This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

NOTA: Etica Dichiarazione: Il protocollo per la gestione degli animali è stato approvato dalla cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) della University of California, Irvine, il protocollo # 2010-2943. 1. Rimozione del midollo spinale <ol><li> Posizionare asciugamani di carta bagnati con ~ 100% di liquido isoflurano, USP di eutanasia da camera e mettere gli asciugamani di carta a secco in cima. Posizionare il mouse nella camera in cima ai tovaglioli di carta a secco in modo da mouse non sia a contatto con l&#39;isoflurano e assicurarsi che la camera è coperto. Attendere almeno un minuto dopo la cessazione della respirazione per garantire che il mouse è eutanasia. </li><li> Eseguire una sezione spinale del collo per assicurare il mouse è eutanasia. Non eseguire una dislocazione cervicale in quanto ciò potrebbe danneggiare il midollo spinale. </li><li> Spruzzare il mouse con etanolo al 70% per bagnare i capelli. </li><li> Utilizzare forbici Belle taglienti per rimuovere i capelli e la pelle dalla parte posteriore del mouse per esporre la colonna vertebrale cervicale lamina da circa 1 (C1) A lamina sacrale 4 (S4). </li><li> Usando un bisturi con lama # 10, fare incisioni a sinistra ea destra della colonna vertebrale per separarlo dal muscolo e grasso. Questo renderà la rimozione del midollo spinale molto più facile. </li><li> optional: a curve rongeurs Luer possono essere utilizzati per scavare via la carne supplementare. </li><li> Tenendo la parte superiore delle vertebre spinali con seghettati pinze Graefe, inserto in titanio curva Vännäs forbici con il lato curvo in alto nella colonna vertebrale esposta a C1, facendo attenzione a non toccare il midollo spinale. </li><li> Far scorrere il titanio curva forbici Vännäs tutta la strada a destra e fare un piccolo taglio. Un piccolo suono crunch deve essere ascoltato e sentito come le forbici tagliano le vertebre. Ripetere questo taglio sul lato più a sinistra del cavo. Fare attenzione a non andare troppo in fretta o fare troppo grande di un taglio come questo rischiare di danneggiare il cavo con le forbici. </li><li> Il singolo lamina deve poter essere sollevato con la pinza una volta che i lati delle vertebre sono tagliate. REPEAT questo passaggio per ogni lamina fino S4, continuando a tenere e tirare indietro le vertebre del midollo spinale. NOTA: le vertebre di sopra del sito di trapianto probabilmente verrà fuori una volta che è tagliato al sito di trapianto. Continuare la procedura di avviamento appena sotto il sito di trapianto. </li><li> optional: gettare le vertebre verso il basso per vedere dove il sito di trapianto è e misura e tagliare ~ 20 millimetri rostrale e caudale al sito di trapianto. NOTA: Quando si trapiantano NPC più non saranno necessari come gli NPC trapiantati in genere non migrano più lontano di 15 mm. La quantità di midollo spinale necessaria dipenderà esperimento e migrazione caratteristiche del tipo di cellula trapiantato. Effettuare tagli rostrale e caudale equidistanti al sito di trapianto consentirà al sito di trapianto di essere poste più facilmente durante l&#39;imaging. </li><li> Usando un bisturi con lama # 11 tagliare accuratamente i gangli a destra e sinistra del lato ventrale del midollo spinale a partire dalla fine rostrale. Questo will consentire il midollo spinale per essere rimosso dalle vertebre ventrale più facilmente. NOTA: Fatto rapidamente (passaggi 1,7-1,11), il midollo spinale non si asciugherà. Tuttavia, 1x PBS può essere somministrato al cavo per mantenerlo umido. </li><li> Capovolgere il mouse e tenere premuto il mouse verso l&#39;alto in modo che il midollo spinale è rivolto verso il basso. Sbucciare cautela il midollo spinale con pinze Graefe seghettate chiusi. Senza intaccare il midollo spinale, tagliare accuratamente ogni gangli rimanenti per consentire al midollo spinale da sollevare in un unico pezzo integro. </li><li> Assicurarsi che il midollo spinale è in RPMI-1640 e posizionarlo sul ghiaccio per il trasporto al microscopio 2P. </li></ol> 2. Preparazione di Spinal Cord for Imaging <ol><li> Incorporare midollo spinale in agarosio <ol><li> Tenere il midollo spinale isolato in refrigerate RPMI-1640 sul ghiaccio prima di imaging. </li><li> Pesare agarosio (bassa temperatura di gelificazione) e preparare una soluzione al 5% in 5 ml di PBS 1x. Microonde la soluzione di agarosio 5% per 15 secondi per sciogliere la agnata e lasciar raffreddare la soluzione a 37 ° C. </li><li> Preparare il midollo spinale appoggiandolo su un foglio di Parafilm con il lato ventrale rivolto verso l&#39;alto. </li><li> Pipettare circa 5 ml di soluzione di agarosio 5% sopra il lato ventrale del midollo spinale. Sia l&#39;agarosio solidificare per raffreddamento a temperatura ambiente. Solidificazione completa richiede circa 5 minuti. </li><li> Applicare un leggero strato di adesivo di tessuto per una copertura di slittamento piazza 22 millimetri. </li><li> Invertire il midollo spinale incorporato, posizionando il lato ventrale su parafilm. Il lato dorsale sarà ora rivolta verso l&#39;alto. Rispettare la