NIH R01MH084894 beteiligte sich an der Finanzierung dieser Studie. Wir möchten Drs danken. Yasmin Hurd und Scott Russo am Mount Sinai School of Medicine für die Spende von Mäusen und die Verwendung ihrer Operation und Verhalten Einrichtungen während der Dreharbeiten zu dieser Arbeit.

HINWEIS: Alle Verfahren für die Tierzucht und Sorgen wurden nach dem Committee (IACUC) Regulierung der Icahn School of Medicine am Berg Sinai Institutional Animal Care und Verwenden durchgeführt. Achten Sie darauf, sterile Handschuhe während des gesamten Verfahrens zu verwenden. 1. Drogen und Virus Vorbereitung <ol><li> Wirkstoffherstellungs <ol><li> Bereiten 15,0 ml Ketamin / Xylazin-Narkose von 1,35 ml von 100 mg / ml Ketamin gelöst und 0,75 ml 20 mg / ml Xylazin in 12,9 ml 0,9% Kochsalzlösung. Gründlich mischen Lösung. </li></ol></li><li> Viruspräparation <ol><li> Klonen Sie die mGlu2 und mGlu2ΔTM4N - Konstrukte in einem bicistronischen Herpes - simplex - Virus (HSV) Vektor nach Standardprotokollen zuvor 6 beschrieben. Paket , um die viralen Partikel wie zuvor beschrieben 6,13,14. Substitution von Resten Ala-677 4,40, Ala-681 4,44 und Ala685 4,48 in mGlu2 für Ser686 4.40, PhE690 4,44 und Gly-694 4,48 in mGlu3 (HA-mGlu2ΔTM4N) wurden bisher 6 beschrieben worden ist . HINWEIS: Es wurde zuvor gezeigt , dass das chimäre Konstrukt HA-mGlu2ΔTM4N an der Plasmamembran mit intaktem G - Protein-abhängige Signalgebung 6 exprimiert wird. </li><li> Speichern von viralen Vektoren in -80 ° C, wenn sie nicht in Gebrauch ist. Thaw viralen Vektor auf Eis, und dann aliquotiert in 10 ul Aliquots. Für den chirurgischen Eingriff, halten auf dem Eis. </li></ol></li></ol> 2. Chirurgie <ol><li> Chirurgie Vorbereitung <ol><li> Wiegen Sie die Maus und injizieren Maus mit geeigneten Dosis von Ketamin / Xylazin-Cocktail (Details siehe 1.1.1). </li><li> Überprüfen Sie die Maus, um zu sehen, wenn sie richtig betäubt, quetschen Fuß und Schwanz für Schmerzreaktion, wenn nicht in der Lage, eine Antwort zu entlocken, wird die Maus richtig betäubt. </li><li> Shave Mauskopf von der Basis des Schädels an der Spitze der Nase mit Klipper. Bewerben ophthalmische Gel auf die Maus afterward Blindheit der Maus zu verhindern. </li><li> Legen Sie jede Spritze auf den Stereotaxierahmen. kippen dann senkrecht Abschnitt jedes Arms des stereotaktischen Rahmen, so dass sie 10 Grad weg von der normal sind. Sicherzustellen, dass die Arme geneigt ist, so daß die Nadeln einander zugewandt sind. </li><li> Reinigen jede Spritze durch die Nadel mit 70% Ethanol gefüllt wird. Füllen die Nadel mindestens drei Mal, um sicherzustellen, dass die Spritze sauber ist. </li><li> Sobald die Nadel gereinigt wurde, spülen Sie die Nadel durch die Nadel mit doppelt destilliertem H 2 O Füllung Sobald gespült, füllen jede Nadel mit 1,3 &amp; mgr; l doppelt destilliertem H 2 O. Drehen Sie den Kolben der Spritze zu lösen 0,3 ul doppelt destilliertem H 2 O. Wenn Wasser Perlen an der Spitze der Nadel vorsichtig abwischen, Wasser entfernt. Wenn nichts aus der Spritze kommt, drücken Sie den Kolben vollständig nach unten und dann Reinigung der Spritze wiederholen. </li><li> Nach dem Befüllen mit Wasser, dann die Spritze nach oben ziehen Befüllen der Spritze mit0,5 l Luft. </li><li> Sobald die Luft und das Wasser in der Spritze sind, sorgfältig die Spritze mit 1,3 &amp; mgr; l der Viruslösung