National Institute of Health Grant DA024009, DK046367 &#38; T32DA007027.

Todo el cuidado de los animales y los procedimientos experimentales se ajustaron y aprobados por el Cuidado de Animales institucional y el empleo Comisión de la Universidad de Virginia Commonwealth. 1. Preparación de cubreobjetos de vidrio estériles de poli-D-lisina y laminina revestida en placas de 24 pocillos <ol><li> Todos los procedimientos para el paso 1 se llevan a cabo en condiciones estériles; debajo de una campana, y con reactivos estériles. Cubreobjetos de vidrio y agua desionizada doble (ddH 2 O) deben ser esterilizados de antemano. Preparación de las placas puede hacerse hasta dos semanas antes del aislamiento neurona. </li><li> Bajo el capó, use pinzas estériles para colocar cubreobjetos de vidrio esterilizados en autoclave de 12 pocillos de una placa de 24 pocillos. </li><li> Prepare Stock poli-D-lisina antes de tiempo. Añadir 50 ml de ddH2O estéril a 5 mg de poli-D-lisina. Mezclar y almacenar en 3 ml alícuotas a -20 ° C. Descongele alícuotas antes de su uso. </li><li> Para obtener una concentración final de 1 ml de caldo de poli-D-lisina por 25 cm 2: pipeta de 80 lde stock poli-D-lisina en la parte superior de cada cubreobjetos de vidrio (2 cm 2). Deje que la solución reposar durante 10 min. </li><li> Retire poli-D-lisina de vacío y enjuague utilizando cubreobjetos de 3x con estéril ddH 2 O. </li><li> Permitir que las placas se sequen durante al menos 2 horas bajo el capó. </li><li> Las placas pueden ser almacenadas a 4 ° C o - 20 ° C antes de proceder al recubrimiento laminina. </li><li> Prepare Stock laminina antes de tiempo. Descongele laminina en hielo y mantener en hielo durante el uso. Diluir a una concentración de 50 g / ml; prestar atención a concentración mucho; cada lote será diferente. Dependiendo de la concentración mucho, se añadieron aproximadamente 20 ml de ddH 2 O a cada vial de laminina. Alícuota (5 ml) y se almacena a -80 ° C. Descongele alícuotas en hielo antes de su uso. </li><li> Cubreobjetos Abrigo con 5 mg / cm 2 laminina; Añadir 200 L de stock laminina en cada portaobjetos. </li><li> Incubar solución de laminina en cubreobjetos durante 1 hora. </li><li> Aspirar solución de laminina restante y enjuagar una vez con cubreobjetosddH 2 O. Evite raspar la superficie del cubreobjetos. </li><li> Placas se almacenan a 4 ° C durante un máximo de dos semanas. </li></ol> 2. Una preparación previa de la neurona Aislamiento Solutions <ol><li> Prepare una solución de Krebs (en mM: 118 NaCl, 4,6 KCl, 1,3 NaH 2 PO 4, 1,2 MgSO4, 25 NaHCO 3, 11 glucosa y 2,5 CaCl2). Place 13,79 g NaCl (FW 58,44), 0,686 g KCl (FW 74,55), 0,312 g NaH 2 PO 4 (FW 120), 0,289 g MgSO4 (FW 120,4), 4,20 g de NaHCO3 (FW 84,01), 3,96 g de glucosa (FW 180,2) y 0.555 g de CaCl2 (FW 111) en 2 l ddH 2 0. Enfriar a 4 ° C. </li><li> Preparar enjuague medios (medio F12 con suero bovino fetal al 10% (FBS) y antibiótico / antimicótico): A una botella ml de medio F12 500, añadir 50 ml de FBS y 5 ml de líquido 100x de antibiótico / antimicótico (Gibco). </li><li> Preparar los medios de comunicación neurona entérica (Neurobasal A los medios de comunicación con B-27, 2 mM de L-glutamina, 1% de FBS, 10 ng / ml, Neurotr derivado de la glíaFactor de ophic (GDNF), y de antibiótico / antimicótico líquido 100x). Para hacer un caldo GDNF, añadir 1 ml ddH2O GDNF a 10 mg y congelar de nuevo en 50 alícuotas, almacenar a -80 ° C. Para un ml frasco de medios de comunicación neurona completos 50, combinar ml Unos medios de comunicación 47.5 Neurobasal, 1 ml de B-27 (50x), 500 l de FBS, 500 mM de L-glutamina (Gibco) 50 de stock GDNF l l, 200 y 500 l antibióticos / Liquid 100x antimicótico. Los medios de comunicación se hace en pequeños lotes de 50 ml para garantizar la frescura de GDNF y para evitar la contaminación de grandes cantidades de esta mezcla de medios de comunicación celular caro. </li></ol> 3. Harvest Longitudinal Plexus (LMMP) Preparación del músculo / mientérico de ratones <ol><li> Éticos nacionales e institucionales apropiadas deben estar en su lugar antes de realizar experimentos con animales. Adultos ratones Swiss Webster que pesan más de 25 g (generalmente más de 8 semanas de edad) se encuentran en grupos de 6 antes de los experimentos. Los tejidos de dos ratones se utilizan para el aislamiento de las neuronas y células gliales entéricas ileales y tres ratones se utilizan paraaislamiento de las células del colon. </li><li> Prepare el área