Wir danken Bakhos A. Tannous, Ph.D., Direktor des Vector Entwicklung und Produktion in der Neuroscience Center of Massachusetts General Hospital in Charlestown, Massachusetts, für die Bereitstellung der rAAV-EGFP Vektor, und John Whang für technische Unterstützung. Diese Arbeit wurde von grant R01 NS050408 (JS) aus dem National Institute of Neurological Disorders and Stroke unterstützt.

Das chirurgische Verfahren beschrieben wurde vom Institutional Animal Care und Use Committee (IACUC) der Columbia University genehmigt worden. Ein. Vorbereitung der Ausrüstung und Virus-Suspension <ol><li> Reinigen und desinfizieren Sie das Gerät, sterilisieren die chirurgischen Instrumente und die V-Kerbe Spikes, die verwendet werden, um Wirbel L1 beheben werden. </li><li> Ziehen und Kegel Glaspipetten. Wir verwenden Pipetten, die einen Durchmesser der Spitze von 40 um aufweisen und in einem Winkel von 20 ° abgeschrägt. Sterilisieren Sie die Glas-Pipetten. </li><li> Richten Sie den stereotaktischen Rahmen, montieren Sie die Mikrospritze Injektor auf dem Manipulator und verbinden Sie den Injektor mit dem Controller. </li><li> Bringen Sie einen der Glaspipetten der Mikrospritze mit der Klemmverschraubung Kit. </li><li> Entfernen Sie den Kolben aus der Mikroliterspritze und füllen Sie die Spritze mit Mineralöl. Oil Red O (1 - (2,5-Dimethyl-4-(2,5-dimethylphenylazo) phenylazo)-2-naphthol), zu dem Mineralöl zugegeben werden, um seine v erhöhenisibility. Setzen Sie den Kolben und schieben Sie es den ganzen Weg bis zur Spitze. Sorgfältig vermeiden damit eine Luftblase erzeugt. </li><li> Bereiten Sie die Viruspartikel Suspension in einer biologischen Sicherheitswerkbank. Auftauen des gefrorenen Virus auf Eis und kurz vor Gebrauch verdünnte mit steriler phosphatgepufferter Kochsalzlösung auf die gewünschte Teilchengröße Konzentration. </li><li> Legen Sie die Mikrospritze in die Halterung an der Einspritzdüse. </li><li> Pipette 5 ul der Virus-Suspension auf eine kleine Kunststoff-Folie, zB Parafilm. Senken Sie das Glas Pipettenspitze in dem Tropfen und ziehen Sie den Kolben, um die Mikrospritze füllen. Erstellen Sie eine kleine Luftblase an der Pipettenspitze, um ein Verstopfen zu verhindern. </li></ol> 2. Laminektomie <ol><li> Bereiten Sie den OP-Bereich durch Abwischen Bank und Heizkissen mit Desinfektionsmittel. </li><li> Anesthetize der Maus. Wir verwenden Inhalationsnarkose mit Isofluran (3% während der Induktion, 2% -3% bei Wartungsarbeiten). </li><li> Zeigen Schmiermittel auf jedes Auge, um die Augen vor dem Austrocknen während der Schutz derBetrieb. </li><li> Rasur das Fell von den unteren Rücken, um den Hals des Maus und desinfizieren die Haut mit wechselnden Tücher einer Antiseptikum wie Chlorhexidin oder PVP-Jod und 70% Ethanol. Isolierung des aseptisch vorbereitete Stelle mit Operationstuch und infiltrieren die Einschnittstelle mit Bupivacain (0,25%, 1:10 mit physiologischer Kochsalzlösung). </li><li> Einzuschneiden die Schale am caudalen Ende des Brustkorbs entlang der Mittellinie (2-3 cm) und trennen die Faszie Abdecken der Wirbelsäule. </li><li> Weil das Rückenmark aufhört zu wachsen früher während der postnatalen Entwicklung außer der Wirbelsäule liegt spinale Segment L4 unterhalb der ersten Lendenwirbel (L1). Wirbel L1 befindet kaudal des Wirbels, der die letzte Paar Rippen hält. Identifizieren und aufzudecken Wirbel L1, indem die kleinen Rückenmuskeln und Bänder an seiner dorsalen Oberfläche. </li><li> Leicht anheben und halten Wirbel L1 mit einer Adson Pinzette. Verwenden Sie einen dedizierten Laminektomie Zangen, um den dorsalen Portio entfernenn des Wirbels (Wirbelsäule und Lamina) und setzen das Rückenmark. Vermeiden Sie Schäden am Rückenmark. </li><li> Übertragen Sie die Maus auf der Heizplatte in der stereotaktischen Rahmen. Überwachen Sie die Temperatur der Maus im späteren Betrieb. </li></ol> 3. Injektion <ol><li> Fix Wirbel L1 mit V-Kerbe Spikes. Die Spitzen müssen stabilisieren die Wirbelsäule, so dass der Wirbel nicht während der Atmung bewegen. </li><li> Bringen Sie die Mikrospritze näher, so dass die Pipettenspitze oberhalb des Laminektomie Website ist. Senken Sie den Kolben, bis Sie die Virussuspension Verlassen der Pipette zu sehen. Nehmen Sie die Tropfen mit einem sterilen Wattestäbchen. </li><li> Positionieren der Pipettenspitze am rostralen äußersten Teil der freiliegenden Rückenmarks. Zentrieren Sie die Pipette über den hinteren Mittelfurche, dann bewegen Sie die Spitze 500 um seitlich. Senken Sie die Spitze an der Oberfläche des Rückenmarks und Punktion der Dura mater oder, wenn Sie mit einem unbeveled Glaspipette arbeiten, verwenden eine abgeschrägte Stahlkanüle zu durchstechen die dura. Senken Sie die Spitze der Glaspipette 300 um in das Rückenmark. </li><li> Inject 1 ul Virussuspension mit einer Rate von 200 nl / min. </li><li> Am Ende der Einspritzung, warten Sie mindestens 2 Minuten vor langsam Zurückziehen der Pipette. </li><li> Wiederholen Schritte 3.3 bis 3,5 bei der kaudalen äußersten Teil der freiliegenden Rückenmarks zur vollständigen Verteilung des viralen Vektors in Wirbelsegment L4 erreichen. Die beiden Injektionsstellen befinden rostralen und kaudal des L4-Segment zu Gewebeschäden in der Zielregion zu vermeiden. </li></ol> 4. Wundverschluss <ol><li> Release Wirbel L1 aus den V-Kerbe Klemmen und entfernen Sie die Maus aus dem stereotaktischen Rahmen. </li><li> Naht der Faszie mit 5,0 Vicryl. Die geschlossene Blende bietet Versicherungsschutz für die Laminektomie Ort. </li><li> Schließen Sie die Haut mit Nylon-Fäden oder chirurgische Klammern. </li></ol> 5. Postoperative Pflege <ol><li> Übertragen Sie die Maus, um eine Erholung Käfig mit weichen, nonparticular Betten. Legen Sie sie auf ter Seite für bequeme Atmung. Überwachen das Tier, bis sie vollständig wach, ambulant ist und fängt an zu trinken. </li><li> Wir bieten postoperativen Analgesie für 72 Stunden mit täglichen subkutanen Injektionen von Carprofen (5,0 mg / kg). </li><li> Überwachung postoperativen Erholung durch tägliche Inspektion für die ersten 3 Tage, dann jeden zweiten Tag oder 3 Tage pro Woche, bis das Experiment abgeschlossen ist. </li><li> Entfernen Sie die Haut Nähte oder Klammern 7 bis 10 Tage nach der Operation, wenn die Wundheilung abgeschlossen ist. </li><li> Euthanize das Tier nach Beendigung des Experiments. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Stereotaktische Injektion ermöglicht Targeting-Vektor Rückenmarksneuronen für Anwendungen wie neuronale Netzwerk-Mapping basierend auf transsynaptische Virusverschleppung 6,7 oder optogenetische Dissektion 8, Axon Führung während der Regenerierung vor Verletzungen 9,10 oder Gentherapie zur Prävention oder Behandlung von Neurodegeneration 11, 12. Virale Vektoren sind für Genmanipulation worden im Rückenmark verwendet, um zu untersuchen somatosensorischen, Motor und vege...

