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

Agradecemos a Jenny Gringer Richards por su minucioso trabajo en la validación de nuestros sujetos de control, a partir de los cuales generamos las iPSC de control. Este trabajo fue apoyado por NIH T32 MH019113 (a D.A.M. y K.A.K.), el Nellie Ball Trust (a T.H.W. y A.J.W.), NIH R01 MH111578 (a V.A.M. y J.A.W.), NIH KL2 TR002536 (a A.J.W.) y Roy J. Carver Charitable Trust (a V.A.M., J.A.W. y A.J.W.). Las figuras fueron creadas con BioRender.com.

La investigación con sujetos humanos fue aprobada bajo el número de aprobación de la Junta de Revisión Institucional de la Universidad de Iowa número 201805995 y el número de aprobación del Comité de Células Madre Pluripotentes Humanas de la Universidad de Iowa 2017-02. Las biopsias de piel se obtuvieron de los sujetos después de obtener el consentimiento informado por escrito. Los fibroblastos se cultivaron en DMEM con 15% de suero fetal bovino (FBS) y 1% de solución de aminoácidos no esenciales MEM a 37 °C...

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La capacidad de modelar el desarrollo cerebeloso humano in vitro es importante para el modelado de enfermedades, así como para promover la comprensión del desarrollo normal del cerebro. Los protocolos menos complicados y rentables crean más oportunidades para la generación de datos replicables y una amplia implementación en múltiples laboratorios científicos. Aquí se describe un protocolo de diferenciación cerebelosa utilizando un método modificado de generación de EB que no requiere enzimas o agentes...

Comprender el desarrollo cerebeloso humano y las ventanas de tiempo críticas de este proceso es importante no solo para decodificar las posibles causas de los trastornos del desarrollo neurológico, sino también para identificar nuevos objetivos para la intervención terapéutica. Modelar el desarrollo cerebeloso humano in vitro ha sido un desafío, sin embargo, con el tiempo, han surgido muchos protocolos que diferencian las células madre embrionarias humanas (hESC) o iPSC con destinos de linaje cerebeloso 1,2,3,4,5,6,7,8 . Además, es importante desarrollar protocolos que generen resultados reproducibles, sean relativamente simples (para reducir el error) y no sean pesados en costos monetarios.
Los primeros protocolos para la diferenciación cerebelosa se generaron a partir de cultivos 2D a partir de cuerpos embrioides (EB) en placas, induciendo el destino cerebeloso con varios factores de crecimiento similares al desarrollo in vivo, incluyendo WNT, BMPs y FGFs 1,9. Los protocolos publicados más recientes indujeron la diferenciación principalmente en el cultivo de organoides 3D con FGF2 e insulina, seguido de FGF19 y SDF1 para estructuras rómbicas similares a labios3,4, o utilizaron una combinación de FGF2, FGF4 y FGF85. Ambos métodos de inducción de organoides cerebelosos dieron como resultado organoides cerebelosos 3D similares, ya que ambos protocolos informaron una expresión similar de marcadores cerebelosos en puntos de tiempo idénticos. Holmes y Heine ampliaron su protocolo 3D5 para mostrar que las células cerebelosas 2D se pueden generar a partir de hESCs e iPSCs, que comienzan como agregados 3D. Además, Silva et al.7 demostraron que las células que representan neuronas cerebelosas maduras en 2D pueden generarse con un enfoque similar al de Holmes y Heine, utilizando un punto de tiempo diferente para cambiar de 3D a 2D y extender el tiempo de crecimiento y maduración.
El protocolo actual induce el destino cerebeloso en iPSC libres de alimentador mediante la generación de cuerpos embrioides (EB) flotantes libres utilizando insulina y FGF2 y luego colocando los EB en platos recubiertos de OLP / laminina el día 14 para el crecimiento y diferenciación 2D. Para el día 35, se obtienen células con identidad cerebelosa. La capacidad de recapitular las primeras etapas del desarrollo cerebeloso, especialmente en un entorno 2D, permite a los investigadores responder preguntas específicas que requieren experimentos con una estructura monocapa. Este protocolo también es susceptible de modificaciones adicionales, como superficies con micropatrones, ensayos de crecimiento axonal y clasificación celular para enriquecer las poblaciones celulares deseadas.

