Questo lavoro è stato sostenuto dal National Multiple Sclerosis Society concedere RG4595A1 / T per DD e il NIH / NINDS concede NS051470, NS052189 e NS066361 alle figure KA e film adattato e / o ristampati da Davalos et al., J Metodi Neurosci. Mar 2008 30; 169 (1) :1-7 Copyright 2008, con il permesso di Elsevier.

1. Costruire il dispositivo di stabilizzazione della colonna vertebrale <ol><li> Ordina la STS-A Narishige Morsetti Compact midollo spinale e la MA-6N Narishige adattatore in mano la testa. </li><li> Design personalizzato e fare una piastra di base in acciaio inox per tenere le due parti Narishige in allineamento in modo che la testa dell&#39;animale è supportato mentre la sua spina dorsale e la coda sono bloccato. Tenete presente che l&#39;intero dispositivo dovrebbe andare bene sotto la lente microscopio di solito su un palco microscopio abbassato. </li></ol> 2. Animal chirurgia <ol><li> Pre-riscaldare la camera di microscopio con un dispositivo di riscaldamento ad aria e mantenerla a 37 ° C. </li><li> Pesare l&#39;animale ed effettuare il mix di anestetico con 100 mg di ketamina, 15 mg e 2,5 xylazina acepromazina mg per kg di peso corporeo, diluito in soluzione di NaCl 0,9% in condizioni di sterilità. </li><li> Anestetizzare l&#39;animale, iniettando l&#39;anestetico mix (KXA) per via intraperitoneale. Pizzicare piedi regolari devono essere eseguiti per garantire l&#39;anestesia profonda in tuttol&#39;esperimento. Supplemento con metà della dose iniziale oraria o, se necessario. </li><li> Utilizzare una piccola piastra elettrica animali per fornire supporto termico durante l&#39;esecuzione della procedura chirurgica. </li><li> Applicare artificiale unguento lacrime sugli occhi per prevenire la disidratazione della cornea e danni. </li><li> Radersi la parte posteriore del primo animale con un dispositivo di taglio e poi con una lama affilata per rimuovere completamente i capelli dalla zona intorno l&#39;incisione. </li><li> Rimuovere i capelli rasati e disinfettare la superficie della pelle rasata con un tampone di alcool. </li><li> Eseguire un piccolo (~ 1,5 cm) incisione longitudinale della pelle sopra il livello desiderato spinale (vertebre toraciche mostrato qui). </li><li> Esporre la muscolatura della schiena ritraendo con cura il tessuto connettivo sottocutaneo. </li><li> Separare con attenzione i muscoli paravertebrali dalla colonna vertebrale al livello desiderato. Eseguire il minor numero di incisioni possibile staccare i muscoli da entrambi i lati di ogni processo spinoso e poi raschiare i muscoli staccò unavia dalla superficie laminare. </li><li> Scegli la lamina che verrà rimosso e preparare i siti di ingresso per i morsetti spinale su entrambi i lati della colonna vertebrale, immediatamente rostrale e caudale alla laminectomia. Con attenzione spostare tessuto muscolare per fare quattro tasche dove i morsetti verrà inserito. </li><li> Sollevare la colonna vertebrale con la dentatura diritta punta pinze per consentire l&#39;inserimento sicuro delle curve punta smussata forbici sotto la lamina senza toccare il midollo spinale. Eseguire la laminectomia lentamente tagliando l&#39;osso da un lato e poi dall&#39;altro lato. </li><li> Utilizzare un tampone di cotone o inserire pretagliati piccoli pezzi Gelfoam assorbibile spugna di gelatina accanto al incisioni di limitare sanguinamento minore. Utilizzare un cauterizer piccolo vaso per controllare l&#39;emorragia ulteriormente se necessario. Usa preriscaldato sterile fluido cerebrospinale artificiale (ACSF) per lavare i tessuti. </li></ol> 3. Stabilizzazione della colonna vertebrale e la preparazione per imaging in vivo <ol><li> Plac<html> e l&#39;animale su una base di elevazione sulla piastra di base del dispositivo di stabilizzazione della colonna vertebrale e fissare la testa l&#39;adattatore capo azienda. </li><li> Posizionare i morsetti spinale della STS-Un dispositivo lungo la asse anteriore-posteriore dell&#39;animale nel pre-preparati tasche che fiancheggiano il laminectomia (Figura 1). I due morsetti deve essere posizionato con un angolo di circa 45 ° rispetto alla dell&#39;animale asse rostro-caudale per consentire spazio sufficiente per abbassare una lente immersione in acqua oltre il midollo spinale esposti (Figura 1). </li><li> Posizionare il morsetto terzo della STS-Un dispositivo alla base della coda così corpo dell&#39;animale possono essere sospesi in aria dopo la rimozione del tampone elevazione per tutta la durata degli esperimenti di imaging (Figura 1). </li><li> Costruire un piccolo pozzo di Gelseal (Amersham Biosciences Corp.) intorno al midollo spinale esposti a facilitare il mantenimento del midollo spinale in ACSF e l&#39;immersione della lente microscopio in questa soluzione per imaging in vivo. </li></ol>jove_title &quot;&gt; 4. Nel imaging in vivo del midollo spinale di topo con la microscopia a due fotoni <ol><li> Trasferire l&#39;animale sul dispositivo di stabilizzazione spinale all&#39;interno della camera preriscaldato che copre il microscopio e fissarlo su un palco microscopio abbassato posizionando il laminectomia direttamente sotto la lente (Figura 1). </li><li> Inferiore ad acqua immersione lente con attenzione nella soluzione ACSF facendo attenzione che non tocchi i morsetti spinale o il Gelseal. </li><li> Usa epifluorescenza per identificare l&#39;area di interesse e concentrarsi su di esso. Passa alla modalità di scansione laser e di eseguire imaging in vivo utilizzando l&#39;apposito a due fotoni laser a lunghezza d&#39;onda di eccitazione, dicroici e filtri passa-banda per la fluorofori presenti nel tessuto fotografato. </li></ol> 5. Immagini ripetitive e assistenza post-operatoria <ol><li> Alla fine degli esperimenti di imaging, togliere il mouse dal dispositivo di stabilizzazione della