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 arbejde er finansieret af CBS Bridging Grant tildelt OOM. OOM, PAA og AS designede undersøgelsen og udførte eksperimenterne. AS og PAA analyserede resultaterne. OOM og PAA forberedte manuskriptet. Vi takker Dr. Karl Disseroth (Stanford University) for at gøre AAV til rådighed for vores brug.

Alle forsøgs- og dyrehåndteringsprocedurer blev godkendt af Institutional Animal Care and Use Committee (IACUC) fra Louisiana State University School of Veterinary Medicine.
1. Forsøgsdyr
<ol><li>Brug 5-6 uger gamle mus.</li>
	<li>Hus 3-5 dyr pr bur under standardbetingelser på 12 timer skiftevis lys og mørk cyklus. Mad og vand skal leveres ad libitum.</li>
</ol>2. Kraniotomi og tilberedning af dyr
BEMÆRK: I dette afsnit beskrives præ-...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72 (6), 938-950 (2011).</li><li>Li, J., Liu, T., Dong, Y., Kondoh, K., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+Neural+Circuit-Tracing+with+Neurotropic+Viruses.">Trans-synaptic Neural Circuit-Tracing with Neurotropic Viruses.</a> Neuroscience bulletin. , 1-12 (2019).</li><li>Kuypers, H., Ugolini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+as+transneuronal+tracers.">Viruses as transneuronal tracers.</a> Trends in neurosciences. 13 (2), 71-75 (1990).</li><li>Atasoy, D., Aponte, Y., Su, H. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+vector:+use+for+transgene+expression+and+anterograde+tract+tracing+in+the+CNS.">Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS.</a> Brain Research. 793 (1-2), 169-175 (1998).</li><li>Zingg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+anterograde+transsynaptic+tagging:+mapping+corticocollicular+input-defined+neural+pathways+for+defense+behaviors.">AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors.</a> Neuron. 93 (1), 33-47 (2017).</li><li>Wang, C., Wang, C., Clark, K., Sferra, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+AAV+serotype+1+transduction+efficiency+and+tropism+in+the+murine+brain.">Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain.</a> Gene therapy. 10 (17), 1528 (2003).</li><li>Ahlgrim, N. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+action+on+hippocampal+pyramidal+cells.">Dopamine action on hippocampal pyramidal cells.</a> Journal of Neuroscience. 2 (4), 415-423 (1982).</li><li>Davidow, J. Y., Foerde, K., Galvan, A., Shohamy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Upside+to+Reward+Sensitivity:+The+Hippocampus+Supports+Enhanced+Reinforcement+Learning+in+Adolescence.">An Upside to Reward Sensitivity: The Hippocampus Supports Enhanced Reinforcement Learning in Adolescence.</a> Neuron. 92 (1), 93-99 (2016).</li><li>Hu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reward+and+Aversion.">Reward and Aversion.</a> Annual Review of Neuroscience. 39, 297-324 (2016).</li><li>Kahn, I., Shohamy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+connectivity+between+the+hippocampus,+nucleus+accumbens,+and+ventral+tegmental+area+in+humans.">Intrinsic connectivity between the hippocampus, nucleus accumbens, and ventral tegmental area in humans.</a> Hippocampus. 23 (3), 187-192 (2013).</li><li>Lisman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+hippocampal+circuitry+to+function:+recall+of+memory+sequences+by+reciprocal+dentate-CA3+interactions.">Relating hippocampal circuitry to function: recall of memory sequences by reciprocal dentate-CA3 interactions.</a> Neuron. 22 (2), 233-242 (1999).</li><li>Lisman, J. E., Grace, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hippocampal-VTA+loop:+controlling+the+entry+of+information+into+long-term+memory.">The hippocampal-VTA loop: controlling the entry of information into long-term memory.</a> Neuron. 46 (5), 703-713 (2005).</li><li>Broussard, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+Regulates+Aversive+Contextual+Learning+and+Associated+In+Vivo+Synaptic+Plasticity+in+the+Hippocampus.">Dopamine Regulates Aversive Contextual Learning and Associated In Vivo Synaptic Plasticity in the Hippocampus.</a> Cell Reports. 14 (8), 1930-1939 (2016).</li><li>Hansen, N., Manahan-Vaughan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+D1/D5+receptors+mediate+informational+saliency+that+promotes+persistent+hippocampal+long-term+plasticity.">Dopamine D1/D5 receptors mediate informational saliency that promotes persistent hippocampal long-term plasticity.</a> Cerebral Cortex. 24 (4), 845-858 (2014).</li><li>Salvetti, B., Morris, R. G., Wang, S. 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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. G., Tejero-Cantero, A., Trouche, S., Campo-Urriza, N., 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=Dopaminergic+neurons+promote+hippocampal+reactivation+and+spatial+memory+persistence.">Dopaminergic neurons promote hippocampal reactivation and spatial memory persistence.</a> Nature Neuroscience. 17 (12), 1658-1660 (2014).</li><li>Cardin, J. 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I det seneste årti er designet af AAV-konstruktioner avanceret betydeligt. Som sådan er mere neuron-specifikke initiativtagere blevet indarbejdet i en række AAV-serotyper for forbedret transfektspecifikitet14. Ved at kombinere gener for fluorescensproteiner, transportører, receptorer og ionkanaler findes der nu biblioteker af AAV til billeddannelse, neuromodulation og synaptisk aktivitetsdetektion. I kommercielt tilgængelige AAV-konstruktioner giver en kombination af en genetisk kodet fluorop...

