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 eksperimentene, Charles Yokoyama for redigering av manuskriptet, Eriko Ohshima og Miyuki Kishino for deres kundestøtte. Dette arbeidet ble støttet av forskningsmidler fra RIKEN th og Grants-in-Aid for vitenskapelig forskning fra Kunnskapsdepartementet, kultur, sport, vitenskap og teknologi (MEXT) i Japan for å th (nyskapende områder "Mesoskopisk Neurocircuitry", 22115004) og SS (25890023).

Alle eksperimentelle prosedyrer ble godkjent av RIKEN ham til dyr eksperimenter komiteen og RIKEN genetisk rekombinant eksperiment sikkerhetskomite og utført i henhold til institusjonelle retningslinjer dyr fasiliteter RIKEN hjernen vitenskap Instituttet.
1. forberedelse Imaging Chambers
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Imaging kammer19
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<li>Bruker silikon gummi ark, forberede et kammer med en tykkelse på ca 5 mm og gulvet plater av ulike tykkelser. Også forberede Pe...

<ol>
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Vi har presentert prosedyrer for å få store tredimensjonale bilder av celle type-spesifikk organisasjonen de store celletyper i musen neocortical lag 5. Sammenlignet med den konvensjonelle skive flekker, er metoden mer nyttig å bestemme tredimensjonale organiseringen av neocortex. Metoden gjør det mulig for image oppkjøpet fra den bredere og dypere hjernen regioner i forhold til den typiske i vivo 2-fotonet mikroskopi eller konvensjonelle AC confocal mikroskopi og, dermed kan tillate av neocortical mobilnet...

