir% 0.9 salin çözeltisi -2-aminopropan hidroklorür (DOI). Ayrıca% 0.9 tuzlu su çözeltisi hazırlayın. </li><li> Herhangi bir yatak olmadan bir ev kafes (28 x 18 x 15 cm) hazırlayın ve video kamera görüntüle doğrudan ana kafes üstünde olacak şekilde bir tri-bölmesini kullanarak, bir kamera ayarlayın. </li><li> Deneyin başlamasından önce en azından 4 saat oda fareler alıştırarak. </li><li> Baş seğirme kaydetmek için kamera ayarlayın. </li></ol></li><li> deneme <ol><li> Bir evde kafes üzerinden doğrudan böylece kamerayı yerleştirin. Tüm ana kafes görüş alanında olacak şekilde kamera kalibre edin. </li><li> fare tartılır ve% 0.9 tuzlu su ya da DOI ya uygun dozu (0.01 ml / g) ile periton içine fare enjekte edilir. Not: Fare 25 gram ağırlığında durumunda, 0.25 ml lik bir toplam hacime dozunun uygulanması. </li><li> fo evlerine kafes geri her fare yerleştirinR 10 dk. Boş ev kafesin merkezine 10 dakika, yer fare sonra kameranın görüş alanında kör noktalar vardır olduğunu doğrulamak. Kamerada basın kaydı. odayı terk. NOT: Fare hareketleri ve orada çeşitli davranışsal tepkileri (... Kafa seğirme, kulak çizik vb DOI tarafından uyarılan davranışsal tepkilerin tam listesi için Gonzalez-Maeso ark 2007 ek veri tablosunun 1&#39;e bakınız) 3, kaydedilmiş olacak 30 dk. Nedenle, kaydedilen görüş alanında kör noktalar vardır önemlidir. </li><li> 30 dakika orijinal ev kafesine geri kamera ve yer fare kayıt durdurduktan sonra. Her fare için bu işlemi tekrarlayın. </li></ol></li><li> gözden geçirmek <ol><li> Bantlar fare deneysel koşullara kör her hakem inceleme var) (yani., Baş sırasında kullanılan ilaçlar deney veya intrakranial enjeksiyon sırasında kullanılan virüs seğirme). El ile her kafa Throug seğirme kayıthout videosu. NOT: Baş-seğirme bir fare (ek videosu) tarafından yapılan hızlı sarsıntı baş hareketi olarak tanımlanır. </li><li> Her fare için, kör hakemlerin üç toplamları nihai HTR yanıtını ortalama. Daha sonra grup, bu deneysel koşulu ile değerler ve istatistiksel analiz (yani., T-testi veya ANOVA) yürütmek. </li></ol></li></ol>