I det sidste årti blev en række genetiske værktøjer udviklet for at øge specificiteten af neuron-type graduering og kortlægning af komplekse neurale netværk1. Især neurotropiske vira med en iboende evne til at inficere og replikere i neuronale celler er blevet indsat for at udtrykke eller ablate specifikke proteiner i neuron sub-typer. Når der huser fluorescens proteiner eller genetisk kodet synaptiske aktivitetsindikatorer, transfected AAV vektorer etiket og afgrænse neurale netværk på tværs af hjernen regioner2,3. Valget af en promotor i AAV-konstruktionen dirigerer vektorens udtryk i neurontyper med en vis specificitet(promotorafhængigt udtryk). Men gennem Cre-lox rekombination, AAV konstruktioner er indsat med større specificitet for neuron mærkning4,5,6,7. Bemærk, fotoaktiverede mikrobielle opsiner og fluorescensproteiner pakket i AAV-vektorer kan udtrykkes i forskellige neuronundertyper8og er ideelle til billeddannelse, neuron-type kredsløbssporing og fotomodulation9,10.
AAV'er konstruerer stereotaktisk injiceres i en hjerne region (eller kerne) drev udtryk for reporter protein i soma, dendrit, og axoner terminaler. Neurale udtryk for AAV huser en reporter gen (eYFP) letter mærkning af neuron celle organer og anatomiske sporing af fremskrivninger til og fra andre hjerne regioner11,12,13,14. AAV-eYFP konstruerer transporterer lys-kontrolleret opsin (f.eks hChR2), kan anvendes som et redskab til billeddannelse6,15 og stimulation-baserede fysiologiske sporing af neurale fremskrivninger til at målrette hjernen områder in vivo16. Afhængigt af AAV-serotypen kan retningen af neuronmærkning være anterograde eller retrograd11,12. Tidligere undersøgelser har fastslået, at AAV5 rejser anterogradely i neuroner12. Således photostimulation af celle organer udtrykke hChR2 producerer præsynaptiske virkninger andre steder i hjernen (mål)17.
Her blev AAV (serotype 5) med en CaMKIIα promotor brugt til at udtrykke eYFP (reporter) og hChR2 (opsin) i VTA glutamat neuroner og axonale fremskrivninger. Resultaterne fra denne undersøgelse viser den lagafhængige fordeling af præsynaptiske VTA-glutamatterminaler i CA1-, CA3- og GD hippocampal-regionerne. Også, photostimulation af VTA glutamat neuroner øget CA1 multi-enhed og enkelt-enhed fyring satser in vivo sammenlignet med baseline værdier. Denne protokol bruger overkommelige værktøjer og kommercielt tilgængelig software, der kan øge kvaliteten af data opnået fra neurale kredsløbssporingseksperimenter.

<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 graduering af neuron subpopulationer i hjernen har gjort det muligt for forskere at dissekere neurale kredsløb in vivo og ex vivo. Dette giver en forudsætning for at bestemme neurontypernes rolle i et neuralt kredsløb og deres betydning i informationskodning i forhold til læring. Ligeledes kan metoden bruges til at teste den fysiologiske betydning af to eller flere forbundne hjerneregioner i vågne og bedøvede dyr. Den nuværende undersøgelse viser, hvordan VTA glutamat neuroner modulere fyring sats formodede pyramide neuroner i CA1 (hippocampus) af bedøvede mus. Denne protokol anvender adeno-associeret virus (AAV) afhængig mærkning af VTA glutamat neuroner til sporing af VTA presynaptic glutamat terminaler i lagene af hippocampus. Ekspression af lysstyret opsin (channelrhodopsin; hChR2) og fluorescensprotein (eYFP), der næres af AAV-vektoren, tilladte anterograde-sporing af VTA glutamatterminaler og fototimulation af VTA glutamat neuroncellelegemer (i VTA). Høj impedans akutte silicium elektroder blev placeret i CA1 til at opdage multi-enhed og enkelt-enhed reaktioner på VTA photostimulation in vivo. Resultaterne af denne undersøgelse viser den lagafhængige fordeling af præsynaptiske VTA glutamatterminaler i hippocampus (CA1, CA3 og GD). Også, den photostimulation af VTA glutamat neuroner øget fyring og brast sats formodede CA1 pyramideenheder in vivo.

Anterograde-sporing
AAV-ekspressionen blev verificeret ved immunfluorescensbilleddannelse af reporterprotein (eYFP) i VTA for C57BL/6-mus 21 dage efter injektionen (figur 2). Vellykket anterograde mærkning af præsynaptiske VTA glutamat fremskrivninger i hippocampus blev også verificeret ved eYFP afsløring i lagene af GD, CA3, og CA1 (Figur 6a-d; Film 2 og 3).
VT...

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 nuværende protokol demonstrerer en enkel metode til sporing af ventral tegmental område (VTA) glutamat fremskrivninger til hippocampus. Photostimulation af VTA glutamat neuroner blev kombineret med CA1 optagelse for at demonstrere, hvordan VTA glutamat terminaler modulere CA1 formodede pyramide fyring sats in vivo.

Kombineret i Vivo Anatomisk og funktionel sporing af Ventral Tegmental Area Glutamat Terminaler 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 ...

Research

JoVE Journal

Neuroscience

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

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Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

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Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge 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 ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex vivo Tilberedninger af den intakte Vomeronasal orgel og tilbehør lugtekolben

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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

Laser Capture mikrodissektion - en demonstration af Isolering af Individual dopaminneuroner og hele Ventral tegmentalområde

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

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Betinget Genetisk transsynaptisk sporing i den embryoniske musehjerne

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Samtidige<em&gt; Ex vivo</em&gt; Functional Test af to nethinder af<em&gt; In vivo</em&gt; Elektroretinogrammet System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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Laserstyrede Neuronal Tracing i Brain Eksplantater

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

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Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...