We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

Todos los procedimientos experimentales descritos en este protocolo fueron aprobados por Cuidado de Animales de la Universidad Hebrea y el empleo. 1. Preparación de las soluciones y herramientas para rebanar <ol><li> Preparar 1 L de solución fisiológica estándar (SPS) que contiene los iones descritos en la Tabla 1. <ol><li> Preparar una solución madre que contiene las sales en 10 veces la concentración final de antemano en una botella de vidrio de almacenamiento lleno de 1000 ml de agua desionizada (conductancia de 0,055 mS / cm). Añadir sales (NaCl, KCl, KH 2 PO 4, y MgSO4), y revuelva hasta que se disuelva por completo. Guarde la solución madre en 4 ° C. </li><li> Hacer la solución final MSF en el día del experimento. Añadir 100 ml de la solución madre a 700 ml de agua purificada, disolver la glucosa (3,6 g) y NaHCO3 (2,18 g) usando una barra de agitación magnética y luego añadir agua purificada hasta 1 L. </li><li> Equilibrar los solutien por gaseamiento con 95% O2 / 5% de CO 2 durante ~ 20 min. Después de esto, añadir 2 ml de solución M CaCl 2 1. </li></ol></li><li> Montar las herramientas siguientes (Figura 1). <ol><li> Prepare grandes tijeras u otra herramienta para la decapitación, pequeñas tijeras quirúrgicas para abrir el cráneo, fórceps de punta fina para levantar el cráneo para exponer el cerebro, con hoja de bisturí para diseccionar la parte deseada del cerebro para ser cortado en lonchas y una pequeña espátula para manipular la partes del cerebro disecados. </li><li> Además, preparar pequeños trozos de papel de filtro para eliminar el exceso de líquido del cerebro, dos pequeñas placas de Petri para el cerebro para ser diseccionados en dos vasos de precipitados de vidrio de 500 ml para contener agua caliente y MSF purificado, y el pegamento de cianoacrilato para pegar el cerebro a la etapa de corte. </li><li> Preparar un pequeño cepillo para el manejo de las rebanadas durante el corte en lonchas y una pipeta Pasteur de vidrio de boca ancha para mover las rebanadas del baño de corte para el cha recuperaciónmbre. Desinfectar las herramientas para prevenir el crecimiento de bacterias en los baños rebanada cálidos. Por otra parte, el uso de guantes de laboratorio limpios. </li></ol></li><li> Preparar una cámara de recuperación rebanada sumergida tal como se describe por Gibb y Edwards 15. Continuamente gas del SPS en ella con un 95% de O 2/5% de CO 2 y mantener a 36 ° C. Asegúrese de que el gaseamiento no da lugar a pequeñas burbujas atrapadas en el baño que podría dañar las rebanadas. </li><li> ~ Caliente de 300 ml de la MSF a 36 ° C en una placa de calefacción con agitador magnético o en un baño de agua. Si se utiliza una placa de calefacción, tenga cuidado para evitar que la solución de un sobrecalentamiento, lo que puede dar lugar a la precipitación de iones. En caso de que suceda, cálidos MSF frescas en lugar de enfriar la antigua. </li><li> Llevar ~ 500 ml de agua purificada a ebullición usando un hervidor de agua eléctrico y, en un vaso de precipitados, se combinan 300 ml de la misma con agua fría para obtener agua ligeramente más caliente que la temperatura de corte. Utilice esta calentó agua purificada durante la decapitación y el uso de la RemaiNing del agua hervida posteriormente para mantener la temperatura fisiológica del baño de corte. </li><li> Anestesiar el ratón mediante la inyección de 0,1 ml de pentobarbital (60 mg / ml) por vía intraperitoneal. Después de unos minutos comprobar que el animal no responde a los fuertes de los pies o la cola pellizcos para determinar la falta de sensación. </li></ol> 2. Disección del cerebro <ol><li> Decapitar el ratón rápidamente cortando el cuello con las grandes tijeras cerca de la parte posterior del cráneo, y dejó que la cabeza caiga en el vaso con agua purificada caliente para enjuagar el exceso de sangre. </li><li> Exponer el agujero occipital en el cráneo debajo de los músculos del cuello y la piel, posiblemente, la eliminación de más del cuello con las tijeras pequeñas. </li><li> Tire de la piel en la parte superior del cráneo para ser capaz de ver claramente las suturas del cráneo con el fin de guiar la abertura del cráneo. </li><li> Cortar el cráneo abierto mediante la inserción de la punta inferior de las tijeras pequeñas a través del agujero magno y de inmediato convertirlos haciael lado lateral. Cortar suavemente a lo largo del borde lateral del hueso parietal hasta un lugar detrás de los ojos y la sutura frontoparietal; a continuación, girar hacia el centro del cráneo y cortar a través de la línea media al otro lado del cráneo (véase la línea discontinua verde en la Figura 2A). </li><li> Con unas pinzas de punta fina, levante el cráneo desde la esquina frontoparietal y tire de ella en diagonal hacia arriba y hacia los lados para exponer el cerebro (ver flecha amarilla discontinua en la Figura 2A). Asegúrese de que el cráneo no está conectado a ningún hueso o la piel de la cabeza, por lo que el cerebro se mantendrá en su lugar