polizza di copertura sul lato dorsale, e immergere l&#39;agarosio incorporato midollo spinale / copertura preparazione slittamento in RMPI per solidificare l&#39;adesivo. Rimuovere l&#39;eccesso solidificato agarosio con una lama di rasoio. </li></ol></li><li> Montaggio del midollo spinale sul palco microscopio <ol><li> Applicare vaselina sul fondo del vetrino. Questo stabilizzerà la preparazione durante la perfusione. </li><li> Posizionare la preparazione del midollo spinale in unl&#39;imaging bene sul palco microscopio con il lato ventrale rivolto verso l&#39;alto verso l&#39;obiettivo immersione 25X. Il pozzo di imaging costruito su misura è di circa 20 mm di profondità, 50 mm di lunghezza, e 50 mm di diametro. </li><li> Immagine mentre superfusing la preparazione con riscaldato (37 ° C), ossigenato (95: 5 ossigeno: anidride carbonica-CarboGen) medium RPMI-1640 senza siero. <ol><li> Supporti Pre-calda a 37 ° C nel bagnomaria e collegarlo alla pompa tubo inserendo il tubo nella bottiglia media. </li><li> Mantenere la temperatura del fluido a 37 ° C un dispositivo riscaldatore al collegamento del tubo alla camera. Mezzi Superfuse attraverso bene a 3 ml / min usando tubo collegato alla pompa tubo (Figura 1B). </li></ol></li></ol></li></ol> 3. 2P Imaging di ventrale del midollo spinale <ol><li> Setup Microscopio <ol><li> Acquisire le immagini da Thy1-EYFP midollo spinale con un Ultra Ti Chameleon: laser Sapphire sintonizzati a 900 nm. Attenuare potenza del laser inesemplare a &lt;5% per garantire la fototossicità minimo 16. Mantenere la temperatura mediante perfusione di ossigeno-perfuso RPMI-1640 ad una costante di 37 ° C utilizzando un unico riscaldatore soluzione di linea di garantire immagini stabili, e da evitare la dispersione dei tessuti e danno cellulare. </li><li> Per separare segnale fluorescente eGFP e EYFP, posizionare una 520 nm dicroico singolo-edge e un nm singolo bordo beam splitter dicroico 560 in serie per separare 2P emissione in tre canali, volutamente dividendo l&#39;emissione verde per migliorare la visibilità. NOTA: Questi canali sono indicati come blu-verde (emissione &lt;520 nm), verde-giallo (520-560 nm), e il rosso (emissione&gt; 560 nm). Tubi fotomoltiplicatori rilevano emessi luce in ogni canale. </li></ol></li><li> Aree di imaging di posizionamento di interesse <ol><li> Utilizzando l&#39;oculare e una fonte di luce campo chiaro, mettere a fuoco l&#39;obiettivo di immersione a bordo ventrale del midollo spinale per impostare un punto di riferimento. </li><li> Assicurarsi fonte di luce ambiente è sicuro spento e swprurito a 2P eccitazione aprendo l&#39;otturatore laser. </li><li> Se disponibile, durante la ricerca dei tessuti per le aree di interesse, di utilizzare un valore inferiore di risoluzione, un volume più alto senza zoom digitale, e velocità di scansione più elevata rispetto a quando l&#39;acquisizione di immagini finali. Le impostazioni tipiche con questo sistema durante la ricerca dei tessuti per una regione di interesse sono: Risoluzione: 256 pixels; Volume: x = 600 micron, y = 600 micron, z = 0-300 micron. </li><li> Osservare gli assoni EYFP vicino al bordo ventrale del midollo spinale. Secondo segnale armonico da collagene (blu) sarà più luminoso sul bordo del tessuto del midollo spinale. Individuare gli assoni EYFP solo dorsale per il secondo segnale armonica dal collagene e il segnale EYFP viene visualizzati nel canale verde-giallo. </li><li> Individuare il sito di trapianto nel centro longitudinale della preparazione del midollo spinale 14. Per i topi 1 giorno dopo eGFP-NPC trapianto, individuare il sito di trapianto puntando il percorso del fascio nel piano z in profondità nei tessuti. Il sito trapianto will variare leggermente tra gli animali, ma è generalmente trova ~ 200 micron dal bordo ventrale. Osservare i gruppi di eGFP-NPC nei tratti di sostanza bianca, più vicino ai bordi ventrali e laterali nei topi dopo giorno 1 post-trapianto. </li></ol></li><li> Acquisizione immagini finali <ol><li> Acquisire risoluzione di 512 pixel; Volume: x = 270 micron, y = 212 micron, e z = 100 micron utilizzando il software Slidebook 6. Compilare z-stack con l&#39;acquisizione di piani focali sequenziali in incrementi di 2,5 micron. </li><li> Eseguire una scansione bidirezionale con adeguata compensazione per garantire una rapida quantificazione di tutti gli oggetti che migrano nel midollo spinale interlacciato. Assicurarsi che il frame rate di acquisizione ideale per determinare la velocità cellulari all&#39;interno del midollo spinale è di circa 1 frame / sec. NOTA: le impostazioni di imaging possono essere modificate per le preferenze dei singoli utenti, quali frame rate di acquisizione, la risoluzione delle immagini e dei volumi di imaging. Volumi di imaging più grandi in genere più tempo per acquisire a un GIVRisoluzione en. </li><li> Analizzare, colture, liscio, e PseudoColor file immagine Slidebook con il software di analisi di immagine, come Bitplane Imaris 7.7. Produrre video finali time-lapse da pettinatura volumi di imaging consecutivi utilizzando un processo automatizzato in Slidebook e Imaris. </li></ol></li></ol>