<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

El desarrollo preciso y oportuno del cerebelo es crucial no solo para la coordinación y el equilibrio motor precisos, sino también para la cognición. Además, la interrupción en el desarrollo cerebeloso se ha implicado en muchos trastornos del desarrollo neurológico, como el autismo, el trastorno por déficit de atención con hiperactividad (TDAH) y la esquizofrenia. Las investigaciones del desarrollo cerebeloso en humanos anteriormente solo han sido posibles a través de estudios post-mortem o neuroimágenes, sin embargo, estos métodos no son suficientes para comprender los cambios moleculares y celulares que ocurren in vivo durante el desarrollo temprano, que es cuando se originan muchos trastornos del desarrollo neurológico. La aparición de técnicas para generar células madre pluripotentes inducidas por humanos (iPSC) a partir de células somáticas y la capacidad de volver a diferenciar aún más las iPSC en neuronas han allanado el camino para el modelado in vitro del desarrollo temprano del cerebro. El presente estudio proporciona pasos simplificados hacia la generación de células cerebelosas para aplicaciones que requieren una estructura monocapa de 2 dimensiones (2D). Las células cerebelosas que representan las primeras etapas del desarrollo se derivan de iPSC humanas a través de los siguientes pasos: primero, los cuerpos embrioides se hacen en cultivo tridimensional (3D), luego se tratan con FGF2 e insulina para promover la especificación del destino cerebeloso, y finalmente, se diferencian terminalmente como una monocapa en sustratos recubiertos de poli-l-ornitina (PLO) / laminina. A los 35 días de diferenciación, los cultivos de células cerebelosas derivadas de iPSC expresan marcadores cerebelosos que incluyen ATOH1, PTF1α, PAX6 y KIRREL2, lo que sugiere que este protocolo genera precursores neuronales cerebelosos glutamatérgicos y GABAérgicos, así como progenitores de células de Purkinje. Además, las células diferenciadas muestran una morfología neuronal distinta y son positivas para marcadores de inmunofluorescencia de identidad neuronal como TUBB3. Estas células expresan moléculas de guía axonal, incluyendo semaforina-4C, plexina-B2 y neuropilina-1, y podrían servir como modelo para investigar los mecanismos moleculares del crecimiento de neuritas y la conectividad sináptica. Este método genera neuronas cerebelosas humanas útiles para aplicaciones posteriores, incluyendo expresión génica, estudios fisiológicos y morfológicos que requieren formatos monocapa 2D.

Descripción general de la diferenciación cerebelosa 3D a 2D Las células cerebelosas se generan a partir de iPSCs. La figura 1A muestra el flujo de trabajo general y la adición de componentes principales para la diferenciación. El día 0, los EB se fabrican levantando suavemente las colonias iPSC (Figura 1B) utilizando una pipeta de vidrio tirado en CDM que contiene SB431542 e Y-27632 y se colocan en placas de fijación ultrabaja. FGF2 se...

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

El presente protocolo explica la generación de una monocapa 2D de células cerebelosas a partir de células madre pluripotentes inducidas para investigar las primeras etapas del desarrollo cerebeloso.

