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>

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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><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% 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

Universidad de Cartagena UDEC

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

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentivirale vermittelte Produktion von transgenen Mäusen: eine einfache und hocheffiziente Methode für die direkte Untersuchung der Gründer

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Forschung

Genetik

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Mosaic Analyse der Genfunktion in Postnatale Mäusehirnen mithilfe von Virus-basierten Cre Rekombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Neurowissenschaften

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus-vermittelte Gen-Manipulation und Visualisierung von Riechzellen In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Viral Tracing von genetisch definierten Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Stereotaktische Injektion eines viralen Vektors für Conditional Gene Manipulation im Maus-Rückenmark

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

Somatic Genom-Engineered Mausmodelle mit In-vivo-Mikroinjektion und Elektroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

Krebsforschung

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Bedingte Genetische transsynaptische Tracing in der embryonalen Maus-Gehirn

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Eine benutzerdefinierte Multiphotonen-Mikroskopie-Plattform für Live-Bildgebung von Maus-Cornea und Konjunktiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Biotechnik

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 ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Multi-Fiber Photometrie zur Aufzeichnung neuronaler Aktivität bei frei beweglichen Tieren

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...