cuando se levantó el cráneo. En caso de que el cerebro se está moviendo con el cráneo, utilizar las tijeras pequeñas para retirar cualquier tejido conectivo entre el cráneo y el cerebro. </li><li> Cuando se expone el cerebro, no permita que el cerebro esté bien seco. Por lo tanto, lleve a cabo los siguientes pasos mientras se mantiene el cráneo y el cerebro en una placa de Petri llena de calentado, gaseados MSF. </li><li> Separe la parte del cerebro que se utiliza para el correoXperiment del resto del cerebro usando un escalpelo y una espátula. En caso de utilizar el cerebelo, desprenda del cerebro anterior por un solo corte a través del mesencéfalo y protuberancia (a lo largo de líneas rojas en las figuras 2B-D) y moverlo a una pequeña placa de Petri que contiene frescos, gaseados MSF. </li><li> Retire el tronco cerebral para formar una base recta y ancha para pegar el cerebro para cortar etapa al rebanar el cerebelo en el plano horizontal. Brevemente colocar el cerebelo en un trozo de papel de filtro húmedo de manera que el lado que fue cortado de la prosencéfalo esté orientada hacia abajo. <ol><li> Cortar el tallo cerebral con un bisturí para formar la base (como se indica con la línea azul en la Figura 2B), y devolver el cerebelo a la MSF. Cuando se necesitan otros planos de corte, recorte el bloque cerebelo en consecuencia (líneas azules en las figuras 2C y D). </li></ol></li></ol> 3. Cortando el Cerebro <ol><li> Preparar la etapa de corte determinando que es dry, antes de aplicar una pequeña gota de pegamento en el centro del escenario. </li><li> Levante el bloque de cerebro de la SPS utilizando una espátula con correcto hacia arriba, y eliminar el exceso de líquido del cerebro utilizando pequeños trozos de papel de filtro. Luego, con un solo movimiento, deslice el cerebro de la espátula sobre la gota de pegamento en el escenario. </li><li> Para evitar que el cerebro de secado y el pegamento de corriendo hacia arriba en los lados del tejido, se aplican unas pocas gotas de SPS con una pipeta Pasteur y colocar la etapa de corte en la cámara de corte. Alinear el bloque de cerebro, de modo que las regiones de interés (por ejemplo, la corteza cerebelar) se enfrentan a la hoja y por lo tanto serán sometidos a menor cantidad de presión mecánica antes de ser cortado. </li><li> Cortar cada rebanada de acuerdo con las instrucciones del fabricante mientras que constantemente gasificación al SPS en la cámara de corte, así como manteniendo la temperatura a 34-37 ° C. Mantener la temperatura de la cámara de corte en este rango llenando el external cámara de la máquina de cortar con agua purificada calentada y reemplazar el agua cuando la temperatura baja demasiado. NOTA: Los parámetros de corte en lonchas deben ser encontrados experimentalmente para cada tipo de tejido y las neuronas que están siendo examinadas. En el caso de la máquina de cortar Campden SMZ 700 con cuchillas de cerámica y células de Golgi del cerebelo, utilice 0,75 mm amplitud de la vibración a 65 Hz y velocidad de avance de 0,05 mm / seg; para las neuronas cerebelosas nucleares mayor amplitud y frecuencia (1 mm y 90 Hz, respectivamente) y velocidad de avance más lenta (0,01-0,02 mm / seg) se recomienda. Por tanto Golgi y las neuronas cerebelosas nucleares, grosor de corte debe ser de 300 micras. </li><li> Durante el corte, no permita que las rebanadas se plieguen sobre sí mismos. Para prevenir esto, apoyando suavemente con un cepillo suave, tocando preferentemente la rebanada sólo en regiones que no son de interés para el experimento. Tenga cuidado para evitar que el cepillo toque la cuchilla vibratome; especialmente en el caso de las hojas de cerámica, incluso un ligero contactopuede dañar la hoja. </li><li> Mueva cada rebanada de la cámara de corte para una recuperación / celebración de cámara utilizando una boca ancha, Pasteur de vidrio pipeta pulida al fuego. </li><li> Deje que las rebanadas de descanso en la cámara durante al menos 1 hora antes de comenzar el experimento. Esto permite la degradación de los tejidos y las células dañadas o moribundas y por lo tanto resulta en una superficie de loncha más limpio. Durante la primera hora, mantener la temperatura de la SPS en la cámara en el intervalo de 34-36 ° C; asegúrese de que las rebanadas no son tocados por las burbujas de aire que pueden dañar las rodajas o hacer flotar. </li></ol> 4. Experimento <ol><li> Después de 1 hora, deje que la temperatura de la cámara de recuperación se enfríe a temperatura ambiente (17-25 ° C). NOTA: Este podría prolongar el período de tiempo durante el cual las rebanadas se pueden utilizar por ralentizar el crecimiento de bacterias, así como el metabolismo celular. Este periodo depende de la región del cerebro y tipo de célula. Por ejemplo, ciertos tipos de células de Golgi se pueden encontrar para estar sano en rodajas8 hr después de cortar mientras que otros tipos durar sólo 6 hr. </li><li> Después de 1 hora ha pasado de la corte, utilizar las rodajas de varios experimentos electrofisiológicos. </li></ol>

