Name: large-scale three-dimensional imaging of cellular organization in the mouse neocortex
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex at JoVE.com

Vi takker Atsushi Miyawaki og Hiroshi Hama for deres råd om AbScale eksperimenter, Charles Yokoyama til redigering af manuskriptet, Eriko Ohshima og Miyuki Kishino for deres tekniske bistand. Dette arbejde blev støttet af forskningsmidler fra RIKEN T.H. og Grants-in-Aid for videnskabelig forskning fra ministeriet for uddannelse, kultur, sport, videnskab og teknologi (MEXT) af Japan til T.H. (Innovative områder "Mesoskopisk Neurocircuitry", 22115004) og S.S. (25890023).

Alle eksperimentelle procedurer blev godkendt af RIKEN Wako dyr eksperimenter udvalget og RIKEN genetiske rekombinant eksperiment sikkerhedsudvalg og udført efter de institutionelle retningslinjer for animalske faciliteter, RIKEN hjernen videnskab Institut.
1. forberedelse af Imaging kamre
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Imaging afdeling19
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<li>Brug af silikone gummi ark, forberede et kammer med en tykkelse på ca 5 mm og gulv plader af forskellige tykkelser. Også, f...

<ol>
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Vi har udarbejdet procedurer for at opnå store tre-dimensionelle billeder af celle type-specifikke organisation af de store celletyper i mus neocortical lag 5. Sammenlignet med konventionelle skive farvning, er metoden mere nyttigt i fastlæggelse af den tredimensionale organisation af neocortex. Metoden gør det billede erhvervelse fra den bredere og dybere hjernen regionerne i forhold til typiske i vivo 2-foton mikroskopi eller konventionelle Konfokal mikroskopi og således kan tillade, at den omfattende anal...

Pattedyr neocortex er sammensat af en lang række celletyper, hver med bestemte gen expression mønstre, elektrofysiologiske og biokemiske egenskaber, synaptiske forbindelser, og i vivo funktioner1,2 ,3,4,5,6,7. Hvorvidt disse celletyper er organiseret i gentagne strukturer har været uklare. Kortikale kolonner, herunder visuel orientering kolonner og somatosensoriske Tønder, har gentaget strukturer, men deres cellulære organisation forbliver uklart8,9. Disse er til stede i de specifikke kortikale områder og er ikke en hjerne-dækkende system.
I neocortical lag 5, er det store flertal af neuroner klassificeret i fire hovedtyper. En større type af excitatoriske neuroner, sub cerebral projektion neuroner, projekter axoner til subkortikale mål herunder pons, rygmarven og overlegen colliculus og derfor repræsenterer store kortikale output vej10. Cortical fremskrivning neuroner, en anden store type excitatoriske neuroner, innerverer cortex10. Hæmmende neuroner også indeholder to store klasser: parvalbumin-udtrykker og somatostatin-udtrykke celler11.
De seneste analyser viser, at de fire celletyper er organiseret i gentagne strukturer12,13,14. Både sub cerebral projektion neuroner12,13,14 og cortical fremskrivning neuroner14 organisere i celletype specifikke microcolumns med en diameter på 1-2 celler. Parvalbumin-udtryk og somatostatin-udtrykke celler Juster specifikt med microcolumns af sub cerebral projektion neuroner, men ikke med microcolumns af cortical fremskrivning neuroner14. Microcolumns sig justere med jævne mellemrum til at danne en sekskantet gittermaster array14 og er til stede i flere kortikale områder herunder visuel, somatosensoriske og motoriske områder i mus hjernen12,14 og sprog områder af menneskets hjerne13. Neuroner i den enkelte microcolumn udviser synkroniseret aktivitet og har lignende sensoriske svar14. Disse observationer viser at lag 5 celletyper organisere i et microcolumn gitterstruktur der repræsenterer den første kendte hjerne-dækkende organisation for at gentage funktionelle moduler.
Microcolumns har en radius på ca. 10 µm og har en rumlig hyppighed af ca 40 µm. Derudover er orienteringen af microcolumns parallel til deres apikale dendritter og skifter, afhængigt af deres placering i cortex14. Microcolumn systemet er derfor vanskeligt at analysere ved hjælp af konventionelle kortikale skiver med en typisk tykkelse på et par snese mikrometer. Derudover analyse af periodicitet kræver tre-dimensionelle data fra en bred vifte af områder i hjernen, og derfor typisk imaging området Konfokal mikroskopi eller i vivo 2-foton imaging er for snæver.
For nylig, teknikker er blevet udviklet for at fjerne tykt væv15,16. Her beskriver vi anvendelsen af disse metoder til at opnå store, tre-dimensionelle billeder af de store celletyper i mus neocortical lag 5, der udgør microcolumn systemet. Subcerebral projektion neuroner er mærket af retrograd mærkningsbestemmelserne eller udtryk for den forbedrede grøn fluorescerende protein i Crym-egfp Transgene mus12og cortical fremskrivning neuroner er mærket af enten den retrograd mærkning eller af tdTomato udtrykket i Tlx3-cre/Ai9 mus17. Parvalbumin-udtryk og somatostatin-udtrykke celler er mærket af Immunhistokemi. (Antistof skala S) AbScale metode18 bruges til antistof farvning eksperimenter, mens (Se Deep Brain) SeeDB metode19 bruges til andre eksperimenter. Disse metoder overvinde de ovennævnte vanskeligheder af de konventionelle Billeddannende metoder og afsløre den nøjagtige cellulære organisation af lag 514.

