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 arbeidet ble støttet med tilskudd fra National Eye Institute R01EY017925 og R21EY020985 og finansiering fra Dana &amp; Whitehall Foundations til PK Vi vil også takke Matthew Petrella for å få hjelp med kirurgiske prosedyrer, Grace Dion for å spore dendritter vist i figur 5A, og Pratik Chhatbar for kommentarer til manuskriptet.

1. Kirurgisk Forberedelse <ol><li> Indusere anestesi og kontinuerlig overvåke hjertefrekvens, endetidal CO 2, EEG og temperatur. Alle prosedyrer ble godkjent av Institutional Animal Care og bruk komité av Medical University of South Carolina og var basert på de vi tidligere utgitt 9,15. </li><li> Utsett framsiden av hodeskallen ved å kutte huden med en skalpell blad. Skjær bindevev overliggende beinet med en Brudon curette. Rengjør bein med bomull tippet applikatorer og bomull gasbind. Påf...

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Vi presenterer en metode for å målrette merking av neuronal celle organer (eller dendritter og aksoner) i forhåndsbestemte funksjonelle mikro-domener neocortex. Sammenslåing egenverdi signal optisk imaging med to-foton mikroskopi tilbyr muligheten for å bestemme hvilke synapser og celler bidrar til mikro-domene noen funksjonell kart, enten nevronale selektivitet korrelerer med plasseringen av nevron i en funksjonell kart, og nevronale kretskomponenter som endrer seg med visuell opplevelse 7 eller anvende...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> 1. Livet 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> Tilbakemelding regulert oppvarming teppe </td><td> Harvard Apparatus </td><td> 50-7079F </td><td></td></tr><tr><td> EKG-monitor </td><td> Digicare Biomedisinsk </td><td> LifeWindow Lite </td><td></td></tr><tr><td> EEG forsterker </td><td> AM Systems </td><td> 1800 </td><td></td></tr><tr><td> EEG skjerm monitor </td&gt; <td> Hewlett Packard </td><td> 78304A </td><td></td></tr><tr><td> Endetidal CO 2 monitor </td><td> Respironics </td><td> Novametrix Capnoguard 1265 </td><td> Optimalisere ventilasjon </td></tr><tr><td> Carbide drill grader for boring bein </td><td> Henry Schein </td><td> fin (0,5 mm spiss) og grov (1,25 mm spiss) </td><td></td></tr><tr><td> Sement for headplate / kammer </td><td> Dentsply </td><td> 675571, 675572 </td><td></td></tr><tr><td> Black Powder Tempera Paint </td><td> Sargent Art Inc. </td><td> 22-7185 </td><td> Legg til sementere å forbedre lys skjerming og redusere refleksjoner </td></tr><tr><td> Agarose - Type III-A </td><td> Sigma </td><td> A9793 </td><td> For å minimalisere pulseringer under iboende signal og to-foton avbildning </td></tr><tr><td> Coverglass: 5 eller 8 mm diameter, 0,17 mm tykkelse </td><td> Verden presisjon iinstrumenter </td><td> 502040, 502041 </td><td> For å minimalisere pulseringer under avbildning, kan coverglass kuttes etter behov </td></tr><tr><td> Brudon curettes </td><td> George Tiemann </td><td> 105-715-0, 105-715-3 </td><td> Rengjøring skull overflate </td></tr><tr><td> Bone voks </td><td> Ethicon </td><td> W31G </td><td> Raskt stanse blødninger </td></tr><tr><td> Bomull tipped applikator </td><td> Elektronmikroskopi Sciences </td><td> 72308-05 </td><td> Ren og tørr bein overflate </td></tr><tr><td> Dumont # 5CO tang </td><td> Fin Science Verktøy </td><td> 11295-20 </td><td> Grab individuelle lag av dura eller pia </td></tr><tr><td> Vännäs Spring saks </td><td> Fin Science Verktøy </td><td> 15000-03 </td><td> Cut dura </td></tr><tr><td> Gelfoam </td><td> Pfizer </td><td> 09-0396-05 </td><td> Å stoppe blødning på dura </td></tr><tr><td> Absorpsjon spyd</td><td> Fin Science Verktøy </td><td> 18105-01 </td><td> Ultra-rask og lofri wicking av 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. Farge forberedelse / injeksjon </td></tr><tr><td> Dimetylsulfoksid (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 Grønn 488 Bapta-1 AM </td><td> Invitrogen </td><td> O6807 </td><td> Kalsium indikator </td></tr><tr><td> Alexa Fluor 594 </td><td> Invitrogen </td><td> A10438 </td><td></td></tr><tr><td> Sentrifugalfilter (0,45 mikrometer porestørrelse) </td><td> Millipore </td><td> UFC30HV00 </td><td> For å fjerne urenheter før injeksjon </td></tr><tr><td> Glass pipette avtrekker </td><td> Sutter Instruments </td><td> P97 </td><td></td></tr><tr><td> Borsilikatglass filamented kapillær (1,5 mm ytre diameter) </td><td> Verdens Precision Instruments </td><td> 1B150F-4 </td><td> Dye utstøting