quirúrgica. Reunir pequeñas tijeras quirúrgicas, pinzas anguladas, hisopos de algodón, tres vasos de vidrio de 200 ml, y una varilla de vidrio / plástico (pincel). Asegúrese que todos los materiales son lavados y enjuagados antes de su uso para minimizar la contaminación de fondo. Este paso puede realizarse el día antes del experimento. </li><li> Coloque solución de Krebs en hielo y la burbuja con carbógeno (95% de oxígeno, 5% de CO 2) durante al menos 30 minutos para estabilizar el pH. </li><li> Encienda diversos equipos y los productos químicos calientes para estabilizar a temperaturas deseadas; baño de agua de calor (s) a 37 ° C, centrífuga fresco a 4 ° C, lugar de solución salina equilibrada de Hank (HBSS) y 0,5% de tripsina en baño de agua (37 ° C ). Medios de neuronas completas y enjuague medios de comunicación deben permanecer refrigerados. </li><li> Colocar 150 ml helada burbujea Krebs en tres vasos de vidrio de 200 ml etiquetados &#39;sucia&#39;, &#39;limpio&#39;, y &#39;LMMP&#39;. Vaso de burbuja etiqueta &#39;LMMP&#39; con carbógeno. </li><li> Tras la aprobación del comité de ética, la eutanasia mouse por dislocación cervical o la asfixia de CO 2. </li><li> Coloque el ratón en decúbito dorsal en la superficie quirúrgica, la piel limpia con un 70% EtOH, y levantar la piel del abdomen con unas pinzas. Con unas tijeras, la cavidad abdominal abierta y revelar órganos digestivos internos. </li><li> Retire la longitud del tracto gastrointestinal mediante el levantamiento de una sección de íleon y revelando su mesenterio. Snip través mesenterio con tijeras para quitar suavemente y desentrañar íleon y colon, con cuidado de no tirar de mesenterio del íleon / colon. </li><li> Después de que se descubrió la longitud completa del intestino grueso, eliminar el íleon cortando a través del intestino distal al estómago y proximal al intestino ciego Nota:. Este procedimiento también se puede realizar con el tejido del colon mediante la eliminación de los dos puntos de distal a la proximal al ciego hasta el ano. </li><li> Los tejidos se pueden dividir entre colon proximal y distal, y yeyuno, e íleon. El resto de este procedimiento se referirá al aislamiento del íleon; tque procedimiento es el mismo para el aislamiento de LMMP de colon. Coloque todo el íleon en un recipiente de vidrio con helado Krebs marcado &quot;sucio&quot;. </li><li> Crear una herramienta para limpiar el íleon; contundente una gran aguja de calibre 20 con una piedra de archivo y adjuntarlo a una jeringa grande (10 ml) que contiene helado Krebs. </li><li> Limpiar la materia fecal desde el íleon mediante el seccionamiento del íleon en tres o más piezas de gran tamaño. Eliminar una sección ileal del vaso de precipitados y colocar la aguja roma en el extremo de una pieza ileal. </li><li> Pase suavemente Krebs a través de la sección de intestino hasta que toda la materia fecal se elimina en un contenedor de residuos por separado. Coloque la sección de limpieza en el recipiente de Krebs marcada &quot;limpia&quot;. Repita hasta que se limpie toda íleon. </li><li> Para quitar el LMMP, corte el íleon en pequeños segmentos, aproximadamente 2-4 cm. Coloque un segmento de íleon en un plástico o varilla de vidrio; íleon debe encajar cómodamente, pero no estar suelto o tenso (~ 2 mm). Comienza la retirada del LMMP suavemente quitando trozos de mesenterio todavía adjuntared en el tracto gastrointestinal (GI) usando un fórceps. Evitar que el tracto GI de girar alrededor de la varilla por el fijar suavemente el tubo para la varilla con el pulgar. </li><li> Separar la LMMP del músculo circular subyacente; primera frotar suavemente el borde de las pinzas a lo largo de toda la línea donde se conecta el mesenterio, desde la parte superior a la parte inferior del segmento, creando suavemente un vacío en el músculo longitudinal. A continuación, se burlan suavemente el músculo longitudinal con un bastoncillo de algodón humedecido con Krebs. </li><li> Comience en la parte superior de la brecha en el músculo longitudinal y se burlan de distancia usando el trazo horizontal ligero mientras se aplica una presión muy ligera hasta que el músculo longitudinal empiece a separarse del músculo circular, hacer esto por toda la franja a lo largo del punto de fijación mesenterio. </li><li> Entonces comienzan suavemente para evitar el tubo gastrointestinal, moviéndose de arriba a abajo y de atrás como el músculo longitudinal se separa lentamente del músculo circular todo el