colonna vertebrale e asciugare con cura il Gelseal dalla zona intorno alla laminectomia.Pulire bene la zona. </li><li> Ripristinare e suturare i muscoli della schiena sopra la laminectomia. </li><li> Ripristinare e suturare la pelle sopra la laminectomia, è tampone con Betadine. </li><li> Fornire 1 ml di soluzione lattato Ringers (Baxter Healthcare) come supplemento nutritivo e l&#39;idratazione, così come il trattamento analgesico per via sottocutanea (0,1 mg / kg di buprenorfina). </li><li> Somministrare per via intraperitoneale antisettico trattamento (0,03 ml per il mouse, 2,27% soluzione iniettabile enrofloxacina antibatterici). </li><li> Animale posto su una piastra elettrica fino a pieno recupero dall&#39;anestesia e successivamente casa individualmente. </li><li> Ripetere la somministrazione antisettico giorno per i primi 3-5 giorni dopo l&#39;intervento chirurgico e un trattamento analgesico ogni 8-12 ore per 2-3 giorni dopo l&#39;intervento. Monitoraggio animali al giorno per garantire un comportamento normale e pieno recupero. </li><li> Per re-imaging attraverso la laminectomia stesso, riaprire la pelle suturata e muscoli e ripetere i passaggi descritti nelle sezioni 2 - 4. </li><li> Ri-localizzare la preceously ripreso zona utilizzando i vasi sanguigni come una mappa come descritto in precedenza 10. </li></ol> 6. Rappresentante risultati Tutte le procedure di animali sono state effettuate in base agli orientamenti fissati dal Animal Care istituzionali e dei Comitati Usa presso la University of California, San Francisco e sono conformi alle normative federali. Una foto del dispositivo di stabilizzazione della colonna vertebrale e uno schema che mostra il posizionamento del mouse sul dispositivo sotto una lente microscopio è mostrato in Figura 1. Lasciando spazio sufficiente per i movimenti respiratori sotto il corpo dell&#39;animale assicura stabile imaging in vivo nel midollo spinale. La figura 2 mostra la stretta relazione tra microglia e la vascolarizzazione come è stato ripreso in vivo nel midollo spinale di CX3CR1 GFP / + topi transgenici 18, microglia, in cui sono endogenamente etichettati con GFP. Figura 3 mostra alcuni esempi di ripetitivoimaging in vivo come è stato eseguito nelle stesse aree del midollo spinale nei topi che esprimono una proteina fluorescente negli assoni spinali (YFP-H linea 3) e microglia (nel CX3CR1 GFP / + topi). <img alt="Figura 1" src="/files/ftp_upload/2760/2760fig1.jpg" /> Figura 1. Stabilizzazione della colonna vertebrale del mouse per imaging in vivo mediante microscopia a due fotoni. (A) Una misura piastra di base in acciaio viene utilizzata per supportare e allineare la STS-A Narishige morsetti compatti del midollo spinale e la MA-6N Narishige testa in possesso di adattatore come mostrato qui. (B) Il corretto posizionamento di topi transgenici adulti anestetizzati con un anestetico KXA sul dispositivo di stabilizzazione della colonna vertebrale. L&#39;inserto viene illustrata la posizione dei morsetti spinale immediatamente rostralmente e caudalmente alla laminectomia e il tessuto esposto midollo spinale. &lt;img alt = &quot;Figura 2&quot; src = &quot;/ files/ftp_upload/2760/2760fig2.jpg&quot; /&gt; Figura 2. Imaging in vivo ad alta densità di cellule microgliali e dei vasi sanguigni nel midollo spinale di topi anestetizzati. Proiezione ma non allineati z-pile di (A) ad alta densità GFP-positive microglia (verde) nel midollo spinale di una GFP CX3CR1 / mouse + in stretta vicinanza con i vasi sanguigni (rosso, marcato con l&#39;iniezione endovenosa di rodamina destrano). (B) ingrandimento dell&#39;immagine ad alta etichettato come in (A) di una singola cellula della microglia attaccato alla parete di un vaso sanguigno con i processi esteso intorno alla nave e verso il parenchima del midollo spinale. Barre di scala, 10μm. <img alt="Figura 3" src="/files/ftp_upload/2760/2760fig3.jpg" /> Figura 3. Ripetuti negli imaging in vivo dei segmenti stessi assonale e microglia come sono stati trasferiti e re-imaging nel midollo spinale di topi anestetizzati in giorni diversi. (A) YFP-lab<html> assoni forme ai giorni 0 e 5. (B). Le stesse strutture vascolari e cellule della microglia che li circonda ripreso in vivo nel midollo spinale di CX3CR1 GFP / mice + ai giorni 0 e 1. Barre di scala, 10μm. Movie 1. Sequenza Timelapse rappresentante acquisito in vivo dal midollo spinale di topo. Questa sequenza mostra in dettaglio la dinamica sottile processo di microglia (verde) e le loro interazioni con il sistema vascolare (rosso, marcato con rodamina iv iniezione di destrano) nel tempo all&#39;interno di un tessuto densamente popolato con strutture fluorescenti. Le immagini RAW sono stati solo corretti per il rumore di fondo, la luminosità e il contrasto e il film timelapse è stato costruito da z-stack proiettando immagini in sequenza acquisita, senza allineamento dell&#39;immagine, con una media o z-selezione di piani individuali. I vasi sanguigni passare attraverso una serie simile di aerei z come le immagini della microglia. Z profondità aereo: 38 micron.oad/2760/Davalos_Movie_1.mov &quot;&gt; Clicca qui per guardare il film. Movie 2. Sequenza Timelapse dimostrare la stabilità prime del metodo di imaging a livello di un singolo piano z. Rapida acquisizione dello stesso singolo piano z nel midollo spinale di topi GFP CX3CR1 / + immessi sul dispositivo di stabilizzazione della colonna vertebrale in anestesia KXA, dimostra cilindrata minima immagine tra fotogrammi consecutivi ad una velocità di scansione di 1 frame / s che è più veloce della frequenza respiratoria del mouse. La cilindrata minore residuo è probabilmente dovuto al battito cardiaco. <a alt="Movie 2" href="http://www.jove.com/files/ftp_upload/2760/Davalos_Movie_2.mov">Clicca qui per guardare il film.</a>