Fortgeschrittenen Technologien bedingter Genmanipulation in der Maus zu ermöglichen vielfältige Ansätze zur Exploration von synaptischen Bahnen und funktionelle Verbindungen im zentralen Nervensystem. Transgene können durch niedermolekulare Effektoren wie Doxycyclin Einwirken auf ein Tetracyclin-gesteuerten Transaktivator, die für den Einsatz als Repressor oder Aktivator der Gentranskription, oder Tamoxifen Erkennen eines mutierten Ligandenbindungsdomäne des Östrogenrezeptors 1 funktionieren kann geregelt werden . Irreversible Modifikation Transgen wird üblicherweise von Desoxyribonukleinsäure (DNA) Rekombinasen erreicht. Cre (Ursachen Rekombination) und Flp (Flippase Rekombinationsenzyms) katalysieren die Exzision, Inversion oder Translokation von DNA-Fragmenten, die von loxP (locus der Kreuzung x über, P1) oder Frt (Flippase Anerkennung target) Seiten bzw. 1 flankiert werden. Zu den Anwendungen gehören Gen-Aktivierung oder Inaktivierung und induzierbare Ribonukleinsäure (RNA) Störungen <sup&gt; 2. Bedingungsausdruck von fluoreszierenden oder enzymatischen Reporter wie β-Galactosidase oder alkalischer Phosphatase verwendet werden, um Neuronen beschriften und prüft ihre topische Organisation und Konnektivität 3 werden. Large-scale Mutagenese-Projekte in Nordamerika ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) und Europa ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) produzieren Bibliotheken embryonalen Stammzellen der Maus Zellklone mit bedingten Gen-Targets und Fallen, die letztlich auf das gesamte Maus-Genom. Mäuse aus diesen Klonen erzeugt werden, können mit einer wachsenden Zahl von Maus Linien gekreuzt werden, daß DNA-Rekombinasen exprimieren unter Promotoren oder Loci für eine bestimmte Population von Neuronen zur selektiven Genmanipulation ( <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . php</a> ). <p class= &quot;Jove_content&quot;&gt; Allerdings kann die Beschränkung auf Genmanipulation unterschiedliche Populationen von Neuronen oder bestimmten Regionen von Interesse nicht durch genetische Targeting allein, wenn ein Promotor spezifisch für die Neuronenpopulation von Interesse nicht bekannt ist und nicht von allen Neuronen in der Region exprimiert erreicht werden von Interesse. Im Rückenmark können experimentellen Designs erfordern räumlichen Beschränkung der Genmanipulation ein oder zwei kraniokaudalen Segmente. Stereotaktische Injektion eines viralen Vektors, Cre oder Flp drückt ermöglicht Begrenzen Genrekombination in Regionen im Rückenmark von Mäusen, in denen DNA-Fragmente von loxP-oder FRT-Stellen flankiert sind, so genannte floxed oder flrted Allelen. Anders als konstitutive DNA-Umlagerung, die würde aus Kreuzungen der Tiere mit Rekombinase exprimierenden Mäusen führen, diese Strategie auch zeitliche Kontrolle über Gen-Aktivierung oder Inaktivierung. Virale Vektoren kodieren gefloxten oder geflirtet Transgene bieten eine umgekehrte Möglichkeit Genmanipulation in Mäusen, die corresponding Rekombinase stromabwärts eines Neuron-spezifischen Promotors. Mehrere rekombinante Vektoren mit Affinität zu Neuronen sind 4. Hochleistungs-(gutless) Adenovirus, Adeno-assoziiertes Virus, Herpes simplex-Virus und Lentivirus-Vektoren werden üblicherweise neurotropen verwendet. Die Auswahl des geeigneten Virus für eine Fragestellung ist ein wichtiger Teil des experimentellen Designs. Größe des Transgens, Lieferroute, Spezifität der Infektion mit Neuronen, um Gliazellen, Infektion Wirksamkeit, entzündlichen und toxische Nebenwirkungen müssen berücksichtigt werden 4 entgegengesetzt. Hier beschreiben wir die stereotaktische Injektion eines viralen Vektors in der dorsalen Horn des Rückenmarks, eine Technik, die wir verwenden zur bedingten Genregulation in unserer Forschung über die Neurobiologie von Schmerz. Das Hinterhorn erhält afferenten Input von primären somatosensorischen Neuronen einschließlich nozizeptiven Neuronen. Lokale Interneurone verarbeiten die Informationen vor der Projektion Neuronen vermitteln sie ausdas Hinterhorn des Gehirns 5. Wir demonstrieren die Infektion von Dorsalhorn spinalen Neuronen an Segmentebene L4 mit einem neurotropen rekombinanten Adeno-assoziierten Virus (rAAV), das verstärkt grün fluoreszierende Protein (EGFP) drückt unter einem konstitutiv aktiven CMV-Promotors.