Pattedyr neocortex består av en rekke celletyper, hver med bestemte genet uttrykk mønstre, elektrofysiologiske og biokjemiske egenskaper, synaptic tilkoblinger, og vivo fungerer1,2 ,3,4,5,6,7. Om disse celletyper organiseres i gjentatte strukturer er uklart. Kortikale kolonner, inkludert visuell retning kolonner og somatosensory fat, har gjentatt strukturer, men organisasjonen mobilnettet forblir uklart8,9. Disse finnes i bestemte kortikale områder og er ikke en hjerne systemoppetid.
I neocortical lag 5, er det store flertallet av neurons klassifisert til fire hovedtyper. En hovedtype av eksitatoriske neurons, sub cerebral projeksjon neurons, prosjekter axons å subkortikal mål inkludert pons, ryggmargen og overlegen colliculus, og representerer derfor den store kortikale utgang sti10. Kortikale projeksjon neurons, en annen stor type av eksitatoriske neurons, innervate cortex10. Hemmende neurons inneholder også to store klasser: parvalbumin-uttrykke og somatostatin-uttrykke celler11.
Siste analyser viser at de fire celletyper organiseres i gjentatte strukturer12,13,14. Både sub cerebral projeksjon neurons12,13,14 og kortikale projeksjon neurons14 organisere i celle-type spesifikke microcolumns med en diameter på 1 – 2 celler. Parvalbumin-uttrykke og somatostatin-uttrykke cellene justeres med microcolumns sub cerebral projeksjon neurons, men ikke med microcolumns av kortikale projeksjon neurons14. Microcolumns seg justerer regelmessig til et Sekskantet gitteret matrise14 og finnes i flere kortikale områder inkludert visuelle, somatosensory og motor i musen hjernen12,14 og språk områder av hjernen13. Nerveceller i den personlige microcolumn utstilling synkronisert aktivitet og har lignende sensoriske svar14. Disse observasjonene indikerer at lag 5 celletyper organisere i en microcolumn gitter struktur representerer den første kjente hjerne hele organisasjonen gjentatte funksjonelle modulene.
Microcolumns har en radius på ca 10 µm og en romlig periodisitet av ca 40 µm. I tillegg er retningen for microcolumns parallell til apikale dendrites og endres avhengig av sin posisjon i cortex14. Microcolumn systemet er derfor vanskelig å analysere ved hjelp av konvensjonelle kortikale skiver med en vanlig tykkelse på noen titalls mikrometer. I tillegg analyse av periodisitet krever tredimensjonale data fra en rekke hjernen områder, og derfor typisk tenkelig området AC confocal mikroskopi eller i vivo 2-fotonet imaging er for smal.
Nylig er teknikker utviklet for å fjerne tykk vev15,16. Her beskriver vi bruk av disse metodene for å få store, tredimensjonale bilder av de store celletyper i musen neocortical lag 5 som utgjør microcolumn systemet. Subcerebral projeksjon neurons er merket retrograd merking eller uttrykk for forbedret grønne fluorescerende proteinet i Crym-egfp transgene mus12og kortikale projeksjon neurons er merket med enten av retrograd merking eller ved det tdTomato uttrykket i Tlx3-grobunn/Ai9 mus17. Parvalbumin-uttrykke og somatostatin-uttrykke celler er merket av immunhistokjemi. (Antistoff skala S) AbScale metoden18 brukes antistoffer flekker eksperimenter, mens (se dypt hjernen) SeeDB metoden19 brukes for andre eksperimenter. Disse metodene overvinne nevnte vanskelighetene av konvensjonelle tenkelig metoder og avsløre nøyaktig mobilnettet organisasjonen 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 består av mange typer eksitatoriske og inhibitory neurons, hver med bestemte elektrofysiologiske og biokjemiske egenskaper, synaptic tilkoblinger, og vivo fungerer, men deres grunnleggende funksjonelle og anatomisk organisasjonen cellular nettverk skala er dårlig forstått. Her beskriver vi en metode for tredimensjonale avbilding av fluorescently-merket neurons over store områder av hjernen for etterforskningen av kortikale mobilnettet organisasjonen. Bestemte typer nerveceller merket av injeksjon av fluorescerende retrograd neuronal tracers eller uttrykk fluorescerende proteiner i transgene mus. Blokkere hjernen prøvene, f.eks en halvkule, utarbeidet etter fiksering, laget gjennomsiktig med vev fjerne metoder og underlegges fluorescent immunolabeling de spesifikke celletyper. Store områder er skannet med AC confocal eller to-fotonet mikroskop utstyrt med store arbeider avstand mål og motorisert stadier. Denne metoden kan løse periodiske organiseringen av celle type-spesifikk microcolumn funksjonelle modulene i musen neocortex. Prosedyren kan være nyttig for studier av tredimensjonale mobilnettet arkitektur i ulike hjernen områder og andre komplekse vev.

Vi merket kortikale projeksjon neurons med uttrykk for tdTomato i Tlx3-grobunn/Ai9 transgene mus og visualisert sub cerebral projeksjon neurons ved å injisere den retrograd tracer CTB488 inn pons. Venstre halvkule av hjernen ble utsatt for metoden SeeDB og skannet med to-fotonet mikroskop utstyrt med en vann-fordypning arbeide lang avstand mål (25 X N.A. 1.1, arbeidsavstand 2 mm) og en motorisert scene. En stabel av 401 bilder (512 x 512 bildepunkter, pikselstørrelsen...

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

Store tredimensjonale Imaging mobilnettet organisasjon i musen Neocortex

Her beskriver vi en prosedyre for vev fjerne fluorescerende merking og store avbilding av musen hjernevev som gir dermed, visualisering av tredimensjonale organisasjonen 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

Storskala opptak av nevroner med bevegelige Silicon Sonder i oppfører Gnagere

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

Disseksjon og kultur Mouse dopaminerg og striatale banene explants i tredimensjonale kollagen matrise 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 av Neuroblast Migrasjon i Akutt Skiver av Adult Mouse forebrain

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 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 ...

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 fotoreseptor Axon terminaler 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

Protokoll for Tredimensjonal Confocal Morfometrisk Analyse av 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

Tredimensjonale Imaging og analyse av mitokondrier i menneskelig Intraepidermal Nerve Fibre

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