Name: modeling human cerebellar development in vitro in 2d structure
Uploaded: 2022-09-16T14:30:00
Description: Watch this Scientific Journal Video about Modeling Human Cerebellar Development In Vitro in 2D Structure at JoVE.com

Wir danken Jenny Gringer Richards für ihre gründliche Arbeit bei der Validierung unserer Kontrollpersonen, aus der wir die Kontroll-iPSCs generiert haben. Diese Arbeit wurde unterstützt von NIH T32 MH019113 (an D.A.M. und K.A.K.), dem Nellie Ball Trust (an T.H.W. und A.J.W.), NIH R01 MH111578 (an V.A.M. und J.A.W.), NIH KL2 TR002536 (an A.J.W.) und dem Roy J. Carver Charitable Trust (an V.A.M., J.A.W. und A.J.W.). Die Figuren wurden mit BioRender.com erstellt.

Die Forschung am Menschen wurde unter der Genehmigungsnummer 201805995 des University of Iowa Institutional Review Board und der Zulassungsnummer 2017-02 des Human Pluripotent Stem Cell Committee der University of Iowa genehmigt. Hautbiopsien wurden von den Probanden nach schriftlicher Einverständniserklärung erhalten. Die Fibroblasten wurden in DMEM mit 15% fetalem Rinderserum (FBS) und 1% MEM-nicht-essentieller Aminosäurenlösung bei 37 °C und 5%CO2 kultiviert. Die Fibroblasten wurden mit einem episomale...

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Die Fähigkeit, die menschliche Kleinhirnentwicklung in vitro zu modellieren, ist wichtig für die Krankheitsmodellierung sowie für das Verständnis der normalen Gehirnentwicklung. Weniger komplizierte und kostengünstige Protokolle schaffen mehr Möglichkeiten für replizierbare Datengenerierung und breite Implementierung in mehreren wissenschaftlichen Labors. Ein Kleinhirndifferenzierungsprotokoll wird hier unter Verwendung einer modifizierten Methode zur Erzeugung von EBs beschrieben, die keine Enzyme oder D...