camino alrededor del tubo. Cuando esté completo, elLMMP forma natural desprenderse del resto del tubo digestivo. </li><li> Coloque la tira fina resultante de músculo longitudinal en el vaso de precipitados con la indicación «LMMP &#39;. Repita este procedimiento para todos los segmentos. </li><li> Después de todas las franjas de LMMP presentadas proceden de un ratón, repita el procedimiento con los otros ratones que se utilizan. Enjuague los vasos &#39;sucias&#39; y &#39;limpio&#39; antes de volver a usarla. </li><li> Enjuague el LMMP 3x para eliminar la contaminación biológica. Llenar tres 2 ml tubos Eppendorf con helada Krebs. Coloque las tiras LMMP en el primer tubo y girar durante 30 segundos a 356 xg en una centrífuga enfriada a 4 ° C. Retirar con cuidado el sobrenadante con una pipeta y mover las tiras a LMMP siguiente tubo lleno de Krebs limpio. Repita este procedimiento para cada tubo restante. </li></ol> 4. Resumen LMMP <ol><li> Preparar la solución de la digestión mediante la colocación de 13 mg de colagenasa de tipo 2 y 3 mg de BSA en 10 ml de solución de Krebs-burbujeó carbógeno. </li><li> Coloque los segmentos de LMMP enjuagado en solución de digestión y uso LICsores para cortar LMMP en pequeños pedazos. </li><li> Resumen LMMP durante 60 min a 37 ° C en baño de agua con agitador mientras se burbujeaba suavemente con carbógeno. </li><li> Después de la digestión es completa, recoger las células por centrifugación durante 8 minutos a 356 xg en centrífuga enfriada a 4 ° C. </li><li> De aquí en adelante todos los procedimientos deben realizarse en condiciones estériles en una campana de cultivo celular! Todos los reactivos y los contenedores deberán ser esterilizados antes de su uso. </li><li> Durante la centrifugación, preparar una solución de tripsina 0,05% mediante la colocación de 1 ml calentó 0,25% de tripsina y 4 ml de HBSS calentado en un tubo de ml de cultivo celular estéril 50. </li><li> Después de la centrifugación, extraer y desechar el sobrenadante. No utilice una aspiradora, lo que probablemente va a chupar el pellet celular debido a la baja velocidad de centrifugación. Retire cuidadosamente el sedimento celular y el lugar en el tubo limpio con 5 ml de solución de tripsina 0,05%. </li><li> Digerir las células en solución de tripsina 0,05% en baño de agua a 37 ° C con agitación durante 7 min. No exceda 7 min del tratamiento total de tripsina o neuronas perecerá. </li><li> Neutralizar la tripsina con 10 ml de enjuague medios fríos después de 7 min de tratamiento con tripsina. </li><li> Centrifugar las células durante 8 min a 356 x g. Retire y deseche los medios de comunicación. </li><li> Durante la centrifugación, equilibrar una sección de malla Nitex esterilizado en la parte superior de un tubo de 15 ml de cultivo celular estéril. </li><li> Después de la centrifugación, extraer y desechar el sobrenadante. Suavemente mezcla de células se resuspende por trituración en ml de medio de neuronas completos 3. Evitar la generación de burbujas de aire durante esta etapa y todos los pasos futuros en los que se tritura mezcla de células. Todas las trituraciones se debe hacer con mucho cuidado. Filtrar a través de solución de células Nitex de malla limpia en 15 ml de tubo de cultivo de células. </li><li> Opcional: tubo de cultivo de células de Cap y el lugar en el mezclador de Hula refrigerado (o cualquier mezclador que proporciona una rotación) con el balanceo y la inclinación suave durante 30 min. Este paso es opcional pero recomendado. </li><li> Opcional: Después de mezclador de Hula, añadir 2 ml de enjuague medios fría para lavar las células. </li><li> Reunir las células por centrifugación (8 min, 356 xg). Quitar y desechar el sobrenadante. </li><li> Resuspender las células en los medios de comunicación 1200 neurona completos l. Triturar las células utilizando suavemente 1 ml de plástico punta de la pipeta. No generar burbujas de aire. Pipetear lenta y suavemente hasta que la mayoría de los bloques se dividen y las células se suspenden en líquido. </li><li> Añadir 750 l de medio completo a cada uno de los 12 pocillos que contenían un cubreobjetos de vidrio pre-recubiertas y añadir 100 ml de la solución de células triturado. </li><li> Incubar las neuronas en cultivo celular incubadora a 37 ° C con 5% de CO 2. </li><li> Cambie la mitad de los medios celulares cada 2 días. </li><li> Las neuronas son listos para las grabaciones electrofisiológicas después de 1 - 2 días en cultivo. </li></ol>

