Name: combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus
Uploaded: 2020-09-09T12:30:00
Description: Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

Dette arbeidet er finansiert av CBS Bridging Grant tildelt OOM. OOM, PAA og AS designet studien og utførte eksperimentene. AS og PAA analyserte resultatene. OOM og PAA utarbeidet manuskriptet. Vi takker Dr. Karl Disseroth (Stanford University) for å gjøre AAV tilgjengelig for vår bruk.

Alle eksperimentelle og dyrehåndteringsprosedyrer ble godkjent av Institutional Animal Care and Use Committee (IACUC) ved Louisiana State University School of Veterinary Medicine.
1. Eksperimentelt dyr
<ol><li>Bruk 5-6 uker gamle mus.</li>
	<li>Hus 3-5 dyr per bur under standardforhold på 12 timer vekslende lys og mørk syklus. Mat og vann bør gis ad libitum.</li>
</ol>2. Kraniotomi og dyreforberedelse
MERK: Denne delen beskriver pre- og p...

<ol>
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R., Aston-Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+context+with+reward:+a+functional+circuit+from+hippocampal+CA3+to+ventral+tegmental+area.">Linking context with reward: a functional circuit from hippocampal CA3 to ventral tegmental area.</a> Science. 333 (6040), 353-357 (2011).</li><li>McNamara, C. G., Dupret, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sources+of+dopamine+for+the+hippocampus.">Two sources of dopamine for the hippocampus.</a> Trends in Neurosciences. 40 (7), 383-384 (2017).</li><li>McNamara, C. 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I løpet av det siste tiåret har utformingen av AAV-konstruksjoner utviklet seg betydelig. Som sådan har mer nevronspesifikke promotorer blitt innlemmet i en rekke AAV-serotyper for forbedret transfeksjonsspesifikkitet14. Ved å kombinere gener for fluorescensproteiner, transportører, reseptorer og ionkanaler, eksisterer det nå biblioteker av AAV for avbildning, nevromodulering og synaptisk aktivitetsdeteksjon. I kommersielt tilgjengelige AAV-konstruksjoner gir en kombinasjon av en genetisk ko...

I det siste tiåret ble en rekke genetiske verktøy utviklet for å øke spesifisiteten til nevron-type modulasjon, og kartlegging av komplekse nevrale nettverk1. Spesielt har nevrotrope virus med en iboende evne til å infisere og replikere i nevronceller blitt utplassert for å uttrykke eller fyre av spesifikke proteiner i nevronundertyper. Når du har fluorescensproteiner eller genetisk kodede synaptiske aktivitetsindikatorer, transfected AAV vektorer etikett og avgrense nevrale nettverk på tvers avhjerneregioner 2,3. Valget av en promotor i AAV-konstruksjonen styrer uttrykket av vektoren i nevrontyper med et visst nivå av spesifisitet(promotoravhengig uttrykk). Gjennom Cre-lox-rekombinasjon distribueres imidlertid AAV-konstruksjoner med større spesifisitet for neuronmerking4,5,6,7. Vær oppmerksom på at fotoaktiverte mikrobielle opsiner og fluorescensproteiner pakket i AAV-vektorer kan uttrykkes i forskjellige neuron-undertyper8, og er ideelle for avbildning, sporing av nevronkrets og fotomodulering9,10.
AAVer konstruerer stereotaktisk injisert i en hjerneregion (eller kjerne) driver uttrykket av reporterproteinet i soma-, dendrite- og axonsterminalene. Nevralt uttrykk for AAV som skjuler et reportergen (eYFP) letter merkingen av nevroncellelegemer og anatomisk sporing av projeksjoner til og fra andre hjerneregioner11,12,13,14. AAV-eYFP-konstruksjoner som bærer lysstyrt opsin (f.eks. hChR2), kan distribueres som et verktøy for avbildning6,15 og stimuleringsbasert fysiologisk sporing av nevrale projeksjoner for å målrette hjerneområder in vivo16. Avhengig av AAV-serotypen kan retningen av neuronmerking være anterograd eller retrograd11,12. Tidligere studier har fastslått at AAV5 reiser anterogradely i nevroner12. Dermed gir fototimulering av cellelegemer som uttrykker hChR2 presynaptiske effekter andre steder i hjernen (målet)17.
Her ble AAV (serotype 5) med en CaMKIIα-promotor brukt til å uttrykke eYFP (reporter) og hChR2 (opsin) i VTA glutamat nevroner og axonale projeksjoner. Resultater fra denne studien viser den lagavhengige distribusjonen av VTA-glutamat presynaptiske terminaler i CA1-, CA3- og DG-hippocampal-regionene. Også fototimulering av VTA glutamat nevroner økte CA1 multi-enhet og single-unit avfyringshastigheter in vivo sammenlignet med baseline verdier. Denne protokollen benytter rimelige verktøy og kommersielt tilgjengelig programvare som kan øke kvaliteten på data hentet fra nevrale kretssporingseksperimenter.

