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

Vi takker Jenny Gringer Richards for hendes grundige arbejde med at validere vores kontrolemner, hvorfra vi genererede kontrol-iPSC'erne. Dette arbejde blev støttet af NIH T32 MH019113 (til D.A.M. og K.A.K.), Nellie Ball Trust (til T.H.W. og A.J.W.), NIH R01 MH111578 (til V.A.M. og J.A.W.), NIH KL2 TR002536 (til A.J.W.) og Roy J. Carver Charitable Trust (til V.A.M., J.A.W. og A.J.W.). Figurerne blev skabt med BioRender.com.

Humanforsøgsforskningen blev godkendt under University of Iowa Institutional Review Board godkendelsesnummer 201805995 og University of Iowa Human Pluripotent Stem Cell Committee godkendelsesnummer 2017-02. Hudbiopsier blev indhentet fra forsøgspersonerne efter indhentning af skriftligt informeret samtykke. Fibroblasterne blev dyrket i DMEM med 15% føtalt bovint serum (FBS) og 1% MEM-ikke-essentiel aminosyreopløsning ved 37 °C og 5% CO2. Fibroblaster blev omprogrammeret ved hjælp af et episomalt omprogra...

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Evnen til at modellere menneskelig cerebellær udvikling in vitro er vigtig for sygdomsmodellering samt fremme forståelsen af normal hjerneudvikling. Mindre komplicerede og omkostningseffektive protokoller skaber flere muligheder for replikerbar datagenerering og bred implementering på tværs af flere videnskabelige laboratorier. En cerebellar differentieringsprotokol er beskrevet her ved hjælp af en modificeret metode til generering af EB'er, der ikke kræver enzymer eller dissociationsmidler, ved hjælp af ...

At forstå menneskelig cerebellær udvikling og de kritiske tidsvinduer i denne proces er vigtig ikke kun for at afkode de mulige årsager til neuroudviklingsforstyrrelser, men også for at identificere nye mål for terapeutisk intervention. Modellering af menneskelig cerebellær udvikling in vitro har været udfordrende, men over tid er der opstået mange protokoller, der differentierer menneskelige embryonale stamceller (hESC'er) eller iPSC'er med cerebellære afstamningsskæbner 1,2,3,4,5,6,7,8 . Desuden er det vigtigt at udvikle protokoller, der genererer reproducerbare resultater, er relativt enkle (for at reducere fejl) og ikke er tunge på monetære omkostninger.
De første protokoller for cerebellær differentiering blev genereret fra 2D-kulturer fra belagte embryoidlegemer (EB'er), hvilket inducerede cerebellær skæbne med forskellige vækstfaktorer svarende til in vivo-udvikling, herunder WNT, BMP'er og FGF'er 1,9. Nyere offentliggjorte protokoller inducerede differentiering primært i 3D-organoidkultur med FGF2 og insulin, efterfulgt af FGF19 og SDF1 til rhombiske læbelignende strukturer 3,4 eller anvendte en kombination af FGF2, FGF4 og FGF85. Begge cerebellære organoide induktionsmetoder resulterede i lignende 3D cerebellar organoider, da begge protokoller rapporterede lignende cerebellære markørekspression på identiske tidspunkter. Holmes og Heine udvidede deres 3D-protokol5 for at vise, at 2D cerebellære celler kan genereres fra hESC'er og iPSC'er, der starter som 3D-aggregater. Derudover viste Silva et al.7, at celler, der repræsenterer modne cerebellære neuroner i 2D, kan genereres med en lignende tilgang til Holmes og Heine ved hjælp af et andet tidspunkt for at skifte fra 3D til 2D og forlænge vækst- og modningstiden.
Den nuværende protokol inducerer cerebellær skæbne i feederfrie iPSC'er ved at generere fritsvævende embryoidlegemer (EB'er) ved hjælp af insulin og FGF2 og derefter plettere EB'erne på PLO / lamininbelagte retter på dag 14 til 2D-vækst og differentiering. På dag 35 opnås celler med cerebellær identitet. Evnen til at rekapitulere de tidlige stadier af cerebellær udvikling, især i et 2D-miljø, gør det muligt for forskere at besvare specifikke spørgsmål, der kræver eksperimenter med en monolagsstruktur. Denne protokol er også modtagelig for yderligere modifikationer såsom mikropatternede overflader, aksonale udvækstassays og cellesortering for at berige de ønskede cellepopulationer.