Das Verständnis der menschlichen Kleinhirnentwicklung und der kritischen Zeitfenster dieses Prozesses ist nicht nur wichtig, um die möglichen Ursachen neurologischer Entwicklungsstörungen zu entschlüsseln, sondern auch um neue Ziele für therapeutische Interventionen zu identifizieren. Die Modellierung der menschlichen Kleinhirnentwicklung in vitro war eine Herausforderung, doch im Laufe der Zeit sind viele Protokolle zur Differenzierung menschlicher embryonaler Stammzellen (hES-Zellen) oder iPS-Zellen mit Kleinhirnlinienschicksalen entstanden 1,2,3,4,5,6,7,8 . Darüber hinaus ist es wichtig, Protokolle zu entwickeln, die reproduzierbare Ergebnisse liefern, relativ einfach sind (um Fehler zu reduzieren) und keine hohen monetären Kosten verursachen.
Die ersten Protokolle für die Kleinhirndifferenzierung wurden aus 2D-Kulturen aus plattierten embryoiden Körpern (EBs) generiert, die das Kleinhirnschicksal mit verschiedenen Wachstumsfaktoren ähnlich der In-vivo-Entwicklung induzierten, einschließlich WNT, BMPs und FGFs 1,9. Neuere veröffentlichte Protokolle induzierten eine Differenzierung hauptsächlich in der 3D-Organoidkultur mit FGF2 und Insulin, gefolgt von FGF19 und SDF1 für rhombische lippenartige Strukturen3,4 oder verwendeten eine Kombination aus FGF2, FGF4 und FGF85. Beide Kleinhirn-Organoid-Induktionsmethoden führten zu ähnlichen 3D-Kleinhirn-Organoiden, da beide Protokolle eine ähnliche Kleinhirnmarkerexpression zu identischen Zeitpunkten berichteten. Holmes und Heine erweiterten ihr 3D-Protokoll5, um zu zeigen, dass 2D-Kleinhirnzellen aus hES-Zellen und iPS-Zellen erzeugt werden können, die als 3D-Aggregate beginnen. Darüber hinaus zeigten Silva et al.7, dass Zellen, die reife Kleinhirnneuronen in 2D repräsentieren, mit einem ähnlichen Ansatz wie Holmes und Heine erzeugt werden können, wobei ein anderer Zeitpunkt für den Wechsel von 3D zu 2D verwendet wird und die Wachstums- und Reifezeit verlängert wird.
Das aktuelle Protokoll induziert das Kleinhirnschicksal in feederfreien iPS-Zellen, indem es frei schwebende embryoide Körper (EBs) mit Insulin und FGF2 erzeugt und dann die EBs am Tag 14 auf PLO / Laminin-beschichtetem Geschirr für 2D-Wachstum und Differenzierung plattiert. Am Tag 35 werden Zellen mit Kleinhirnidentität erhalten. Die Fähigkeit, die frühen Stadien der Kleinhirnentwicklung, insbesondere in einer 2D-Umgebung, zu rekapitulieren, ermöglicht es Forschern, spezifische Fragen zu beantworten, die Experimente mit einer Monoschichtstruktur erfordern. Dieses Protokoll ist auch für weitere Modifikationen wie mikrostrukturierte Oberflächen, axonale Auswuchsassays und Zellsortierung zugänglich, um die gewünschten Zellpopulationen anzureichern.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26D</td>
			<td></td>
		</tr>
		<tr>
			<td>1-thio-glycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26B</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Filter Unit, 0.2 &#181;m aPES, 50 mm Dia</td>
			<td>Fisher Scientific</td>
			<td>FB12566502</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Easy Grip Tissue Cluture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>276885</td>
			<td>Nucleofector</td>
		</tr>
		<tr>
			<td>5 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-25D</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Easy Grip Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well ultra-low attachment plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Cytiva</td>
			<td>SH30529.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Cayman</td>
			<td>34681</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type A2, Biological safety Cabinet</td>
			<td>NuAire, Inc.</td>
			<td>NU-540-600</td>
			<td>Hood, UV light</td>
		</tr>
		<tr>
			<td>Costar 24-well plate, TC treated</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well plate, TC treated</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethly sulfoxide)</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS+/+</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Cayman</td>
			<td>22204</td>
			<td></td>
		</tr>
		<tr>
			<td>Epi5 episomal iPSC reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A15960</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8-Flex</td>
			<td>Gibco</td>
			<td>A2858501</td>
			<td>PSC medium with heat-stable FGF2</td>
		</tr>
		<tr>
			<td>EVOS XL Core Imaging system</td>
			<td>Life Technologies</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum - Premium Select</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanine-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell VIOS 160i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Anti-EN2, mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-293311</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-Ki67/MKI67, rabbit</td>
			<td>R&#38;D Systems</td>
			<td>MAB7617</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-PTF1a, rabbit</td>
			<td>Novus Biologicals</td>
			<td>NBP2-98726</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-TUBB3, mouse</td>
			<td>Biolegend</td>
			<td>801213</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Gibco</td>
			<td>12585</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Labnet International</td>
			<td>C1310</td>
			<td>Benchtop mini centrifuge</td>
		</tr>
		<tr>
			<td>Monarch RNA Cleanup Kit (50 &#181;g)</td>
			<td>New England BioLabs</td>
			<td>T2040</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>Monarch Total RNA Miniprep Kit</td>
			<td>New England BioLabs</td>
			<td>T2010</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamine supplement</td>
			<td>Sigma</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PLO)</td>
			<td>Sigma</td>
			<td>3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma</td>
			<td>54317</td>
			<td></td>
		</tr>
		<tr>
			<td>See through self-sealable pouches</td>
			<td>Steriking</td>
			<td>SS-T2 (90x250)</td>
			<td>Autoclave pouches</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, sterile, PES 0.22 &#181;m, 30 mm Dia</td>
			<td>Research Products International</td>
			<td>256131</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-ISRIB</td>
			<td>Cayman</td>
			<td>16258</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>Phenol and guanidine isothiocyanate</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x)</td>
			<td>Gibco</td>
			<td>12604039</td>
			<td>Cell dissociation reagent&#160;</td>
		</tr>
		<tr>
			<td>Vapor pressure osmometer</td>
			<td>Wescor, Inc.</td>
			<td>Model 5520</td>
			<td>Osmometer</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biogems</td>
			<td>1293823</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling human cerebellar development in vitro in 2d structure

