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

Wij danken Atsushi Miyawaki en Hiroshi Hama voor hun advies over de AbScale experimenten, Charles Yokoyama voor het bewerken van het manuscript, Eriko Ohshima en Miyuki Kishino voor hun technische bijstand. Dit werk werd gesteund door onderzoeksgelden van RIKEN T.H. en Grants-in-Aid voor wetenschappelijk onderzoek van het ministerie van onderwijs, cultuur, sport, wetenschap en technologie (MEXT) van Japan naar T.H. (innovatieve gebieden "Mesoscopische Neurocircuitry"; 22115004) en ZB (25890023).

Alle experimentele procedures werden goedgekeurd door de RIKEN Wako dier experimenten Commissie en RIKEN genetische Recombinant Experiment veiligheidscommissie en uitgevoerd volgens de richtlijnen van de dierlijke faciliteiten van het hersenonderzoek RIKEN van institutionele Instituut.
1. bereiding van Imaging Chambers
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Imaging kamer19
	<ol>
<li>Met behulp van silicone rubber sheets, bereiden een kamer met een dikte van ongeveer 5 mm en vloer ...

<ol>
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Wij hebben de procedures voor het verkrijgen van grootschalige driedimensionale beelden van de cel type-specifieke organisatie van de grote celtypes in muis neocortical laag 5 ingediend. Vergeleken met de conventionele segment kleuring, is de methode meer nuttig bij het bepalen van de driedimensionale organisatie van de neocortex. De methode kunt image aquisition van de bredere en de diepere hersengebieden in vergelijking met de typische in vivo 2-foton microscopie of conventionele confocale microscopie en, dus,...

De zoogdieren neocortex is samengesteld uit een groot aantal celtypes, elk met de specifiek gen-expressiepatronen, elektrofysiologische en biochemische eigenschappen, synaptic verbindingen, en functies in vivo 1,2 ,3,4,,5,,6,7. Of deze celtypes herhaalde structuren worden geordend is onduidelijk. Corticale kolommen, met inbegrip van visuele oriëntatie kolommen en somatosensorische vaten, structuren hebben herhaald, maar hun cellulaire organisatie blijft onduidelijk8,9. Deze zijn aanwezig in de specifieke corticale gebieden en niet een hersenen-omspannend rechtssysteem.
In neocortical laag 5, worden de grote meerderheid van de neuronen ingedeeld in vier grote typen. Een belangrijk type van excitatory neuronen, sub cerebrale projectie neuronen, axonen bij subcorticale doelen, met inbegrip van de pons, ruggenmerg en superieure colliculus projecten, en daarom, vertegenwoordigt de grote corticale uitvoer traject10. Projectie van de corticale neuronen, een andere belangrijke soorten excitatory neuronen, innervate de cortex10. Remmende neuronen bevatten ook twee grote klassen: parvalbumin-uitdrukken en het uiten van Somatostatine cellen11.
Recente analyses geven aan dat de vier celtypes herhaalde structuren12,13,14worden geordend. Zowel sub cerebrale projectie neuronen12,13,14 en projectie van de corticale neuronen14 indelen in celtype specifieke microcolumns met een diameter van 1-2 cellen. Parvalbumin-uitdrukken en het uiten van Somatostatine cellen uitlijnen specifiek met microcolumns van sub cerebrale projectie neuronen maar niet met de microcolumns van projectie van de corticale neuronen14 Microcolumns zelf uitlijnen periodiek op vorm een zeshoekige rooster matrix14 en aanwezig zijn in meerdere corticale gebieden met inbegrip van visuele, somatosensorische en motor in12,14 van de hersenen van de muis en in taal gebieden van menselijke hersenen13. Neuronen in de individuele microkolom gesynchroniseerde activiteit vertonen en hebben soortgelijke sensorische reacties14. Deze opmerkingen geven aan dat de laag 5 celtypes organiseren in een microkolom rooster structuur vertegenwoordigen de eerste bekende hersenen bestrijkende organisatie functionele modules te herhalen.
Microcolumns hebben een straal van ongeveer 10 µm en een ruimtelijke frequentie van ongeveer 40 µm. Daarnaast is de richting van microcolumns parallel aan hun apicale dendrites en wijzigingen afhankelijk van hun positie in de cortex-14. De microkolom systeem is daarom moeilijk om te analyseren met behulp van conventionele corticale segmenten met een typische dikte van enkele tientallen micrometers. Bovendien, de analyse van de periodiciteit driedimensionale gegevens uit een groot aantal hersengebieden, en dus de typische opnamegebied van confocale microscopie vereist of in vivo 2-photon imaging is te smal.
Onlangs, technieken zijn ontwikkeld om te wissen van dikke weefsels15,16. Hier beschrijven we de toepassing van deze methoden voor het verkrijgen van grootschalige, drie-dimensionale beelden van de belangrijkste celtypen in muis neocortical laag 5 waaruit de microkolom systeem. Subcerebral projectie neuronen worden aangeduid door de retrograde labelen of door de uitdrukking van de verbeterde groen fluorescent proteïne in Crym-egfp transgene muizen12, en corticale projectie neuronen worden aangeduid door hetzij de retrograde labeling of door de expressie van de tdTomato in Tlx3-cre/Ai9 muizen17. Parvalbumin-uitdrukken en het uiten van Somatostatine cellen worden aangeduid door immunohistochemie. De methode (antilichaam schaal S) AbScale18 wordt gebruikt voor het antilichaam kleuring van experimenten, terwijl de methode (Zie diepe brein) SeeDB19 wordt gebruikt voor andere experimenten. Deze methoden de bovengenoemde moeilijkheden van de conventionele beeldvormende methoden en onthullen de nauwkeurige cellulaire organisatie van laag 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

