Questo studio è stato supportato anche da finanziamenti della Vanderbilt-Ingram Cancer Center Support Grant (P30 CA068485), il Brain Tumor Foundation Infanzia e il National Institutes of Health (NS042205).

1. Micro-dissezione del tumore-cuscinetto cervelletto e dissociazione del tessuto tumorale <ol><li> Recupero del tessuto tumorale <ol><li> Topi malati cuscinetto medulloblastoma sono spesso runted e visualizzare idrocefalo e tipici sintomi neurologici, tra cui paralisi posteriore e l&#39;incapacità di riacquistare la postura in caso di ribaltamento. Per recuperare tessuto tumorale, eutanasia topi per inalazione di anidride carbonica. E &#39;troppo importante per noi non eseguire dislocazione cervicale, una procedura che genera una pressione al cranio posteriore e può compromettere l&#39;integrità del tessuto tumorale. </li><li> Decapitazione viene eseguita subito dopo la morte con un paio di forbici, la rimozione dei capelli e tessuto muscolare il più possibile per una buona visualizzazione del cranio. Pulire la superficie del cranio con Kimwipe imbevuto di etanolo al 95%. </li><li> Usare le forbici per tagliare bene un&#39;apertura lungo la linea mediana del cranio, e rimuovere il tessuto cranio con una pinzetta sottile, a questo punto l&#39;intero cervello compreso portatori di tumore cervelletto è esposto. </li><li> Mentre il cervello dei degli adulti sani visualizzare ben definite emisferi e del verme, il tumore del cervello dei topi portatori sono spesso allargata, amorfo con una superficie liscia e vasi sanguigni cospicua. Usando tecniche sterili, recuperare il tumore del cervelletto con delle pinzette e mettere in ghiaccio freddo tampone fosfato (PBS) senza Mg 2 + e Ca 2 +. </li></ol></li></ol> Nota: tutti gli strumenti sono disinfettati in etanolo al 95% e del vapore in autoclave prima dell&#39;uso. <ol start="2"><li> Dissociazione del tessuto tumorale <ol><li> Trasferire il tessuto tumorale da PBS al 50% Accutase (diluito in PBS) che è circa 4 volte il volume del tessuto tumorale, tritare il tessuto con le forbici multa per 3 minuti a temperatura ambiente, seguita da incubazione a 37 ° C per 4 minuti , dopo di che il tessuto subisce pipeting ripetitivi con un 1 mL Pipetman per altri 3 minuti. Questo metodo dovrebbe produrre un insieme di celle singole e piccoli aggregati cellulari. </li><li> Diluire la sospensione cellulare di 3 volte con PBS e centrifugare per 5 minuti a 1000xg per agglomerare le cellule. (Le procedure di cui sopra sono stati descritti in &quot;isolamento, l&#39;arricchimento e il mantenimento delle cellule staminali medulloblastoma&quot;, Giove in stampa) </li></ol></li></ol> Risospendere il pellet di cellule in appena preparato medio di cellule staminali neurali composto medio Neurobasal con la glutamina, penicillina-streptomicina, N2, B27, umano EGF (25 ng / ml) e di base FGF (25 ng / mL). Calcolare la densità delle cellule della soluzione e le opportune diluizioni ulteriormente con l&#39;aggiunta di cellule staminali neurali medio tale che si può carico di 4 ml di soluzione con un numero adeguato di cellule tumorali dissociata, ad esempio 5x10 5, in una sterile conici a punta siringa 10μL. Il numero esatto di cellule da inoculare deve essere determinato dal ricercatore in base alle specifiche esigenze sperimentali. Conservare la soluzione di cellule in una provetta da microcentrifuga mini 200μL (tubo PCR) su ghiaccio. 2. Procedure di anestetico per il Mice destinatario Per la procedura chirurgica, una zona disinfettata dedicata alla chirurgia roditori è necessario per la durata della procedura. Idealmente, impostare una lunga panca con aree separate ma adiacenti per la preparazione degli animali, campo operatorio e il recupero degli animali. Guanti chirurgici, non guanti esame, sono necessari per l&#39;intervento chirurgico. Tecnica asettica è mantenuta tenendo le mani guantate di sopra della vita e utilizzato solo per gestire gli oggetti sterili da una superficie asciutta sterile. <ol><li> Una miscela di ketamina (100 mg di ketamina per 1 kg di peso del mouse) e xilazina (10 mg xylazina per 1 kg di peso del mouse) è utilizzato per l&#39;anestesia. Amministrare il intraperitoneale anestetici. </li><li> Di solito, occorrono 3-5 minuti per il anestetici abbiano pieno effetto. Determinare se il mouse è completamente anestetizzato da tep / coda pizzico. Mettere una piccola quantità di pomata oftalmica sugli occhi del mouse. Portare il mouse completamente anestetizzati per l&#39;area operativa, monitorando costantemente