AK è sostenuta dalla Fondazione Oppenheim e RH è supportato dalla Fondazione Velux. Ringraziamo I. Krumbein per Buder tecnica e K. per il sostegno istologico, e J. Goldschmidt (Istituto Leibniz di Neurobiologia, Magdeburgo, Germania) per un parere tecnico su TIMM colorazione.

Tutti gli interventi di animali vengono eseguite in conformità con la Convenzione europea per la cura degli animali ed uso di animali da laboratorio e la Dichiarazione ARVO per l&#39;uso di animali in Oftalmica e Vision Research. Tutti gli esperimenti sono approvate dal comitato etico locale. La procedura di lesioni ON nei topi è descritto altrove 9. 1. Intravitreali Manganese Injection <ol><li> Eseguire la Mn 2 + iniezione di 24 ore prima della MR scansione con l&#39;aiuto di un assistente. Anestetizzare gli animali mediante iniezione intraperitoneale di una soluzione di cloralio idrato 5% (420-450 mg / kg di peso corporeo in PBS sterile). Per ulteriori anestesia topica, applicare una goccia di liquido conjuncain (0,4% ossibuprocaina cloridrato) alla cornea prima puntura occhio. Per iniettare 15 nmol Mn 2 + per occhio preparare una MnCl mM soluzione 7.5 2, ad esempio, diluendo 1 L di 1 M MnCl 2 stock soluzione in 132 LH 2 &lt;/ Sub&gt; O. Carico 5 microlitri della soluzione finale in una siringa da 5 microlitri Hamilton collegato ad un piccolo mozzo dell&#39;ago rimovibile 34 G (RN ago). </li><li> Quando si avvia con l&#39;occhio destro, posizionare il mouse a sinistra lati sotto un microscopio binoculare e delicatamente aperto e fissare l&#39;occhio destro tra il pollice e l&#39;indice della mano sinistra. Raccogliete la siringa con la mano destra e prendere in mano l&#39;ago vicino alla punta. Per puntura atraumatica del bulbo oculare, inserire con attenzione l&#39;ago nel corpo vitreo alla circonferenza infero-temporale di circa 1 mm distale al limbus, risparmiando quindi i vasi sclerali. </li><li> Successivamente, l&#39;assistente applica lentamente il volume totale di 2 ml controllando la scala della siringa Hamilton. Durante questa procedura, controllare il posizionamento dell&#39;ago ottimale sotto il microscopio ed evitare la puntura della lente o fuoriuscita del liquido. Tenere l&#39;ago staticamente inserito, per altri 30 sec, quindi estrarre lentamente per minimizzare liquidofuoriuscite dal sito di iniezione. </li><li> Durante tutta la procedura, particolare attenzione deve essere presa per evitare la pressione per l&#39;occhio. Allo stesso modo, evitare i tentativi aggressivi o numerosi per bucare il bulbo oculare. Dal Mn 2 + assorbimento in RGC e di trasporto lungo l&#39;ON è già saturo a 15 nmol MnCl 2, questo riduce al minimo le variazioni di segnale da volumi di iniezione leggermente imprecisi. Per il potenziamento del segnale bilaterale della proiezione visiva, ripetere la procedura di iniezione per l&#39;occhio sinistro. </li><li> Applicare