<table>
	<tbody>
		<tr>
			<td>3% Hydrogen peroxide</td>
			<td>Fisher chemical</td>
			<td>H312</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV-CaMKII&#945;-ChR2-eGYP</td>
			<td>Addgene</td>
			<td>Plasmid #26969</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC cable</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BNC Splitter</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Split Mating Sleeve for &#216;1.25mm Ferrules.</td>
			<td>Thorlabs</td>
			<td>ADAL1-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>LR 39098</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin coated slides</td>
			<td>Fisher scientific</td>
			<td>OBSLD01CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton&#39;s syringe (Neuros)</td>
			<td>WPI Inc.</td>
			<td>06H</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage adapter</td>
			<td>Neuronexus</td>
			<td>Adpt-Q4-OM32</td>
			<td></td>
		</tr>
		<tr>
			<td>High impedance silicon probe</td>
			<td>Neuronexus</td>
			<td>Q1x1-tet-5mm-121-CQ4</td>
			<td></td>
		</tr>
		<tr>
			<td>INTAN 512ch Recording Controller</td>
			<td>INTAN</td>
			<td>RHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution</td>
			<td>Dynarex</td>
			<td>1425</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Spectrum</td>
			<td>K1068</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light source (470 nm)-blue light</td>
			<td>Thorlabs</td>
			<td>M470F3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M0-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber</td>
			<td>Thorlabs</td>
			<td>CFMLC14L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Pan head philips screw (M0.6 X 2mm)</td>
			<td>Amazon</td>
			<td>M0.6 X 2mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-amplifier headstage (32 Channel)</td>
			<td>INTAN</td>
			<td>C3314</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Kopf</td>
			<td>1530</td>
			<td></td>
		</tr>
		<tr>
			<td>TTL pulser</td>
			<td>Prizmatix</td>
			<td>4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alfa Aesar</td>
			<td>J61430</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intan Recording Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroexplorer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plexon Offline Spike Sorter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACSF Composition:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>oxygenated ACSF with 95% Oxygen/5%CO2 constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2 and 25 Glucose).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus

Optogenetisk modulering av nevron subpopulasjoner i hjernen har tillatt forskere å dissekere nevrale kretser in vivo og ex vivo. Dette gir et premiss for å bestemme rollen til nevrontyper i en nevral krets, og deres betydning i informasjonskoding i forhold til læring. På samme måte kan metoden brukes til å teste den fysiologiske betydningen av to eller flere tilkoblede hjerneregioner hos våken og bedøvet dyr. Den nåværende studien demonstrerer hvordan VTA glutamat nevroner modulerer avfyringshastigheten til putative pyramidale nevroner i CA1 (hippocampus) av bedøvede mus. Denne protokollen bruker adeno-assosiert virus (AAV)-avhengig merking av VTA glutamat nevroner for sporing av VTA presynaptiske glutamat terminaler i lagene i hippocampus. Uttrykk for lyskontrollert opsin (channelrhodopsin; hChR2) og fluorescensprotein (eYFP) som ligger i AAV-vektoren tillot anterogradsporing av VTA-glutamatterminaler, og fototimulering av VTA glutamat neuroncellelegemer (i VTA). Akutte silisiumelektroder med høy impedans ble plassert i CA1 for å oppdage multienhets- og enkeltenhetsresponser på VTA-fototimulering in vivo. Resultatene av denne studien viser den lagavhengige fordelingen av presynaptiske VTA-glutamatterminaler i hippocampus (CA1, CA3 og DG). Også fototimuleringen av VTA glutamat nevroner økte avfyrings- og bristehastigheten til putative CA1 pyramidale enheter in vivo.

Anterograde sporing
AAV-uttrykk ble verifisert ved immunfluorescensavbildning av reporterprotein (eYFP) i VTA av C57BL/6 mus 21 dager etter injeksjon (Figur 2). Vellykket anterogradmerking av presynaptiske VTA-glutamatprojeksjoner i hippocampus ble også verifisert ved eYFP-deteksjon i lagene i DG, CA3 og CA1 (Figur 6a–d; Film 2 og 3).
VTA glutamat projeksjoner ti...

Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Den nåværende protokollen demonstrerer en enkel metode for sporing av ventral tegmental område (VTA) glutamat projeksjoner til hippocampus. Fototimulering av VTA glutamat nevroner ble kombinert med CA1-opptak for å demonstrere hvordan VTA glutamatterminaler modulerer CA1 putativ pyramidal avfyringshastighet in vivo.

Kombinert in Vivo anatomisk og funksjonell sporing av ventrale tegmental område glutamatterminaler i Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

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