pipette </td></tr><tr><td> Microloader </td><td> Eppendorf </td><td> 5242 956 003 </td><td> For lasting fargestoff inn pipette </td></tr><tr><td> Micromanipulator </td><td> Sutter Instruments </td><td> MP-285 </td><td> Hvis du vil plassere pipette </td></tr><tr><td> Trykkpuls kontrolleren </td><td> Parker Hannifin </td><td> PicoSpritzer III </td><td> For trykk injeksjon av fargestoffet </td></tr><tr><td> Encellede electroporator </td><td> Molecular Devices </td><td> Axoporator 800A </td><td> For elektroporering av fargestoffet </td></tr><tr><td></td><td></td><td></td><td> 3. Intrinsic 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 programvare brukes for episodisk bildebehandling </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> JAE 15-15M </td><td></td></tr><tr><td> Lyskilde </td><td> Optisk Imaging Inc. </td><td> HAL 100 </td><td> Lysintensiteten på kortikale overflaten er 3-5 mW </td></tr><tr><td> Grønt filter (for vaskulær bilde) </td><td> Optisk Imaging Inc. </td><td> λ = 546 nm (Båndpassdesign 30 nm) </td><td> For referanse bilde av overflaten vasculature </td></tr><tr><td> Rødfilter (for indre signal) </td><td> Optisk Imaging Inc. </td><td> λ = 630 nm (Båndpassdesign 30 nm) </td><td> Å samle indre 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 rigg / bildebehandling </td></tr><tr><td> To-foton mikroskop og programvare </td><td> Prairie Technologies </td><td></td><td> Se Shen et al. 2012 for lysvei, filtre og laser makt </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 blir kun brukt til å samkjøre pipette over kraniotomi (ikke for to foton imaging) </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> Air tabellen </td><td> Newport </td><td> ST-200 </td><td> Isolerer forberedelse fra eksterne vibrasjoner </td></tr><tr><td> xy scenen </td><td> Mike Machine Co (Attleboro, MA) </td><td></td><td> Forsøkspersonen og Sutter micromanipulator plassert på xy scenen </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td colspan="2"> Oppskrifter </td></tr><tr><td> Kunstig cerebrospinalvæsken </td><td> NaCl (135 mM), KCl (5,4 mM), 2 MgCl (1,0 mM), 2 CaCl (1,8 mM), HEPES (5 mM), pH 7,4 </td></tr><tr><td> Pipette Solution 14 </td><td> NaCl (150 mM), KCl (2,5 mM), HEPES (10 mM), pH 7,4 </td></tr></tbody>
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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 av ikke-gnagere pattedyr, er nervecellene gruppert i henhold til deres preferanser for stimulans funksjoner som 1-4 orientering, retning 5-7, okulær dominans 8,9 og kikkert 9 ulikhet. Orientering selektivitet er den mest studerte funksjonen og en kontinuerlig kart med en kvasi-periodisk oppsett for foretrukket retning er tilstede over hele primær visuelle cortex 10,11. Integrering av synaptiske, mobil og nett bidrag som fører til stimulans selektive tiltak i disse funksjonelle kart krever hybridisering av imaging teknikker som spenner sub-micron til millimeter romlige skalaer. Med konvensjonell egenverdi signal optisk avbildning, kan den totale utformingen av funksjonelle kartene over hele overflaten av den visuelle cortex bestemmes 12. Utvikling av in vivo to-foton mikroskopi hjelp kalsium sensitive fargestoffer muliggjør en å bestemme synaptic inngang ankommer enkelte dendrittiske spines 13 eller registrere aktivitet samtidig fra hundrevis av individuelle nevrale celle organer 6,14. Følgelig kombinerer egenverdi signal avbilding med sub-mikron romlig oppløsning av to-foton mikroskopi tilbyr muligheten for å bestemme nøyaktig hvilken dendrittiske segmenter og celler bidrar til mikro-domene noen funksjonell kart i neocortex. Her viser vi en high-yield metode for å raskt få en cortical orientering kart og rettet mot en bestemt mikro-domene i denne funksjonelle kart for merking nevroner med fluorescerende fargestoffer i en ikke-gnagere pattedyr. Med samme mikroskop brukes til to-foton bildebehandling, må vi først generere en orientering kart ved hjelp av iboende signal optisk imaging. Da viser vi hvordan å målrette en mikro-domenet av interesse ved hjelp av en mikropipette lastet med fargestoff til enten etiketten en befolkning på neuronal celle organer eller etikett en enkelt nervecelle slik at dendritter, spines og aksoner er synlige ivivo. Våre avgrensninger i forhold til tidligere metoder rette for en undersøkelse av neuronal struktur-funksjon relasjoner med sub-mobilnettet oppløsning innenfor rammen av neocortical funksjonelle arkitekturer.