ofloxacina contenenti (3 mg / ml) collirio e pomata-pantenolo contenente una volta dopo la procedura per prevenire le infezioni oculari e asciugatura dell&#39;occhio. Restituire i topi nelle loro gabbie ma in condizioni normali fino all&#39;inizio della scansione MR. </li></ol> 2. Preparazione degli animali MRI <ol><li> Anestetizzare il mouse mediante somministrazione di una miscela di gas isoflurano / ossigeno 2% / 98%. Montare il mouse su un supporto per mouse in una posizione torti quasi orizzontale. Insert in bobina MR, e viene modificata all&#39;interno dello scanner MR. Monitorare la respirazione e la frequenza cardiaca da sistemi adeguati. Per i dettagli tecnici, consultare Herrmann et al 20. </li><li> Durante MRI, deposito di anestesia mediante insufflazione continua di un 1,5% / 98,5% miscela di gas inizialmente isoflurano / ossigeno tramite un evaporatore collegato al supporto della testina di topo da un tubo integrato. Durante la scansione, regolare la profondità di anestesia in base ai parametri vitali registrati (cioè puntare ad una frequenza respiratoria stabile di circa 40 respiri al minuto). Utilizzando un dispositivo di riscaldamento a mantenere la temperatura della superficie corporea stabile tra 35 e 37 ° C, come misurato da un sensore termico posizionato al sito addominale del mouse. Per i dettagli tecnici, consultare Herrmann et al 17. </li><li> Dopo la scansione, di liberare il mouse dal supporto e la fornitura di ossigeno puro per accelerare il recupero dall&#39;anestesia. Inoltre, mantenere la temperatura corporea stabile utilizzandouna fonte di calore luce rossa. </li></ol> 3. MRI protocollo <ol><li> Il protocollo è validato per un Tesla scanner 3, dotata di una SNR-efficiente, piccola bobina animale dedicato (bobina Litz linearmente polarizzata) con un campo efficace di vista di 35 mm × 38 mm di diametro. Azionare la bobina in modalità di trasmissione-ricezione. </li><li> Con l&#39;animale nella sua posizione finale, regolare la sintonia e corrispondenza della bobina con l&#39;ausilio di un analizzatore di frequenza. Regolare manualmente la tensione di riferimento del trasmettitore e le correnti spessori per ottimizzare l&#39;omogeneità di immagine e qualità. </li><li> Acquisire T 1 pesate immagini 2D TSE con una risoluzione di 0,5 mm × 0,5 millimetri × 2 mm di vista sagittale e trasversale per la pianificazione. Utilizzando i pianificazione esami di risonanza magnetica, acquisire le immagini MEMR in direzione di misura coronale, ruotato di essere parallela alla testa dell&#39;animale con la codifica di fase lungo la direzione sinistra-destra. Per minimizzare il tempo di acquisizione, utilizzare un campo rettangolare di vista adeguato allaDimensioni della testina reali. Impiegare una sequenza FLASH 3D viziato (VIBE 3D) utilizzando i seguenti parametri: matrice di base 256, campo visivo 54 millimetri × 