Este trabajo fue financiado por el NIH subvenciones DA15169 y DA035224 en HEM

Todo el manejo de animales y la recolección de tejido se realizó de acuerdo con las directrices de la Universidad de Massachusetts Medical School Comité de Uso Institucional Cuidado de Animales (IACUC), siguiendo el protocolo aprobado # A1506 (Melikian, PI). Soluciones requeridas Líquido cefalorraquídeo artificial (ACSF) - Hacer fresco todos los días NaCl 125 mM, KCl 2,5 mM, 1,2 mM NaH 2 PO 4, 1,2 mM de MgCl 2, 2,4 mM CaCl 2, 26 mM NaHCO 3, y 11 mM de glucosa Nota: Prepare ACSF como una solución madre de 10 veces, con exclusión de NaHCO3 y glucosa. Hacer las soluciones de trabajo 1x todos los días de la 10x, que complementa con frescos glucosa NaHCO3 y. ACSF sacarosa suplementados (SACSF) - Hacer fresco todos los días Suc 250 mMrosa, KCl 2,5, 1,2 mM NaH 2 PO 4, 1,2 mM de MgCl 2, 2,4 mM CaCl 2, 26 mM NaHCO3 y glucosa 11 mM Nota: Prepare SACSF como una solución madre de 10 veces, con exclusión de NaHCO3 y glucosa. Hacer las soluciones de trabajo 1x todos los días de la 10x, que complementa con frescos glucosa NaHCO3 y. Sulfo-N-hidroxisuccinilo-SS-biotina (sulfo-NHS-SS-biotina, Pierce Chemical Company) Las soluciones madre deben ser de 200 mg / ml en DMSO y son resistentes a múltiples ciclos de congelación / descongelación. Las alícuotas se almacenaron a -20 ° C. El éster de succinil se hidroliza rápidamente en solución acuosa, por lo que las soluciones de trabajo debe prepararse inmediatamente antes de aplicar a las rebanadas. Slice Solución Quench ACSF suplementado con glicina 100 mM Lisis RIPA Buffer Tris 10 mM, pH 7,4, NaCl 150 mM, EDTA 1,0 mM, 1% de Triton-X-100, 0,1% de SDS, 1% de Na desoxicolato RIPA con inhibidores de la proteasa (RIPA / PI) - Hacer fresco todos los días RIPA suplementado con leupeptina 1 mM, pepstatina 1 mM, 1 mM de aprotinina, y 1 mM fenilmetil sulfonil fluoruro. 1. Preparar cerebrales Slices <ol><li> Hacer SACSF 1x fresco y 1x ACSF </li><li> Chill SACSF en el hielo en un vaso pequeño. Esto se utiliza para mantener el cerebro de ratón recién cosechado. </li><li> Saturar la ACSF y SACSF con oxígeno mediante burbujeo con 95% / 5% de O 2 / CO 2, 20 min en hielo. </li><li> P30-38 ratones se deben utilizar para la viabilidad del tejido óptimo. Sacrificar animales por dislocación cervical y decapitación, y eliminar rápidamente los cerebros en previamente enfriado, SACSF oxigenada. </li><li> El uso de un microtomo vibrante, hacer 300 micras secciones cerebrales en la región de interés. </li><li> Si se desea, las rebanadas se pueden disecados más antes de la recuperación para enriquecer en regiones particulares del cerebro o derecho por separado y los hemisferios izquierdo para utilizar como control y rebanadas experimentales, respectivamente. </li><li> Utilizando una pipeta Pasteur pulida al fuego, transferir las rebanadas a la malla de fondo en las cámaras establecidas en placas de 24 pocillos. </li><li> Permitir que las rebanadas se recuperen durante 40 min, 31 ° C en ACSF oxigenada, con continua, suave burbujeo. </li></ol> 2. Tratamiento de Drogas (si procede) y Slice Biotinilación <ol><li> Después de la recuperación, lavar 3 veces en rodajas precalentado (37 º C), burbujeante ACSF oxigenada constantemente con un 95% / 5% O 2 / CO 2. </li><li> Añadir los compuestos de ensayo y se incuba con oxigenación continua. <ol><li> Para mayor comodidad, agregue un th volumen de 10x fármaco concentrado 1/10, y mezclar por inversión suave. </li><li> Agitar las placas suavemente en un baño de agua a la temperatura deseada. </li></ol></li><li> Después de tratamiento de drogas, rápidamenterodajas de frío por lavar 3 veces en el hielo frío ACSF. </li></ol> 3. Las proteínas de superficie biotinilar <ol><li> Preparar 1,0 mg / ml de sulfo-NHS-SS-biotina en hielo frío ACSF inmediatamente antes de