Ringraziamo Bakhos A. Tannous, Ph.D., Direttore dello Sviluppo e Produzione vettore nel Centro Neuroscienze del Massachusetts General Hospital, Charlestown, Massachusetts, per averci fornito il rAAV-EGFP vettore, e John Whang per l&#39;assistenza tecnica. Questo lavoro è stato sostenuto dalla concessione R01 NS050408 (JS), presso l&#39;Istituto nazionale dei disordini neurologici e colpo.

La procedura chirurgica descritta è stato approvato dalla cura degli animali e del Comitato Istituzionale uso (IACUC) della Columbia University. 1. Preparazione delle apparecchiature e delle particelle sospensione di virus <ol><li> Pulire e disinfettare le attrezzature, sterilizzare gli strumenti chirurgici e le punte notch V che verranno utilizzati per fissare vertebra L1. </li><li> Tirare e pipette di vetro conici. Usiamo pipette che hanno un diametro di punta di 40 micron e sono smussati con un angolo di 20 °. Sterilizzare le pipette di vetro. </li><li> Impostare il telaio stereotassico, montare l&#39;iniettore microsiringa sulla manipolatore e collegare l&#39;iniettore al controllore. </li><li> Collegare una delle pipette di vetro per la microsiringa utilizzando il kit di raccordo a compressione. </li><li> Rimuovere lo stantuffo dalla microsiringa e riempire la siringa con olio minerale. Oil Red O (1 - (2,5-dimetil-4-(2,5-dimethylphenylazo) fenilazo)-2-naftolo) può essere aggiunto al olio minerale per aumentare la sua visibility. Reinserire lo stantuffo e spingerla per tutta la strada fino alla punta. Accuratamente evitare di creare una bolla d&#39;aria. </li><li> Preparare la sospensione di particelle di virus in un armadio biosicurezza. Scongelare il virus congelato su ghiaccio e, immediatamente prima dell&#39;uso, diluire con sterile PBS alla concentrazione di particelle desiderato. </li><li> Inserire la microsiringa nel supporto dell&#39;iniettore. </li><li> Pipettare 5 microlitri della sospensione di virus su un foglio di plastica, ad esempio Parafilm. Abbassare la punta della pipetta di vetro nella goccia e tirare lo stantuffo per riempire la microsiringa. Creare una piccola bolla d&#39;aria sulla punta della pipetta per evitare che si intasi. </li></ol> 2. Laminectomia <ol><li> Preparare l&#39;area di intervento pulendo panchina e piastra elettrica con disinfettante. </li><li> Anestetizzare il mouse. Usiamo l&#39;anestesia per inalazione con isoflurano (3% durante l&#39;induzione, 2% -3% durante la manutenzione). </li><li> Posizionare lubrificante su ciascun occhio per proteggere gli occhi dalla disidratazione durante l&#39;funzionamento. </li><li> Accorciare il pelo della schiena al collo del mouse e disinfettare la pelle con salviette alternata di un antisettico topico come clorexidina o polivinilpirrolidone-iodio e il 70% di etanolo. Isolare il sito in modo asettico preparato con telo chirurgico e infiltrarsi nel sito di incisione con bupivacaina (0,25%, diluito 1:10 con soluzione fisiologica). </li><li> Incidere la pelle all&#39;estremità caudale della gabbia toracica lungo la linea mediana (2-3 cm) e separare la fascia che copre la colonna vertebrale. </li><li> Poiché il midollo spinale smette di crescere in precedenza durante lo sviluppo postnatale rispetto alla colonna vertebrale, segmento spinale L4 si trova sotto la prima vertebra lombare (L1). Vertebra L1 si trova caudalmente alla vertebra che contiene l&#39;ultimo paio di costole. Ed esporre vertebra L1, eliminando i piccoli muscoli della colonna vertebrale e dei legamenti attaccati alla superficie dorsale. </li><li> Sollevare leggermente e tenere premuto L1 vertebra con una pinza Adson. Utilizzare una pinza laminectomia dedicato per rimuovere la dorsale portion della vertebra (colonna vertebrale e lamina) ed esporre il midollo spinale. Evitare di danneggiare il midollo spinale. </li><li> Trasferire il mouse sulla piastra riscaldante nel telaio stereotassico. Monitorare la temperatura del mouse durante la successiva operazione. </li></ol> 3. Iniezione <ol><li> Fissare vertebra L1 con punte tacca V. Le punte devono stabilizzare la colonna in modo che la vertebra non si sposta durante la respirazione. </li><li> Portare il microsiringa più vicino in modo che la punta della pipetta è al di sopra del sito di laminectomia. Abbassare lo stantuffo fino a visualizzare la sospensione di virus in uscita la pipetta. Rimuovere la goccia con la punta di cotone