Die präzise und rechtzeitige Entwicklung des Kleinhirns ist nicht nur für eine genaue motorische Koordination und Balance, sondern auch für die Kognition von entscheidender Bedeutung. Darüber hinaus wurde eine Störung der Kleinhirnentwicklung mit vielen neurologischen Entwicklungsstörungen in Verbindung gebracht, darunter Autismus, Aufmerksamkeitsdefizit-Hyperaktivitätsstörung (ADHS) und Schizophrenie. Untersuchungen der Kleinhirnentwicklung beim Menschen waren bisher nur durch Post-mortem-Studien oder Neuroimaging möglich, doch reichen diese Methoden nicht aus, um die molekularen und zellulären Veränderungen in vivo während der frühen Entwicklung zu verstehen, wenn viele neurologische Entwicklungsstörungen entstehen. Das Aufkommen von Techniken zur Erzeugung von humaninduzierten pluripotenten Stammzellen (iPS-Zellen) aus somatischen Zellen und die Fähigkeit, iPS-Zellen weiter in Neuronen umzudifferenzieren, haben den Weg für die In-vitro-Modellierung der frühen Gehirnentwicklung geebnet. Die vorliegende Studie bietet vereinfachte Schritte zur Erzeugung von Kleinhirnzellen für Anwendungen, die eine 2-dimensionale (2D) Monoschichtstruktur erfordern. Kleinhirnzellen, die frühe Entwicklungsstadien repräsentieren, werden aus menschlichen iPS-Zellen über die folgenden Schritte abgeleitet: Zuerst werden embryoide Körper in 3-dimensionaler (3D) Kultur hergestellt, dann werden sie mit FGF2 und Insulin behandelt, um die Spezifikation des Kleinhirnschicksals zu fördern, und schließlich werden sie terminalerweise als Monoschicht auf Poly-L-Ornithin (PLO) / Laminin-beschichteten Substraten differenziert. Nach 35 Tagen Differenzierung exprimieren iPSC-abgeleitete Kleinhirnzellkulturen Kleinhirnmarker wie ATOH1, PTF1α, PAX6 und KIRREL2, was darauf hindeutet, dass dieses Protokoll glutamaterge und GABAerge zerebelläre neuronale Vorläufer sowie Purkinje-Zellvorläufer erzeugt. Darüber hinaus zeigen die differenzierten Zellen eine ausgeprägte neuronale Morphologie und sind positiv für Immunfluoreszenzmarker der neuronalen Identität wie TUBB3. Diese Zellen exprimieren axonale Leitmoleküle, einschließlich Semaphorin-4C, Plexin-B2 und Neuropilin-1, und könnten als Modell für die Untersuchung der molekularen Mechanismen des Neuritenwachstums und der synaptischen Konnektivität dienen. Diese Methode erzeugt menschliche Kleinhirnneuronen, die für nachgelagerte Anwendungen nützlich sind, einschließlich Genexpression, physiologischer und morphologischer Studien, die 2D-Monoschichtformate erfordern.

Überblick über die 3D-zu-2D-Kleinhirndifferenzierung Kleinhirnzellen werden ausgehend von iPS-Zellen erzeugt. Abbildung 1A zeigt den gesamten Workflow und die Hinzufügung wichtiger Komponenten zur Unterscheidung. Am Tag 0 werden EBs hergestellt, indem die iPSC-Kolonien (Abbildung 1B) vorsichtig mit einer gezogenen Glaspipette aus CDM mit SB431542 und Y-27632 angehoben und in extrem niedrig befestigte Platten gelegt werden. FGF2 wird an Tag...