50,65 millimetri x 14,08 millimetri, con il 93,8% del campo rettangolare di vista in fase di direzione codificare, e 128 fette di 0,11 mm spessore della fetta con una risoluzione fetta impostata a 61%. </li><li> Attivare l&#39;interpolazione nel piano per creare immagini finali con 512 × 480 × 128, fornendo una risoluzione effettiva di 0,21 mm x 0,21 millimetri × 0,18 millimetri (0.1 mm × 0,1 millimetri × 0,09 millimetri interpolati), tempo di eco T E = 6.51 msec, tempo di ripetizione T R = 16 msec, larghezza di banda = 160 Hz / px, flip angle = 22 °. Applicare due medie e tre ripetizioni per ottenere un tempo totale di acquisizione (T A) di circa 30 min. </li></ol> 4. MRI Analisi dei dati <ol><li> Analizzare i dati utilizzando il software fastView syngo. Per il potenziamento del segnale quantitativa, selezionare le regioni definite of interesse in 2D planari registrazioni MRI e determinare intensità di segnale (SI) della struttura migliorata (SI MEMRI), sfondo di tessuto (SI Priorità), e la deviazione standard del rumore (SD N). Se necessario, utilizzare un atlante del cervello mouse per facilitare l&#39;orientamento neuro-anatomico per LGN e le strutture SC. Calcolare il rapporto di contrasto-rumore (CNR) mediante la formula: </li><li> CNR = (SI MEMRI - SI Priorità) / SD N </li><li> Quantificare tre immagini consecutive per il calcolo della media CNR per ogni campione. Negli animali bilateralmente iniettati, analizzare ogni emisfero indipendente. </li><li> Per la rappresentazione di immagini orizzontali, coronali e sagittali, calcolare ricostruzioni multiplanari dal set di dati 3D MRI originale. Queste immagini elaborate non sono raccomandati per l&#39;analisi quantitativa. Per creare ricostruzioni 3D animati (proiezioni di massima intensità, MIPS) del retino-Tectal proiezione, utilizzare un modulo software di post-elaborazione angiografia. </li></ol> 5. Mn 2 + Autometallography (TIMM colorazione) <ol><li> Per TIMM colorazione di Mn 2 strutture cerebrali + tracciate seguendo MEMRI, iniettare una dose di 15-150 nmol Mn 2 + ivit 24 ore prima di imaging. </li><li> Dopo la scansione del MR, profumato gli animali con 30 ml ghiacciata 0,325% Na 2 S in PBS (pH 7,4). Sezionare retine e congelare i campioni nella sezione congelata. </li><li> Tagliare sequenziali, sezioni equatoriali di spessore 15 micron su un cryotome. </li><li> Eseguire TIMM colorazione 21 in assenza di fissativo e crioprotezione secondo Angenstein et al 22. </li></ol> 6. Analisi statistica Effettuare analisi statistiche utilizzando il test t di Student per singoli confronti, seguita da post hoc ANOVA. I dati sono presentati come media ± errore standard. Numeri individuali N sonoindicati separatamente per ciascun esperimento. Risultati raggiungendo P ≤ 0,05 sono considerati statisticamente significativi (P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0,001, ***).