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The authors declare that no major competing interests exist.

Animals Used
This protocol has been optimized for Swiss Webster mice. However, this method is easily adaptable to other small-sized mammals such as rats and to other strains of mice. We have successfully performed preliminary isolations with C57 mice and &mu;-opioid receptor knock-outs. However, it is also possible that other strains of mice may be problematic due to morphological variations in the GI tract. Furthermore, there are known differences between mouse strains (C57Bl/6 vs. Balb/c) in the neuronal circuitry of the LMMP preparation 13, which can affect the resulting mix of neurons in the final isolation. Additionally, age should be taken into consideration when using this method, as age-related neuronal losses occur in the myenteric plexus that are specific to cholinergic neurons and their corresponding glia, and can be seen as early as 12 months of age 14. Finally, when adapting this protocol to other species of animals, care should be taken to ensure that the myenteric plexus comes away with the longitudinal muscle rather than remaining attached to the circular muscle.
Tissue Used
Neurons and glia can be cultured from both the ileum and colon using this protocol. Ileal isolations are easier to perform due to a greater amount of available tissue and the ease at which the longitudinal muscle separates from the thick circular muscle. A minimum of two animals are needed to occupy 12 wells of the 24-well plate for applications such as electrophysiology or immunocytochemistry. The isolation of colonic material is more problematic. The thick colonic longitudinal muscle is more difficult to separate from the circular muscle. Furthermore, the underlying circular muscle is thinner in the colon, making it more susceptible to tears. Also, the colon is significantly shorter than the ileum so three animals, at minimum, are required to occupy 12 wells of a 24-well plate. Both the ileum and the colon can be harvested and plated separately from the same animal, but additional animals or the use of less cell plating area will be required for the colon isolation.
Duration of Cell Culture
Electrophysiological recordings are optimally performed in isolated enteric neurons/glia after 2 to 6 days in culture; at this time cells are rounded and pliant, ideal conditions for making a whole-cell seal. After a week in culture cells become flatter and more visually differentiated as the cells adhere to the plate. Immunocytochemical experiments are best done when cells are more firmly attached to the plate, around day 10, to prevent cell loss during numerous wash steps. Mouse enteric cells have been maintained in culture for up to three weeks. Patterns of receptor and neurotransmitter expression over time have yet to be studied in these cultures. However, previous studies on neuronal survival and biochemical/morphological differentiation in traditional guinea pig enteric neuron isolations have found that neurons survived up to 15 days and maintain a high degree of histochemical and morphological properties up to three weeks 8, which suggests that neurons derived from other animals sources may do the same.
Cell Yield
The overall yield of cells from this technique is 12 wells of a 24-well plate from two mice when isolating from the ileum. This is a relatively low number of neurons compared to tissue volume, as the myenteric plexus consists of less than 1% of the total gut wall. One drawback to this method is that to increase the number of cells obtained, one must increase the number of animals used. When animal numbers are increased, it is suggested that several lab members collaborate in the first step of the isolation of the LMMP. This reduces the amount of time the cells are in a disrupted state before final plating and this increases cell survival.
Cell Density
As written, this protocol uses two animals to yield an ileal isolation of low-density cell confluence (~10 - 40% measured after one day in culture). Low-density cell plating is ideal in this methodology to reduce the risk of contamination in the final culture. It is also useful when single cell electrophysiology is performed. Cell number can be increased by using more animals or reducing the final plating area, but risk of contamination increases. To off-set this problem when higher cell densities are required, either increased washing steps or the increased use of antibiotics is suggested.
Cell Heterogeneity
Neurons acquired using these protocols are comprised of at least two functionally distinct populations, as indicated by electrophysiological characterization (Figure 3). However, there are many known types of neurons with specific neurochemical coding patterns found in the guinea pig 15 and the same is probably true of the mouse. This cell heterogeneity is both an advantage and disadvantage to this methodology. Cell heterogeneity is beneficial when performing single-cell functional studies, observing cell-cell interactions or in immunocytochemistry, among others. Cell heterogeneity may be particularly advantageous when studying neuron/glia interactions in culture (Figure 3). However, cell heterogeneity is a hindrance to methodologies such as immunoblotting, where changes in protein expression, etc., cannot be contributed to any one cell type or neuron type. Examination of glia will be less problematic as only two subtypes are shown to exist in the LMMP preparation: intraganglionic and intramuscular enteric glia 16. Other cell types may also be present in this culture, such as interstitial cells of Cajal or fibroblasts.
Plating Cells
In this methodology, cells are plated in twelve wells. Occasionally, one or two of these wells will develop contamination in the first day after isolation. If this occurs, the slides from the contaminated wells are promptly discarded and the well is rinsed with 70% ethanol to kill the remaining contamination. This protocol can be modified to plate all of the isolated cells in fewer wells or in a single dish, and as a result, the risk of contamination will increase. Again, when the risks of contamination increase, extra wash steps or increased antibiotic use is suggested. The antibiotic/antimycotic liquid (Gibco) used in the protocol consists of Amphotericin B, Streptomycin, and Penicillin. Other antibiotic combinations or increased concentrations can be used to optimize the reduction of contamination while preparing the rinse and complete neuron media.
Attaching Cells
Cells are plated on coverslips coated with laminin and poly-D-lysine. Laminin is important in the survival of enteric neurons and glia, and encourages neurite outgrowth and neuronal development 17, and as such, is considered essential to this isolation. However, alternative attachment factors to poly-D-lysine, such as ornithine or Matrigel, may be tried as desired.
In conclusion, this isolation provides a mixed culture of enteric neurons and glia suitable for a wide range of study techniques. This methodology is easy to master, inexpensive, and time efficient. It is our hope that this cell model will contribute to the understanding of various pathologies associated with the ENS.

