We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1. Etichettatura assonale <ol><li> Fibre rampicanti possono essere etichettati iniettando né coloranti organici coniugati con destrano ad alto peso molecolare o plasmidici / virus che inducono l&#39;espressione di proteine ​​fluorescenti 26-29. In questo protocollo, il colorante organico Alexa Fluor destrano 488 viene iniettato nel oliva inferiore per etichettare fibre rampicanti e visualizzarli nella corteccia cerebellare (Figura 1). Tutte le procedure descritte qui sono stati approvati dal Ministero della Salute italiano. </li><li> Preparare il tubo capillare di vetro tirandolo su un estrattore micropipetta. Tagliare la punta del tubo capillare di vetro con le forbici fino al diametro esterno è di circa 30 micron. </li><li> Caricare il capillare con 1-2 ml di colorante destrano coniugato. </li><li> Prima di iniziare, sterilizzare tutti gli strumenti chirurgici in autoclave. Durante la procedura, ridurre i rischi di contaminazione utilizzando uno sterilizzatore perle di vetro. </li><li> Anestetizzare il mouse (9-12settimane) mediante iniezione intraperitoneale di ketamina (90 mg / kg) e xilazina (9 mg / kg). Assicurarsi che l&#39;animale è completamente sedato con coda e / o pizzichi punta. Utilizzare veterinario unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Radersi i peli sopra il collo sia con una lama di rasoio o crema depilatoria. Disinfettare il sito chirurgico due volte con betadine, alternati con etanolo al 70%. </li><li> Posizionare il mouse su un supporto stereotassico. Tenere il caldo animale con una piastra elettrica. </li><li> Spingere delicatamente le earbars osso accanto alle orecchie in modo che la testa è tenuto saldamente. Ruotare la testa a formare un angolo di 140 ° con il corpo. L&#39;orientamento desiderato può essere ottenuta modificando leggermente l&#39;altezza dell&#39;asta titolare naso sul supporto stereotassica. </li><li> Applicare alcune gocce di lidocaina sulla pelle tra le orecchie. Usare pinze o una lama di rasoio per effettuare una incisione verticale di circa 1 cm sulla pelle sotto all&#39;occipite. </li><li> Separare delicatamente il tessuto sottocutaneo e muscoli pinzetta smussatas sotto il microscopio di dissezione. Spingere verso il basso il fascio muscolare orizzontale e tenere a parte i due muscoli verticali fascicoli al fine di esporre la dura sopra il forame magno. </li><li> Usare estrema cautela, sezionare la dura con un ago da siringa o pinze molto affilati per esporre il tronco encefalico. Una piccola quantità di liquido cerebro-spinale potrebbe fuoriuscire quando la dura è stato tagliato. </li><li> Sulla micromanipolatore stereotassica, ruotare il titolare del capillare 45 ° rispetto alla verticale. Posizionare il capillare nella linea mediana, nel punto mediano tra il bordo caudale della corteccia cerebellare e la prima vertebra cervicale. Inserire la pipetta ad una profondità di 2 mm dalla superficie. </li><li> Fornire un volume di 0,5-1,5 ml di tintura su 10-15 min. Un sistema di erogazione microiniezione consente l&#39;erogazione controllata pressione del liquido nel capillare (pressione Pulse = 20 psi; durata dell&#39;impulso = 3-4 msec; frequenza di impulsi consegna = 12 Hz). </li><li> Lasciare il capillare in vigore per 15 minuti, poi slowly (in 2-3 min) rimuoverlo. Delicatamente riallineare i muscoli e suturare la pelle sopra. Tenere i muscoli ben idratata applicando alcune salina durante l&#39;intera procedura. </li><li> Per evitare la