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			<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>
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		<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>
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			<td>Chemically defined lipid concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
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			<td>Chroman 1</td>
			<td>Cayman</td>
			<td>34681</td>
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			<td>Class II, Type A2, Biological safety Cabinet</td>
			<td>NuAire, Inc.</td>
			<td>NU-540-600</td>
			<td>Hood, UV light</td>
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		<tr>
			<td>Costar 24-well plate, TC treated</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
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		<tr>
			<td>Costar 6-well plate, TC treated</td>
			<td>Corning</td>
			<td>3516</td>
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		<tr>
			<td>DAPI solution</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
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		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
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		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
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		<tr>
			<td>DMSO (Dimethly sulfoxide)</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
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		<tr>
			<td>DPBS+/+</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
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		<tr>
			<td>Emricasan</td>
			<td>Cayman</td>
			<td>22204</td>
			<td></td>
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		<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>
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		<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

Den præcise og rettidige udvikling af lillehjernen er afgørende ikke kun for nøjagtig motorkoordinering og balance, men også for kognition. Derudover har forstyrrelser i cerebellar udvikling været impliceret i mange neurodevelopmental lidelser, herunder autisme, opmærksomhedsunderskud-hyperaktivitetsforstyrrelse (ADHD) og skizofreni. Undersøgelser af cerebellær udvikling hos mennesker har tidligere kun været mulige gennem post mortem-undersøgelser eller neuroimaging, men disse metoder er ikke tilstrækkelige til at forstå de molekylære og cellulære ændringer, der forekommer in vivo under tidlig udvikling, hvilket er når mange neuroudviklingsforstyrrelser stammer fra. Fremkomsten af teknikker til at generere menneskeskabte pluripotente stamceller (iPSC'er) fra somatiske celler og evnen til yderligere at differentiere iPSC'er til neuroner har banet vejen for in vitro-modellering af tidlig hjerneudvikling. Denne undersøgelse giver forenklede trin i retning af at generere cerebellære celler til applikationer, der kræver en 2-dimensionel (2D) monolagsstruktur. Cerebellarceller, der repræsenterer tidlige udviklingsstadier, stammer fra humane iPSC'er via følgende trin: først fremstilles embryoidlegemer i 3-dimensionel (3D) kultur, derefter behandles de med FGF2 og insulin for at fremme cerebellar skæbnespecifikation, og endelig differentieres de terminalt som et monolag på poly-l-ornithin (PLO) / lamininbelagte substrater. Ved 35 dages differentiering udtrykker iPSC-afledte cerebellære cellekulturer cerebellære markører, herunder ATOH1, PTF1α, PAX6 og KIRREL2, hvilket tyder på, at denne protokol genererer glutamatergiske og GABAergiske cerebellære neuronale forstadier samt Purkinje-celleforfædre. Desuden viser de differentierede celler tydelig neuronal morfologi og er positive for immunfluorescensmarkører for neuronal identitet såsom TUBB3. Disse celler udtrykker aksonale vejledningsmolekyler, herunder semaphorin-4C, plexin-B2 og neuropilin-1, og kan tjene som en model til undersøgelse af de molekylære mekanismer for neuritudvækst og synaptisk forbindelse. Denne metode genererer humane cerebellære neuroner, der er nyttige til downstream-applikationer, herunder genekspression, fysiologiske og morfologiske undersøgelser, der kræver 2D-monolagsformater.

Oversigt over 3D til 2D cerebellar differentiering Cerebellar celler genereres startende fra iPSC'er. Figur 1A viser den overordnede arbejdsgang og tilføjelsen af hovedkomponenter til differentiering. På dag 0 fremstilles EB'er ved forsigtigt at løfte iPSC-kolonierne (figur 1B) ved hjælp af en trukket glaspipette i CDM indeholdende SB431542 og Y-27632 og placeres i plader med ultralav fastgørelse. FGF2 tilføjes på dag 2. På dag 7 ænd...

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

Den nuværende protokol forklarer dannelsen af et 2D-monolag af cerebellære celler fra inducerede pluripotente stamceller til undersøgelse af de tidlige stadier af cerebellær udvikling.

Modellering af menneskelig cerebellær udvikling in vitro i 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, ...