El sistema nervioso entérico (ENS) es vasta red de nervios y glía que recorre toda la longitud del tracto gastrointestinal (GI). La ENS controla funcionalmente todos los aspectos de la digestión, incluyendo el peristaltismo, la absorción / secreción de fluidos, sensación de estímulos, etc (para revisión ver 1). Contiene más de 500 millones de neuronas, más que las encontradas en la médula espinal, y contiene todas las clases neurotransmisor encontrado en el cerebro. Por otra parte, la ENS es única, ya que puede funcionar sin reflejo de entrada desde el sistema nervioso central 2. La comprensión de la ENS es crucial, no sólo para comprender su papel fisiológico normal, pero para comprender su participación en una variedad de neuropatías que pueden ser congénito (enfermedad de Hirschsprung), adquirida (enfermedad de Chagas), secundaria a estados de enfermedad (gastroparesia diabética), drogas inducida (síndrome de intestino narcótico), o debido a una lesión (íleo posoperatorio) 1. Además, las neuronas entéricas pueden ser un reSERVOIR para la infección viral (varicela zoster) 3. Debido a sus similitudes con el cerebro y los altos niveles de serotonina en el intestino, medicamentos destinados a tratar los defectos del sistema nervioso central a menudo tienen efectos secundarios no deseados en la ENS 2. También es digno de mención que muchas neuropatías tales como la enfermedad de Alzheimer y la enfermedad de Parkinson muestran cambios celulares similares en las neuronas entéricas mucho antes de su aparición en las neuronas centrales, por lo que la ENS un modelo accesible para estudiar la patogénesis de estas enfermedades 4. Por lo tanto, un conocimiento profundo de la ENS es una necesidad en la comprensión de los estados de enfermedad y la prevención / la predicción de efectos secundarios farmacológicos. Las neuronas de la ENS se han estudiado tradicionalmente en el conejillo de indias usando preparaciones de wholemount 5-7 o neuronas cultivadas 8. A pesar de la facilidad con la que las neuronas pueden ser estudiados en este gran animal, este modelo tiene muchas limitaciones comofalta de cepas modificadas genéticamente, la falta de reactivos específicos para esta especie, y el alto costo asociado con el pedido y albergar estos temas. El desarrollo de un modelo de sistema nervioso entérico murino tiene la ventaja única de diversos sistemas de golpe hacia fuera, una amplia gama de otras metodologías establecidas que pueden ser utilizados en conjunción con la técnica de cultivo celular, y la capacidad de proporcionar una validación para el modelo de cobaya . La ENS se compone de tres plexi que corren a lo largo del tracto gastrointestinal: el plexo mientérico exterior (entre el músculo longitudinal y circular), que es principalmente responsable de las acciones peristálticos del intestino, así como los plexos submucosa y la mucosa, ( encontrado en y dentro de la mucosa, respectivamente), que controla en gran medida la absorción / secreción de fluido y la detección de los estímulos 1. Este método comienza con el aislamiento de la preparación del plexo muscular / mientérico longitudinal (LMMP) por peladofuera de la capa muscular exterior del tracto GI. Esto reduce drásticamente los problemas de contaminación que surgen cuando la capa de la mucosa está involucrado en el aislamiento. Como resultado, este proceso es ideal para el estudio del control neuronal de la motilidad en lugar de acciones de secreción de la ENS. El método que aquí se presenta resultados en un cultivo mixto de las neuronas y células gliales entéricas. Al menos dos tipos diferentes de neuronas se basan presente en las anteriores observaciones electrofisiológicas y inmunocitoquímico 9. La presencia de la glía es muy ventajoso, ya que no sólo son un importante tipo de células a estudiar en su propio derecho, pero que contribuyen a la supervivencia de las neuronas entéricas 10 y mantener la expresión del receptor nativo en la superficie celular neuronal 11. Por otra parte, las deficiencias de la glía entérica pueden conducir a estados de enfermedad motilidad gastrointestinal anormal, acuñadas 12 &#39;neuro-gliopathies. Por lo tanto, la cultura ENS presenta aquí resultados en varios tipos de células que están maduras para la investigación. Las ventajas de este método son la facilidad de aislamiento, requerimientos de herramientas de bajo costo y muy poco tiempo para dominar la técnica por personal de laboratorio con experiencia. Limitaciones de la metodología incluyen el rendimiento general de la célula baja de volúmenes de los tejidos y la exclusión de las neuronas del plexo ENS mucosa y submucosa. Este procedimiento será muy ventajoso para los estudiosos especializados en electrofisiología, inmunohistoquímica, PCR de una sola célula, y otras metodologías.