Watch this Scientific Journal Video about Modeling Human Cerebellar Development In Vitro in 2D Structure at JoVE.com

Modeling Human Cerebellar Development In Vitro in 2D Structure

Das vorliegende Protokoll erklärt die Erzeugung einer 2D-Monoschicht von Kleinhirnzellen aus induzierten pluripotenten Stammzellen zur Untersuchung der frühen Stadien der Kleinhirnentwicklung.

Modellierung der menschlichen Kleinhirnentwicklung in vitro in 2D-Struktur

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, ...

Diese Methode kann bei der Untersuchung der frühen menschlichen Kleinhirnentwicklung helfen und wie sie bei neuropsychiatrischen Störungen verändert werden kann. Der entscheidende Vorteil dieser Methode besteht darin, entwicklungsfrühe menschliche Kleinhirnzellen in einer 2D-Schichtstruktur zu erzeugen, ohne komplizierte Schritte durchzuführen. Es ist auch kosteneffizient.Die Herstellung von Zugpipetten kann am Anfang schwierig sein, aber mit Übung und Zeit wird es einfacher, die Pipetten auf eine Weise zu ziehen, mit der man leichter arbeiten kann. Beginnen Sie die experimentelle Vorbereitung, indem Sie eine 22,9 Zentimeter große Pasteur-Pipette etwa zwei Zentimeter unter dem Hals über den Bunsenbrenner ziehen, um zwei Glaspipetten herzustellen. Die dünnere Seite wird etwa vier Zentimeter kürzer sein als die andere.Biegen Sie die Spitzen der gezogenen Seite auf der Flamme, um ein glattes R zu erzeugen, legen Sie dann die gezogenen Pipetten in Autoklavhülsen und sterilisieren Sie sie in einem Autoklaven. Am Tag Null fügen Sie einen Milliliter Kleinhirndifferenzierungsmedium, ergänzt mit 10 Mikromolaren Y-27632 und SB-431542, zu jeder Vertiefung einer Sechs-Well-Ultra-Low-Attachment- oder ULA-Platte hinzu und legen Sie es in den Inkubator, bis die angehobenen Kolonien bereit sind, in die Vertiefungen gegeben zu werden. Reinigen Sie die differenzierten Zellen mit einer gezogenen Glaspipette.Das Medium absaugen und einen Millimeter iPSC-Passaging-Lösung für jede 35-Millimeter-Schale hinzufügen. Nach drei Minuten Inkubation bei 37 Grad Celsius das Medium aspirieren und zwei Millimeter Kleinhirndifferenzierungsmedium hinzufügen, ergänzt mit 10 Mikromolaren Y-27632 und SB-431542 Unter 4-facher Vergrößerung eines Durchlicht-Inversmikroskops die Kolonien mit dem gebogenen Rand der gezogenen Glaspipette anheben. Sobald jede Kolonie angehoben ist, übertragen Sie alle Kolonien vorsichtig mit einer 10-Milliliter-serologischen Pipette in eine Vertiefung der ULA-Platte mit sechs Vertiefungen.Wiederholen Sie diesen Vorgang für jede iPSC-Platte und inkubieren Sie die Zellen bei 37 Grad Celsius mit 5% Kohlendioxid. Am zweiten Tag fügen Sie FGF2 zu jeder Vertiefung hinzu, um eine Endkonzentration von 50 Nanogramm pro Milliliter einzustellen. Am siebten Tag wechseln Sie ein Drittel des Mediums, indem Sie einen Milliliter verbrauchtes Medium absaugen und durch einen Milliliter frisches Kleinhirndifferenzierungsmedium ersetzen.Inkubieren Sie die embryoiden Körper für sieben Tage. Schwenken Sie am 14. Tag die Platte, um alle embryoiden Körper in der Mitte der Platte zu sammeln. Dann kippen Sie die Platte und saugen Sie das gesamte verbrauchte Medium mit einem 1000-Mikroliter-Pipetter vom Rand ab.Wenn die mittlere Menge abnimmt, legen Sie die Platte langsam flach und saugen Sie weiter ab, wobei genügend Medium übrig bleibt, um das Herausziehen der embryoiden Körper zu vermeiden. Als nächstes fügen Sie drei Milliliter frisches