per assicurarsi che il mouse sta respirando normalmente. </li></ol> 3. Intracranica innesto delle cellule tumorali <ol><li> Radere la regione dorsale posteriore del capo del mouse, utilizzando un tagliatore di capelli, per rivelare il cranio posteriore sopra il mesencefalo e cervelletto. </li><li> Applicare la soluzione di Betadine sul cuoio capelluto esposto utilizzando un cotton-fioc, seguiti strofinando con un tampone di alcool. Ripetere questi due passaggi di sterilizzazione per due volte. </li><li> Fare un quarto di pollice incisione nel cuoio capelluto posteriore utilizzando un bisturi sterilizzato. Praticare un foro mm 0,5 bava 2 mm a destra e 2 mm posteriormente al lambda con un trapano sterile dentale. </li><li> Posizionare il mouse su un telaio stereotassico agganciando la sua incisivi sul possesso telaio. Con attenzione scendere e posizionare il bisello a punta siringa da 10 microlitri caricata con 4 ml di soluzione delle cellule tumorali nel foro bava. Una volta che la smussatura dei SyriESN è l&#39;ago sotto la superficie del cranio, si scende per un altro mm 3 e poi risalire per 0,5 mm. Iniettare lentamente la quantità desiderata di cellule tumorali, con una forza costante in un fotogramma 30 la seconda volta, nel cervelletto del destinatario. Lasciare l&#39;ago al suo posto dopo il completamento di iniezione per altri due minuti. Quindi rimuovere l&#39;ago e siringa. Clip della ferita utilizzando un autoclip. </li><li> Tenere il mouse su una coperta riscaldamento dopo l&#39;intervento per aiutare a mantenere la sua temperatura corporea. Modelli di mobilità e respiratorie verrà osservato continuamente. Una volta che il mouse recupera da anestesia, è posto di nuovo in una gabbia alloggiamento sterile. Monitorare giorno per l&#39;assunzione di cibo e acqua, comportamento, governare, e la colorazione rossa degli occhi. Controllare eventuali segni di infezione nel sito di incisione. Rimuovere il autoclips al giorno 7 dopo l&#39;intervento chirurgico. Mantenere i topi per una media di 2-6 mesi e di sacrificio quelle che espongono i sintomi tipici medulloblastoma per ulteriori studi. </li></ol> 4. Rappresentante Risultati Medulloblastomi secondario sviluppato in topi riceventi molto simile a quelle del tumore primario. Il cervello dei portatori di tumori secondari erano spesso allargata, amorfo con i vasi sanguigni ectopica evidente se visto nel suo complesso-monti. Analisi immunoistochimica del tessuto tumorale secondaria rivelato che le cellule tumorali sono altamente proliferative, esprimere forte SmoM2-YFP e marcatori dei precursori neuronali dei granuli cerebellari, come Math1 e Pax6. Quando i metodi con dissociato e colto riportato (&quot;L&#39;isolamento, l&#39;arricchimento e il mantenimento delle cellule staminali medulloblastoma&quot;, Giove in stampa), il secondario cellule tumorali medulloblastoma possono essere espanse rapidamente, esprimere molteplici marcatori di cellule staminali, sono clonogenica e possono ulteriormente avviare la formazione di tumori quando trapiantate in ulteriori destinatari immunocompromessi. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="Figura 1" /> Figura 1. Tipica con tutto il supporto e viste in sezione di un tessuto medulloblastoma secondaria Un tipico esempio di tessuto medulloblastoma secondario sviluppato 26 giorni dopo l&#39;iniezione di 5x10 5 cellule del tumore primario. Colorazione ematossilina rivela la tipica morfologia cellulare del medulloblastoma, tra cui alti nucleare / citoplasma rapporto e il polimorfismo nucleare. Queste cellule tumorali secondarie sono altamente proliferative come indicato da Ki67 espressione e anche robusta esprimere membranose SmoM2-YFP. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="Figura 2" /> Figura 2. Cellule di medulloblastoma secondarie possono essere coltivate ed espanse in vitro Utilizzando il protocollo descritto per le cellule primarie medulloblastoma del mouse (&quot;L&#39;isolamento, l&#39;arricchimento e il mantenimento delle cellule staminali medulloblastoma&quot;, Giove in stampa), queste cellule secondarie medulloblastoma possono essere coltivate ed espanse per passaggi multipli. L&#39;immagine mostra la stessa colonia tumore rappresentante colto in 4 giorni e 14 giorni. Le cellule tumorali in coltura sono bipolari, di forma allungata con citoplasma scarso e spesso si irradiano da un nucleo denso di aggregati cellulari. Essi non presentano contatto inibizione della crescita. Le piccole cellule rotonde sono globuli rossi.