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

MEMRI del sistema visivo si estende tecniche neurobiologiche convenzionali per valutare la funzionalità in condizioni ingenue e patologiche. Oltre ad una visione unica l&#39;integrità di un isolato fibra tratto CNS, MEMRI può essere facilmente integrato con test comportamentali, per esempio, optometria e compiti a base di acqua visivamente, di indagare le conseguenze immediate di un dato paradigma per la percezione visiva. Si collega anche elettrofisiologici e le indagini istologiche con caratterizzazione vi...

Sulla base della sua struttura neuro-anatomica favorevole il sistema visivo roditore offre possibilità uniche per valutare composti farmacologici e la loro capacità di mediare neuroprotezione 1 o effetti pro-rigenerativi 2,3. Inoltre, per studi sulle caratteristiche funzionali e neuro-anatomica di mutanti di topo, come recentemente esemplificato per topi privi della proteina ponteggio presinaptica Bassoon 4. Inoltre, un ampio spettro di strumenti supplementari permette supplementare con delle cellule gangliari retiniche (RGC) e numeri assoni RGC nonché attività RGC, ad esempio, da elettroretinografia e test comportamentali, e la determinazione di riarrangiamenti corticali mediante imaging ottico di segnali intrinseci. Gli ultimi sviluppi tecnici in microscopia laser consentono di visualizzazione situ di RGC rigenerazione profonda dei tessuti imaging di fluorescenza in interi esemplari di montaggio delle nervo ottico (ON) e il cervello. In questo histologapproccio iCal, tetraidrofurano basato radura del tessuto in combinazione con foglio leggero microscopia a fluorescenza permette la risoluzione delle singole fibre che ri-entrare nel deafferented ON e tratto ottico 5. Mentre tali tecniche possono essere superiori in risoluzione e determinazione della curva di crescita, non consentono analisi ripetitive e longitudinali di eventi di crescita individuali, particolarmente desiderati per valutare il processo di rigenerazione lungo termine. Contrasto-enhanced MRI è stata impiegata per la visualizzazione minima invasività della proiezione retino-tectal in topi e ratti 6,7. Ciò può essere ottenuto mediante consegna intraoculare diretta di ioni paramagnetici (ad esempio, Mn 2 +) di cellule retiniche. Come un analogo calcio, Mn 2 + è incorporato in RGC somata tramite i canali del calcio voltaggio-dipendenti e attivamente trasportati lungo il citoscheletro assonale della ON intatta e tratto ottico. Mentre si accumula nei nuclei cerebralidella proiezione visiva, cioè il nucleo genicolato laterale (LGN) e collicolo superiore (SC), la propagazione transsynaptic nella corteccia visiva primaria appare trascurabile 8,9, anche se può verificarsi 10,11. Sotto MR sequenziamento, paramagnetica Mn 2 + Potenziati MR contrasto principalmente accorciando T 1 spin-reticolo tempo di rilassamento 12. Tale Mn 2 + migliorato MRI (MEMRI) è stato applicato con successo in diversi studi neuro-anatomici e funzionali di ratti, compresa la valutazione della rigenerazione assonale e la degenerazione dopo sul pregiudizio 13,14, la precisa mappatura anatomica della proiezione 15 retino-tectal , nonché la determinazione delle caratteristiche trasporto assonale dopo il trattamento farmacologico 16. Recenti ricerche nel dosaggio, tossicità, e la cinetica dei protocolli MRI neuronali Mn 2 + assorbimento e il trasporto, nonché il miglioramento hanno esteso la sua applicazione agli studi sui transgenicitopi 9 con 3 scanner Tesla comunemente utilizzati nella pratica clinica 17. Qui vi presentiamo un protocollo MEMRI adatto longitudinale imaging in vivo del mouse retino-tectal proiezione ed esemplificare la sua applicabilità valutando Mn 2 + dipendente potenziamento del segnale in condizioni di neurodegenerazione ingenue e vari. Il nostro protocollo pone particolare enfasi sulla acquisizione dati MR in un moderato 3 T campo magnetico che è generalmente più accessibile rispetto agli scanner di animali dedicati. Nei topi naive, illustriamo come intensità di segnale specifico tratto può essere sostanzialmente e riproducibile diventare aumentata dopo intravitreale (ivit) Mn 2 + applicazione. Quantitativamente, Mn 2 + propagazione lungo la proiezione visiva si verifica indipendentemente dal normale processo di invecchiamento (misurata tra 3 e 26 mesi di età topi) e aumento è refrattario alla stimolazione visiva e l&#39;adattamento all&#39;oscurità. Al contrario, Mn <sup&gt; 2 + arricchimento nei centri talamo e mesencefalo è diminuita a seguito acuta ON lesione da schiacciamento 18, così come in NFKB1 topi knock-out (p50 KO) affetti da spontanea morte apoptotica RGC e sulla degenerazione 19. Così, in espansione per analisi istologica convenzionale, longitudinale analisi MEMRI singoli animali ha la profilatura della cinetica uniche di processi neurodegenerativi. Questo dovrebbe rivelarsi utile per gli studi sulla neuroprotezione e rigenerazione assonale associati a interventi farmacologici o genetici.

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			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">207434/00</td>
			<td>removable needle RN, 34 gauge, lenght 38.1 mm, point style 4</td>
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			<td style="width:269px;">Zeiss, Oberkochen, Germany</td>
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			<td>Doty Scientific Inc., Columbia, SC, USA</td>
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			<td rowspan="5">for TIMM staining</td>
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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