De zoogdieren neocortex is samengesteld uit vele soorten excitatory en remmende neuronen, elk met specifieke elektrofysiologische en biochemische eigenschappen, synaptic verbindingen, en in vivo functies, maar hun basic functionele en anatomische organisatie van cellulaire netwerk schaal wordt slecht begrepen. Hier beschrijven we een methode voor de driedimensionale beeldvorming van fluorescently-geëtiketteerden neuronen over grote gebieden van de hersenen voor het onderzoek van de corticale cellulaire organisatie. Bepaalde soorten neuronen worden aangeduid door de injectie van fluorescerende retrograde neuronale verklikstoffen of expressie van fluorescente proteïnen in transgene muizen. Blok hersenen monsters, bijvoorbeeld een halfrond, zijn bereid na fixatie, transparant gemaakt met weefsel van het ontruimen van de methoden, en onderworpen aan fluorescerende immunolabeling van de specifieke celtypes. Grote gebieden worden gescand met behulp van de confocal of twee-foton microscopen uitgerust met grote werken afstand doelstellingen en gemotoriseerde stadia. Deze methode kan de periodieke organisatie van de cel type-specifieke microkolom functionele modules in de neocortex muis is opgelost. De procedure kan nuttig zijn voor de studie van drie-dimensionale cellulaire architectuur in de verschillende hersengebieden en andere complexe weefsels.

We label projectie van de corticale neuronen door expressie van tdTomato in Tlx3-cre/Ai9 transgene muizen en sub cerebrale projectie neuronen gevisualiseerd door het injecteren van de retrograde tracer CTB488 in de pons. De linker hemisfeer van de hersenen werd blootgesteld aan de SeeDB-methode en gescand met behulp van een twee-foton microscoop voorzien van een water-onderdompeling lange afstand doelstelling werken (25 X, N.A. 1.1, afstand 2 mm) en een gemotoriseerde po...

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

Grootschalige driedimensionale beeldvorming van cellulaire organisatie in de Neocortex muis

Hier beschrijven we een procedure voor het weefsel wissen, fluorescerende benoemen, en grootschalige beeldvorming van muis hersenweefsel, waardoor, zodat, visualisatie van de driedimensionale organisatie van celtypes in de 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

Multiple-muis neuroanatomische 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

Opname Grootschalige Neuronale Ensembles met Silicon Probes in de verdoofde 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

Grootschalige Opname van Neuronen door Movable Silicon Probes in Behaving Knaagdieren

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

Dissectie en Cultuur van de muis Dopaminerge en het striatum Explantaten in drie-dimensionale Collageen Matrix Assays

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 van neuroblast migratie in Acute Schijfjes van de volwassen muis voorhersenen

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

Twee-foton Imaging van Cellular Dynamics in de Muis 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; Dynamiek in Cone fotoreceptorcellen Axon Terminals van de 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

Protocol voor drie-dimensionale confocale Morfometrische Analyse van Astrocytes

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

Driedimensionale beeldvorming en analyse van Mitochondria binnen menselijke Intraepidermal zenuwvezels

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