Name: targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging
Uploaded: 2012-12-12T11:00:00
Description: Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

Dette arbejde blev støttet af tilskud fra National Eye Institute R01EY017925 og R21EY020985 og finansiering fra Dana &amp; Whitehall Foundations til PK Vi takker også Matthew Petrella for assistance med kirurgiske procedurer, Grace Dion til sporing dendriterne vist i figur 5A og Pratik Chhatbar for kommentarer til manuskriptet.

1. Kirurgisk forberedelse <ol><li> Inducere anæstesi og løbende overvåge puls, ende tidevandsenergi CO2, EEG og temperatur. Alle procedurer blev godkendt af Institutional Animal Care og brug Udvalg det medicinske universitet i South Carolina og var baseret på dem, vi tidligere har udgivet 9,15. </li><li> Blotlægge den dorsale overflade af kraniet ved at skære i huden med et skalpelblad. Dissekere bindevævet ligger over knoglen ved hjælp af en Brudon curette. Rengør knoglen ved hjælp af ...

<ol>
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Vi præsenterer en fremgangsmåde til at målrette mærkning af neuronale cellelegemer (eller dendritter og axoner) i forudbestemte funktionelle mikro-domæner i neocortex. Sammenfletning iboende signal optisk billeddannelse med to-foton mikroskopi giver mulighed for bestemmelse af, hvilke synapser og celler bidrager til mikro-domænet af et funktionelt kort, hvad enten neuronale selektivitet korrelerer med placeringen af ​​neuron i et funktionelt kort, og det neuronale kredsløbskomponenter denne ændring med visue...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> 1. Life support / eksperiment prep </td></tr><tr><td> Isofluran </td><td> Webster Vet </td><td> NDC 57319-474-05 </td><td></td></tr><tr><td> Isofluran vaporizer </td><td> Midmark </td><td> VIP 3000 </td><td></td></tr><tr><td> Feedback reguleret varmetæppe </td><td> Harvard Apparatus </td><td> 50-7079F </td><td></td></tr><tr><td> EKG-monitor </td><td> Digicare Biomedical </td><td> LifeWindow Lite </td><td></td></tr><tr><td> EEG-forstærkeren </td><td> AM Systems </td><td> 1800 </td><td></td></tr><tr><td> EEG display monitor </td&gt; <td> Hewlett Packard </td><td> 78304A </td><td></td></tr><tr><td> Afslut tidevandsenergi CO 2 monitor </td><td> Respironics </td><td> Novametrix Capnoguard 1265 </td><td> Optimer ventilation </td></tr><tr><td> Carbide bor grater til boring knogle </td><td> Henry Schein </td><td> bøde (0,5 mm spids) og grov (1,25 mm spids) </td><td></td></tr><tr><td> Cement til hjelm / kammer </td><td> Dentsply </td><td> 675.571, 675.572 </td><td></td></tr><tr><td> Black Powder Tempera Paint </td><td> Sargent Art Inc. </td><td> 22-7185 </td><td> Tilføj til cement til forbedring af lysafskærmningsorganer og reducere refleksioner </td></tr><tr><td> Agarose - type III-A </td><td> Sigma </td><td> A9793 </td><td> For at minimere pulsationer under iboende signal og to-foton imaging </td></tr><tr><td> Dækglas: 5 eller 8 mm i diameter, 0,17 mm tykkelse </td><td> Verden præcision iinstrumenter </td><td> 502.040, 502.041 </td><td> For at minimere pulsationer under billeddannelse, kan dækglas skæres efter behov </td></tr><tr><td> Brudon curettes </td><td> George Tiemann </td><td> 105-715-0, 105-715-3 </td><td> Rengøring kranium overflade </td></tr><tr><td> Knoglevoks </td><td> Ethicon </td><td> W31G </td><td> Hurtigt standse blødninger </td></tr><tr><td> Cotton spids applikator </td><td> Elektronmikroskopi Sciences </td><td> 72.308-05 </td><td> Ren og tør knogleoverflade </td></tr><tr><td> Dumont # 5co Tang </td><td> Fin Science Tools </td><td> 11.295-20 </td><td> Grab individuelle lag af dura eller pia </td></tr><tr><td> Vannas Spring Sakse </td><td> Fin Science Tools </td><td> 15.000-03 </td><td> Cut dura </td></tr><tr><td> Gelfoam </td><td> Pfizer </td><td> 09-0396-05 </td><td> For at stoppe blødningen på dura </td></tr><tr><td> Absorption spears</td><td> Fin Science Tools </td><td> 18.105-01 </td><td> Ultra-hurtig