sterile. </li><li> Posizionare la punta della pipetta in più a parte rostrale del midollo spinale esposto. Centro la pipetta sopra il solco posteriore mediana, quindi spostare la punta 500 pm lateralmente. Abbassare la punta sulla superficie del cavo e la puntura della dura madre o, se si lavora con una pipetta di vetro unbeveled, utilizzare una cannula smussata in acciaio per forare il dura. Abbassare la punta della pipetta di vetro 300 micron nel midollo spinale. </li><li> Iniettare 1 ml di sospensione di virus a una velocità di 200 nl / min. </li><li> Al termine dell&#39;iniezione, attendere almeno 2 minuti prima lentamente ritraendo la pipetta. </li><li> Ripetere i passaggi 3,3-3,5 al-caudale più porzione del midollo spinale esposta per ottenere una distribuzione completa del vettore virale in L4 segmento spinale. I due siti di iniezione si trovano rostrale e caudale del segmento L4, per evitare danni ai tessuti nella regione di destinazione. </li></ol> 4. Chiusura della ferita <ol><li> Rilasciare vertebra L1 dalle fascette tacca le V e togliere il mouse dal telaio stereotassico. </li><li> Suturare la fascia con 5,0 Vicryl. La fascia chiuso prevede la copertura per il sito laminectomia. </li><li> Chiudere la pelle con punti di sutura in nylon o graffette chirurgiche. </li></ol> 5. Cura postoperatoria <ol><li> Trasferire il mouse a una gabbia di recupero con morbido, biancheria da letto nonparticular. Mettere sul tegli laterali per la respirazione confortevole. Monitorare l&#39;animale fino a quando non è completamente vigile, ambulatoriale e inizia a bere. </li><li> Forniamo analgesia post-chirurgica per 72 ore al giorno con iniezioni sottocutanee di carprofen (5,0 mg / kg). </li><li> Monitorare il recupero post-chirurgico mediante ispezione giornaliera per i primi 3 giorni, poi a giorni alterni o 3 giorni a settimana fino a quando l&#39;esperimento è completo. </li><li> Rimuovere le suture cutanee o graffe 7 a 10 giorni dopo l&#39;operazione, quando la guarigione della ferita è completa. </li><li> Euthanize l&#39;animale al termine dell&#39;esperimento. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sensory+axon+regeneration+in+adult+spinal+cord.">Targeting sensory axon regeneration in adult spinal cord.</a> J. Neurosci. 27, 6068-6078 (2007).</li><li>Cameron, A. A., Smith, G. M., Randall, D. C., Brown, D. R., Rabchevsky, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+intraspinal+plasticity+after+spinal+cord+injury+alters+the+severity+of+autonomic+dysreflexia.">Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.</a> J. Neurosci. 26, 2923-2932 (2006).</li><li>Passini, M. 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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=dsAAV+type+2-mediated+gene+transfer+of+MORS196A-EGFP+into+spinal+cord+as+a+pain+management+paradigm.">dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 20096-20101 (2007).</li><li>South, S. 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=A+conditional+deletion+of+the+NR1+subunit+of+the+NMDA+receptor+in+adult+spinal+cord+dorsal+horn+reduces+NMDA+currents+and+injury-induced+pain.">A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.</a> J. Neurosci. 23, 5031-5040 (2003).</li><li>Tappe, 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=Synaptic+scaffolding+protein+Homer1a+protects+against+chronic+inflammatory+pain.">Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.</a> Nat. Med. 12, 677-681 (2006).</li><li>Colle, M. 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=Efficient+intracerebral+delivery+of+AAV5+vector+encoding+human+ARSA+in+non-human+primate.">Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.</a> Human Molecular Genetics. 19, 147-158 (2010).</li><li>Carbajal, K. S., Weinger, J. G., Whitman, L. M., Schaumburg, C. S., Lane, T. 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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Iniezione stereotassica vettore permette il targeting neuroni del midollo spinale per applicazioni come la mappatura della rete neuronale basata sul virus transsynaptic diffondere 6,7 o dissezione optogenetic 8, la guida durante la rigenerazione assonale da lesioni 9,10, o la terapia genica per la prevenzione o il trattamento di neurodegenerazione 11, 12. Vettori virali sono stati utilizzati per la manipolazione del gene nel midollo spinale di studiare somatosensoriali, motori...