<table>
	<tbody>
		<tr>
			<td>Crym-egfp transgenic mice</td>
			<td>MMRRC</td>
			<td>012003-UCD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tlx3-cre transgenic mice</td>
			<td>MMRRC</td>
			<td>36547-UCD</td>
			<td></td>
		</tr>
		<tr>
			<td>ROSA-CAG-flox-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td>JAX #7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber sheet</td>
			<td>AS ONE</td>
			<td>6-611-01</td>
			<td>0.5 mm thickness</td>
		</tr>
		<tr>
			<td>Silicone rubber sheet</td>
			<td>AS ONE</td>
			<td>6-611-02</td>
			<td>1.0 mm thickness</td>
		</tr>
		<tr>
			<td>Silicone rubber sheet</td>
			<td>AS ONE</td>
			<td>6-611-05</td>
			<td>3.0 mm thickness</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Matsunami</td>
			<td>C022241</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit B (recombinant), Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>C22841</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit B (recombinant), Alexa Fluor 555 conjugate</td>
			<td>Invitrogen</td>
			<td>C22843</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>C22842</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit B (recombinant), Alexa Fluor 647 conjugate</td>
			<td>Invitrogen</td>
			<td>C34778</td>
			<td></td>
		</tr>
		<tr>
			<td>26G Hamilton syringe</td>
			<td>Hamilton</td>
			<td>701N</td>
			<td></td>
		</tr>
		<tr>
			<td>Injector pump</td>
			<td>KD Scientific</td>
			<td>KDS 310</td>
			<td>Pons injection</td>
		</tr>
		<tr>
			<td>Injector pump</td>
			<td>KD Scientific</td>
			<td>KDS 100</td>
			<td>Superior colliculus injection</td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Narishige</td>
			<td>SM-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Kyoritsu Seiyaku</td>
			<td>Somnopentyl</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>AstraZeneca</td>
			<td>Xylocaine injection 1% with epinephrine</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Toyo Associates</td>
			<td>HP-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Avitene microfibrillar hemostat</td>
			<td>Davol Inc</td>
			<td>1010090</td>
			<td></td>
		</tr>
		<tr>
			<td>Alonalfa</td>
			<td>Daiichi-Sankyo</td>
			<td>Alonalpha A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical silk</td>
			<td>Ethicon</td>
			<td>K881H</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>UVP</td>
			<td>HB-1000 Hybridizer</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipette</td>
			<td>Drummond Scientific Company</td>
			<td>2-000-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Liquid, light</td>
			<td>Nacalai tesque</td>
			<td>26132-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Otsuka</td>
			<td>1326</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Nacalai tesque</td>
			<td>26126-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td>Inter medical</td>
			<td>&#934;0.1 *L200 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL plastic tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-thioglycerol</td>
			<td>Nacalai tesque</td>
			<td>33709-62</td>
			<td></td>
		</tr>
		<tr>
			<td>D(-) Fructose</td>
			<td>Nacalai tesque</td>
			<td>16315-55</td>
			<td></td>
		</tr>
		<tr>
			<td>BluTack</td>
			<td>Bostik</td>
			<td>CKBT-450000</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscope</td>
			<td>Nikon</td>
			<td>A1RMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-immersion long working distance objectives</td>
			<td>Nikon</td>
			<td colspan="2">CFI Apo LWD 25XW, NA 1.1, WD 2 mm</td>
		</tr>
		<tr>
			<td>Water-immersion long working distance objectives</td>
			<td>Nikon</td>
			<td>CFI LWD 16XW, NA 0.8, WD 3 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized stage</td>
			<td>COMS</td>
			<td>PT100C-50XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Semrock</td>
			<td>FF01-492/SP-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Semrock</td>
			<td>FF03-525/50-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Semrock</td>
			<td>FF03-575/25-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Semrock</td>
			<td>FF01-629/56</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma</td>
			<td>D605/55m</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL plastic tube</td>
			<td>AS ONE</td>
			<td>VIO-5B</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL plastic tube</td>
			<td>Eppendorf&#160;</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Nacalai tesque</td>
			<td>35905-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyserol</td>
			<td>Sigma-aldrich</td>
			<td>191612</td>
			<td></td>
		</tr>
		<tr>
			<td>D(-)-sorbitol</td>
			<td>Wako</td>
			<td>191-14735</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl-&#946;-cyclodextrin</td>
			<td>Tokyo chemical industry</td>
			<td>M1356</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-Cyclodextrin</td>
			<td>Wako</td>
			<td>037-10643</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-hydroxyproline</td>
			<td>Skin Essential Actives</td>
			<td>33996-33-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Nacalai tesque</td>
			<td>13445-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (1.1 g/mL)</td>
			<td>Nacalai tesque</td>
			<td>35624-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A21422</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A21428</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A21235</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-immersion long working distance objectives</td>
			<td>Olympus</td>
			<td>XLUMPLFLN 20XW, NA 1.0, WD 2 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NeuN</td>
			<td>Millipore</td>
			<td>MAB377</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NeuN</td>
			<td>Millipore</td>
			<td>ABN78</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CTIP2</td>
			<td>Abcam</td>
			<td>ab18465</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Statb2</td>
			<td>Abcam</td>
			<td>ab51502</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAD67</td>
			<td>Millipore</td>
			<td>MAB5406</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GABA</td>
			<td>Sigma</td>
			<td>A2052</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parvalbumin</td>
			<td>Swant</td>
			<td>235</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parvalbumin</td>
			<td>Frontier Institute</td>
			<td>PV-Go-Af460</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parvalbumin</td>
			<td>Sigma</td>
			<td>P3088</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parvalbumin</td>
			<td>Abcam</td>
			<td>ab11427</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Somatostatin</td>
			<td>Peninsula Laboratories</td>
			<td>T-4103</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-c-Fos</td>
			<td>CalbioChem</td>
			<td>PC38</td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale three-dimensional imaging of cellular organization in the mouse neocortex

