Name: visualization of cortical modules in flattened mammalian cortices
Uploaded: 2018-01-22T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

Dette arbejde blev støttet af Humboldt Universität zu Berlin, Bernstein Center for Computational Neuroscience Berlin, det tyske Center for Neurodegenerative sygdomme (DZNE), den tyske føderale ministerium for uddannelse og forskning (BMBF, Förderkennzeichen 01GQ1001A), NeuroCure og Gottfried Wilhelm Leibniz-prisen af DFG. Vi takker Shimpei Ishiyama for fremragende grafisk design og Juliane Diederichs for fremragende teknisk bistand.

Alle eksperimentelle procedurer blev udført efter tyske retningslinjer vedrørende dyrs velfærd under tilsyn af lokale etiske komitéer (LaGeSo). Menneskelige og bat brain data blev afledt fra Naumann et al. 5 følgende procedure er udført på en mandlig voksen Wistar rotter (stamme: RJHan:WI).
1. perfusion og hjernen udvinding
Bemærk: For at opnå en homogen måde fast og blod-gratis hjerne, transcardial perfusion af dyret er stæ...

<ol>
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Modularitet i hjernebarken er blevet identificeret ved hjælp af en række teknikker. De tidligste studier der typisk kortikale moduler af enten visualisere celle tætte områder, eller fravær af fibre1. Efterfølgende metoder har udnyttet tilstedeværelsen af dendritiske bundter24, afferenter fra en bestemt region25eller berigelse af neurotransmittere26. Her viser vi to teknikker, (i) cytokrom oxidase histokemi og (ii) immun...

Nogle af de mest markante ændringer i hjernens struktur på tværs af fylogeni kan observeres i hjernebarken. Trods betydelige forskelle, cortex af dyr følger et fælles mønster og kan groft inddeles i to særskilte måder, af lag og områder1. Kortikale lag ligger parallelt med overfladen af hjernen og variere i antal fra 3 lag i krybdyrs cortex2 til 6 lag i pattedyr cortex1. Kortikale områder på den anden side er forskellige regioner af cortex, som stort set svarer til forskellige funktioner, f.eks., den somatosensoriske cortex er involveret i følelsen af touch eller den visuelle cortex ved behandling af visuelle input. Disse kortikale områder kan ofte opdeles i patches eller moduler3, som regelmæssigt gentaget anatomiske strukturer, hovedsagelig fundet parallelt med pial overfladen af hjernen. Kortikale moduler kan begrænses til et bestemt lag4eller strækker sig over flere lag5.
Skæring standardmetoder af hjernen inddrage afsnit vinkelret på overfladen af hjernen, som koronal og sagittal. Mens disse metoder kan bruges til at visualisere kortikale moduler, kan et væld af interessante funktioner blive afsløret når de kortikale moduler er visualiseret tangentielt, i et plan parallelt med overfladen af hjernen. For eksempel, somatosensoriske moduler i gnavere hjernen repræsenterer whiskers, vises som Tønder når visualiseret normal hjerne overflade, og dermed Regionsudvalget udlede navnet tønde cortex. Dog på visualisere tønder i en tangential orientering, afslører de en bakkenbart-kort, med tønder er lagt ud i en topografisk orientering spejling den nøjagtige layout af knurhår på eksterne kroppens overflade. I visse tilfælde, modulære arrangement har endnu undsluppet påvisning i store perioder, når visualiseret i en ikke-tangential måde. Den mediale entorhinal cortex, er kendt for tilstedeværelsen af gitterceller, neuroner, som brand i en regelmæssig sekskantede mønster, når et dyr gennemkører en miljø. Selv om det er et stærkt undersøgte område, indtil for nylig, tilstedeværelsen af patches eller moduler af celler i den mediale entorhinal cortex, var som fysisk er lagt ud i en sekskantet mønster6, undsluppet påvisning. Tilstedeværelsen og placeringen af disse moduler, i hjernen, rotte, blev fremmet ved at gøre tangential dele af mediale entorhinal cortex og undersøge cytoarchitecture på en layer-wise måde.
Efter skæring, indså det særlige aspekt af visualisering af kortikale moduler kan også på flere måder. Klassisk, har undersøgelser afgrænset moduler baseret på celle tæthed eller fiber layout1. En anden populær metode er brugen af cytokrom oxidase histokemi, som afslører områder af højere aktivitet8. Nyere tilgange omfatte kigger på genetisk bestemt celletyper, udmærker sig på grundlag af deres protein udtryk profiler6,8.
I denne undersøgelse fremhæve vi metoder for at isolere cortex fra pattedyr hjerner, få fladtrykte tangential sektioner og visualisere kortikale moduler baseret på cytokrom oxidase histokemi og Immunhistokemi celletype specifikke proteiner.

