Ringraziamo N. Sawtell (Centro Medico dell&#39;Ospedale Pediatrico di Cincinnati, Cincinnati, Ohio, USA), S. Efstathiou (Università di Cambridge, UK) e James Hill (LSU Health Sciences Center, New Orleans, USA) per la fornitura di campioni provenienti da HSV-1 topi e conigli infettati, rispettivamente, e per i reagenti; H. Masumoto (Kazusa DNA Research Institute, Chiba, Giappone) e S. Khochbin (Institut Albert Bonniot, Grenoble, Francia) per le discussioni utili. Questo lavoro è stato finanziato da sovvenzioni dal Centre National de la Recherche Scientifique (CNRS) (programma ATIP, a PL, http://www.cnrs.fr), l&#39;Agenzia Nazionale Francese di Ricerca (ANR) (ANR-05-MIIM- 008-01, CENTROLAT, <a href="http://www.agencenationale-recherche.fr">http://www.agencenationale-recherche.fr</a> ), la Fondazione FINOVI ( <a href="http://www.finovi.org/:fr:start">http://www.finovi.org/:fr:start</a> ), il LABEX DEVweCAN (ANR-10-labx-61 ), della Université de Lyon, nell&#39;ambito del programma &quot;Investissements d&#39;Avenir &quot;(ANR-11-IDEX-0007) gestito dalla ANR ( <a href="http://www.agence-nationale-recherche.fr">http://www.agence-nationale-recherche.fr</a> ), l&#39;Association pour la Recherche contre le Cancer (ARC-7979 e ARC- 4910, <a href="http://www.arc-cancer.net">http://www.arc-cancer.net</a> ), la Ligue Nationale Contre le Cancer (LNCC, <a href="http://www.ligue-cancer.net">http://www.ligue-cancer.net</a> ) e Inca (programma EPIPRO, <a href="http://www.e-cancer.fr">http://www.e -cancer.fr</a> ). FC e PL sono ricercatori CNRS.

Gli autori dichiarano interessi finanziari concorrenti.

Il protocollo qui descritto permette il rilevamento di HSV-1 genoma latente all&#39;interno dei neuroni di sezioni di tessuto neuronali di topo. La nostra comprensione dei percorsi che regolano l&#39;espressione genica virale è stata limitata dalla mancanza di metodo per rilevare HSV-1 DNA genomico in situ all&#39;interno dei tessuti neuronali. Informazioni sul numero di copie del genoma e la proporzione dei neuroni infetti è venuto soprattutto da PCR sui neuroni dissociati 11,12. Nel chiarire il r...