Å illustrere presisjonen våre fargestoff merking metoder, målrettet vi den minste mikro-domene av enhver kjent funksjonell kartet i den ikke-gnager neocortex. Sparsomt krydres hele orientering kartet i den primære visuelle cortex er singulariteter. Disse forekommer på punkter hvor alle foretrukne orientering konvergerer slik at i falske farger kartene over foretrukket retning, regionene rundt singularitet ligne &quot;pinwheels&quot; (figur 2A-B). En pinwheel per kraniotomi er valgt for dye merking ...

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

En fremgangsmåte er beskrevet for merking nevroner med fluorescerende fargestoffer i forutbestemte funksjonelle mikro-domener av neocortex. Først, egenverdi signal optisk imaging brukes for å oppnå en funksjonell kart. Deretter to-foton mikroskopi brukes for å merke og image nevroner innenfor mikro-domene på kartet.

Målrettet Merking av nevroner i en bestemt Funksjonell Micro-domenet neocortex ved å kombinere Intrinsic Signal 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, ...

Research

JoVE Journal

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 av Mouse Spinal Cord Bruke 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 av Microregional Hypoksi i Mus Cerebral Cortex av to-foton Imaging av endogen NADH Fluorescence

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

Retrograde Fluorescent Merking Tillater målrettet Ekstracellulær Single-enhet Opptak fra Identifiserte Nerveceller<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

I Vivo to-foton avbildning av erfaring-avhengige molekylære endringer i kortikale Neurons

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-Photon<em&gt; In vivo</em&gt; Imaging av dendrittutløperne i Mouse Cortex Bruke en Tynnet-skalle 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 av Cellular 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-fotonet kalsium Imaging i Neuronal Dendrites 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

I Vivo To-fotonet avbilding av kortikale nevroner 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

Avansert Diffusion Imaging i hippocampus hos 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 merking av pyramidale nevroner i hjerneskiver og i primær cellekultur

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