disidratazione, iniettare per via sottocutanea 0,5 ml di NaCl allo 0,9%. Tenere l&#39;animale in una gabbia riscaldata fino al pieno recupero dall&#39;anestesia. </li><li> Sottocutanea amministrare Carprofen (5 mg / kg) al giorno per 2-3 giorni dopo l&#39;intervento chirurgico. </li></ol> 2. Finestra ottica sulla cerebellare Cortex NOTA: Il colorante viene trasportato in 2 settimane a partire dal olivary nucleo inferiore fino alla corteccia cerebellare ai terminali CFS (vedere Figura 1). CFS scarsamente etichettati possono essere visualizzati sotto la finestra cranica permanente che può essere eseguita come segue: <ol><li> Sterilizzare tutti gli strumenti chirurgici. </li><li> Anestetizzare il mouse come descritto sopra. Utilizzare pizzichi punta per controllare l&#39;animale è completamente sedato. </li><li> Radersi i capelli sopra la testa, pulire la skcon alternanza di colpi di 70% di alcol e betadine e applicare pomate oftalmiche. Posizionare il mouse in un frame stereotassico e posizionare le barre di orecchio in modo da tenere saldamente la testa. </li><li> Sottocutanea somministrare desametasone (0,2 mg / kg) e Carprofene (5 mg / kg) per prevenire il gonfiore del cervello e possibili infiammazioni al sito finestra cranica. </li><li> Applicare una goccia di lidocaina sulla pelle sopra il cranio e tagliare un lembo, da tra le orecchie sopra gli occhi. Applicare una soluzione di lidocaina nuovamente sul periostio prima raschiando con un bisturi. </li><li> Utilizzare il trapano dentistico per assottigliare una zona semicircolare del teschio di sopra del cervelletto. La finestra è di solito posto centrale e verso la sutura posteriore, in modo che la profondità cranio è pressoché costante per tutto il perimetro del semicerchio. Posizionando la finestra troppo vicino alla sutura lambda e foratura ripetitivo in questa regione molto spessa può causare il surriscaldamento del cervello e formazione di edemi. </li><li> Applicare emostaticospugna sul cranio in caso di sanguinamento. </li><li> Tagliare il coprioggetti in due metà con l&#39;aiuto di una lama di rasoio e pinzette. Controllare che il coprioggetti è leggermente più grande della isola di osso ed ha una forma simile prima di rimuovere il lembo di osso con la pinzetta; se non, rimodellare il coverglass o scegliere un altro. Non toccare la dura madre con le pinzette mentre si disegna l&#39;osso. Adagiare il coverglass sulla dura. NOTA: L&#39;aderenza di tutta la superficie del vetro dalla durata è predicendo di lunghe finestre cranici durata. </li><li> Rimuovere le bolle d&#39;aria sotto la lente ripetutamente risciacquo con soluzione salina e asciugatura con bastoncini di cotone. Questo aiuterà anche a rimuovere alcuni residui di sangue che può riversarsi fuori dai piccoli capillari dopo il posizionamento del vetro. Sigillare la finestra con una miscela di colla acrilica e cemento dentale 30. </li><li> Tenere l&#39;animale su una piastra elettrica in una camera di recupero fino alla completa guarigione dall&#39;anestesia. Non riutilizzaregirare topi in gabbia a casa fino alla completa guarigione; non lasciarli incustoditi fino a quando ripresero conoscenza sufficiente per mantenere decubito sternale. </li><li> Sottocutanea amministrare Carprofen (5 mg / kg) al giorno per 2-3 giorni dopo l&#39;intervento chirurgico. </li></ol> 3. In Vivo multi-fotone laser assotomia <ol><li> Aspettare 2-7 giorni da un intervento chirurgico prima di eseguire la prima sessione di imaging. L&#39;personalizzato costruito microscopio a due fotoni