Le tecnologie avanzate di manipolazione genica condizionale nel topo consentono molteplici approcci per l&#39;esplorazione di sinapsi e collegamenti funzionali del sistema nervoso centrale. Transgeni può essere regolata da piccole molecole come effettori doxiciclina agenti su un transattivatore della tetraciclina controllato, che può essere progettato per funzionare come un repressore o un attivatore della trascrizione genica, o tamoxifene riconoscere un mutato ligando dominio di legame del recettore di estrogeno 1 . Modifica irreversibile transgene è comunemente ottenuto da acido desossiribonucleico (DNA) ricombinasi. Cre (ricombinazione cause) e Flp (enzima ricombinazione flippase) catalizzare l&#39;, inversione escissione o la traslocazione di frammenti di DNA che vengono affiancati da loxP (luogo di x crossing over, P1) o Frt (target riconoscimento flippase) siti, rispettivamente, 1. Le applicazioni includono l&#39;attivazione del gene o silenziamento inducibile e acido ribonucleico (RNA) ingerenza <sup&gt; 2. Espressione condizionale reporter fluorescente o enzimatica come β-galattosidasi o fosfatasi alcalina può essere usato per etichettare neuroni ed esaminare la loro organizzazione topica e connettività 3. Grandi progetti di mutagenesi in Nord America ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) e in Europa ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) producono librerie di cloni di cellule staminali embrionali di topo con obiettivi gene condizionali e le trappole che alla fine coprono l&#39;intero genoma del mouse. Topi generati da questi cloni possono essere attraversata con un numero crescente di topi che esprimono ricombinasi DNA sotto promotori o loci specifici per una particolare popolazione di neuroni di manipolazione genica selettiva ( <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; Tuttavia, limitando la manipolazione genetica di popolazioni distinte di neuroni o regioni particolari di interesse non può essere ottenuto genetica mira solo se un promotore specifico per la popolazione neurone di interesse non è noto o non espresso da tutti i neuroni della regione di interesse. Nel midollo spinale, disegni sperimentali possono richiedere restrizione spaziale della manipolazione genetica di uno o due segmenti cranio-caudale. Iniezione stereotassica di un vettore virale che esprime Cre o Flp consente di limitare ricombinazione genica per regioni nel midollo spinale di topi in cui si affiancano frammenti di DNA da loxP o siti FRT, cosiddetti alleli floxed o flrted. A differenza di riarrangiamenti del DNA costitutivo, con la conseguenza di incroci animali con topi che esprimono ricombinasi, questa strategia consente anche il controllo temporale su attivazione genica o silenziamento. Vettori virali codificanti floxed o flirtato transgeni offre un&#39;opzione di inversione di manipolazione del gene nei topi esprimere la corresponding valle ricombinasi di un neurone-specifica promotore. Diversi vettori ricombinanti con affinità per i neuroni sono disponibili 4. Alta capacità (gutless) adenovirus, virus adeno-associati, virus dell&#39;herpes simplex e lentivirus sono comunemente utilizzati vettori neurotropici. Selezione del virus appropriato per una domanda di ricerca è una parte cruciale del disegno sperimentale. Dimensione del transgene, percorso di consegna, specificità dell&#39;infezione di neuroni a differenza di cellule gliali, efficacia infezione, effetti collaterali tossici infiammatori e devono essere considerati 4. Qui descriviamo l&#39;iniezione stereotassica di un vettore virale nel corno dorsale del midollo spinale, una tecnica che usiamo per la regolazione genica condizionale nella nostra ricerca sulla neurobiologia del dolore. Il corno dorsale riceve l&#39;input afferente da primari neuroni somatosensoriali inclusi i neuroni nocicettivi. Interneuroni locali elaborare le informazioni prima di neuroni di proiezione che trasmettono dail corno dorsale al cervello 5. Dimostriamo l&#39;infezione di neuroni del corno dorsale a livello segmentale spinale L4 con neurotropico ricombinante virus adeno-associato (rAAV) che esprime migliorato proteina fluorescente verde (EGFP) sotto un promotore di citomegalovirus costitutivamente attivo.