Pattedyr neocortex er sammensat af mange typer af excitatoriske og hæmmende neuroner, hver med specifikke elektrofysiologiske og biokemiske egenskaber, synaptiske forbindelser, og i vivo fungerer, men deres grundlæggende anatomiske og funktionelle organisation fra cellular netværk målestok er dårligt forstået. Her beskriver vi en metode for den tre-dimensionelle billeddannelse af fluorescently mærket neuroner på tværs af store områder af hjernen for undersøgelsen af de kortikale cellulære organisation. Specifikke typer for neuroner er mærket af injektion af fluorescerende retrograd neuronal røbestoffer eller udtryk for fluorescerende proteiner i Transgene mus. Blok hjernen prøver, f.eks. en halvkugle, er udarbejdet efter fiksering, lavet gennemsigtig med væv clearing metoder og underkastes fluorescerende immunolabeling af de specifikke celletyper. Store områder er scannet ved hjælp af Konfokal eller to-foton mikroskoper udstyret med store arbejde afstand mål og motoriseret faser. Denne metode kan løse den periodiske organisation af celle typespecifikke microcolumn funktionelle moduler i mus neocortex. Proceduren kan være nyttig for studiet af tre-dimensionelle cellulære arkitektur i de forskellige hjerne områder og andre komplekse væv.

Vi mærket cortical fremskrivning neuroner ved ekspression af tdTomato i Tlx3-cre/Ai9 Transgene mus og visualiseret sub cerebral projektion neuroner ved at indsprøjte den retrograd tracer CTB488 i pons. Den venstre hjernehalvdel blev udsat for SeeDB metoden og scannes ved hjælp af en to-foton mikroskop udstyret med en vand-fordybelse længe arbejde afstand mål (25 X, N.A. 1.1, arbejde afstand 2 mm) og en motoriseret fase. En stak af 401 billeder (512 x 512 pixel, pixe...

Watch this Scientific Journal Video about Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex at JoVE.com

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Storstilet tredimensional billeddannelse af cellulære organisation i mus Neocortex

Her beskriver vi en procedure for væv clearing, fluorescerende mærkning og storstilet billeddannelse af musen hjernevæv, som dermed giver mulighed for visualisering af den tredimensionale organisation af celletyper i neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

Storstilet Registrering af neuroner ved Flytbare Silicon Probes at opføre sig Gnavere

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Dissektion og kultur Mouse dopaminerge og Striatale eksplantater i Tredimensionelle kollagenmatrix Analyser

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Time-lapse Imaging af neuroblast Migration i Akutte Skiver af den voksne Mouse forhjerne

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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 ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca<sup&gt; 2+</sup&gt; Dynamics i Cone Lysmodtageren Axon Terminals i Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Protokol til tredimensionel Konfokal Morfometrisk Analyse af Astrocytter

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Tre-dimensionelle billedbehandling og analyse af mitochondrier i menneskelige Intraepidermal nervefibre

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...