<ol>
	<li>Fribourg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+Signaling+of+a+GPCR+Heteromeric+Complex+Reveals+a+Unifying+Mechanism+of+Action+of+Antipsychotic+Drugs.">Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs.</a> Cell. 147 (5), 1011-1023 (2011).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+serotonin/glutamate+receptor+complex+implicated+in+psychosis.">Identification of a serotonin/glutamate receptor complex implicated in psychosis.</a> Nature. 452 (7183), 93-97 (2008).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens+Recruit+Specific+Cortical+5-HT(2A)+Receptor-Mediated+Signaling+Pathways+to+Affect+Behavior.">Hallucinogens Recruit Specific Cortical 5-HT(2A) Receptor-Mediated Signaling Pathways to Affect Behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+fingerprints+distinguish+hallucinogenic+and+nonhallucinogenic+5-hydroxytryptamine+2A+receptor+agonist+effects+in+mouse+somatosensory+cortex.">Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.</a> J Neurosci. 23 (26), 8836-8843 (2003).</li><li>Moreno, J. L., Holloway, T., Albizu, L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+mGlu2+receptor+is+necessary+for+the+pharmacological+and+behavioral+effects+induced+by+hallucinogenic+5-HT2A+receptor+agonists.">Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.</a> Neurosci Lett. 493 (3), 76-79 (2011).</li><li>Moreno, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Three+Residues+Essential+for+5-HT2A-mGlu2+Receptor+Heteromerization+and+its+Psychoactive+Behavioral+Function.">Identification of Three Residues Essential for 5-HT2A-mGlu2 Receptor Heteromerization and its Psychoactive Behavioral Function.</a> J Biol Chem. 287, 44301-44319 (2012).</li><li>Geyer, M. A., Vollenweider, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+research:+contributions+to+understanding+psychoses.">Serotonin research: contributions to understanding psychoses.</a> Trends Pharmacol Sci. 29 (9), 445-453 (2008).</li><li>Nichols, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens.">Hallucinogens.</a> Pharmacol Ther. 101 (2), 131-181 (2004).</li><li>Hanks, J. B., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+serotonergic+psychedelics.">Animal models of serotonergic psychedelics.</a> ACS Chem Neurosci. 4 (1), 33-42 (2013).</li><li>Fiorella, D., Rabin, R. A., Winter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+5-HT2A+and+5-HT2C+receptors+in+the+stimulus+effects+of+hallucinogenic+drugs.+II:+Reassessment+of+LSD+false+positives.">Role of 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. II: Reassessment of LSD false positives.</a> Psychopharmacology (Berl). 121 (3), 357-363 (1995).</li><li>Vollenweider, F. X., Vollenweider-Scherpenhuyzen, M. F., Babler, A., Vogel, H., Hell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psilocybin+induces+schizophrenia-like+psychosis+in+humans+via+a+serotonin-2+agonist+action.">Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action.</a> Neuroreport. 9 (17), 3897-3902 (1998).</li><li>Moreno, J. L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+II+metabotropic+glutamate+receptors+and+schizophrenia.">Group II metabotropic glutamate receptors and schizophrenia.</a> Cell Mol Life Sci. 66 (23), 3777-3785 (2009).</li><li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC2+regulates+atypical+antipsychotic+responses+through+the+modulation+of+mGlu2+promoter+activity.">HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.</a> Nat Neurosci. 15 (9), 1245-1254 (2012).</li><li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repressive+Epigenetic+Changes+at+the+mGlu2+Promoter+in+Frontal+Cortex+of+5-HT2A+Knockout+Mice.">Repressive Epigenetic Changes at the mGlu2 Promoter in Frontal Cortex of 5-HT2A Knockout Mice.</a> Mol Pharmacol. 83 (6), 1166-1175 (2013).</li><li>Rives, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+GABAB+and+mGlu1a+receptors+reveals+new+insight+into+GPCR+signal+integration.">Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration.</a> Embo J. 28 (15), 2195-2208 (2009).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prevalence,+Maintenance+and+Relevance+of+GPCR+Oligomerization.">The Prevalence, Maintenance and Relevance of GPCR Oligomerization.</a> Mol Pharmacol. (84), 158-169 (2013).</li><li>Ferre, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptor+oligomerization+revisited:+functional+and+pharmacological+perspectives.">G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives.</a> Pharmacol Rev. 66 (2), 413-434 (2014).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+oligomers+in+pharmacology+and+signaling.">GPCR oligomers in pharmacology and signaling.</a> Mol Brain. 4 (1), 20 (2011).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Family+a+GPCR+heteromers+in+animal+models.">Family a GPCR heteromers in animal models.</a> Front Pharmacol. 5, 226 (2014).</li><li>Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+resonance+energy+transfer+(BRET)+imaging+of+protein-protein+interactions+within+deep+tissues+of+living+subjects.">Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (29), 12060-12065 (2011).</li><li>Calebiro, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization.">Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.</a> Proc Natl Acad Sci U S A. 110 (2), 743-748 (2013).</li><li>Fonseca, J. M., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+a+class+a+G+protein-coupled+receptor+oligomer+interface.">Instability of a class a G protein-coupled receptor oligomer interface.</a> Mol Pharmacol. 75 (6), 1296-1299 (2009).</li><li>Hern, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+dissociation+of+M1+muscarinic+receptor+dimers+seen+by+total+internal+reflection+fluorescence+imaging+of+single+molecules.">Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.</a> Proc Natl Acad Sci U S A. 107 (6), 2693-2698 (2010).</li><li>Hlavackova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+inter-+and+intrasubunit+rearrangements+during+activation+of+dimeric+metabotropic+glutamate+receptor+1.">Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1.</a> Sci Signal. 5 (237), 59 (2012).</li><li>Irannejad, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+biosensors+reveal+GPCR+signalling+from+endosomes.">Conformational biosensors reveal GPCR signalling from endosomes.</a> Nature. 495 (7442), 534-538 (2013).</li><li>Calebiro, D., Nikolaev, V. O., Persani, L., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+internalized+G-protein-coupled+receptors.">Signaling by internalized G-protein-coupled receptors.</a> Trends Pharmacol Sci. 31 (5), 221-228 (2010).</li><li>Celada, P., Puig, M. V., Diaz-Mataix, L., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex:+reversal+by+antipsychotic+drugs.">The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs.</a> Biol Psychiatry. 64 (5), 392-400 (2008).</li><li>B&#233;&#239;que, J. -. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).</li></ol>