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The authors have no competing interests.

In tempo reale 2P imaging tessuto intatto è necessario per indagare cinetica NPC e interazioni seguenti trapianto nel topo demyelinated midollo spinale. L&#39;imaging intravitale 2P è comunemente usato per determinare le dinamiche cellulari sul lato dorsale del midollo spinale nei topi viventi, ed è stato utilizzato per studiare demielinizzazione dorsale in malattia demielinizzante 17-19. Tuttavia, poiché NPC trapiantate migrano alla materia bianca ventrale, che si trova troppo in profondità all&#39;imma...

Modelli murini di demielinizzazione, tra cui l&#39;encefalomielite sperimentale autoimmune (EAE) e l&#39;infezione intracranica con neuroadapted mouse virus dell&#39;epatite (MHV), sono ottimi strumenti per studiare percorsi molecolari e interazioni cellulari associati con la malattia. Essi hanno portato e sostenuto l&#39;efficacia della FDA ha approvato terapie farmacologiche, rivolti principalmente la cessazione di autoimmunità e infiammazione 1. Tuttavia, una volta che la rimielinizzazione endogena ha fallito, le terapie attualmente approvate non riparano efficacemente lesioni demielinizzati nel sistema nervoso centrale. Pertanto, le terapie di riparazione focalizzato in questa fase della malattia sono fondamentali per la riduzione dei sintomi cronici e il miglioramento della qualità della vita. Recentemente, le cellule precursori neurali (NPC) sono venuti alla ribalta come un potenziale rigenerativo modalità terapeutica per aree di infiammazione e demielinizzazione bersaglio. Diversi studi hanno evidenziato la capacità di NPC di indurre endogenonoi rimielinizzazione e partecipare direttamente rimielinizzazione 2-8. Perché NPC sono coinvolti in rimielinizzazione diretta, è fondamentale per capire le loro cinetiche e le interazioni con le cellule endogene successivi al trapianto. Dopo il trapianto, NPC migrano ventralmente ad aree di danno sostanza bianca, poi rostralmente e caudalmente relativi al sito di trapianto 5,9. La cinetica della migrazione differiscono in risposta a stimoli ambientali; NPC trapiantati in un midollo spinale danneggiato non hanno velocità maggiori rispetto NPC trapiantate in un midollo spinale danneggiato 6. Dopo un periodo migratorio, trasferiti NPC proliferano ampiamente, ad un tasso più elevato in un cavo danneggiato rispetto spinale per un midollo spinale intatto 6. Infine, la maggior parte degli NPC differenziarsi in oligodendrociti e avviare 4,6,9 rimielinizzazione diretta. La lesione demyelinated è complesso e può includere una popolazione eterogenea di cellule in diverse fasi di unctivation. Ad esempio, un sclerosi multipla (MS) lesione attiva può includere una frazione significativa di cellule T attivate, microglia e dei macrofagi M1 M1, ma un MS lesione cronica silente può essere costituito principalmente da astrociti reattivi con poche cellule infiammatorie 10-13. A causa della diversità delle cellule effettrici, a due fotoni (2P) per immagini in modelli murini di demielinizzazione è uno strumento estremamente utile per aiutare a capire le interazioni cellulari locali all&#39;interno della lesione. In MS e molti modelli di ricerca MS ampiamente utilizzati, la maggior parte delle lesioni si trovano sul lato ventrale del midollo spinale, una regione inaccessibile per l&#39;imaging intravitale 2P grazie alla profondità della lesione e l&#39;alto contenuto lipidico del midollo spinale. Per aggirare questi problemi e interazioni studio cellula-cellula all&#39;interno lesioni lungo il midollo spinale ventrale abbiamo sviluppato un semplice vivo 2P preparazione di imaging 6 ex. Questo studio fa seguito ad