This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

NOTA: Tutti i volontari di ricerca in questo studio sono stati somministrati i documenti di consenso informato approvato dal Institutional Review Board di Howard University e la Johns Hopkins University. Solo ai partecipanti che hanno acconsentito, firmando documenti consenso informato sono stati arruolati nello studio. La base di studio per questa analisi consisteva in: 20 soggetti (12 BD e 10 controlli, 30% maschi) ed età (sd) di 38,2 (14,1) anni significa. NOTA: Spray tutte le superfici con RNase Zap per eliminare la contaminazione RNasi dalle superfici di vetro e plastica. 1. Biopsia Procedura <ol><li> Spray topico lidocaina (xylocaina) liquido 4% e ossimetazolina HCl 0,05% nella cavità nasale del partecipante allo studio e permettere 15 minuti di tempo paralizzante. </li><li> Impregnano cottonoids chirurgici ½ x 3 pollici in una miscela di lidocaina e ossimetazolina e inserire nella cavità nasale lungo la porzione superiore del setto nasale e lasciar riposare per 15 min. </li><li> Utilizzando un 30 ° rigidoendoscopio nasale per individuare la parte superiore della cavità nasale, rimuovere mucosa nasale con up-mordere pinze nasali dalla parte superiore del setto nasale, prendendo una serie di piccoli campioni. </li></ol> 2. Preparazione di blocchi di tessuto <ol><li> Immediatamente prima biopsia, compilare cryomolds di dimensioni standard con tessuto congelamento media. </li><li> Con una pinzetta in autoclave, montare tessuto fresco nel mezzo di congelamento e congelare in ghiaccio secco. Riempire resto cryoblock con il mezzo di congelamento, congelare cryoblock in ghiaccio secco, e memorizzare i blocchi a -80 ° C. </li></ol> 3. criosezionamento <ol><li> Preparare polietilennaftalato (PEN) vetrini membrana immediatamente prima criosezionamento. Spruzzare diapositive con soluzione RNase Zap per 5 min. Quindi sciacquare i vetrini in dietilpirocarbonato (DEPC) acqua e lasciare asciugare per 30 minuti. Attivare diapositive secchi sotto la luce UV per 30 minuti. </li><li> Cryosection ogni blocco interamente a 30 micron. Tenere i vetrini su asciuttoce e sezioni congelate per quanto possibile. Conservare i vetrini a -80 ° C al termine. </li></ol> 4. Laser-Capture Microdissection <ol><li> Inizia disidratazione diapositive immediatamente prima microdissezione. Lasciate scongelare i vetrini congelati per 10 sec, e quindi applicare la seguente serie di etanolo per ogni diapositiva: 60% (2 ml, 15 sec), l&#39;80% (2 ml, 15 sec), il 95% (3 ml, 25 sec), 100 % (7 ml, 60 sec). </li><li> Girare microscopio la localizzazione fotoattivato (PALM) microscopio e impostare le seguenti condizioni: Per 10X di ingrandimento impostato l&#39;energia per 82, si concentrano a 74, e la velocità di 25 - 30. Per 20X di ingrandimento, impostare l&#39;energia per 63, si concentrano a 67, e la velocità di 5 - 6. Regolare come necessario per ottenere risultati ottimali di taglio. </li><li> Visivamente identificare neuroni utilizzando 20X o 10X di ingrandimento. Distinguere la superficie sottile strato neurone olfattivo sottostante sub-mucosa al microscopio senza l&#39;uso di procedura di colorazione. Osservare i neuroni come una fitta aspetto colonnare 18.</li><li> Utilizzando la funzione Pencil al microscopio citata in fase 4.2, tracciare intorno allo strato neuronale. Traccia un po &#39;lontano dai neuroni per evitare di bruciare dei neuroni da laser. Ciò è essenziale per ottenere RNA alta qualità. Poi avviare guida laser di taglio. </li><li> Pick up sezioni sezionati con punta fine pinze microdissezione e mettere il tessuto in una provetta contenente 100 microlitri tampone di lisi su ghiaccio. Ripetere l&#39;operazione per tutte le sezioni neuronali ottenute da un singolo campione. Tempo dissezione totale per campione