füllen. An dieser Stelle dafür sorgen, dass das gesamte Volumen in der Spritze 2,8 &amp; mgr; l ist. Wieder drehen Sie die Spitze der Spritze 0,3 ul Virus freizusetzen. Wenn Flüssigkeit Perlen an der Spitze der Nadel vorsichtig abwischen, Flüssigkeit entfernt. Wenn nichts aus der Spritze kommt, drücken Sie den Kolben vollständig nach unten und dann Reinigung der Spritze wiederholen. </li></ol></li><li> Chirurgie <ol><li> Bringen Sie die Maus an den Stereotaxierahmen, um sicherzustellen, das Stereotaxierahmen so einzustellen, dass der Schädel Ebene und flach ist. Bewerben povodine-Jod auf die exponierte Kopfhaut. Mit einem Skalpell, machen Sie einen sagittalen Schnitt entlang der Mittellinie des Schädels innerhalb des belichteten rasierten Bereich. Dann fügen Sie die Bürette Klemmen an die Haut an der Einschnittstelle, um sicherzustellen, dass der Schädel ausgesetzt bleibt. </li><li> Verwenden Sie H 2 O 2 , um die Knochenhaut herauslösen , um die Nähte des ExposesSchädel. Nun, da die Bregma und Nähte sichtbar sind, sollten Sie die Stereotaxierahmen anzupassen, um sicherzustellen, dass der Schädel Ebene ist. </li><li> Richten Sie die Nadelspitzen der Spritzen mit dem Bregma und notieren Sie die Koordinaten des Bregma. Berechnen Sie die Koordinaten des in denen die Nadeln werden eingefügt. <ol><li> Für die Rostra-Cauldal (RC) Ebene, fügen Sie 1,6 mm auf die aufgezeichneten RC Bregmas Koordinaten (+1,6 von Bregma). Für die Dorsal-Ventral (DV) -Ebene, subtrahieren 2,4 mm von den DV Bregmas Koordinaten aufgezeichnet (-2,4 von Bregma). </li><li> Schließlich wird für die Medial Lateral (ML) Ebene, fügen Sie 2,6 mm auf die aufgezeichneten ML Bregmas Koordinaten (+2,6 von Bregma). Für alle Koordinaten, müssen Sie beide linken und rechten Koordinaten aufzeichnen, da dies eine bilaterale Injektion ist. </li></ol></li><li> Bringen Sie die Nadeln auf die gewünschten Koordinaten. Markieren Sie die Orte, wo die Nadeln werden eingefügt werden und mit einem Bohrer, bohren Sie die markierten Bereiche. </li><li> Mit einem Wattestäbchen wipe überschüssiges Blut oder Knochenfragment entfernt. </li><li> Bringen die Nadeln an den Schädel, wo die Spitzen der Nadeln die Oberfläche des Gehirns berühren. Dann senken Sie die Nadeln auf die gewünschten Koordinaten sie langsam zu senken. </li><li> Sobald die Nadeln an den gewünschten Koordinaten sind, langsam um den Inhalt der Spritze zu injizieren, indem der Kolben der Nadel 0,1 &amp; mgr; l pro Minute über einen Zeitraum von 5 min (insgesamt 0,5 &amp; mgr; l) zu drehen. </li><li> Sobald die Injektion vorgenommen wurde verlassen die Spritze in der Rinde für weitere 5 min. </li></ol></li><li> Closing Up / Pflege <ol><li> Entfernen Sie die Nadeln von der Maus Cortex stetig und langsam. Dann entfernen Sie die Maus vom Stereotaxierahmen. </li><li> Bewerben Cyanacrylat (dermale Kleber) an die Basishautlappen aus dem Einschnitt und dann mit einer Pinzette die Hautlappen greifen und legen Sie sie zusammen. </li><li> Lassen Sie den Cyanacrylat zu trocknen. Platzieren Sie die Maus im Käfig über ein Heizkissen (Heizkissen, wenn der surg optionalische Suite ist unter RT von 37 ° C gehalten - ansonsten nicht notwendig). Achten Sie darauf, mit der Maus auf einem Papiertuch zu setzen, um sicherzustellen, dass Bettwäsche nicht auf Operationsstelle haftet. </li><li> In Abhängigkeit von der