Modelado del desarrollo cerebeloso humano in vitro en estructura 2D

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

Este método puede ayudar a estudiar el desarrollo cerebeloso humano temprano y cómo podría alterarse en los trastornos neuropsiquiátricos. La ventaja clave de este método es generar células cerebelosas humanas tempranas en desarrollo en una estructura de capa 2D sin realizar pasos complicados. También es rentable.Hacer pipetas de tracción puede ser complicado al principio, pero con la práctica y el tiempo, es más fácil tirar de las pipetas de una manera que sea más fácil de trabajar. Comience la preparación experimental tirando de una pipeta Pasteur de 22,9 centímetros aproximadamente dos centímetros por debajo del cuello, por encima del quemador bunsen para crear dos pipetas de vidrio. El lado más delgado será aproximadamente cuatro centímetros más corto que el otro.Doble las puntas del lado tirado sobre la llama para crear una R suave, luego coloque las pipetas tiradas en las mangas del autoclave y esterilícelas en un autoclave. En el día cero, agregue un mililitro de medio de diferenciación cerebelosa suplementado con 10 micromolares Y-27632 y SB-431542 a cada pocillo de una placa de accesorio ultra bajo de seis pocillos, o ULA, y colóquelo en la incubadora hasta que las colonias levantadas estén listas para agregarse a los pocillos. Limpie las células diferenciadas con una pipeta de vidrio tirado.Aspire el medio y agregue un milímetro de solución de paso iPSC por cada plato de 35 milímetros. Después de tres minutos de incubación a 37 grados centígrados, aspirar el medio y añadir dos milímetros de medio de diferenciación cerebelosa suplementado con 10 micromolares Y-27632 y SB-431542 Bajo un aumento de 4x de un microscopio invertido de luz transmitida, levante las colonias utilizando el borde doblado de la pipeta de vidrio tirado. Una vez que se levante cada colonia, transfiera suavemente todas las colonias a un pocillo de la placa ULA de seis pocillos utilizando una pipeta serológica de 10 mililitros.Repita este proceso para cada placa iPSC e incube las células a 37 grados centígrados con 5% de dióxido de carbono. En el segundo día, agregue FGF2 a cada pocillo para ajustar una concentración final de 50 nanogramos por mililitro. En el séptimo día, cambie un tercio del medio aspirando un mililitro de medio gastado y reemplazándolo con un mililitro de medio de diferenciación cerebelosa fresca.Incubar los cuerpos embrioides durante siete días. El día 14, agita la placa para reunir todos los cuerpos embrioides en el centro de la placa. Luego incline la placa y aspire todo el medio gastado usando un pipetter de 1000 microlitros desde el borde.A medida que la cantidad media disminuye, coloque lentamente la placa plana y continúe aspirando, dejando suficiente medio para evitar sacar los cuerpos embrioides. A continuación, agregue tres mililitros de medio de diferenciación cerebelosa fresca suplementado con 10 micromolares Y-27632. Luego transfiera los cuerpos embrioides usando una pipeta serológica de 10 mililitros a un plato recubierto de poli-L-ornitina laminina.El día 15, aspirar el medio y reemplazarlo con un medio de diferenciación cerebelosa fresca. En el día cero, las colonias de iPSC se limpiaron y se elevaron en un medio de diferenciación cerebelosa que contenía SB-431542 e Y-27632 y se colocaron en placas de fijación ultra bajas para generar cuerpos embrioides. La adición de FGF2 en el segundo día y un cambio de 1/3 de medio en el séptimo día dio como resultado cuerpos embrioides que mostraron agrandamiento en el día 14.Las células comenzaron a migrar hacia afuera desde los cuerpos embrioides a lo largo de la superficie recubierta en el día 15. El cambio completo del medio al medio de maduración cerebelosa a partir del día 21 dio como resultado una monocapa de células con morfología y complejidad neuronales para el día 35. El análisis de expresión génica en el día 35 reveló que las células expresan marcadores progenitores cerebelosos tempranos como progenitores glutamatérgicos, ATOH1, progenitores GABAérgicos, PTF1 alfa y los marcadores celulares progenitores de Purkinje KIRREL2 y SKOR2.Además, también se observó la expresión del marcador rómbico del labio OTX2 y en su mayoría de la neurona anterior específica SIX3. El marcaje inmunofluorescente de las células recolectadas en el día 35 mostró tinción positiva para los marcadores cerebelales EN2 y PTF1 alfa, el marcador neuronal beta tubulina III y el marcador de proliferación KI67. La tinción nuclear con 4'6-diamidino-2-fenilindol, o DAPI, mostró núcleos celulares.Para una diferenciación exitosa, es crucial comenzar con colonias de iPSC sanas e indiferenciadas y usar reactivos que estén lo más recién reconstituidos posible.

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.

Sensación operante Buscando en el ratón

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

Investigación

Neurociencias

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.

Time-lapse de imágenes en vivo de la relación clonal con células progenitoras neurales en el cerebro anterior El desarrollo de pez cebra

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.

Modelado neuronal de señalización inmune de la migraña episódica y crónica mediante la depresión propagada 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.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

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 Nanocirugía del cerebelo Los axones<em&gt; En 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.

Modelado astrocitoma Patogénesis<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Uso cortical astrocitos o células troncales nerviosas de ratones condicionales, Genéticamente Manipulados

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.

En Vitro Modelado de canceroso Neural Invasion: Modelo del ganglio de la raíz dorsal

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.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

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.

Modelado enfermedad del Charcot-Marie-diente In Vitro por transferencia primaria Motoneurons de ratón

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.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

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