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Il metodo qui descritto consente di stabile e ripetitivo nel imaging in vivo di densamente popolate strutture fluorescenti cellulari nel midollo spinale di topi anestetizzati utilizzando la microscopia a due fotoni. La stabilità raggiunta è il risultato di un dispositivo su misura di stabilizzazione della colonna vertebrale e un regime di anestetico che riduce respiratoria indotta artefatto movimento. Il dispositivo di stabilizzazione della colonna vertebrale permette di respiro sotto il corpo del mo...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Rodamina B destrano </td><td> Invitrogen </td><td> D1841 </td><td> 70 kDa, diluito in ACSF (3% w / v) </td></tr><tr><td> Ketamina HCl </td><td> Bionichepharma </td><td> NDC No: 67457-001-10 </td><td> Iniettabili, 50mg/ml </td></tr><tr><td> Anased </td><td> Lloyd Labs </td><td> NADA No: 139-236 </td><td> Xylazina iniettabili, 20mg/ml </td></tr><tr><td> Acepromazina </td><td> Vedco </td><td> NADA No: 117-531 </td><td> Iniettabili, 10mg/ml </td></tr><tr><td> Lacrime artificiali unguento </td><td> Fenice farmaceutico </td><td> NDC No: 57319-760 - 25 </td><td> Lubrificante </td></tr><tr><td> Betadine </td><td> Pescatore </td><td> 19-061617 </td><td></td></tr><tr><td> McPherson-Westcott Forbici </td><td> Mondo di precisione Strumenti </td><td> 555500S </td><td> Curvato, smussato-tip forbici </td></tr><tr><td> Pinza dritta </td><td> Mondo di precisione Strumenti </td><td> 555047FT </td><td> Pinza punta dentata </td></tr><tr><td> Cauterizzare piccoli vasi </td><td> Strumenti Scienza multa </td><td> 18000-00 </td><td></td></tr><tr><td> Gelfoam </td><td> Pharmacia, Pfizer Inc. </td><td> Mixer Mill MM400 </td><td></td></tr><tr><td> Compatta del midollo spinale morsetti </td><td> Narishige </td><td> STS-A </td><td></td></tr><tr><td> Adattatore tenendo testa </td><td> Narishige </td><td> MA-6N </td><td></td></tr><tr><td> Gelseal </td><td> Amersham Biosciences Corp. </td><td> 80-6421-43 </td><td></td></tr><tr><td> Ringer lattato </td><td> Baxter Healthcare &lt;/ Td&gt; <td> 2B8609 </td><td></td></tr><tr><td> Buprenex </td><td> Reckit Benckiser Pharmaceuticals Inc. </td><td> NDC No: 12496 - 6757-1 </td><td> Buprenorfina, iniettabili </td></tr><tr><td> Baytril </td><td> Bayer </td><td> NADA 140-913 </td><td> Enrofloxacina, iniettabili antibatterico 2,27% (20ml) </td></tr><tr><td> Riscaldamento pad - Grande </td><td> Strumenti Scienza multa </td><td> 21060-10 </td><td></td></tr></tbody></table>