Länge des chirurgischen Verfahrens, sicherzustellen, dass die Mäuse innerhalb von 30 aus der Narkose ist - 60 min nach dem Verfahren. Nach der Operation wird das Tier in seinem eigenen Käfig gesetzt und überwacht, bis er bewusst wird, bevor sie ihre Zimmer zurückkehren zu erholen. Keine Analgetika sind postoperativ eingesetzt, weil sie die Ergebnisse unserer Experimente verändern können, die von einigen der Gehirn biochemische Wege zu modifizieren. Allerdings Tiere überwacht werden, bis sie aus der Narkose am Tag der Operation erholen und täglich nach der Operation auf Anzeichen einer Infektion und Bewertung von Schmerz / Not. </li></ol></li></ol> 3. Leiter Zuckungsreaktion Experiment <ol><li> Konfiguration <ol><li> Führen Sie alle Verhaltenstests von 10.00 Uhr bis 14.00 Uhr, 2 - 3 Tage nach stereotaCTIC Injektion viraler Partikel. </li><li> Auflösen von (±) -1- (2,5-Dimethoxy-4-iodphenyl) -2-aminopropan-hydrochlorid (DOI) in eine 0,9% ige Kochsalzlösung auf 2,0 mg / kg. Auch bereiten eine 0,9% ige Kochsalzlösung. </li><li> Bereiten Sie einen Käfig (28 x 18 x 15 cm) ohne Betten und ein Tri-Pod verwenden, stellen Sie eine Kamera, so dass der Blick auf die Videokamera direkt über dem Käfig ist. </li><li> Habituate die Mäuse in den Raum für mindestens 4 Stunden vor dem Beginn des Experiments. </li><li> Stellen Sie einen Camcorder, um den Kopf zucken aufzunehmen. </li></ol></li><li> Experiment <ol><li> Positionieren Sie die Kamera so, dass sie direkt über einen eigenen Käfig ist. Kalibrieren Sie die Kamera so, dass das gesamte Heimkäfig im Blickfeld ist. </li><li> Wiegen Maus und Maus intraperitoneal injizieren mit geeigneten Dosis von entweder 0,9% Kochsalzlösung oder DOI (0,01 ml / g). HINWEIS: Wenn eine Maus 25 Gramm wiegt, zu verwalten Dosis bis zu einem Gesamtvolumen von 0,25 ml. </li><li> Platzieren Sie jede Maus zurück in ihre Heimat Käfig for 10 min. Nach 10 min Platz Maus in die Mitte des leeren Käfig nach Hause, und bestätigen Sie, dass es in dem Sichtfeld der Kamera keine blinden Flecken sind. Drücken Sie Aufzeichnung am Camcorder. Verlasse den Raum. HINWEIS: Mausbewegungen und verschiedene darin Verhaltensreaktionen (... Kopf zucken, Ohr kratzen, etc. verweisen auf zusätzliche Datentabelle 1 von Gonzalez-Maeso et al 2007 für die vollständige Liste der Verhaltensreaktionen induziert durch DOI) 3, wird aufgezeichnet 30 Minuten. Daher ist es wichtig, keine toten Winkel in dem Sichtfeld aufgezeichnet sind. </li><li> Nach 30 Minuten stoppen Sie den Camcorder und Platz Maus wieder in Original-Käfig Aufnahme. Wiederholen Sie diesen Vorgang für jede Maus. </li></ol></li><li> Überprüfung <ol><li> Lassen Sie jeden Schiedsrichter Überprüfung die Bänder an die experimentellen Bedingungen der Maus blind machen (dh., Drogen während der Kopf verwendet zucken Experiment oder Virus während der intrakranielle Injektion verwendet wird )). Aufzeichnung manuell jeder Kopf zucken throughout das Video. HINWEIS: Kopfzucken als schnelle Schütteln Kopfbewegung durch eine Maus (ergänzende Video) durchgeführt definiert. </li><li> Für jede Maus, im Durchschnitt die endgültige HTR Antwort von den drei Summen der blinden Schiedsrichter. Dann Gruppe diese Werte durch experimentellen Bedingungen und Durchführung von statistischen Analyse (dh., T-Test oder ANOVA). </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.