<html> Tipo virus herpes simplex 1 (HSV-1) è un virus umano neurotropo persistente, stabilendo una infezione latente a lungo termine nei neuroni dei gangli del trigemino (TG) del sistema nervoso periferico, da cui si riattiva periodicamente per replicare e diffondersi. La HSV-1 è un genoma 150 kb dsDNA localizzazione nel nucleo del neurone ospitante dove rimane plasmidi chromatinized come multicopia, che non si integrano nel genoma della cellula ospite 1,2. Durante la latenza, il programma genetico HSV-1 ciclo replicativo è fortemente represso, e l&#39;espressione genica è limitata alla trascrizione latenza associata (LAT) locus, da stabilimento latenza apertura di riattivazione 3. LAT produce un lungo 8.5 kb non codificante RNA trasformato in un importante 2 kb lariat stabile, e molti miRNA 4-7. HSV-1 latenza è quindi caratterizzato dalla presenza del DNA genomico virale, LAT RNA, e l&#39;assenza di proteine ​​del ciclo replicativi rilevabili. ONTENUTO &quot;&gt; I modelli animali, prevalentemente topo e coniglio, sono modelli sperimentali riassumono diverse caratteristiche di latenza umana. Uno dei principali interessi di tali modelli è che essi consentono studiare aspetti fisiologici di HSV-1 latenza in ospiti immunocompetenti. Negli ultimi decenni , molti strumenti sperimentali, quali virus e topi geneticamente modificati, sono stati sviluppati per studiare la fisiologia, genetica e biologia cellulare di HSV-1 latenza, da tessuti animali. Finora, DNA genomico virale è stata rilevata e quantificata mediante Southern blot e qPCR da dissociato TG. Tuttavia, attualmente non esiste un metodo per rilevare il HSV-1 genoma mediante ibridazione in situ su sezioni di tessuto 8. conseguenza, la latenza viene normalmente valutata su sezioni istologiche attraverso la rilevazione di RNA LAT da RNA ibridazione in situ anziché rilevazione del genoma virale. Poiché non è stato possibile caratterizzare cellule infette in base alla presenza di genomi virali, this limitazione tecnica è stato un grave inconveniente per l&#39;analisi di molti aspetti delle interazioni ospite-virus, come il rapporto tra il genoma virale e l&#39;espressione genica cellulare e virale o cellulo-mediata risposta immunitaria dell&#39;ospite 9,10. Soprattutto, l&#39;eterogeneità cellula-cellula dell&#39;infezione latente rimane relativamente inesplorato e ha dimostrato di essere un elemento chiave della latenza nei topi e nei neuroni dei gangli sensoriali umani impiantati in topi SCID 11-17. Tipicamente, è stato dimostrato da qPCR che il HSV-1 genoma numero di copie per cellula varia da 5 a diverse centinaia. Anche se LAT appare come un regolatore chiave della latenza e la riattivazione, i dati qPCR sui neuroni isolati e in situ PCR indicato che solo un sottoinsieme di neuroni latentemente infette, a partire da 30%, esprime il locus LAT 11,12,18-21. Come la cellula ospite e l&#39;ambiente cellulare all&#39;interno del tessuto impatto sulla costituzione di latenza virus el&#39;espressione genica virale rimane poco chiaro. Qui si descrive un robusto ibridazione in situ fluorescente (FISH) metodo per la rilevazione efficiente di bassa copia HSV-1 DNA genomico entro sezioni di tessuto neuronali animali. Questo metodo è stato progettato e da noi utilizzato per ottenere l&#39;accesso a microscopia ad alta risoluzione di immagini che è necessario studiare l&#39;interazione del genoma virale con la cellula ospite componenti intra-nucleari 22. Inoltre, si descrive un metodo di colorazione multipla per la rilevazione simultanea del DNA virale con RNA e proteine, che è uno strumento unico per descrivere le interazioni virus-ospite che regolano l&#39;espressione genica virale. Il metodo può essere applicato anche per una vasta gamma di analisi che richiedono la rilevazione di HSV-1 genoma latente, come quantificazione neuroni infetti in gran numero di sezioni. Un passo fondamentale è quello di applicare antigene trattamento di recupero per rendere il DNA virale accessibile a ibridazione. Così, questo protocollo potrebbe anche essere efficace per il rilevamentodi altri virus dsDNA, che attualmente non rilevabili da approcci DNA-FISH convenzionali all&#39;interno di tessuti animali.