<table border="0" cellpadding="0" cellspacing="0" width="676">
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			<td height="22" style="height:22px;width:291px;"></td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Fisherbrand Coverglass for Growth Cover Glasses (12 mm diameter)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">12-545-82</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Poly-D-lysine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:137px;">P6407- 5 mg</td>
			<td style="width:79px;"> </td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">24-well cell culture plate</td>
			<td style="width:171px;">CELLTREAT</td>
			<td style="width:137px;">229124</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="22" style="height:22px;width:291px;">Laminin</td>
			<td style="width:171px;">BD Biosciences</td>
			<td style="width:137px;">354 232</td>
			<td style="width:79px;"> </td>
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		<tr height="64">
			<td height="64" style="height:64px;width:291px;">ddH2O</td>
			<td style="width:171px;"> </td>
			<td style="width:137px;"> </td>
			<td style="width:79px;">Can prepare in lab </td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">15 ml Sterile Centrifuge Tube</td>
			<td style="width:171px;">Greiner Bio-one</td>
			<td style="width:137px;">188261</td>
			<td style="width:79px;">May use any brand</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">50 ml Sterile Centrifuge Tube</td>
			<td style="width:171px;">Greiner Bio-one</td>
			<td style="width:137px;">227261</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="22" style="height:22px;width:291px;">NaCl</td>
			<td style="width:171px;">Fisher BioReagents</td>
			<td style="width:137px;">BP358-212</td>
			<td style="width:79px;">MW 58.44</td>
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			<td style="width:79px;">MW 74.55</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:291px;">NaH2PO4 .2H2O</td>
			<td style="width:171px;">Fisher Chemicals</td>
			<td style="width:137px;">S369-3</td>
			<td style="width:79px;">MW137.99</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:291px;">MgSO4</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">M7506-500G</td>
			<td style="width:79px;">MW 120.4</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:291px;">NaHCO3</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">S6014-5KG</td>
			<td style="width:79px;">MW 84.01</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">glucose</td>
			<td style="width:171px;">Fisher Chemicals</td>
			<td style="width:137px;">D16-1</td>
			<td style="width:79px;">MW 180.16</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">CaCl22H2O</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">C5080-500G</td>
			<td style="width:79px;">MW 147.02</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:291px;">F12 media</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">11330</td>
			<td style="width:79px;"> </td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Fetal Bovine Serum</td>
			<td style="width:171px;">Quality Biological Inc.</td>
			<td style="width:137px;">110-001-101HI</td>
			<td style="width:79px;">May use any brand</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antibiotic/antimycotic 100x liquid</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">15240-062</td>
			<td style="width:79px;"> </td>
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			<td height="22" style="height:22px;width:291px;">Neurobasal A media</td>
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			<td style="width:137px;">10888</td>
			<td style="width:79px;"> </td>
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			<td height="22" style="height:22px;width:291px;">200 mM L-glutamine</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">25030164</td>
			<td style="width:79px;"> </td>
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			<td height="22" style="height:22px;width:291px;">Glial Derived Neurotrophic Factor (GDNF)</td>
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			<td style="width:137px;">PR27022</td>
			<td style="width:79px;"> </td>
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			<td height="43" style="height:43px;width:291px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">8940</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="43" style="height:43px;width:291px;">Dissecting Tissue Forceps</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">13-812-41</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="43" style="height:43px;width:291px;">Cotton-Tipped Applicators</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">23-400-101</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="43" style="height:43px;width:291px;">250 ml Graduated Glass Beaker</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">FB-100-250</td>
			<td style="width:79px;">May use any brand</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">2 L Glass Erlenmyer flask</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">FB-500-2000</td>
			<td style="width:79px;">May use any brand</td>
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			<td height="43" style="height:43px;width:291px;">Plastic rod (child&#39;s paint brush)</td>
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			<td style="width:137px;">05 3516</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">10 ml Leur-lock Syringe</td>
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			<td style="width:137px;">309604</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">21 G x 1 1/2 in. Hypodermic Needle</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:137px;">305167</td>
			<td style="width:79px;">May use any brand</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Collagenase type 2</td>
			<td style="width:171px;">Worthington</td>
			<td style="width:137px;">LS004174</td>
			<td style="width:79px;"> </td>
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		<tr height="63">
			<td height="84" rowspan="2" style="height:84px;width:291px;">Bovine Serum Albumin</td>
			<td rowspan="2" style="width:171px;">American Bioanalytical</td>
			<td rowspan="2" style="width:137px;">AB00440</td>
			<td rowspan="2" style="width:79px;"> </td>
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		<tr height="21">
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">2 ml Microcentrifuge Eppendorf tubes</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">13-864-252</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Nitrex Mesh 500 &#181;M</td>
			<td style="width:171px;">Elko Filtering Co</td>
			<td style="width:137px;">100560</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Pipette Set</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">21-377-328</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Sharpeining Stone</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">NC9681212</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0" width="722">
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			<td style="width:108px;"></td>
			<td style="width:137px;"></td>
			<td style="width:195px;">Equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:283px;">LabGard ES 425 Biological Safety Cabinet (cell culture hood)</td>
			<td style="width:108px;">Nuaire</td>
			<td style="width:137px;">NU-425-400</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">10 L Shaking Waterbath</td>
			<td style="width:108px;">Edvotek</td>
			<td style="width:137px;">5027</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:283px;">Microcentrifuge 5417R</td>
			<td style="width:108px;">Eppendorf</td>
			<td style="width:137px;">5417R</td>
			<td style="width:195px;">May use a single larger centrifuge with size adapters</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:283px;">Allegra 6 Series Centrifuge</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:137px;">366816</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">HuluMixer Sample Mixer</td>
			<td style="width:108px;">Invitrogen</td>
			<td style="width:137px;">15920D</td>
			<td style="width:195px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">AutoFlow Water Jacket CO2 Incubator</td>
			<td style="width:108px;">Nuiare</td>
			<td style="width:137px;">NU-4750</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Analytical Balance Scale</td>
			<td style="width:108px;">Mettler Toledo</td>
			<td style="width:137px;">XS104</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
	</tbody>
</table>