og fnugfri wicking af CSF </td></tr><tr><td> Blackout materiale </td><td> Thorlabs </td><td> BK5 </td><td> Shield kraniotomi </td></tr><tr><td></td><td></td><td></td><td> 2. Dye forberedelse / indsprøjtning </td></tr><tr><td> Dimethylsulfoxid (DMSO) </td><td> Sigma </td><td> D2650 </td><td></td></tr><tr><td> Pluronic </td><td> Sigma </td><td> P2443 </td><td></td></tr><tr><td> Oregon Green 488 BAPTA-1:00 </td><td> Invitrogen </td><td> O6807 </td><td> Calciumindikator </td></tr><tr><td> Alexa Fluor ® 594 </td><td> Invitrogen </td><td> A10438 </td><td></td></tr><tr><td> Centrifugal filter (0,45 um porestørrelse) </td><td> Millipore </td><td> UFC30HV00 </td><td> At fjerne urenheder før injektion </td></tr><tr><td> Glaspipette aftrækker </td><td> Sutter Instruments </td><td> P97 </td><td></td></tr><tr><td> Borosilikatglas filamenteret kapillær (1,5 mm ydre diameter) </td><td> Verden Precision Instruments </td><td> 1B150F-4 </td><td> Dye udslyngning pipette </td></tr><tr><td> Microloader </td><td> Eppendorf </td><td> 5242 956 003 </td><td> Til læsning farvestof i pipette </td></tr><tr><td> Mikromanipulator </td><td> Sutter Instruments </td><td> MP-285 </td><td> For at placere pipette </td></tr><tr><td> Trykimpuls controller </td><td> Parker Hannifin </td><td> PicoSpritzer III </td><td> Til trykindsprøjtning af farvestoffet </td></tr><tr><td> Encellede electroporator </td><td> Molecular Devices </td><td> Axoporator 800A </td><td> Til elektroporering af farvestoffet </td></tr><tr><td></td><td></td><td></td><td> 3. Iboende imaging </td></tr><tr><td> 4x Mål (0,13 NA, 17 mm WD) </td><td> Olympus </td><td> UPLFLN4X </td><td></td></tr><tr><td> Intrinsic hardware / software </td><td> Optisk Imaging Inc. </td><td> Imager 3001 / VDAQ </td><td> VDAQ software bruges til episodisk billeddannelse </td></tr><tr><td> CCD-kamera </td><td> Adimec </td><td> Adimec-1000 </td><td></td></tr><tr><td> Lyskilde strømforsyning </td><td> KEPCO </td><td> ATE 15-15M </td><td></td></tr><tr><td> Lyskilde </td><td> Optisk Imaging Inc. </td><td> HAL 100 </td><td> Lysintensiteten på den kortikale overflade er 3-5 mW </td></tr><tr><td> Grønt filter (for vaskulær image) </td><td> Optisk Imaging Inc. </td><td> λ = 546 nm (båndpas 30 nm) </td><td> For reference billede af overfladen vaskulatur </td></tr><tr><td> Rødt filter (for iboende signal) </td><td> Optisk fantasing Inc. </td><td> λ = 630 nm (båndpas 30 nm) </td><td> At indsamle iboende signaler </td></tr><tr><td> Heat filter </td><td> Optisk Imaging Inc. </td><td> KG-1 </td><td></td></tr><tr><td></td><td></td><td></td><td> 4. To-foton rig / imaging </td></tr><tr><td> To-foton mikroskop og software </td><td> Prærie Technologies </td><td></td><td> Se Shen et al. 2012 for lysbanen, filtre og lasereffekt </td></tr><tr><td> Ti: Sapphire laser </td><td> Spectra-Physics </td><td> Mai Tai XF </td><td></td></tr><tr><td> 20x (0,5 NA, 3,5 mm WD) </td><td> Olympus </td><td> UMPLFLN20X </td><td> 0,5 NA mål udelukkende anvendes til justering pipette over kraniotomi (ikke for to foton billeddannelse) </td></tr><tr><td> 20x (1,0 NA, 2,0 mm WD) </td><td> Olympus </td><td> XLUMPLFLN20X </td><td></td></tr><tr><td> 40x (0,8 NA, 3.3 mm WD) </td><td> Olympus </td><td> LUMPLFLN40X/IR </td><td></td></tr><tr><td> Luftbord </td><td> Newport </td><td> ST-200 </td><td> Isolerer præparat fra eksterne vibrationer </td></tr><tr><td> XY-scene </td><td> Mikes Machine Co (Attleboro, MA) </td><td></td><td> Eksperimentel emne og Sutter mikromanipulator placeret på XY-scene </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td colspan="2"> Opskrifter </td></tr><tr><td> Kunstig cerebrospinalvæske </td><td> NaCI (135 mM), KCl (5,4 mM), MgCl2 (1,0 mM), CaCl2 (1,8 mM), HEPES (5 mM), pH 7,4 </td></tr><tr><td> Pipette Solution 14 </td><td> NaCI (150 mM), KCl (2,5 mM), HEPES (10 mM), pH 7,4 </td></tr></tbody>
			</table>
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targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging

I den primære visuelle cortex af ikke-gnavere pattedyr, er neuroner klynger efter deres præference for stimulus funktioner såsom orientering 1-4, retning 5-7, okulær dominans 8,9 og kikkert ulighed 9. Orientering selektivitet er den mest undersøgte funktion og en kontinuerlig kort med en kvasi-periodisk layout for foretrukken orientering er til stede over hele den primære visuelle cortex 10,11. Integration de synaptiske, cellulære og netværk bidrag, der fører til stimulus selektive svar i disse funktionelle maps kræver hybridisering af billeddannende teknikker, der spænder over sub-mikron til millimeter rumlige skalaer. Med konventionel iboende signal optisk billeddannelse, kan det generelle layout af de funktionelle kort over hele overfladen af den visuelle cortex bestemmes 12. Udviklingen af in vivo to-foton mikroskopi under anvendelse af calcium-følsomme farver gør det muligt at bestemme synaptic-indgang ankommer til de enkelte dendritiske Torner 13 eller optage aktivitet samtidig fra hundredvis af individuelle neuronale cellelegemer 6,14. Derfor kombinerer iboende signal billeddannelse med sub-micron rumlig opløsning af to-foton mikroskopi giver mulighed for bestemmelse af præcis hvilke dendritiske segmenter og celler bidrager til mikro-domænet af et funktionelt kort i neocortex. Her viser vi et højt udbytte fremgangsmåde til hurtig opnåelse af en cortical orientering kort og med et bestemt mikro-domænet i denne funktionelle kort til mærkning af neuroner med fluorescerende farvestoffer i en ikke-gnaver pattedyr. Med samme mikroskop bruges til to-foton billedbehandling, vi først generere en orientering kortet ved hjælp af iboende signal optisk billeddannelse. Så vi vise, hvordan man målrette en mikro-domæne af interesse ved hjælp af en mikropipette lastet med farvestof til enten etiket en population af neuronale cellelegemer eller etiket en enkelt neuron, således at dendritter, pigge og axoner er synlige ivivo. Vores finjusteringer i forhold til tidligere metoder letter en undersøgelse af neuronale struktur-funktion relationer med sub-cellulær beslutning inden for rammerne af neocortical funktionelle arkitekturer.

For at illustrere præcisionen af ​​vores farvestof mærkningsmetoder, vi målrettet den mindste mikro-domæne for enhver kendt funktionelt kort i den ikke-gnaver neocortex. Tyndt præget igennem orienteringen kortet i den primære visuelle cortex er singulariteter. Disse forekommer ved punkter, hvor alle foretrukne orienteringer konvergerer således, at i falske farve kort af foretrukken orientering, regionerne omkring singularitet ligne &quot;vejrhaner&quot; (fig. 2A-B). Én mølle pr kraniotomi e...

Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

Der beskrives en fremgangsmåde til mærkning af neuroner med fluorescerende farvestoffer i forudbestemte funktionelle mikro-domæner i neocortex. For det første er iboende signal optisk billeddannelse anvendes til at opnå et funktionelt kort. Derefter to-foton mikroskopi anvendes til at mærke og billed neuroner i en mikro-domæne af kortet.

Målrettet Mærkning af neuroner i en specifik funktionel Micro-domæne af neocortex Ved at kombinere Intrinsic-signalet og to-foton Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

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Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging af Mouse Spinal Cord ved hjælp af to-foton mikroskopi

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Påvisning af Microregional Hypoxia i mus hjernebarken ved to-foton Imaging af endogent NADH Fluorescens

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograd fluorescensmærkning Tillader Målrettet Extracellular Single-unit Optagelse fra Identificerede Neuroner<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In vivo to-foton billedbehandling af oplevelsesbaseret afhængige molekylære ændringer i kortikal neuroner

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

To-foton<em&gt; In vivo</em&gt; Imaging af dendritiske spidser i Mouse Cortex Ved hjælp af en tyndere-kranium Forberedelse

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

To-foton Imaging for Cellulær Dynamics i Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

To-foton Calcium Imaging i Neuronal dendritter i hjernen skiver

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo To-foton billeddannelse af kortikale neuroner i Neonatal mus

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Avanceret diffusion Imaging i hippocampus af rotter med mild traumatisk hjerneskade

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

Ballistisk mærkning af pyramideformede neuroner i hjerneskiver og i primærcellekultur

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...