etiquetado. </li><li> Añadir 0,75 ml de sulfo-NHS-SS-biotina a rodajas y se incuba rodajas de hielo, 45 min. </li><li> Lavar rebanadas tres veces rápidamente con ACSF enfriado en hielo, a continuación se incuba durante 10 min en hielo ACSF frío en hielo. </li><li> Lávese las rebanadas tres veces con tampón de hielo de enfriamiento rebanada fría e incubar con 0,75 ml de tampón de enfriamiento corte dos veces, 25 min, en el hielo para calmar libre sulfo-NHS-SS-biotina. </li></ol> 4. Preparar lisados ​​tisulares <ol><li> Lavar rebanadas tres veces en hielo frío ACSF y transferir cada sector a un tubo de microcentrífuga utilizando una pipeta Pasteur pulida al fuego. </li><li> Pellet suavemente rebanada centrifugando 200 xg, 1 min y cuidadosamente aspirado restante ACSF. </li><li> Añadir 400 l de hielo frío RIPA / PI y romper el tejido de la pipeta hacia arriba y hacia abajo una vez a través de un pip P200ette. </li><li> Transferencia disociado rebanada / RIPA a un tubo fresco y se incuba 30 min, 4 ° C, rotación, para completar la lisis. </li><li> Se precipitan los desechos celulares por centrifugación, 18.000 xg, 15 min, 4 º C. </li><li> Determinar las concentraciones de proteína lisado utilizando el ensayo de proteína BCA, con albúmina de suero bovino (BSA) como estándar. </li></ol> 5. Aislar Biotinylated Proteínas <ol><li> Optimizar bolas relación proteína / total. Nota: Los niveles de expresión de proteínas individuales pueden variar ampliamente entre las diferentes regiones del cerebro. A menos que la totalidad de la proteína biotinilada en una cantidad dada de lisado de tejido es capturado, no es posible detectar con precisión los cambios potenciales en la expresión de superficie. Por lo tanto, es imperativo determinar empíricamente la relación óptima de proteínas del grano / total de una región de la proteína / particular del cerebro antes de embarcarse en un nuevo estudio biotinylation rebanada. <ol><li> Incubar 25 mu l de estreptavidina perlas de agarosa con el aumento de cantidads de lisado tisular (rango aproximado de 25-200 mg), y luego proceder como se describe a continuación para la unión, las etapas de lavado y elución. </li><li> Cuantificar las bandas inmunorreactivas resultantes y elegir una relación grano / lisado en el rango lineal de la unión que va a permitir la cuantificación exacta de aumento o disminución de la expresión de proteínas de superficie. </li></ol></li><li> Preparar perlas de estreptavidina-agarosa <ol><li> Determinar el volumen de perla total es necesario para todas las muestras. Preparar un volumen de grano suficiente para esa cantidad más uno extra (es decir, 4 muestras de 25 l / muestra = 100 l beads + uno = 125 mu l adicionales cuentas. </li><li> Vortex Estreptovidina grano de valores y pipeta de volumen fuera deseaban. Si se utiliza una pipeta P200, cortar el extremo de la punta para evitar daños obstrucción o perla. </li><li> Lávese las perlas 3 veces en 0,5-1,0 ml RIPA / PI para eliminar conservantes, vórtex después de cada adición RIPA y recogiendo los granos entre lavados por centrifugación 18.000 xg, 1 min, al temperamento habitaciónratura. </li><li> Aspirar fuera de amortiguación entre lavados utilizando una pipeta Pasteur de vidrio unido a un matraz de vacío. Una punta de P200 de plástico unida al extremo de la pipeta Pasteur de vidrio proporciona un control más preciso cuando la aspiración. No es necesario quitar absolutamente toda la memoria intermedia para los dos primeros lavados, ya que corre el riesgo de perlas de aspiración en la pipeta. Después del lavado final, quitar tanto el exceso de tampón como sea posible y sin aspiración de las perlas. </li><li> Añadir RIPA / PI para llevar cuentas de nuevo a su volumen original, la pipeta hacia arriba y hacia abajo para volver a suspender. Evitar la agitación con vórtex en este punto, como perlas se adhieren