Zusammen mit früheren Ergebnissen in mGlu2-KO - Mäusen 5 sind die Ergebnisse mit und mGlu2 mGlu2 / mGlu3 chimären Konstrukten , die nicht bilden schlagen die 5-HT 2A Rezeptor - Komplex -mGlu2 in kultivierten Zellen, die die 5-HT 2A -mGlu2 heteromeren Rezeptor - Komplex in Maus frontalen Kortex ist erforderlich Kopfzucken Verhalten von LSD-ähnlichen halluzinogene 5-HT 2A -Rezeptor - Agonisten zu induzieren. Eine Einschränkung dieses Verfahrens ist, dass es nicht zu einem s...

Halluzinogene wie LSD, Psilocybin und Meskalin zu erheblichen Veränderungen im menschlichen Bewusstsein, Kognition und Emotion 7-9. Die Inaktivierung von Serotonin 5-HT 2A - Rezeptor - Signal entweder durch genetische oder pharmakologische Ansätze verursacht deutlich Verhaltensreaktionen auf Halluzinogene in beiden Tiermodellen 3,10 und Menschen 11 gedämpft. Obwohl Halluzinogene anderen Rezeptor - Subtypen binden , 8, die 5-HT 2A -Rezeptor ist als die für die einzigartige Verhaltensaktivität dieser Chemikalien betrachtet. Gruppe II metabotropen Glutamatrezeptoren (dh., MGlu2 und mGlu3) haben das Ziel beträchtlicher Aufmerksamkeit in Bezug auf den molekularen Mechanismus von Halluzinogenen und deren integrale Rolle Psychose zugrunde liegenden 12. Zuvor wurde gezeigt, dass Mäuse ohne Expression mGlu2 Protein (mGlu2-KO-Mäuse) unempfindlich sind, auf die zelluläre und Verhaltenswirkungen von hallucinogens 5. Es wurde auch vorgeschlagen, dass der 5-HT 2A -Rezeptoren und die mGlu2 einen bestimmten heteromeren Komplex bilden , durch den Serotonin und Glutamat - Liganden um das Muster der G - Protein - Kopplung in lebenden Zellen 1,2 modulieren. Strukturell Trans (TM) Domänen 4 und 5 der mGlu2 eine fundamentale Rolle in heteromeren Bildung spielen mit dem 5-HT 2A -Rezeptor - 5. Zusätzlich zeigten weitere Untersuchungen , daß drei Reste am intrazellulären Ende TM4 von mGlu2 angeordnet sind notwendig , um die 5-HT 2A -Rezeptor -mGlu2 Hetero in lebenden Zellen 6 zu bilden. Auf Basis dieser Erkenntnisse in heterologen Expressionssystemen beobachtet, beschreiben wir die Verwendung von HSV-vermittelte Expression von Wildtyp mGlu2 und mGlu2 / mGlu3 chimären Konstrukten im frontalen Kortex von mGlu2-KO - Mäuse , ob heteromeren Bildung zu testen , zwischen 5-HT 2A und mGlu2 ist notwendig, dass dieKopf-Twitch - Verhalten von halluzinogenen 5-HT 2A -Rezeptor - Agonisten induziert.

<table border="0" cellpadding="0" cellspacing="0" width="2329">
	<tbody>
		<tr height="21">
			<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.

Vorherige Ergebnisse zeigen , dass die Kopf zucken murine Verhaltensreaktion zuverlässig und robust durch Halluzinogene hervorgerufen, und es fehlt die in 5-HT 2A -KO Mäuse 3. Darüber hinaus hat es sich gezeigt , dass der Kopf-Zuckungsreaktion durch die halluzinogene 5-HT 2A -Agonisten DOI und LSD ausgelöst deutlich in mGlu2-KO - Mäusen 5 verringert wurde. Obwohl jedoch frühere Ergebnisse überzeugend zeigen , dass 5-HT 2A und ...

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

Dieser Artikel beschreibt, wie virale Vektoren in die Maus frontalen Kortex zu injizieren Verhaltenstests zu testen, die GPCR heteromeren Bildung erfordern.

HSV-Mediated Transgene Expression von chimären Konstrukten Behavioral Funktion von GPCR Heteromeren bei Mäusen zu studieren

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 Aufrufe.  Icahn School of Medicine at Mount Sinai.  Dieser Artikel beschreibt, wie virale Vektoren in die Maus frontalen Kortex zu injizieren Verhaltenstests zu testen, die GPCR heteromeren Bildung erfordern. 

Serie: 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.

Eine allgemeine Methode zur Bewertung der Inkubation von Saccharose Craving in Ratten

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 ...

Forschung

Neurowissenschaften

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.

Lokale Anwendung von Medikamenten zur nikotinischen Acetylcholin-Rezeptor-Funktion in Maus Hirnschnitten Studie

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.

Eine einfache Verhaltenstest zur Prüfung von Visual Function in<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.

Bewertung der dendritischen Verzweigung im Gyrus dentatus des Hippocampus-Region an der Maus

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.

Quantifizierung von filamentösen Aktin (F-Aktin) 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 ...

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.

Eine frei atmende fMRI-Methode zur Untersuchung der menschlichen olfaktorischen Funktion

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.

Adeno-assoziierten Virus-vermittelten Transgene Ausdruck in genetisch definierten Nervenzellen des Rückenmarks

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.

Die Macht der Interstimulus Intervall für die Beurteilung der zeitlichen Verarbeitung bei Nagern

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.

3D Kinematische Analyse zur funktionellen Bewertung im Rattenmodell von Ischias-Nerven-Crush-Verletzungen

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.

Ballistische Kennzeichnung von Pyramidal neurons in Gehirnscheiben und in der primären Zellkultur

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