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The authors declare that they have no competing financial interests.

Se demuestra un método para la preparación de cortes de cerebro de ratones agudas en lugar de la temperatura fisiológica enfriada con hielo. Se ha demostrado 11 que la calidad de las rebanadas obtenidas en condiciones de calor es superior en comparación con los preparados con condiciones de frío, a condición de que la cuchilla rebanadora tiene vibración vertical mínima. El rebanar de la temperatura fisiológica puede evitar artefactos fisiológicos causados ​​por la ba...

El objetivo del método presentado es conseguir cortes de cerebro agudos de alta calidad para experimentos electrofisiológicos in vitro, especialmente cuando se utiliza adulto o incluso los animales de edad. El método de corte cerebral agudo, tal como se describe por Skrede y Westgaard 1 en dos frases elegantes, se ha convertido en uno de los fundamentos de la investigación de la neurociencia moderna y está empleado en innumerables variaciones en todo el mundo. La calidad de las rodajas se refleja en el número de neuronas por rebanada viviente, el período de tiempo durante el cual las células mantienen sus propiedades electrofisiológicas y morfológicas, así como en la integridad del tejido. Además, la duración máxima para las grabaciones estables depende de la calidad de las rodajas. Por lo tanto, a lo largo de las décadas, el método de la división original ha sido desarrollado por los grupos individuales de investigación para mejorar la recuperación después de cortar la rebanada 2-10, a menudo por modificaciones complejas de la composición de corte osoluciones de recuperación r (tales como la adición de ascorbato, tiourea o incluso H 2 O 2), así como pre-perfusión intra-cardiaca del animal con soluciones fisiológicas enfriado. Como se ha demostrado recientemente 11, temperatura fisiológica durante el corte parece ser más beneficiosa que la refrigeración para la salud neuronal; la mejora es más notable cuando se trabaja con adultos (2-8 meses) los roedores. Evitar cambios drásticos de temperatura evita artefactos debido a los procesos dependientes de la temperatura en las células, tales como la plasticidad y 13 canales de iones cinética de 13,14. Tales cambios podrían influir en la tensión de la membrana y la señalización del calcio intracelular, el umbral de pico y pico de forma. El método &quot;caliente&quot; rebanada aguda preparación que aquí se presenta es un procedimiento general para la obtención de cortes de cerebro agudos de alta calidad desde cualquier región del cerebro, incluyendo el cerebelo, la corteza y el hipocampo, núcleos de helio tronco cerebral 16 &lt;/ Sup&gt; así como el bulbo olfatorio, tanto en ratas y ratones. En particular, el procedimiento de corte en lonchas temperatura fisiológica requiere que la cuchilla de corte vibra casi perfectamente horizontal y es sin defectos estructurales. Tal precisión podría no ser alcanzable con los modelos de máquina de cortar de edad avanzada; en tales casos, se recomienda la realización de la preparación rebanada en condiciones de congelación frío como la temperatura baja parece tener el tejido más resistente a los daños mecánicos, aunque a costa de las aberraciones metabólicas.