non deve superare i 50 minuti. </li><li> Una volta che la dissezione è completa, immediatamente lisati campione vortice e mantenere in ghiaccio secco. Conservare i campioni a -80 ° C per l&#39;isolamento totale di RNA. </li></ol> 5. Isolamento di RNA totale e analisi Controllo Qualità <ol><li> Lisati campione Scongelare su ghiaccio. Effettuare totale isolamento di RNA con RNAqueous Micro Kit con DNase-I inattivazione seguendo il protocollo del produttore senza modifiche (vedi &lt;strong&gt; Figura 1). Il volume totale di eluizione è di circa 20 microlitri. </li><li> Misurare la concentrazione di RNA e valutare la qualità RNA con l&#39;RNA Integrity Number (RIN) mediante Bioanalyzer. I campioni che soddisfano i criteri di controllo della qualità della Figura 2 possono essere utilizzati per la sintesi di cDNA. </li></ol> 6. cDNA Synthesis <ol><li> Sintetizzare cDNA utilizzando un kit commerciale convalidato (vedi Tabella dei reagenti). Eseguire la fase di ricottura combinando 0,1 - 1,0 ng di RNA (a seconda della disponibilità) e RNasi acqua libera ad un volume totale di 8 microlitri per provetta. Quindi aggiungere 1 ml di oligo d (T) 20 primer e 1 ml di miscela dNTP 10 mM per provetta, per un volume totale di 10 microlitri. </li><li> Delicatamente campioni vortex e centrifugare e campioni ricottura a 65 ° C per 5 min. Dopo che la reazione è completa, i campioni immediatamente fresco su ghiaccio. </li><li> Preparare la soluzione master mix in base alle specifiche di cui alla tabella 1. Quindi aggiungere 9,5 μl master mix e 0,5 ml di preparati commercialmente trascrittasi inversa (RT) in ciascun tubo. Aggiungere 0,5 acqua priva di RNasi microlitri per controllare tubo invece di RT. </li><li> Eseguire una reazione RT con la prima incubazione a 50 ° C per 50 minuti, poi 85 ° C per 5 minuti e una finale attesa 4 ° C, senza il ciclismo. </li><li> Diluire tutti i campioni di 5x con acqua e aliquota. Conservare i campioni a -80 ° C. </li></ol> 7. Real Time PCR - Confermare neuronale Enrichment <ol><li> Preparare la soluzione master mix secondo le norme di cui alla tabella 2. Utilizzare un controllo interno, come GAPDH. Confronta olfattiva proteina marker (OMP) espressione nel tessuto microdissezione con espressione OMP nel tessuto undissected per determinare arricchimento neuronale. NOTA: OMP è un marcatore per i neuroni olfattivi. OMP sequenza: in avanti, 5&#39;-CTGTCGGACCTGGCCAAG-3 &#39; inversa, 5&#39;-CACCCTCGCCAAAGGTGA-3 &#39; Sequenza GAPDH: avanti, 5&#39;-CATGAGAAGTATGACAACAGCCT-3 &#39; inversa, 5&#39;-AGTCCTTCCACGATACCAAAGT-3 &#39;. </li><li> Istituito piastra PCR aggiungendo 7 ml di master mix in ciascun pozzetto e 3 ml di cDNA. </li><li> Piatto di centrifuga per 5 minuti a 300 xg (1.300 rpm). Reazione Run Real Time-PCR utilizzando le condizioni di ciclo termico predefiniti specificati per supermix commerciale come SYBR Greener qPCR. </li><li> Analizzare i dati (vedi figura 2 per le specifiche). </li></ol> 8. Real Time PCR - espressione di geni di interesse <ol><li> Eseguire su campioni che mostrano arricchimento neuronale di 2 volte o più. </li><li> Preparare master mix in ghiaccio per ogni sonda (sonda FAM-marcata di interesse e VIC-etichettati GAPDH) in tubi separati secondo le specifiche di cui alla tabella 3. </li><li> Impostare un piatto PCR con 8 microlitri master mix in ciascun pozzetto e 2 ml di cDNA. </li><li> Centrifugare la piastra per 5 min a 300 xg (1.300 rpm). Eseguire reale reazione di Time-PCR utilizzando il defAult condizioni di ciclo termico come da protocollo master mix espressione genica fornito dal produttore. Analizzare i risultati di espressione (vedi figura 2 per le specifiche). </li></ol>

<ol>
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Gli autori non segnalano rapporti finanziari con interessi commerciali.