Denne metode kan hjælpe med at studere tidlig menneskelig cerebellar udvikling og hvordan det kan ændres i neuropsykiatriske lidelser. Den vigtigste fordel ved denne metode er at generere udviklingsmæssigt tidlige humane cerebellære celler i en 2D-lagstruktur uden at udføre komplicerede trin. Det er også omkostningseffektivt.At lave trækpipetter kan være vanskeligt i starten, men med øvelse og tid bliver det lettere at trække pipetterne på en måde, der bliver lettere at arbejde med. Begynd det eksperimentelle præparat ved at trække en 22,9 centimeter Pasteur-pipette cirka to centimeter under halsen over bunsenbrænderen for at skabe to glaspipetter. Den tyndere side vil være cirka fire centimeter kortere end den anden.Bøj spidserne på den trukne side på flammen for at skabe en glat R, placer derefter de trukne pipetter i autoklavehylstre og steriliser dem i en autoklave. På dag nul tilsættes en milliliter cerebellært differentieringsmedium suppleret med 10 mikromolære Y-27632 og SB-431542 til hver brønd i en seks-brønds ultra-lav fastgørelse eller ULA-plade og læg den i inkubatoren, indtil de løftede kolonier er klar til at blive tilføjet til brøndene. Rengør de differentierede celler ved hjælp af en trukket glaspipette.Aspirer mediet og tilsæt en millimeter iPSC-passaging-opløsning for hver 35 millimeter skål. Efter tre minutters inkubation ved 37 grader Celsius, aspirer mediet og tilsæt to millimeter cerebellært differentieringsmedium suppleret med 10 mikromolære Y-27632 og SB-431542 Under 4x forstørrelse af et transmitteret lys omvendt mikroskop, løft kolonierne ved hjælp af den bøjede kant af den trukne glaspipette. Når hver koloni er løftet, overføres alle kolonierne forsigtigt til en brønd i ULA-pladen med seks brønde ved hjælp af en 10 milliliter serologisk pipette.Gentag denne proces for hver iPSC-plade, og inkuber cellerne ved 37 grader Celsius med 5% kuldioxid. På dag to tilsættes FGF2 til hver brønd for at justere en endelig koncentration på 50 nanogram pr. Milliliter. På dag syv ændres en tredjedel af mediet ved at aspirere en milliliter brugt medium og erstatte det med en milliliter frisk cerebellar differentieringsmedium.Inkuber embryoidlegemerne i syv dage. På dag 14 hvirvles pladen for at samle alle embryoidlegemerne i midten af pladen. Vip derefter pladen og opsug alt det brugte medium ved hjælp af en 1000 mikroliter pipetter fra kanten.Når den mellemstore mængde falder, skal du langsomt lægge pladen fladt og fortsætte med at aspirere, hvilket efterlader nok medium til at undgå at trække embryoidlegemerne ud. Derefter tilsættes tre milliliter frisk cerebellar differentieringsmedium suppleret med 10 mikromolære Y-27632. Overfør derefter embryoidlegemerne ved hjælp af en 10 ml serologisk pipette til en Poly-L-Ornithin Laminin-belagt skål.På dag 15 skal du aspirere mediet og erstatte det med frisk cerebellært differentieringsmedium. På dag nul blev iPSC-kolonier renset og løftet til cerebellært differentieringsmedium indeholdende SB-431542 og Y-27632 og blev anbragt i ultralave fastgørelsesplader til generering af embryoide kroppe. Tilføjelsen af FGF2 på dag to og en ændring på 1/3 medium på dag syv resulterede i, at embryoide kroppe viste udvidelse på den 14. dag.Celler begyndte at migrere udad fra embryoidlegemerne langs den belagte overflade på den 15. dag. Den fuldstændige ændring af mediet til cerebellar modningsmedium fra den 21. dag resulterede i et monolag af celler med neuronallignende morfologi og kompleksitet på den 35. dag. Genekspressionsanalysen på dag 35 afslørede, at cellerne udtrykker tidlige cerebellære stammarkører såsom glutamaterge forfædre, ATOH1, GABAergiske forfædre, PTF1 alfa og Purkinje stamfadercellemarkører KIRREL2 og SKOR2.Derudover blev ekspressionen af rhombisk læbemarkør OTX2 og for det meste forreste neuronspecifik SIX3 også observeret. Den immunfluorescerende mærkning af cellerne høstet på den 35. dag viste positiv farvning for cerebellummarkørerne EN2 og PTF1 alfa, neuronal markør beta tubulin III og spredningsmarkøren KI67. Nuklear farvning med 4'6-diamidino-2-phenylindol eller DAPI viste cellekerner.For en vellykket differentiering er det afgørende at starte med sunde og udifferentierede iPSC-kolonier og bruge reagenser, der er så friskrekonstituerede som muligt.

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.

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

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.

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.

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.

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

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 nanosurgery af cerebellare Axoner<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.

Modeling astrocytom Patogenese<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; Brug Kortikal Astrocyter eller neurale stamceller fra Betinget, gensplejset Mus

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 Modellering af Kræft Neural Invasion: Dorsal rodganglie 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 ...

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.

Modeling Neuronal død og Degeneration i musen primære cerebellare granulet neuroner

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.

Modellering Charcot-Marie-Tooth sygdom In Vitro af Transfecting musen primære Motoneurons

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.

Vurdering af langvarig depression induktion i voksne cerebellar skiver

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