<table border="1"><tbody><tr><td> Material Name </td><td> Firma </td><td> Katalog-Nummer </td><td></td></tr><tr><td> Spinal Grundplatte </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Kleintier stereotaktischen Instrument </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Maus Gas Anästhesie Kopfhalter </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Verstellbare Basis mounts </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> V-Kerbe Spikes </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Kleintier-Temperaturregelung </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson Pinzette </td><td> Fine Science Tools </td><td> 11006-12 </td><td></td></tr><tr><td> Laminektomie Zangen </td><td> Fine Science Tools </td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump (ein) mit SYS-Micro4-Controller </td><td> World Precision Instruments </td><td> UMP3-1 </td><td></td></tr><tr><td> Mikroliterspritze, 65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> RN Klemmverschraubung, 1 mm </td><td> Hamilton </td><td> 55750-01 </td><td></td></tr><tr><td> Borosilikatglas Kapillaren </td><td> World Precision Instruments </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Intraparenchymalen Injektion eines viralen Vektors ermöglicht bedingten Genmanipulation in unterschiedliche Populationen von Neuronen oder bestimmte Bereiche des zentralen Nervensystems. Wir demonstrieren eine stereotaktische Injektion Technik, gezielt Genexpression oder Silencing im dorsalen Horn des Rückenmarks Maus ermöglicht. Der chirurgische Eingriff ist kurz. Es erfordert Laminektomie eines einzelnen Wirbel, bietet für eine schnelle Wiederherstellung des Tieres und ungestörte Beweglichkeit der Wirbelsäule. Kontrollierte Injektion eines kleinen Vektor Suspensionsvolumen bei niedriger Drehzahl und die Verwendung eines Mikrospritze mit abgeschrägten Glaskanüle minimieren Gewebeläsion. Die lokale Immunantwort auf den Vektor, hängt von den spezifischen Eigenschaften des Virus eingesetzt; in unserer Erfahrung ist es kleinen und kurzlebigen wenn ein rekombinantes Adeno-assoziierten Virus verwendet wird. Ein Reportergen wie verstärkt grün fluoreszierende Protein erleichtert die Beobachtung räumliche Verteilung des Vektors oder der Wirksamkeit und zellulären Spezificity der Transfektion.