in vivo imaging of the mouse spinal cord using two-photon microscopy

Imaging in vivo mediante microscopia a due fotoni 1 nei topi che sono stati geneticamente modificati per esprimere proteine ​​fluorescenti in specifici tipi cellulari 03/02 ha notevolmente ampliato la nostra conoscenza dei processi fisiologici e patologici in numerosi tessuti in vivo 4-7. Negli studi del sistema nervoso centrale (SNC), c&#39;è stata una vasta applicazione di imaging in vivo del cervello, che ha prodotto una pletora di nuove e spesso inattese scoperte sul comportamento delle cellule come i neuroni, astrociti, microglia, in condizioni fisiologiche o patologiche 8-17. Tuttavia, le complicanze per lo più tecnici hanno limitato l&#39;attuazione di imaging in vivo negli studi del midollo spinale del mouse vivente. In particolare, la vicinanza anatomica del midollo spinale ai polmoni e al cuore genera artefatti significativo movimento che rende l&#39;imaging del midollo spinale che vivono un compito impegnativo. </p&gt; Abbiamo sviluppato un nuovo metodo che supera i limiti intrinseci di imaging del midollo spinale mediante la stabilizzazione della colonna vertebrale, la riduzione delle vie respiratorie indotta da movimenti e facilitando così l&#39;utilizzo della microscopia a due fotoni per l&#39;immagine del midollo spinale del mouse in vivo. Questo si ottiene combinando un dispositivo personalizzato di stabilizzazione vertebrale con un metodo di anestesia profonda, con una conseguente significativa riduzione delle vie respiratorie indotta movimenti. Questo protocollo video mostra come esporre una piccola zona del midollo spinale vivente che può essere mantenuto in condizioni fisiologiche stabili per lunghi periodi di tempo, mantenendo danno tissutale e sanguinamento al minimo. Rappresentante immagini RAW acquisite in dettaglio vivo in alta risoluzione la stretta relazione tra microglia e la vascolarizzazione. Una sequenza timelapse mostra il comportamento dinamico dei processi microgliali nel midollo spinale del mouse vivente. Inoltre, una scansione continua della stessa z-frame dimostrares la stabilità eccezionale che questo metodo può raggiungere per generare pile di immagini e / o filmati timelapse che non richiedono l&#39;allineamento dell&#39;immagine post-acquisizione. Infine, ci mostrano come questo metodo può essere usato per rivisitare e re-imaging stessa area del midollo spinale in seguito timepoints, consentendo studi longitudinali in corso di processi fisiologici o patologici in vivo.

Watch this Scientific Journal Video about In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy at JoVE.com

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

Un protocollo minimamente invasiva per stabilizzare la colonna vertebrale del mouse ed eseguire ripetitivo<em&gt; In vivo</emL&#39;imaging del midollo&gt; spinale utilizzando la microscopia a due fotoni è descritto. Questo metodo combina un dispositivo di stabilizzazione della colonna vertebrale e un regime di anestetico per ridurre al minimo i movimenti respiratori indotti e produrre dati di immagini grezzi che non richiedono l&#39;allineamento o altre post-elaborazione.

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 ...

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Research

JoVE Journal

Neuroscience

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

23.8K Views.  University of California, San Francisco. Un protocollo minimamente invasiva per stabilizzare la colonna vertebrale del mouse ed eseguire ripetitivo In vivo

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

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

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

In Vivo microscopia a due fotoni di Endings singolo nervo in pelle

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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 ...

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 ...

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Due-fotone Imaging dei neuroni corticali in topi neonatali

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...