Il sistema visivo roditore comprende cellule gangliari della retina e dei loro assoni che formano il nervo ottico per entrare nei centri talamo e mesencefalo, e proiezioni postsinaptici alla corteccia visiva. Sulla base della sua struttura anatomica distinta e comoda accessibilità, è diventato la struttura preferita per gli studi sulla sopravvivenza neuronale, rigenerazione assonale, e la plasticità sinaptica. Recenti progressi in RM hanno permesso la visualizzazione in vivo della parte retino-tectal di questa proiezione utilizzando manganese mediata aumento del contrasto (MEMRI). Qui vi presentiamo un protocollo MEMRI per l&#39;illustrazione della proiezione visiva nei topi, da cui risoluzioni (200 micron) 3 possono essere raggiunti utilizzando comuni 3 scanner Tesla. Dimostriamo come l&#39;iniezione intravitreale di una singola dose di 15 nmol MnCl 2 porta ad un miglioramento saturo della proiezione intatto entro 24 ore. Con eccezione della retina, cambiamenti di intensità del segnale sono indisidente della coincisa stimolazione visiva o invecchiamento fisiologico. Applichiamo ulteriormente questa tecnica per monitorare longitudinalmente degenerazione assonale in risposta al danno del nervo ottico acuto, un paradigma con il quale Mn 2 + trasporto completamente arresti presso il sito di lesione. Al contrario, attivo Mn 2 + trasporto è quantitativamente proporzionale alla vitalità, il numero e l&#39;attività elettrica delle fibre assoni. Per tale analisi, rappresentiamo Mn 2 + cinetiche di trasporto lungo il percorso visivo in un modello di topo transgenico (NF-kB p50 KO) visualizzando atrofia spontanea di sensoriali, tra cui visivi, proiezioni. In questi topi, MEMRI indica ridotta ma non ritardato Mn 2 + trasporto rispetto ai topi wild type, rivelando così segni di alterazioni strutturali e / o funzionali da mutazioni NF-KB. In sintesi, MEMRI ponti comodamente in vivo e post mortem istologia per il characterizatisu integrità e l&#39;attività delle fibre nervose. E &#39;molto utile per studi longitudinali sulla degenerazione assonale e rigenerazione, e ricerche di topi mutanti per fenotipi originali o inducibili.

La capacità di questa tecnica di imaging per valutare con precisione la vitalità e la funzionalità della proiezione visiva si basa su una precisa applicazione di un tossico Mn 2 + dosaggio al corpo vitreo e il suo assorbimento da RGC. Questo importante assunzione viene testato in Figura 1, dove strato specifico Mn 2 + assorbimento è dimostrato dal autometallography (TIMM colorazione) 21. Sezioni retina sono state analizzate a 24 ore dopo l&#39;applicazione di una <em...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Questo video illustra un metodo, utilizzando un clinico 3 scanner T, per contrasto-enhanced RM del mouse ingenua proiezione visiva e per studi ripetitivi e longitudinali in vivo di degenerazione del nervo ottico associati a lesioni acute ottico schiacciamento del nervo e la degenerazione del nervo ottico cronica topi knock-out (p50 KO).

In vivo imaging di Optic Nerve Fiber Integrity by MRI Contrast-Enhanced nei topi

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K Views.  Jena University Hospital.  Questo video illustra un metodo, utilizzando un clinico 3 scanner T, per contrasto-enhanced RM del mouse ingenua proiezione visiva e per studi ripetitivi e longitudinali in vivo di degenerazione del nervo ottico associati a lesioni acute ottico schiacciamento del nervo e la degenerazione del nervo ottico cronica topi knock-out (p50 KO). 

Video: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Metodi per manipolazioni sperimentali dopo resezione del nervo ottico nel sistema nervoso centrale dei mammiferi

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Ricerca

Neuroscienze

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta risoluzione<em&gt; In Vivo</em&gt; Manuale Protocollo Segmentazione for Human ippocampale Sottocampi Utilizzando 3T Risonanza Magnetica

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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 ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Registrazione potenziale evocato visivo in un modello murino di sperimentale del nervo ottico demielinizzazione

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Ad alta risoluzione strutturale Risonanza Magnetica del subcortex Umana<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Gene Transfer di cellule di Schwann nel nervo sciatico roditori da elettroporazione

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.