<table>
	<tbody>
		<tr>
			<td>Cytochrome oxidase staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;Diaminobenzidine tetrahydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Saccharose</td>
			<td>Carl Roth&#160;</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium nickel(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A1827</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antigen retrieval</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phosphate buffer/phosphate-buffered saline/prefix/PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-Sodium hydrogen phosphate dihydrate</td>
			<td>Carl Roth</td>
			<td>4984.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON-X 100</td>
			<td>Carl Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k puriefied from chicken gut, Mouse monoclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000347</td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k from recombinant rat calbindin D-28k, Rabbit polyclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000340</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V, biotin free</td>
			<td>Carl Roth</td>
			<td>0163.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mounting or freezing media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount (immunofluorescence)</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt (histochemistry)</td>
			<td>Sigma-Aldrich</td>
			<td>03989</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>Leica Biosystems</td>
			<td>NC0696746</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Alcohol dehydration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Carl Roth</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth</td>
			<td>P075.3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth</td>
			<td>6752.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>Fluka</td>
			<td>78475</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Devices/tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microm HM 650V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jung RM2035</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps - Inox</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Inox Biology Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps - Inox Super Fine Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Shears - 24 cm</td>
			<td>Fine Science Tools</td>
			<td>16150-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16000-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Scissors</td>
			<td>Fine Science Tools</td>
			<td>14000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Large Loops</td>
			<td>Fine Science Tools</td>
			<td>14101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of cortical modules in flattened mammalian cortices

Cortex af pattedyr hjerner er parcellated i særskilte delstrukturer eller moduler. Kortikale moduler typisk ligge parallelt med det kortikale ark, og kan være afgrænset af visse histokemiske og immunhistokemiske metoder. I denne undersøgelse fremhæve vi en metode til at isolere cortex fra pattedyr hjerner og tromle dem for at få sektioner parallelt med det kortikale ark. Vi yderligere fremhæve valgt histokemiske og immunhistokemiske metoder til at behandle disse fladtrykt tangential sektioner for at visualisere kortikale moduler. I den somatosensoriske cortex af forskellige pattedyr udføre vi cytokrom oxidase histokemi for at afsløre krop kort eller kortikale moduler som repræsenterer forskellige dele af kroppen af dyret. I den mediale entorhinal cortex, et område, hvor gitterceller genereres, udnytter vi immunhistokemiske metoder for at fremhæve moduler af genetisk bestemt neuroner, der er arrangeret i et gitter-mønster i det kortikale ark på tværs af flere arter. Samlet set, vi leverer en ramme til at isolere og forberede layer-wise fladtrykt kortikale sektioner og visualisere kortikale moduler bruger histokemiske og immunhistokemiske metoder i en bred vifte af pattedyr hjerner.

Vi opnåede fladtrykte kortikale sektioner af somatosensoriske cortex i en række forskellige hjerner, og behandlet dem for cytokrom oxidase histokemi at visualisere de somatotopic moduler repræsenterer forskellige kropsdele. Denne komparative fremgangsmåde giver mulighed for at studere de evolutionære styrker denne figur cortex, f.eks., viser meget bevaret repræsentation af mystacial vibrissae i gnavere og hareagtige som Tønder21 (<strong class="xfig...

Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

Visualization of Cortical Modules in Flattened Mammalian Cortices

I denne artikel beskrives en detaljeret metode til at opnå fladtrykte tangential sektioner fra pattedyr cortex og visualisere kortikale moduler bruger histokemiske og immunhistokemiske metoder.

Visualisering af kortikale moduler i fladtrykte pattedyr cortex

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

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