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

Diversi fattori critici di successo per garantire la cellula medulloblastoma allotrapianto che la formazione di tumori rendimenti secondarie sono le seguenti: in primo luogo, utilizzare solo Accutase 50% per il tessuto dissociazione e non oltre il digest del tessuto come trattamento enzimatico prolungata porta ad una significativa riduzione della vitalità delle cellule. Anche utilizzare solo il Pipetman taglia 1 ml più piccole punte può anche generare danni fisici alle cellule dissociate. E &#39;bene avere una miscela di celle singole e piccoli aggregati per allotrapianto. In secondo luogo, mescolare ed equilibrare la soluzione delle cellule tumorali ogni volta prima di caricare la siringa Hamilton. Non utilizzare più di 4 microlitri per ogni iniezione, come un volume maggiore genererebbe troppa pressione intracranica e la resistenza. Infine, è importante aspettare altri 2 minuti per consentire la soluzione iniettata dissipano nel tessuto cerebrale dopo ogni iniezione. Le procedure descritte consentono la determinazione del tipo di cellule geneticamente marcato (s) in grado di avviare e propagare i tumori. Questo protocollo si applica anche alle situazioni in cui le cellule da iniettare non derivano direttamente dalla scuola primaria tessuto tumorale, ma da linee cellulari in coltura. Si può preparare un numero adeguato di cellule da uno digestione enzimatica e / o pipettaggio ripetitivo, e ripartire dal punto 2 del presente protocollo. Quindi, possiamo anche testare le funzioni dei geni candidati e l&#39;effetto inibitorio di composti chimici con un tumore in vivo di crescita di lettura.

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<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
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<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
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<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
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<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
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<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
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<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50% </td>
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<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
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<td>Stereotaxic instrument</td>
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<td>Stoelting </td>
<td>51730</td>
<td>&nbsp;</td>
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<td>Foredom flexible shaft drill, hang-up style</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>58650</td>
<td>&nbsp;</td>
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<td>Large probe holder</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51633</td>
<td>&nbsp;</td>
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<td>10 &mu;L syringe, 26 ga., bevel tip</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51105</td>
<td>&nbsp;</td>
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<td>Drill bit .75mm</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

Il medulloblastoma è il tumore più comune pediatrici del sistema nervoso. Una grande quantità di studi sugli animali si è concentrata sui precursori neuronali dei granuli del cervelletto (CGNPs) come la cellula di origine per il medulloblastoma 1-4. Tuttavia, le diverse presentazioni cliniche dei sottotipi medulloblastoma nei pazienti umani (nodulare, desmoplastico, classica e grandi cellule / anaplastico), e il fatto che il medulloblastoma si trova in un sottogruppo di pazienti umani senza espressione ectopica di CGNP marcatore 5, suggeriscono che la origini cellulare e molecolare di medulloblastoma sono più complessi e lungi dall&#39;essere completamente decifrato. Pertanto, è essenziale per determinare se vi è un tumore medulloblastoma alternativa di cellule di origine sulla base di quale tipo cellulare specifico modalità terapeutica può essere sviluppato. A tal fine, ortotopico intracranica allotrapianto di organismi geneticamente segnato tipi di cellule tumorali seguita da successive analisi secondaria di sviluppo del tumore nei pazienti consentirà di determinare l&#39;origine cellulare delle cellule tumorali iniziare. Qui descriviamo il protocollo sperimentale per ortotopico intracranica allotrapianto di cellule derivate da medulloblastoma primario tessuto tumorale, e questa procedura può essere utilizzata anche per il trapianto di cellule provenienti da linee cellulari stabilizzate.

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Questo protocollo descrive l&#39;isolamento e la dissociazione del tessuto medulloblastoma mouse e allotrapianto successive delle cellule tumorali in topi immunocompromessi destinatario al fine di avviare medulloblastoma secondario.

Intracranica allotrapianto ortotopico di cellule di medulloblastoma in topi immunocompromessi

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.1K Views.  Vanderbilt University. Questo protocollo descrive l'isolamento e la dissociazione del tessuto medulloblastoma mouse e allotrapianto successive delle cellule tumorali in topi immunocompromessi destinatario al fine di avviare medulloblastoma secondario.

Video: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Isolamento, di arricchimento e manutenzione delle cellule staminali Medulloblastoma

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Ricerca

Neuroscienze

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Iniezione intracranica di adeno-associato vettori virali

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Un metodo semplice e poco costoso per la determinazione fredda sensibilità e adattamento nei topi

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Dosaggi di dolore nei roditori e farmacoterapia intracranica

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Il rilevamento di 5-Hydroxytethylcytosine in cellule staminali neurali e cervelli di topi

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Misurazione quantitativa delle proteine intratecalliamente sintetizzate nei topi

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Registrazione elettrocorticografica delle aree della corteccia cerebrale manipolate utilizzando un virus adeno-associato che prende di mira Cofilin nei topi

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.