Kleinhirndifferenzierungsmedium hinzu, ergänzt mit 10 Mikromolar Y-27632. Anschließend werden die embryoiden Körper mit einer 10-Milliliter-serologischen Pipette in eine mit Poly-L-Ornithin-Laminin beschichtete Schale überführt.Am Tag 15 saugen Sie das Medium ab und ersetzen Sie es durch ein frisches Kleinhirndifferenzierungsmedium. Am Tag Null wurden iPSC-Kolonien gereinigt und in ein Kleinhirndifferenzierungsmedium mit SB-431542 und Y-27632 gehoben und in extrem niedrige Befestigungsplatten zur Erzeugung embryoider Körper gelegt. Die Zugabe von FGF2 am zweiten Tag und eine Veränderung von 1/3 Medium am siebten Tag führten dazu, dass embryoide Körper am 14. Tag eine Vergrößerung zeigten.Die Zellen begannen am 15. Tag von den embryoiden Körpern entlang der beschichteten Oberfläche nach außen zu wandern. Der vollständige Wechsel des Mediums zum Kleinhirnreifungsmedium ab dem 21. Tag führte am 35. Tag zu einer Monoschicht von Zellen mit neuronaler Morphologie und Komplexität. Die Genexpressionsanalyse am Tag 35 ergab, dass die Zellen frühe zerebelläre Vorläufermarker wie glutamaterge Vorläufer, ATOH1, GABAerge Vorläuferzellen, PTF1 alpha und die Purkinje-Vorläuferzellmarker KIRREL2 und SKOR2 exprimieren.Darüber hinaus wurde auch die Expression des rhombischen Lippenmarkers OTX2 und des meist vorderen Neuronen-spezifischen SIX3 beobachtet. Die immunfluoreszierende Markierung der am 35. Tag geernteten Zellen zeigte eine positive Färbung für die Kleinhirnmarker EN2 und PTF1 alpha, den neuronalen Marker Beta Tubulin III und den Proliferationsmarker KI67. Die Kernfärbung mit 4'6-Diamidino-2-phenylindol oder DAPI zeigte Zellkerne.Für eine erfolgreiche Differenzierung ist es entscheidend, mit gesunden und undifferenzierten iPSC-Kolonien zu beginnen und möglichst frisch rekonstituierte Reagenzien zu verwenden.

modeling-human-cerebellar-development-in-vitro-in-2d-structure

Research

JoVE Journal

Neuroscience

Modeling Human Cerebellar Development In Vitro in 2D Structure

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operante Sensation Seeking in der Maus

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Forschung

Neurowissenschaften

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Zeitraffer-Live-Imaging von klonal verbundenen neuronalen Vorläuferzellen in der Entwicklung von Zebrafisch Vorderhirnneuronen

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Modeling Neural Immune Signalisierung von episodischen und chronischen Migräne mit Spreading Depression In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Laser-Nanochirurgie der Kleinhirn-Axone<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modellierung Astrozytom Pathogenese<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt; Verwenden kortikale Astrozyten oder neuralen Stammzellen aus dem Bedingten, gentechnisch veränderte Mäuse

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In - vitro - Modellierung von Cancerous Neural Invasion: The Dorsalwurzelganglion Modell

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellierung von neuronalen Tod und Degeneration in Maus primären zerebelläre Granulat Neuronen

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Charcot-Marie-Zahn Krankheit In Vitro Modellierung durch Transfecting Maus primären Motoneurone

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Bewertung der Langzeit-Depressionsinduktion bei erwachsenen Cerebellarscheiben

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...