a la pared del tubo. </li><li> Perlas de alícuotas en tubos de microcentrífuga con un P200 con la punta cortada. Pipetear hacia arriba y abajo varias veces entre el muestreo para asegurar los granos permanecen suspendidas uniformemente y dividido entre los tubos. </li></ol></li><li> Proteínas con biotina se unen a estreptavidina cuentas <ol><li> Distribuir los lisados ​​celulares a los tubos que contienen las perlas de agarosa. Para los experimentos whe<html> re múltiples muestras están siendo comparados, asegúrese de utilizar la misma cantidad de proteína de cada muestra para las comparaciones precisas. </li><li> Añadir RIPA / PI adicional para llevar a muestras de un volumen mínimo de 200 l. Esto asegura que las muestras se mezclan adecuadamente durante la incubación y también unifica las concentraciones de proteínas a través de muestras. </li><li> Coloque los tubos en un rotador de tubo y mezcle durante la noche a 4 ° C. </li><li> En tubos separados, dispensar un equivalente del volumen de lisado total utilizado para cada muestra. Alternativamente, si los volúmenes de muestra son elevados, una fracción del volumen total de lisado se puede reservar en lugar, para acomodar los volúmenes de carga máxima sobre geles de SDS-PAGE. Estos serán utilizados para normalizar la expresión de la superficie a la cantidad total de proteína. </li><li> Añadir ya sea un volumen igual de 2x o quinto volumen de 6x tampón de muestra de SDS-PAGE y, o bien se incuba a 4 ° C, en paralelo con muestras de perlas, o almacenar a -20 ° C hasta que se analizaron las muestras. </li></ol></li></ol>ove_title &quot;&gt; 6. Elute y analizar muestras <ol><li> Pellet perlas por centrifugación 18.000 x g, 2 min, a temperatura ambiente. </li><li> Aspirar el sobrenadante y lavar perlas tres veces con 0,75 ml RIPA. Para reducir al mínimo la pérdida de grano, dejar un pequeño volumen jefe del tampón anterior cuentas entre lavados. Después del lavado final, eliminar la mayor cantidad de RIPA como sea posible, sin alterar el sedimento de perlas. </li><li> Eluir proteínas biotiniladas a partir de perlas de estreptavidina mediante la reducción del enlace disulfuro. Añadir 25 l de 2x tampón de muestra reductor, así vórtice SDS-PAGE y girar muestras 30 min, temperatura ambiente. Nota: Muchas proteínas de membrana tienen una alta tendencia a agregarse cuando se hierve en tampón de muestra de SDS-PAGE, perjudicando gravemente su movilidad electroforética. Si este es el caso para la proteína que se investiga, evitar calentamiento de las muestras y, en cambio, eluir lentamente a temperatura ambiente. Si la ebullición es absolutamente necesario (por ejemplo, si otra proteína evaluada en paralelo requiere de ebullición), sampltampón E puede ser complementada con 2 M de urea (concentración final) para reducir al mínimo la agregación. Aunque es eficaz en la reducción de la agregación en algunos casos, la urea a menudo compromete apariciones de la banda. </li><li> Analizar las muestras por inmunoblot. </li><li> Descongelar total de muestras de lisado y girar en paralelo con muestras de perlas, 30 min, temperatura ambiente. </li><li> Proteínas separadas en geles de SDS-PAGE. </li><li> Identificar la proteína (s) de interés por inmunotransferencia. <ol><li> Asegúrese de que las bandas se detectan en el intervalo lineal de detección para la cuantificación adecuada. </li><li> Para asegurar que el reactivo de biotinilación no ha tenido acceso a las proteínas intracelulares a través de las células dañadas / comprometidos. Esto se logra mejor por inmunotransferencia en paralelo para una proteína intracelular específica del tipo de célula que se está investigado. </li><li> Cuantificar banda densidades y calcular la densidad relativa superficie de la proteína como un porcentaje del nivel total de expresión de la proteína. </li></ol></li></ol>