The authors have nothing to disclose.

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...

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.

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			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">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)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>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)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>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)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>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</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>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</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

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 mGlu2&#916;TM4N (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.

Önceki bulgular halusinojenler tarafından güvenilir ve sağlam ortaya bir fare davranış cevabı baş-seğirme olduğunu göstermektedir ve bu 5-HT 2A -KO fareler 3 yoktur. Ayrıca, DOI ve LSD halüsinojenik 5-HT2A agonistlerin kafa seğirme yanıtı anlamlı mGlu2-KO farelerde 5 azaldığını göstermiştir. Bununla birlikte, her ne kadar önceki bulgular ikna edici çözülemediğini canlı farelerde gibi bu yapısal düzenleme hareket i...

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Bu makalede, GPCR heteromerik oluşumunu gerektiren davranış testleri test etmek için fare frontal korteks içine viral vektörlerin enjekte açıklamaktadır.

Kimerik Yapılar HSV-aracılı Transgen İfade Farelerde GPCR Heteromers Davranışsal Fonksiyon Eğitim için

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Research

JoVE Journal

Neuroscience

7.2K Görüntüleme.  Icahn School of Medicine at Mount Sinai.  Bu makalede, GPCR heteromerik oluşumunu gerektiren davranış testleri test etmek için fare frontal korteks içine viral vektörlerin enjekte açıklamaktadır. 

Video: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Nörobilim

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Fare Beyin dilimleri içinde Nikotinik Asetilkolin Reseptör Fonksiyon Okumak Uyuşturucu Yerel Uygulama

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Test Görsel Fonksiyonu için Basit Bir Davranış Testi<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Farelerde Hipokampal Bölgesi Dentate Gyrus dendritik arborization Değerlendirilmesi

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Rat Kortikal Nöronlar Filamentli Aktin (F-aktin) puncta Niceleme

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

İnsanın Koku Fonksiyonunu İncelemek İçin Free-breathing fMRI Yöntemi

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Omurilik genetik olarak tanımlanmış nöronlar Adeno ilişkili virüs-aracılı Transgene ifade

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Interstimulus Aralık kemirgenler zamansal işleme değerlendirilmesi için güç

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Siyatik Sinir Ezilme Hasarının Sıçan Modelinde Fonksiyonel Değerlendirme için 3D Kinematik Analiz

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Beyin Dilimlerinde ve Birincil Hücre Kültüründe Piramidal Nöronların Balistik Etiketlemesi

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...