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	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
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		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
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			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
			<td style="width:292px;"></td>
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			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

Aquí se presenta un protocolo para la preparación de cortes de cerebro agudas. Este procedimiento es un elemento crítico para los experimentos de patch-clamp electrofisiológicos que determina en gran medida la calidad de los resultados. Se ha demostrado que la omisión de la etapa de enfriamiento durante el procedimiento de corte es beneficioso en la obtención de rebanadas y las células sanas, especialmente cuando se trata de estructuras cerebrales altamente mielinizadas de animales maduros. Aunque el mecanismo exacto por el que la temperatura elevada favorece la salud de los nervios sólo puede especular sobre, es lógico pensar que, siempre que sea posible, la temperatura en la que se realiza el corte debe estar cerca de las condiciones fisiológicas para evitar los artefactos relacionados con la temperatura. Otra ventaja importante de este método es la simplicidad del procedimiento y por lo tanto el tiempo de preparación corto. En el método demostrado se utilizan ratones adultos pero el mismo procedimiento se puede aplicar con ratones más jóvenes, así como las ratas. También, las siguientes cl parcheamp experimento se realiza en rebanadas horizontales cerebelosos, pero el mismo procedimiento se puede utilizar también en otros planos, así como otras áreas posteriores del cerebro.

Rebanadas preparadas en la forma descrita se puede utilizar para diversos experimentos electrofisiológicos y optogenética. En la Figura 3A y 3C, se muestra un ejemplo representativo de una rebanada horizontal cerebelosa cortical y una rebanada coronal cerebral, respectivamente, se observa bajo (DIC) óptica de interferencia diferencial. En el trozo del cerebelo, varios tipos de neuronas del cerebelo pueden ser fácilmente reconocidos por su forma y localización de células del cuerpo...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo cerebrales en adultos rebanar en la temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

26.1K vistas.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Video: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

La dependencia estatal Efectos sobre la EMT: Una mirada a la conducta motriz fosfeno

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Investigación

Neurociencias

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imágenes de detección de feromonas en una preparación de ratón Vomeronasal aguda lámina de tejido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Time-lapse de imágenes de Migración neuroblasto en rodajas agudas del cerebro anterior ratón adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

La cirugía estereotáxica para la lesión excitotóxica de áreas específicas del cerebro en la rata adulta

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

La detección de palmitoilación proteínas en neuronas del hipocampo cultivadas por inmunoprecipitación e Intercambio Acyl-Biotina (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cerebro Slice Biotinilación: Una<em&gt; Ex Vivo</em&gt; Enfoque medir tráfico de proteínas específicas Región membrana plasmática en adultos las neuronas

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramación de células derivadas de pericitos del cerebro humano adulto en neuronas inducidas

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Análisis fisiológico en profundidad de poblaciones de células definidas en el infarto agudo del tejido Rebanadas del ratón órgano vomeronasal

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Grabación de la plasticidad sináptica en rebanadas Hippocampal agudas mantenidas en un pequeño volumen reciclado - perfusión- y sistema de cámara de tipo inmersión

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Un análisis de gradiente de temperatura para determinar las preferencias térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...