Una nuova piattaforma per l&#39;ottenimento arricchiti strati di neuroni olfattivi combinando biopsia nasale e LCM è presentato ed è stato convalidato in questo studio. Questa tecnica può avere implicazioni diffuse. Può essere applicato verso studi biomarcatori, compresi quelli per la risposta al trattamento, e gli sforzi di scoperta di farmaci per altre condizioni neuropsichiatriche, avere un impatto più ampio nel campo. Per ottenere i neuroni di alta qualità suscettibili di applicazi...

Il disturbo bipolare (BD) è un grave disturbo neuropsichiatrico, caratterizzato da alterazioni patologiche di umore, guidare e cognizione 1. Litio usato per il trattamento di BD ha dimostrato di alterare costanti i livelli di mRNA stato di un gran numero di geni in studi su animali 2, ma rimane sconosciuta se uno qualsiasi di queste molecole è associato con risposta clinica nell&#39;uomo 2. Comprendere il meccanismo di risposta litio richiederebbe indagare i cambiamenti molecolari litio-indotta nei tessuti neuronali. Purtroppo, non è pratico di ottenere biopsie cerebrali di pazienti BD prenatale e della post-litio per identificare le firme molecolari di risposta litio. Post-mortem tessuti cerebrali sono stati utilizzati per studiare biomarcatori in BD, tuttavia, non possono essere utilizzati per valutare marcatori molecolari associati con i cambiamenti dinamici in emozioni, cognizione e guidare; e la validità di retrospettivamente accertato risposta al trattamento di litio può essere problematico 4. I linfociti e le altre cellule del sangue possono essere utili, ma i cambiamenti molecolari nelle cellule del sangue non possono riflettere le alterazioni neuronali 3-5. Liquido cerebrospinale potrebbe essere insufficiente per ottenere informazioni su molecole intracellulari che possono riflettere associate alla malattia e farmaci reversibile modifiche intrinseche. L&#39;epitelio olfattivo (OE) è una parte unica del sistema nervoso centrale (SNC), embriologicamente legati alle strutture limbiche 6; ed è facilmente accessibile attraverso biopsie nasali. Si compone di gliali come cellule di supporto celle (cioè, sustentacular), basali proliferanti, e neuroni olfattivi a diversi stadi di sviluppo 7-9. Pertanto, OE offre un&#39;opportunità unica per studiare accessibilmente cambiamenti dinamici nel sistema nervoso centrale dei pazienti con malattie neuropsichiatriche 7. Gli studi stanno dimostrando l&#39;utilità del OE come un tessuto surrogato per studiare la malattia associata eventi che riflettono tali occurring in neuroni cerebrali 8,9. Ad esempio, gli studi hanno utilizzato OE per indagare i profili molecolari associati alle condizioni psichiatriche 10-14. Sistema olfattivo serve anche ad identificare endophenoytpes clinici come deficit odore che sono associati con i sintomi negativi della schizofrenia 15. Inoltre, i processi di sviluppo neurologico continuano nella OE per tutta la vita, fornendo un viale utile per modellare la fisiopatologia di condizioni psichiatriche 8,9. Tuttavia, un inconveniente per l&#39;uso di questo tessuto è la sostanziale contaminazione biopsie olfattivi con cellule non-neuronali 16. Per esempio, l&#39;RNA totale utilizzato per studi di espressione genica in studi precedenti OE conteneva RNA estratto da nasale intera biopsia tissutale compreso RNA da cellule non-neuronali 17. Pertanto, gli approcci precedenti sono stati limitati dalla qualità delle cellule. Per ovviare a questo problema, un nuovo approccio per ottenere<html> arricchito popolazioni di cellule neuronali combinando biopsie nasali con laser-capture microdissezione (LCM) è stato sviluppato 18. LCM è una tecnica che consente l&#39;isolamento selettivo di cellule usando taglio laser UV combinato con infrarossi laser 19-21. Combinando