an in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse

El sistema nervioso entérico es una vasta red de neuronas y glía que recorren la longitud del tracto gastrointestinal que funcionalmente controla la motilidad gastrointestinal. Se describe un procedimiento para el aislamiento y cultivo de una población mixta de neuronas y células gliales del plexo mientérico. Los cultivos primarios se pueden mantener durante más de 7 días, con conexiones en desarrollo entre las neuronas y la glía. La tira de músculo longitudinal con el adjunto del plexo mientérico se extrae del músculo circular subyacente del íleon o colon del ratón y se sometió a digestión enzimática. En condiciones estériles, la población de neuronas y glía aislado se conservan dentro del sedimento después de la centrifugación y se sembró en cubreobjetos. Dentro de 24 a 48 horas, neuritas se produce y las neuronas se pueden identificar por marcadores pan-neuronal. Después de dos días en cultivo, aisladas neuronas potenciales de acción del fuego como se observa en los estudios de patch clamp. Por otra parte, la glía entérico también puede ser identifdiado por tinción GFAP. Una red de neuronas y células gliales en estrecha aposición se forma dentro de 5 - 7 días. Neuronas entéricas pueden ser individualmente y estudiado directamente utilizando métodos tales como la inmunohistoquímica, la electrofisiología, imágenes de calcio, y la PCR de una sola célula. Por otra parte, este procedimiento se puede realizar en animales modificados genéticamente. Esta metodología es simple de realizar y de bajo costo. En general, este protocolo expone los componentes del sistema nervioso entérico de una manera fácil de manipular por lo que podemos descubrir mejor la funcionalidad de la ENS en estados normales y de enfermedad.