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Numero di catalogo </td><td></td></tr><tr><td> Piastra di base della colonna vertebrale </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Piccolo animale strumento stereotassico </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Mouse gas anestesia testa porta </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Supporti di base regolabili </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> V notch picchi </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Piccolo animale temperatura del sistema di controllo </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson forcipe </td><td> Strumenti Scienza Belle </td><td> 11006-12 </td><td></td></tr><tr><td> Laminectomia pinza </td><td> Strumenti Scienza Belle </td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump (uno) con SYS-Micro4 controller </td><td> Mondo Strumenti di precisione </td><td> UMP3-1 </td><td></td></tr><tr><td> Microsiringa, 65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> RN raccordo a compressione, 1 mm </td><td> Hamilton </td><td> 55750-01 </td><td></td></tr><tr><td> Vetro borosilicato capillari </td><td> Mondo Strumenti di precisione </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

Iniezione intraparenchimale di un vettore virale consente la manipolazione condizionale gene in distinte popolazioni di neuroni o regioni specifiche del sistema nervoso centrale. Dimostriamo una tecnica stereotassica iniezione che permette l&#39;espressione del gene bersaglio o silenziamento nel corno dorsale del midollo spinale del mouse. La procedura chirurgica è breve. Richiede laminectomia di una singola vertebra, che prevede il recupero rapido dell&#39;animale e motilità perfetta della colonna vertebrale. Iniezione controllata di un piccolo volume di sospensione vettore a bassa velocità e l&#39;uso di una cannula microsiringa con vetro smussato minimizzare la lesione tissutale. La risposta immunitaria locale al vettore dipende dalle proprietà intrinseche del virus impiegati, nella nostra esperienza, è minore e di breve durata, quando un ricombinante adeno-associated virus viene utilizzato. Un gene reporter come migliore proteina fluorescente verde facilita distribuzione spaziale monitoraggio del vettore, e l&#39;efficacia e cellulare specificity della trasfezione.

Rendimenti di trasfezione di successo l&#39;espressione genica robusta nei neuroni del corno dorsale iniettato (Figura 1), risparmiando il corno dorsale del lato controlaterale, il corno ventrale e gangli delle radici dorsali. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figura 1" /> Figura 1. La trasfezione di neuroni del corno dorsale. (A) Espressione della EGFP reporter fluorescente (verde) nel corno sinistro dorsale del midollo spinale L4, ...

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

Vettori virali per consentire la manipolazione genica mirata. Abbiamo dimostrato un metodo per l&#39;espressione genica condizionale o ablazione nel midollo spinale topo, utilizzando iniezione stereotassica di un vettore virale nel corno dorsale, un luogo di rilievo di contatto sinaptico tra afferenze somatosensoriali primarie e neuroni del sistema nervoso centrale.

Iniezione stereotassica di un vettore virale per la manipolazione del gene condizionale nel midollo spinale del mouse

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

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30.1K Views.  Centre National de la Recherche Scientifique (CNRS). Vettori virali per consentire la manipolazione genica mirata. Abbiamo dimostrato un metodo per l'espressione genica condizionale o ablazione nel midollo spinale topo, utilizzando iniezione stereotassica di un vettore virale nel corno dorsale, un luogo di rilievo di contatto sinaptico tra afferenze somatosensoriali primarie e neuroni del sistema nervoso centrale.

Video: 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 Elettrofisiologia II: extracellulare fabbricazione di elettrodi di aspirazione

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

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Neuroscienze

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.

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

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.

Ampliamento della embrionali e cellule staminali neurali adulte per<em&gt; In Utero</em&gt; Elettroporazione o Viral iniezione stereotassica

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 Iniezione virale del cervello neonatale mouse per persistente e diffusa trasduzione neuronale

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.

Una procedura di impianto del Camera spinale per longitudinale<em&gt; In Vivo</em&gt; Imaging del cavo del mouse spinale

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.

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

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.

Chirurgia stereotassica per la manipolazione genetica in cellule striatali di cervelli di topo neonatale

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.

Laminectomia e impianto della finestra del cavo spinale nel 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 ...

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.

Iniezione diretta di un vettore lentivirale evidenzia più vie motorie del midollo spinale del ratto

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.

Consegna intra-arteriosa di cellule staminali neurali al cervello di ratto e topo: applicazione all'ischemia cerebrale

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