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Los autores no tienen intereses financieros que estén en conflicto con esta obra.

A pesar del conocimiento de muchos años que el tráfico endocítica críticamente impactos de señalización sináptica en el cerebro, que ha resultado ser un desafío para medir cuantitativamente los cambios en la expresión de superficie de la proteína en las neuronas adultas. En este trabajo, se presenta un enfoque fiable para etiquetar proteína de la superficie ex vivo en rodajas de cerebro agudas. Preparaciones de cortes de cerebro tienen una larga historia de utilidad para los registros electrofisioló...

Endocítica tráfico es un mecanismo celular ubicua que afina la presentación membrana plasmática de una variedad de proteínas integrales de membrana. Endocitosis proporciona nutrientes vitales para el medio intracelular 1 y desensibiliza la señalización del receptor en respuesta a la activación del receptor 2. Endocítica reciclar de nuevo a la membrana de plasma puede mejorar adicionalmente la señalización celular mediante el aumento de los niveles de expresión de proteínas en la superficie celular 3. Por otra parte, las perturbaciones de transporte de membrana están implicadas en numerosas enfermedades y condiciones patológicas 4,5, haciendo hincapié en la necesidad de investigar los mecanismos moleculares que regulan el tráfico de proteínas endocítica. Mientras que muchas proteínas utilizan mecanismos de internalización clásicos clatrina dependiente, pruebas de montaje en los últimos años demuestra que los múltiples mecanismos de endocitosis clathrin independiente gobiernan el potencial endocítica de un conjunto cada vez mayor deproteínas 6,7. Por lo tanto, la necesidad de investigar los mecanismos de endocitosis que favorecen la trata en los sistemas fisiológicos relevantes ha crecido considerablemente. En el cerebro, el tráfico endocítica de receptores, canales iónicos y transportadores de neurotransmisores tiene un papel principal en el establecimiento de la plasticidad sináptica 8-11 y la respuesta a drogas de abuso 12-15, en última instancia afectan a la excitabilidad neuronal y las respuestas sinápticas. Hasta la fecha la mayoría de los estudios de tráfico neuronales, apoyarse en los sistemas de expresión heterólogos o neuronas en cultivo primario, ninguno de los cuales puede reflejar de forma fiable los mecanismos en juego en las neuronas adultas. Aquí, se presenta un enfoque que utiliza biotinilación superficie para medir cuantitativamente los niveles de proteína de superficie en rodajas de cerebro agudas derivadas de roedores adultos. Con este enfoque, se presentan datos que demuestran que el ratón estriatal de dopamina transportador internaliza rápidamente en rerespuesta a forbol éster mediada por la activación de la proteína quinasa C (PKC).

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Endocítica tráfico regulado es el mecanismo central facilitar una variedad de eventos neuromoduladores, mediante el control de forma dinámica del receptor, canal de iones, transportador y presentación de la superficie celular en una escala de tiempo de minutos. Hay una gran diversidad de mecanismos que controlan el tráfico endocítica de las proteínas individuales. Los estudios que investigan las bases moleculares de la trata se han basado principalmente en la superficie de biotinilación para medir cuantitativamente los cambios en la expresión de proteínas de superficie de membrana en respuesta a estímulos exógenos y la manipulación genética. Sin embargo, este enfoque se ha limitado principalmente a las células cultivadas, que pueden no reflejar fielmente los mecanismos fisiológicamente relevantes en juego en las neuronas adultas. Por otra parte, los enfoques de células cultivadas pueden subestimar las diferencias específicas de la región en los mecanismos de la trata. Aquí se describe un enfoque que se extiende biotinilación superficie de la célula a la preparación de cortes de cerebro aguda. Nosotrosdemuestran que este método ofrece un enfoque de alta fidelidad para medir los cambios rápidos en los niveles de proteínas de superficie de membrana en las neuronas adultas. Este enfoque es probable que tenga una amplia utilidad en el ámbito del tráfico neuronal endocítica.

El transportador de dopamina neuronal se internaliza en respuesta a la activación de PKC en líneas celulares de 16-20. A pesar de muchos informes que demuestran las pérdidas de superficie DAT inducidas por la PKC en una variedad de líneas celulares y sistemas de expresión, ha sido un reto para confirmar este hallazgo en las neuronas dopaminérgicas cultivadas 21-23. Se utilizó rodajas de cuerpo estriado de ratón para probar directamente si DAT internaliza en respuesta a la activación de PKC...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

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

El tráfico de membrana neuronal controla dinámicamente la membrana plasmática y la disponibilidad de proteína significativamente impactos neurotransmisión. Hasta la fecha, ha sido un desafío para medir el tráfico endocítica neuronal en las neuronas adultas. Aquí se describe un método altamente efectivo, cuantitativa para medir los cambios rápidos en la superficie de la proteína expresión ex vivo en rodajas de cerebro agudas.

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 ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

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

12.8K vistas.  University of Massachusetts Medical School.  El tráfico de membrana neuronal controla dinámicamente la membrana plasmática y la disponibilidad de proteína significativamente impactos neurotransmisión. Hasta la fecha, ha sido un desafío para medir el tráfico endocítica neuronal en las neuronas adultas. Aquí se describe un método altamente efectivo, cuantitativa para medir los cambios rápidos en la superficie de la proteína expresión ex vivo en rodajas de cerebro agudas. 

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

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Investigación

Neurociencias

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

El uso de un enlace del sitio Tag α-Bungarotoxina para el Estudio de GABA A receptor de membrana localización y el Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo Optogenética Disección de circuitos del miedo en rodajas de cerebro

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imágenes en vivo de la proteína GLUT4 trata de ratón las neuronas hipotalámica primaria utilizando microscopía de deconvolución

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un enfoque alternativo para estudiar acontecimientos primarios en neurodegeneración con rodajas de cerebro de rata Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Proyección de imagen de calcio para la visualización de las respuestas cerebrales a la señalización endocrina en Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Uso de fluorescencia de infrarrojo cercano y escaneo de alta resolución para medir la expresión de proteína en el cerebro de roedores

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...