LCM con approccio OE minimizza sostanziale contaminazione OE da cellule non-neuronali, migliorando così l&#39;arricchimento delle cellule neuronali 18. Inoltre, lo strato neuronale può essere distinto dallo strato sottomucosa al microscopio, eliminando così la necessità di colorazione. Tipi di cellule neuronali possono più essere distinti da altre popolazioni cellulari utilizzando anticorpi primari che sono espressi dal tipo di cellula di interesse 7. Pertanto, questa procedura prevede un metodo più semplice per l&#39;arricchimento di popolazione cellulare quasi puramente neuronale che può essere utilizzato per studi di espressione genica, immunoistochimica e altre indagini morfologiche. ve_content &quot;&gt; Questo studio mira a istituire una piattaforma sperimentale per studiare i cambiamenti molecolari nei neuroni olfattivi associati stati di malattia e la risposta al trattamento. Per risolvere questo problema, un piccolo gruppo di pazienti non-fumatori che hanno incontrato i criteri diagnostici del DSM-IV per BD basa su Intervista diagnostica per studi genetici (DIG) 22 è stato reclutato per subire due biopsie nasali: una biopsia pre-trattamento con litio e la seconda biopsia, dopo 6 settimane di terapia quotidiana litio orale Inoltre, i pazienti BD ammissibili devono essere:. sintomatico per la depressione , basata sul punteggio ≥10 su 60 nella clinico somministrato Montgomery-Asberg Rating Scale (MADRS) 23; sintomatica per ipomania o mania, sulla base di un punteggio ≥10 su 56 sul medico somministrato Young-Mania Rating Scale (YMRS) 24;. o ≥10 su entrambi MADRS e YMRS coefficiente Rater-Inter-Rater di un accordo tra i medici sia per le scale è&gt; 0.96 Dopo biopsie, i neuroni erano Enri.ched da OE da LCM. A seguito di misure di controllo supplementari di qualità, al fine di garantire l&#39;estrazione di RNA di alta qualità dal tessuto e arricchimento neuronale, Real Time RT PCR è stato condotto per indagare i livelli di pre e post-trattamento di espressione dei geni di interesse. Le sezioni successive contengono una descrizione della convalida di questo approccio, mettendo in evidenza l&#39;ottimizzazione del protocollo e le strategie che sono state applicate per la risoluzione dei problemi del protocollo.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Reagent</td>
			<td style="width:143px;">Manufacturuer&#160;</td>
			<td style="width:175px;">Manufacturer Catalog #</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold Molds</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryosectioning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Membrane Slide 1.0 PEN (D)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>415190-9041-000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase Zap</td>
			<td>Ambion</td>
			<td>AM9780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC Treated Water</td>
			<td>Quality Biological</td>
			<td>351-068-131</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software: Robo v3.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No.5 Dumont Microdissction Forceps</td>
			<td>Roboz</td>
			<td>RS-49085</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous Micro Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cDNA Synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III First Strand Synthesis Kit</td>
			<td>Invitrogen&#160;</td>
			<td>18080-051</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OMP qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR GreenER qPCR SuperMix</td>
			<td>Invitrogen&#160;</td>
			<td>11760-500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Expression Assay Probes</td>
			<td>Applied Biosystems</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
		</tr>
	</tbody>
</table>

olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

Il disturbo bipolare (BD) è un grave disturbo neuropsichiatrico con fisiopatologia poco compreso e in genere trattate con lo stabilizzatore dell&#39;umore, carbonato di litio. Gli studi sugli animali e studi genetici umani indicano che il litio colpisce bersagli molecolari che sono coinvolti nella crescita neuronale, la sopravvivenza e la maturazione, e in particolare le molecole coinvolte nella segnalazione Wnt. Data la sfida etica di ottenere biopsie cerebrali per studiare variazioni dinamiche molecolari associati con litio-risposta nel sistema nervoso centrale (CNS), si può considerare l&#39;uso di neuroni ottenuti da tessuti olfattive per raggiungere questo goal.The epitelio olfattivo contiene neuroni recettrici a diversi stadi di sviluppo e gliali come cellule di supporto. Questo offre un&#39;opportunità unica per studiare i cambiamenti dinamici nel sistema nervoso centrale dei pazienti con malattie neuropsichiatriche, utilizzando il tessuto olfattivo ottenuto in modo sicuro da biopsie nasali. Per superare l&#39;inconveniente posta dal substla contaminazione antial di tessuto olfattiva biopsia con cellule non neuronali, un nuovo approccio per ottenere popolazioni di cellule neuronali arricchito è stato sviluppato combinando biopsie nasali con laser-capture microdissezione. In questo studio, un sistema per studiare variazioni dinamiche molecolari associati al trattamento nel tessuto neuronale è stato sviluppato e convalidato, con un piccolo campione pilota di pazienti BD reclutati per lo studio dei meccanismi molecolari di risposta al trattamento litio.

La strategia di protocollo e la risoluzione dei problemi nello studio delle firme molecolari sono state ottimizzate con successo. Qualità dell&#39;RNA e criteri di arricchimento neuronali per campioni da utilizzare in una fase successiva analisi reale Time-PCR sono stati standardizzati. RIN e concentrazione di RNA sono stati esaminati i fattori di confondimento a livelli di espressione rilevati. Sulla base dell&#39;analisi di diversi campioni con RIN da 1 - 10, è stato determinato che un RIN minimo di ~ 3,0 e sopra è...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

In questo studio, una nuova piattaforma per indagare le firme molecolari intraneuronali di risposta al trattamento nel disturbo bipolare (BD) è stato sviluppato e validato. Epitelio olfattivo da pazienti BD è stato ottenuto attraverso biopsie nasali. Poi laser-capture microdissezione è stato combinato con Tempo reale RT PCR per indagare la firma molecolare della risposta litio in BD.

I neuroni olfattivi ottenuti attraverso nasale biopsia combinato con laser-Capture Microdissection: un approccio potenziale per studiare risposta al trattamento in Disturbi Mentali

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

Research

JoVE Journal

Neuroscience

9.7K Views.  Johns Hopkins University.  In questo studio, una nuova piattaforma per indagare le firme molecolari intraneuronali di risposta al trattamento nel disturbo bipolare (BD) è stato sviluppato e validato. Epitelio olfattivo da pazienti BD è stato ottenuto attraverso biopsie nasali. Poi laser-capture microdissezione è stato combinato con Tempo reale RT PCR per indagare la firma molecolare della risposta litio in BD. 

Video: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Microdissezione Cattura di Drosophila Neuroni periferici

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Ricerca

Neuroscienze

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isolare cellule staminali olfattivo nasale da roditori o Umani

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

Targeting neuroni bulbo olfattivo utilizzo combinato In Vivo Elettroporazione e GAL4-Based Enhancer Trappola Linee Zebrafish

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non Laser Capture Microscopy approccio per la Microdissezione delle Regioni discreti cervello di topo per la totale isolamento di RNA e valle Next-Generation Sequencing e profili di espressione genica

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Microdissezione Cattura di popolazioni Arricchito di neuroni singoli neuroni o per analisi di espressione genica dopo trauma cranico

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Microdissezione laser di neuroni da cellule differenziate Neuroprogenitor umani in coltura

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Produzione di RNA per l'analisi trascritomica dai corpi cellulari dei motoneuroni del midollo spinale del topo mediante microdisezione di cattura laser

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Capture Microdissezione - Una dimostrazione dell&#39;isolamento individuale dopamina neuroni e l&#39;intera area ventrale tegmentale

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Atlas-guida stereotassica del Laser Capture Microdissection di regioni del cervello colpite da lesione traumatica

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.