<table border="0" cellpadding="0" cellspacing="0" width="822">
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	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:203px;">Balb/c mice</td>
			<td style="width:189px;">Janvier, France</td>
			<td style="width:93px;"> </td>
			<td style="width:337px;">6 week-old females</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">HSV-1 strains</td>
			<td>SC16 strain (wild type)</td>
			<td></td>
			<td style="width:337px;">See Labetoule, M. et al. (2003) Invest Ophthalmol Vis Sci 44: 217&#8211;225, for details on HSV-1 strain and virus stock preparation.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine hydrochloride</td>
			<td>Sigma</td>
			<td>K2753</td>
			<td rowspan="2" style="width:337px;">Intraperitoneal injection of a solution containing Ketamine (100mg/kg) and Xylazine (10mg/kg)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine hydrochloride</td>
			<td>Sigma</td>
			<td>X1251</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>158127</td>
			<td style="width:337px;">Suspend 4g of PFA in 90mL of water. Add 50&#181;L of 1N NaOH, and heat at 60&#176;C in a water bath with agitation. PFA dissolves in about 30min. Add 10mL of 10X PBS. This solution can be prepared in advance and stored at -20 &#176;C in 5mL tubes. Caution. Manipulate under a fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Physiological Saline</td>
			<td>Sigma</td>
			<td colspan="2">07982-100TAB-F</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS, pH 7.4 (sterile)</td>
			<td>Life Technologies</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>Sigma</td>
			<td>84100</td>
			<td>Prepare a 20% sucrose solution in 1X PBS.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound -</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Large vector DNA purification kit</td>
			<td>Qiagen</td>
			<td>12462</td>
			<td style="width:337px;">To purify Cosmid or BAC vector containing HSV-1 genome and store at -20&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nick translation kit</td>
			<td>Roche Applied Sciences</td>
			<td>10 976 776 001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA53021</td>
			<td>Protect from light</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.5M EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">G50 Mini spin column</td>
			<td>GE Healthcare</td>
			<td>27-5330-01 </td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA 10mg/mL</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol molecular biology grade</td>
			<td>Sigma</td>
			<td>87047</td>
			<td>Prepare a 70% solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamid Molecular biology grade</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td>Caution. Manipulate under fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HSV-1 biotinylated commercial probe </td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-40838</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ImmEdge hydrophobic pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20X Saline Sodium Citrate (SSC)</td>
			<td>Sigma</td>
			<td>S6639</td>
			<td>Prepare a 2X SSC solution in ddH20.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td style="width:337px;">Prepare a 10% stock solution in water and store at +4&#176;C. Prepare the 0.5% solution in 1X PBS right before use.</td>
		</tr>
		<tr height="102">
			<td height="102" style="height:102px;">10mM sodium citrate pH 6.0</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td style="width:337px;">Prepare a 100mM stock solution (10X). Weigh 10,5g of citric acid (MW 210.14. Caution, irritant and toxic, wear appropriate mask and gloves), and dissolve in 400mL water. Adjust pH at 6.0 with 1N NaOH (caution, irritant, wear gloves). Adjust to 500mL with distilled water. Dilute 10 times in distilled water before use.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic Acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Methanol, molecular biology grade</td>
			<td>Sigma</td>
			<td>322415</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dextran sulfate - MW 500 000</td>
			<td>Euromedex</td>
			<td>EU0606-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Denhardt&#39;s solution (100X)</td>
			<td>Euromedex</td>
			<td>1020-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rubber Cement &#34;FixoGum&#34;</td>
			<td>Marabut</td>
			<td>290110000</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">DNA purification kit - Qiaquick PCR purification kit -</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 in vitro transcription kit</td>
			<td>Ambion / Life Technologies</td>
			<td>AM1314</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biotin-16-UTP</td>
			<td>Roche Applied Sciences</td>
			<td>11388908910</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNA purification mini-column</td>
			<td>Qiagen</td>
			<td>73404</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ribonucleoside Vanadyl Complex</td>
			<td>New England Biolabs</td>
			<td>S1402S</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">H2O2</td>
			<td>Sigma</td>
			<td>H3410</td>
			<td style="width:337px;">Prepare a 3% solution in distilled water. Store at +4 &#176;C and protect from light.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401011</td>
			<td style="width:337px;">prepare a 10mg/mL solution in RNAse free water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Normal Goat Serum</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Primary antibodies</td>
			<td>Any supplier</td>
			<td></td>
			<td style="width:337px;">The following primary antibodies were used in the result section: anti-mouse CENP-A (rabbit mAb C51A7, Cell Signaling Technologies), and anti-ATRX H-300 (Santa Cruz Biotechnology)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Secondary fluorescent antibodies</td>
			<td>Invitrogen / Life Technologies</td>
			<td></td>
			<td style="width:337px;">The fluorescent secondary antibodies routinely used in our protocol are AlexaFluor labeled goat antibodies (IgG H+L). The antibody used in the result section is an anti-rabbit goat antibody labaled with AlexaFluor 488 (reference A11001)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 350 (blue fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20937</td>
			<td rowspan="2" style="width:337px;">TSA kits are also available from Perkin Elmer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 488 (green fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20932</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hoechst 33342</td>
			<td>Invitrogen / Life Technologies</td>
			<td>H3570</td>
			<td style="width:337px;">Prepare a 0.5&#181;g/mL solution in 1X PBS immediatly before use. Discard the remaining solution.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22x50mm coverslip. n&#176;1.5 glass.</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-04</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;"> Mounting medium with anti-fading agent - Vectashield -</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:337px;">Another conventional product is Fluoromount G from electron microscopy Science</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfrost glass slides</td>
			<td>FisherScientific</td>
			<td>12-550-15</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Equipment / material</td>
			<td>Company</td>
			<td>Reference</td>
			<td>Note</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle for infection</td>
			<td></td>
			<td></td>
			<td>Glass micropipette hot drawn. Home made.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dissection equipement</td>
			<td>Moria, France</td>
			<td></td>
			<td>Microsurgical scissors and forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Peristaltic pump</td>
			<td style="width:189px;">Cole Palmer Instruments</td>
			<td></td>
			<td>Easyload Masterflex</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-syringe pump device (Nano Pump)</td>
			<td>kdScientific</td>
			<td>KDS310</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryostat</td>
			<td>Leica France</td>
			<td>CM 1510-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">-80 &#176;C freezer</td>
			<td>Sanyo</td>
			<td></td>
			<td>Ultra Low -80&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Domestic microwave oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry block heater</td>
			<td>Eppendorf</td>
			<td>022670204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Incubator Slide moat</td>
			<td>Boekel Scientific</td>
			<td>240000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin Jar</td>
			<td>Dominique Dutscher</td>
			<td>68512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining glass container</td>
			<td>Dominique Dutscher</td>
			<td>68506</td>
			<td></td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:337px;">The images presented in the result section were collected with a Zeiss AxioObserver with objective x40 LD NeoFluor N.A 0.6, and x100 PlanApochromat N.A 1.3. Filter set #38, #43 and #43. HXP 120 fluorescence light source. Photometrics CoolSNAP HQ2 CCD camera. Signal will be more easily observed on a recent high efficiency microscope such as Zeiss AxioImager/AxioObserver series, Nikon Ti-E/Ni-E series or Leica DM/DMI6000 series</td>
		</tr>
	</tbody>
</table>

detection of the genome and transcripts of a persistent dna virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining

Codetection singola cella di un gene, il suo prodotto RNA e proteine ​​regolatrici cellulari è fondamentale per studiare regolazione dell&#39;espressione genica. Questa è una sfida nel campo della virologia, in particolare per persistenti virus a DNA nucleare replicanti che coinvolgere modelli animali per il loro studio. Tipo virus herpes simplex 1 (HSV-1) stabilisce un&#39;infezione latente permanente nei neuroni periferici. Latent virus funge da serbatoio, da cui riattiva e induce un nuovo episodio erpetica. La biologia cellulare di HSV-1 latenza rimane poco compresa, in parte a causa della mancanza di metodi per rilevare HSV-1 genomi in situ in modelli animali. Descriviamo un DNA-ibridazione in situ fluorescente (FISH) approccio efficiente rilevazione low-copia genomi virali all&#39;interno di sezioni di tessuti neuronali provenienti da modelli animali infetti. Il metodo si basa sul-based calore antigene smascheramento, e direttamente etichettati sonde di DNA fatti in casa, o sonde disponibili in commercio. Abbiamo sviluppato un Stai triplaapproccio ning, combinando DNA-FISH con RNA-FISH e immunofluorescenza, utilizzando perossidasi basato amplificazione del segnale per soddisfare ogni esigenza di colorazione. Un miglioramento importante è la possibilità di ottenere, entro 10 micron sezioni di tessuto, segnali a bassa priorità bassa che possono essere esposte ad alta risoluzione mediante microscopia confocale e grande campo epifluorescenza convenzionale. Inoltre, la colorazione tripla lavorato con una vasta gamma di anticorpi diretti contro proteine ​​cellulari e virali. Il protocollo completo dura 2,5 giorni per accogliere anticorpo e la penetrazione della sonda all&#39;interno del tessuto.

Dopo diversi mesi di test approfonditi, abbiamo scoperto che a base di calore chimico smascheramento fatto latente HSV-1 genoma disponibili per ibridazione in situ fluorescente. Durante il processo, abbiamo provato varie procedure smascheramento, e trattamenti a base di solo calore (cioè il riscaldamento delle sezioni fino a temperatura sub-di ebollizione in un forno a microonde) apparso efficiente. Abbiamo poi testato diversi buffer di sale che vengono abitualmente utilizzati in immunoistochimica (IH...

Watch this Scientific Journal Video about Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining at JoV

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Abbiamo stabilito un protocollo di ibridazione fluorescente in situ per la rilevazione di un genoma del virus DNA persistente all&#39;interno di sezioni tissutali di modelli animali. Questo protocollo permette studiando processo di infezione da codetection del genoma virale, i suoi prodotti di RNA e proteine ​​virali o cellulari all&#39;interno delle cellule singole.

La rilevazione del genoma e Trascrizioni di un virus DNA persistente in tessuti neuronali da fluorescente In situ Ibridazione In combinazione con Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

detection-genome-transcripts-persistent-dna-virus-neuronal-tissues

Research

JoVE Journal

Neuroscience

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

18.2K Views.  CNRS UMR 5534.  Abbiamo stabilito un protocollo di ibridazione fluorescente in situ per la rilevazione di un genoma del virus DNA persistente all'interno di sezioni tissutali di modelli animali. Questo protocollo permette studiando processo di infezione da codetection del genoma virale, i suoi prodotti di RNA e proteine ​​virali o cellulari all'interno delle cellule singole. 

Video: Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Doppia fluorescenza In situ</emIbridazione> nelle sezioni cervello fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Ricerca

Neuroscienze

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Intracerebroventricular Iniezione virale del cervello neonatale mouse per persistente e diffusa trasduzione neuronale

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Rilevazione di assoni localizzato mRNA nelle sezioni del cervello Utilizzando ad alta risoluzione<em&gt; In Situ</em&gt; Ibridazione

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizzando metodologie combinati per definire il ruolo della membrana plasmatica di consegna Durante Axon ramificazione e neuronale morfogenesi

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracellulare La registrazione di un neurone attività combinata con Microiontophoretic applicazione di sostanze neuroattivi nei topi Awake

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

La combinazione di doppio Fluorescenza<em&gt; In Situ</em&gt; Ibridazione con Immunolabelling per il rilevamento della Manifestazione di tre geni nel cervello di topo Sezioni

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localizzazione dei geni del recettore degli odoranti nell&#39;antenna di Locust da RNA<em&gt; In Situ</em&gt; Ibridazione

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificazione della dopamina D1-alfa del ricevitore all'interno del roditore Nucleus Accumbens da un innovativo RNA In Situ Detection Technology