Immediately following isolation of LMMP-derived cells, neurons and other cell types will not be readily evident. Living, round cells of indistinct phenotype can be seen as well as tissue detritus from incompletely digested tissue fragments and connective tissue. This flotsam is of no concern and will be largely removed with the first media change in two days. Do not attempt to clean the slides before this as the healthy, viable cells will be removed as well.
After one day in culture, neurons will begin to show neurite outgrowth. Specific identification of neurons may still be indistinct at this time. Provided the cells are adherent enough to transfer to an experimental chamber, they will be ready for electrophysiological study after one day of culture. However, the cells are more adherent after two days in culture and ideal for function studies (electrophysiology, calcium imaging) from approximately days 2 - 5.
Morphological features of neuron become distinct after approximately a week in culture (Figure 1). Ideal immunocyctochemical features can be identified after about ten days, when neurons display long projections and grow in an almost 'ganglionic' like fashion interspersed with glia (Figure 2). This staining suggests it may be possible to study the synaptic interactions of these cells using this methodology.
After one day in culture, neurons are recognized by their sine que non or 'defining feature', the action potential (Figure 3). Electrophysiologically, they can be functionally classified into at least two neuronal types: neurons that contain an after-hyperpolarization and increased current/density relationships of sodium and potassium and neurons that do not have an after-hyperpolarization and significantly less sodium and potassium conductance (Figure 4). AHP positive and negative neurons appear to correlate with AH and S neurons seen in previous guinea work, respectively. AHP positive neurons (AH neurons) have multiple long projections originating from around the cell body, while the AHP negative neurons (S neurons) have one long projection which branches many times. This, in conjunction with the immunocytochemical coding in Figure 1, suggests that neuronal population is very heterogeneous.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig1highres.jpg" src="/files/ftp_upload/50688/50688fig1.jpg" /> 
 Figure 1. Immunohistochemical characterization of enteric neurons and glia isolated from the mouse longitudinal muscle. Confocal microscopy revealed neuronal-specific &beta;-III-tubulin (Abcam, rabbit, 1:1,000) staining in whole mount ileal longitudinal muscle (A) preparation from the mouse. Cells isolated from longitudinal muscle/myenteric plexus (LMMP) preparations contain neurons (B; &beta;-III-tubulin, Abcam, rabbit, 1:1,000) that stain positively for calbindin (C) (Chemicon, rabbit, 1:1,000) and calretinin (D) (Swant, rabbit, 1:2,000). Glia cells (E) were visualized with the glia-specific marker GFAP (Chemicon, mouse, 1:500). Antibodies were visualized via appropriate goat secondary antibody Alexa 488 (green, Molecular Probes, 1:1,000)0. Nuclei were visualized using Hoescht 33342 (blue, C-G, 1 &mu;g/ml). No staining was seen when primary antibody was omitted (F). Modified and reprinted from Smith, T.H., et al. Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels. PLoS One., doi:10.1371/journal.pone.0045251.g001 (2012). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50688/50688fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig2highres.jpg" src="/files/ftp_upload/50688/50688fig2.jpg" /> 
 Figure 2. Neurons and glia isolated from the mouse longitudinal muscle grow in close proximity to one another. Confocal microscopy images indicate the neurons (green, &beta;-III-tubulin, Abcam, rabbit, 1:1,000) and glia (red, GFAP, Chemicon, mouse, 1:500) readily grow adjacent to one another and appear to interact in vitro. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50688/50688fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig3highres.jpg" src="/files/ftp_upload/50688/50688fig3.jpg" /> 
 Figure 3. Electrophysiology of cultured enteric neurons and glia. In current clamp mode, all neurons (A) displayed action potentials upon current injection. Glia (B) do not have action potentials but do display large electrotonic potentials in response to current injection. Protocols in A &amp; B start with a current injection of -0.01 nA and increase to 0.09 nA in eleven 0.01 nA steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50688/50688fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig4highres.jpg" src="/files/ftp_upload/50688/50688fig4.jpg" /> 
 Figure 4. Neurons cultured from the mouse ileum are an electrophysiologically heterogeneous population. In current clamp mode, a current injection of 0.09 nA into neurons results in action potentials. S neurons (A), immediately return to the resting membrane potential following stimulation. AH-type neurons (B) display an afterhyperpolarization (AHP) following stimulation in which the resting membrane potential falls below baseline before slowly returning to the initial value. Modified and reprinted from Smith, T.H., et al. Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels. PLoS One., doi:10.1371/journal.pone.0045251.g001 (2012).

Watch this Scientific Journal Video about An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse at JoVE.com

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Un<em&gt; In-vitro</em&gt; Preparación de las neuronas entéricas aisladas y células gliales del plexo mientérico del ratón adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

an-vitro-preparation-isolated-enteric-neurons-glia-from-myenteric

Nanyang Technological University

Research

JoVE Journal

Neuroscience

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

26.7K vistas.  Virginia Commonwealth University. We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Video: An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

El estudio de la integración de los adultos nacidos en las neuronas

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigación

Neurociencias

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparación y tejido de fibras inmunotinción ratón nerviosas sensoriales que inervan la piel y los huesos de extremidades

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La especificación de telencefálicas neuronas glutamatérgicas a partir de células madre pluripotentes

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Una preparación semi-intacta aislada del ratón epitelio sensorial vestibular de Electrofisiología y de alta resolución de la microscopía de dos fotones

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Un<em&gt; In Vitro</em&gt; Mouse Adulto Preparación músculo-nervio para estudiar las propiedades de cocción de los aferentes musculares

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulaciones Ambientales del Número de dopamina mesencéfalo neuronas en ratones adultos

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

La indirecta neurona-astrocito Coculture Ensayo: Un<em&gt; In Vitro</em&gt; Puesta en marcha de la investigación detallada de las interacciones neurona-glía

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Preparación de la superficie coclear en el ratón adulto

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...