usato qui è fornito di un Ti: zaffiro sorgente laser (120 fs di larghezza, 90 MHz frequenza di ripetizione) che viene prima acquisito da una coppia di specchi galvanometrici quindi focalizzato sul campione da una immersione in acqua 20X Obiettivo (NA 0.95, WD 2 mm). Una fase piezoelettrico a circuito chiuso esegue spostamenti assiali fino a 400 micron dell&#39;obiettivo. Tubi fotomoltiplicatori raccolgono il segnale di fluorescenza. </li><li> Anestetizzare l&#39;animale, poi posizionare la testina nel supporto stereotassica modo che la finestra cranica pone parallelo al obiettive piano focale. </li><li> Esplorare con TPF di imaging corteccia sotto la finestra cranica. Scegliere l&#39;assone di essere danneggiati tra quelli più brillanti. </li><li> Acquisire una Z-stack (solitamente il campo visivo è di 100 x 100 micron 2, a 512 x 512 pixel, 2 micron passo asse z) per ottenere un 3D-ricostruzioni del assone mirato. All&#39;interno del volume illustrato, scegliere il sito della lesione assonale su un ramo che si trova principalmente parallelo al piano focale. Questo orientamento consente di segnare rapidamente cambiamenti morfologici, che sono predittivi di una dissezione di successo. </li><li> Irradiare con un&#39;alta dose di energia nel punto selezionato una porzione distale di un CF. Questo viene fatto attraverso il software Labview: aumentare la potenza del laser 5-10x potenza superiore a quella utilizzata per l&#39;imaging (≈ 300 mW, misurata dopo la lente obiettivo), impostare la posizione degli specchi galvanometrici per fare una scansione linea (~ 4 - 10 micron) sul sito della lesione e aprire l&#39;otturatore laser per 400-600 msec. Orientare la linea di scansioneperpendicolare al piano CF transezione completamente l&#39;assone. Irradiando macchie luminose (ad es., Varici), di solito aiuta a sezionare l&#39;assone. La lunghezza d&#39;onda per assotomia laser è la stessa utilizzata per l&#39;imaging (920 nm). </li><li> Solitamente la fluorescenza dell&#39;assone nella zona irradiata riduce transitoriamente causa photobleaching. Acquisire una pila di stessa regione pochi minuti dopo l&#39;irradiazione di garantire l&#39;ablazione è stata ben eseguita. Se la regione ha iniziato il gonfiore e l&#39;assone formata strutture branello-like, il neurone è stato sezionato con successo. In questo caso, in poche ore (da poche decine di minuti a 24 ore) la porzione distale dell&#39;assone degenerare e scomparire completamente. </li><li> Purtroppo la dose di energia necessaria per produrre una assotomia completo non è sempre prevedibile. Se l&#39;unico risultato dell&#39;irraggiamento è Fotodecolorazione, la fluorescenza deve essere rapidamente recuperate grazie per tingere diffusione, e l&#39;irradiazione deve essere ripetuto nuovamente efficacely sezionare l&#39;assone. Inoltre, in caso segni di gonfiore non compaiono entro 5 - 20 minuti, o il gonfiore è seguito da recupero (. Analogamente a quanto descritto per dendrotomy laser, vedere Allegra Mascaro et al 23) Prova di nuovo con nuove scansioni aumentando la tempo di permanenza e / o la potenza del laser. </li><li> Controllare l&#39;eventuale rimodellamento dell&#39;assone nei giorni seguenti al assotomia laser con due fotoni imaging in vivo (figure 2 e 3). Il pattern vascolare è utilizzato come modello di riferimento per recuperare l&#39;assone sezionato nei giorni successivi. </li><li> Sottocutanea amministrare Carprofen (5 mg / kg) ogni sessione di imaging per tutta la durata dell&#39;esperimento. </li></ol>