una precedente pubblicazione metodi, che ha mostratola procedura per il trapianto di proteine ​​maggiore fluorescente verde (eGFP) NPC esprimente nel midollo spinale dei topi dopo JHMV ceppo di demielinizzazione MHV-indotta 14. Cinque settimane di età i topi sono infettati con JHMV e trapiantati con eGFP-NPC a livello toracico 9 al giorno 14 dopo l&#39;infezione. Il protocollo presentato qui fornisce la procedura dettagliata su come estrarre il midollo spinale, fare un ex vivo agarosio preparazione, e di immagine trapiantato interazioni eGFP-NPC con proteine ​​maggiore giallo fluorescente (EYFP) assoni esprimente. Mice esprimenti EYFP sotto il promotore Thy1 specifico-neuronale sono stati utilizzati in questa procedura 15. Solo alcuni degli assoni esprimono EYFP, rendendolo utile per l&#39;imaging singoli assoni. Qui vi mostriamo midollo spinale rimossi a 7 giorni dopo il trapianto; tuttavia, midollo spinale possono essere estratti in qualsiasi punto nel tempo dopo il trapianto. Mentre noi mostriamo interazioni di NPC con assoni danneggiati, il nostro protocollo può essere utilizzato in combinazione con marcatori fluorescenti geneticidi altri tipi di cellule per indagare una moltitudine di interazioni cellulari che si verificano durante il mouse midollo spinale.

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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.

<html> Mentre il protocollo di imaging midollo spinale espiantato può essere utilizzato per visualizzare alcuna fluorescenza all&#39;interno del midollo spinale, i nostri risultati dimostrano rappresentativi interazioni eGFP-NPC con EYFP-assoni. In primo luogo, noi mostriamo la preparazione ventrale del midollo spinale incorporato nella Figura 1A. Avanti, mostriamo la configurazione 2P microscopio e componenti chiave nella Figura 1B. Figura 2 dimostra eGFP e EYFP fluorescenza in una sin...

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

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

Un nuovo ex vivo preparazione per l&#39;imaging del midollo spinale di topo. Questo protocollo consente a due fotoni imaging interazioni cellulari vivi tutto il midollo spinale.

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 ...

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.1K Views.  University of California, Irvine.  Un nuovo ex vivo preparazione per l'imaging del midollo spinale di topo. Questo protocollo consente a due fotoni imaging interazioni cellulari vivi tutto il midollo spinale. 

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo a due fotoni di imaging dei cambi molecolari esperienza-dipendenti nei neuroni corticali

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Ricerca

Neuroscienze

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon<em&gt; In vivo</em&gt; Imaging di spine dendritiche nella corteccia mouse Uso un teschio Diluito-Preparazione

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Diffusion Imaging nel midollo spinale cervicale Rat

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultanea a due fotoni<em&gt; In Vivo</em&gt; Imaging di Synaptic ingressi e degli obiettivi postsinaptici nella corteccia mouse retrosplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imaging Fibre serotoninergici nel midollo spinale mouse Uso del CHIAREZZA / Tecnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imaging di attività neurale nella corteccia somatosensoriale primaria utilizzando Thy1-topi transgenici GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

In Vivo Duo-colore metodo di Imaging vascolare dinamica dopo la lesione del midollo spinale contusivi

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...