Erfolgreiche Transfektion ergibt robuste Genexpression in Nervenzellen des eingespritzten Dorsalhorn (Abbildung 1), Schonung der dorsalen Horn von der Gegenseite, Vorderhorn und der Spinalganglien. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Transfektion von Hinterhorn-Neuronen. (A) Expression des fluoreszierenden Reporter eGFP (grün) in der linken Hinterhorn des L4 Rückenmark, zwei Wochen nach der stereotaktisch...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

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

Virale Vektoren können für gezielte Genmanipulation. Wir demonstrieren, ein Verfahren zur bedingten Genexpression oder Ablation im Rückenmark der Maus, stereotaktische Injektion unter Verwendung eines viralen Vektors in der dorsalen Horn, ein bekannter Ort der synaptischen Kontakt zwischen primären somatosensorischen Afferenzen und Neuronen des zentralen Nervensystems.

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 ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

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30.1K Aufrufe.  Centre National de la Recherche Scientifique (CNRS). Virale Vektoren können für gezielte Genmanipulation. Wir demonstrieren, ein Verfahren zur bedingten Genexpression oder Ablation im Rückenmark der Maus, stereotaktische Injektion unter Verwendung eines viralen Vektors in der dorsalen Horn, ein bekannter Ort der synaptischen Kontakt zwischen primären somatosensorischen Afferenzen und Neuronen des zentralen Nervensystems.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elektrophysiologie II: Extrazelluläre Saugelektrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Forschung

Neurowissenschaften

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In-vivo-Imaging von der Maus Spinal Cord mit Zwei-Photonen-Mikroskopie

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Erweiterung der embryonalen und adulten neuralen Stammzellen<em&gt; In Utero</em&gt; Elektroporation oder Viral stereotaktische Injektion

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Intracerebroventricular Viral Injektion der Neonatal Maus Gehirn für anhaltenden und verbreiteten Neuronale Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Ein Verfahren für die Implantation eines Spinal Kammer für Längs<em&gt; In-vivo-</em&gt; Imaging der Maus Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Zwei-Photonen-Imaging von Zelluläre Dynamik in der Maus Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Stereotaktischen Chirurgie für Genmanipulation in Striatale Zellen des Neugeborenen Mäusegehirnen

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminektomie und Rückenmarksfenster-Implantation in der Maus

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Direkter Einspritzung von Lentivirale Vektor zeigt mehrere motorische Bahnen im Rückenmark Ratte

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Intra-arterielle Lieferung von neuronalen Stammzellen an das Ratten- und Maushirn: Anwendung auf zerebrale Ischämie

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...