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We have nothing to disclose.

Questo protocollo mostra come etichettare neuroni dell&#39;oliva inferiore con un colorante fluorescente. Successivamente, il metodo per eseguire una finestra del cranio sulla corteccia cerebellare è descritto. Questa tecnica fornisce accesso ottico alla porzione terminale dei neuroni olivocerebellar, le fibre rampicanti. Purtroppo, il risultato sia di etichettatura e chirurgia craniotomia è piuttosto basso anche nelle mani di operatori specializzati (di solito 1 su 3 topi è etichettato, e 1 su 3 finestre cranici res...

Transezione assonale derivanti da lesioni meccaniche, insulto tossico o malattie neurodegenerative è di solito seguita da degenerazione della parte distale dell&#39;assone che si stacca dal corpo cellulare 10-13. Con poche eccezioni 2,7,14,15, assoni mozzate nel sistema nervoso centrale degli animali adulti di solito sono in grado di attivare un programma di ricrescita 16. Poco si sa circa le dinamiche in tempo reale di eventi degenerativi a livello cellulare e subcellulare. Lo sviluppo di nuove strategie per limitare il danno neuronale e promuovere la ricrescita neuronale richiede, come primo passo, chiarire il meccanismo con cui le cellule neuronali singolarmente feriti degenerano e rigenerano. Questo studio è più direttamente indirizzata monitorando le dinamiche di un singolo neurone in vivo. Mentre le tecniche di un fotone di imaging di fluorescenza sono limitati dalla intensa diffusione della luce visibile, eccitazione a due fotoni raggiunge profondi strati corticali in live mouse con subcellulare risoluzione di 3,4,17. Approfittando di topi transgenici in cui proteine ​​fluorescenti sono selettivamente espressi in sottopopolazioni di neuroni 18-20, microscopia TPF è stato applicato alla esplorazione di plasticità sinaptica e allungamento assonale durante lo sviluppo in vivo 21,22. L a capacità di neuroni singolarmente danneggiate ricrescere dopo l&#39;infortunio può essere indagata mediante accoppiamento nel monitoraggio vivo da due fotoni di imaging con un modello di danno specificamente mirato al assone di interesse. Assorbimento multi-fotone di impulsi a femtosecondi è stato usato per distruggere singoli dendriti o anche singole spine 5,23. Inoltre, questo paradigma lesioni permette di tagliare rami singoli assonale senza interrompere il dendrite contattando 6. Nel contesto di sezionare le caratteristiche che permettono specifica popolazione neuronale per rigenerare i loro assoni, le fibre di arrampicata cerebellari una volta danneggiati (CFS) sono un modello SI utilence conservano notevoli proprietà plastiche dopo l&#39;infortunio anche negli animali adulti 24,25. Recentemente, imaging a lungo termine di CFS ha mostrato che questi assoni sono in grado di ricrescita nei giorni successivi assotomia laser 6. Questo protocollo descrive come etichettare i neuroni olivocerebellar e il loro allungamento assonale attraverso anterograda tracing. Una volta che i neuroni di interesse sono fluorescente, possono essere monitorati ripetutamente in momenti arbitrari per settimane o mesi in una finestra del cranio. La procedura di sezionare singoli rami assonali da assotomia laser in vivo verrà illustrato. Le tecniche qui presentate offrono nuove intuizioni nel meccanismo di rimodellamento assonale in vivo e possono aiutare lo sviluppo di strategie terapeutiche per limitare la degenerazione neuronale e promuovere la ricrescita assonale.

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			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
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			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
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			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
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			<td></td>
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			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
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			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
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laser nanosurgery of cerebellar axons in vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Questo protocollo descritto come eseguire etichettatura assonale, imaging in vivo e assotomia laser su singoli neuroni. La linea temporale dell&#39;esperimento è mostrato in Figura 1. Un esempio di CF marcati con Alexa Fluor 488 destrano e visualizzati sotto la finestra cranica in vivo microscopia a due fotoni è riportato in Figura 2. Come precedentemente riportato 6,27, i rami ascendenti mostrano una elevata stabilità durante...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

Due fotoni di imaging, accoppiato ad nanodissection laser, sono strumenti utili per studiare processi degenerativi e rigenerativi nel sistema nervoso centrale con risoluzione subcellulare. Questo protocollo viene illustrato come etichetta, immagine e sezionare singole fibre di arrampicata nella corteccia cerebellare in vivo.

Laser nanochirurgia di cerebellari Assoni<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K Views.  University of Florence.  Due fotoni di imaging, accoppiato ad nanodissection laser, sono strumenti utili per studiare processi degenerativi e rigenerativi nel sistema nervoso centrale con risoluzione subcellulare. Questo protocollo viene illustrato come etichetta, immagine e sezionare singole fibre di arrampicata nella corteccia cerebellare in vivo. 

Video: Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Nella visualizzazione in vivo delle vescicole sinaptiche In Drosophila assoni larvali segmentale

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Ricerca

Neuroscienze

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo Laser assotomia in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging di postnatale cerebellare granuli migrazione cellulare Utilizzando confocale macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellazione di morte neuronale e degenerazione in neuroni cerebellari del granello primario del Mouse

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

In Vivo Imaging multimodale e l'analisi di Mouse indotta da Laser modello di Neovascularization coroidico

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Microscopia spettrale riflettometrica su assoni Myelinated In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Valutazione dell'induzione della depressione a lungo termine nelle fette di cerebellar adulto

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...